Learn about 4Life patents

4Life was recently awarded another patent. That makes 11 in all as of my last count. See the list at the bottom of this page for more information about how they do it.



Salt Lake City, Utah (January 3, 2011) 4Life Research™ announced today that the United States Patent & Trademark Office (USPTO) has awarded the company a composition patent that protects the 4Life Transfer Factor® Cardio formula until 2025. This is the company's sixth worldwide patent. 4Life has numerous international patents and dozens of patent applications pending.

4Life’s progressive science and pursuit of patent protection strengthens the business opportunity for 4Life distributors with the exclusive right to market 4Life Transfer Factor® products. 4Life President Steve Tew: “We invest a great deal of resources into patent protection on behalf of our distributors because we know that the opportunity they share must accompany the kind of exclusivity, credibility, and longevity that patents exist to provide.”

4Life's commitment to patents includes an emphasis on research and development to generate formulas, compositions, and processes that are patentable. While 4Life's legal team concentrates on patent acquisition, the International Product Registration department focuses on registrations around the world to support distributor growth now and in the future. In 2010, 4Life completed 101 product registrations in 30 countries.

4Life Founder and CEO David Lisonbee: "Patent protection is just one way that we work to empower 4Life distributors with exclusivity and credibility in the marketplace. 4Life patents provide our distributors with security for decades to come.

4Life has offices on five continents to serve a global network of independent distributors through science, success, and service.



According to Patent Storm US* these are the Patents given to 4Life:

7815943 Cardiovascular therapy composition including transfer factor and therapeutic methods including use of the composition ... of 4698 ) United States Patent 7,815,943 Hennen October 19, 2010 Cardiovascular therapy composition including transfer factor and therapeutic methods including use of the composition Abstract A composition for use in cardiovascular ... 10/19/2010

6468534 Methods for obtaining transfer factor from avian sources, compositions including avian-generated transfer factor, and methods of use ... solution 26 of FIG. 3, was administered, the delayed-type hypersensitivity that causes such swelling is attributed to the administered transfer factor. The following examples are merely illustrative of embodiments of methods for generating, obtaining, and ... 10/22/2002

6866868 Compositions including different types of transfer factor, methods for making the compositions, and methods of treatment using the compositions ... from 4Life Research, LLC, of Sandy, Utah, Transfer Factor Plus.TM. (TFP), also available from 4Life Research, avian transfer factor available in a lyophilized (ie., freeze-dried) whole egg powder, and mixtures of TF and TFP (both the formula marketed in ... 03/15/2005

20070053919 Compositions including different types of transfer factor, methods for making the compositions, and methods of treatment using the compositions ... from 4Life Research, LLC, of Sandy, Utah, Transfer Factor Plus.TM. (TFP or TF ), also available from 4Life Research, avian transfer factor available in a lyophilized (i.e., freeze-dried) whole egg powder, and mixtures of TF and TFP (both the ... 03/08/2007

20050058716 COMPOSITIONS INCLUDING DIFFERENT TYPES OF TRANSFER FACTOR, METHODS FOR MAKING THE COMPOSITIONS, AND METHODS OF TREATMENT USING THE COMPOSITIONS ... 4Life Research, LLC, of Sandy, Utah, Transfer Factor Plus.TM. (TFP), also available from 4Life Research, avian transfer factor available in a lyophilized (i.e., freeze-dried) whole egg powder, and mixtures of TF and TFP (both the formula ... 03/17/2005

20090053197 Transfer Factor Compositions and Methods ... which serve as a source of transfer factor. The avian transfer factor may be obtained from commercial sources such as, for example, 4Life.RTM. Research; ... factor 50.00 0.05 16.00 250.00 Vitamin C (ascorbic acid) 21.62 0.2162 2.162 33.73 ... 02/26/2009

20090074751 GROWTH FACTOR FRACTION COMPOSITIONS AND METHODS ... 2, and Day 12. Amounts are given per ounce of formulation. This formulation is available from 4Life.RTM. Research. TABLE-US-00002 Transfer factor 1450 mg (Proprietary mixture from avian and mammalian sources) Proprietary Blend ... 03/19/2009

20080081076 NANOFRACTION IMMUNE MODULATORS, PREPARATIONS AND COMPOSITIONS INCLUDING THE SAME, AND ASSOCIATED METHODS ... colostrum were: 250 Da to 2,000 Da, 2,000 Da to 4,000 Da, 4,000 Da to 8,000 Da (which includes transfer ... fraction having a MWCO of about 3,000 Da that had been spray dried; (c) 4Life Transfer Factor.RTM. XF, which is currently available ... 04/03/2008

20100119531 NUTRICEUTICAL GELS ... et al. (hereinafter "Lisonbee"), the disclosure of which is hereby incorporated herein, it its entirety, by this reference. [0013]Transfer factor is known or believed to improve the oxidative balance of a living being, as well as to enhance the ... 05/13/2010

20050233967 Compositions, systems, and methods for focusing a cell-mediated immune response ... second group was initiated one week prior to surgery. The treated individuals were each provided with two capsules of TRANSFER FACTOR from 4Life Research, LC, of Sandy, Utah, three times daily, throughout the course of the evaluation. [0042] ... 10/20/2005

20090170774 COMPOSITIONS AND METHODS FOR ENHANCING FERTILITY ... which serve as a source of transfer factor. In various embodiments, the avian transfer factor may be obtained from commercial sources such as, for example, 4LIFE.RTM. Research; Labelle, Inc., Bellingham, Wash.; Troue; and Ghen Corporation, ... 07/02/2009

You are about to learn the secret technical benefits of immune education

Abstract

A composition for use in cardiovascular therapy includes transfer factor. The transfer factor may be nonmammalian transfer factor, such as that derived from eggs, or mammalian transfer factor, such as that derived from colostrum. The composition may also include one or more of the following: an LDL receptor-binding element; a blood flow-enhancing element; a cholesterol reducing element; a fat oxidation prevention element, and an antioxidant. Treatment methods include enlisting the immune system of a subject receiving therapy to attack pathogens that cause inflammation of blood vessels or to otherwise reduce inflammation of blood vessels. Claims

What is claimed is:

1. A nutritional supplement consisting of at least one transfer factor, Butcher's Broom, ginkgo biloba, hawthorn, garlic, Coenzyme Q10, red yeast rice extract,reservatrol, ginger oil, vitamin A, vitamin C, vitamin E, niacin, vitamin B6, folate, vitamin B12, magnesium, zinc, selenium, copper, and potassium.

2. A nutritional supplement consisting of at least one transfer factor; Butcher's Broom; ginkgo biloba; hawthorn, garlic; Coenzyme Q10; red yeast rice extract; reservatrol; ginger oil; beta carotene; vitamin C selected from thegroup consisting of magnesium dehydroascorbate, ascorbyl palmitate, and ascorbic acid; d-alpha tocopherol succinate; niacinamide; pyridoxine hydrochloride; folic acid; cyancobalamin; magnesium selected from the group consisting of magnesiumdehydroascorbate, magnesium chloride, magnesium arginate, and magnesium lysinate; zinc arginate; selenomethionine; copper glycinate; and potassium citrate.

3. The nutritional supplement of claim 1, wherein the at least one transfer factor comprises at least a part of an egg extract or at least a part of a colostrum extract.

4. The nutritional supplement of claim 1, wherein the at least one transfer factor comprises at least a part of an egg extract and at least a part of a colostrum extract.

5. The nutritional supplement of claim 1, wherein the at least one transfer factor is an egg-derived transfer factor.

6. The nutritional supplement of claim 1 contained within a capsule.

7. The nutritional supplement of claim 2, wherein the at least one transfer factor comprises at least a part of an egg extract or at least a part of a colostrum extract.

8. The nutritional supplement of claim 2, wherein the at least one transfer factor comprises at least a part of an egg extract and at least a part of a colostrum extract.

9. The nutritional supplement of claim 2, wherein the at least one transfer factor is an egg-derived transfer factor.

10. The nutritional supplement of claim 2 contained within a capsule. Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to compositions, including nutritional supplements, for use in improving cardiovascular health and, more specifically, to compositions that may be useful for preventing arteriosclerosis.

2. Background of Related Art

The term "cardiovascular disease," as used herein, is intended to refer to all pathological states leading to a narrowing and/or occlusion of blood vessels throughout the body. In particular, the term "cardiovascular disease" refers toconditions including atherosclerosis, thrombosis and other related pathological states, especially within arteries of the heart and brain. Accordingly, the term "cardiovascular disease" encompasses, without limitation, various types of heart disease, aswell as Alzheimer's disease and vascular dimension.

For some time, conventional medical treatment of cardiovascular disease has focused on low density lipoprotein, or "LDL," the so called "bad cholesterol," and strategies for lowering its concentration in the bloodstream. A great many studieshave been published ostensibly linking cardiovascular disease with elevated levels of LDL. As a result, most therapies for the prevention and treatment of cardiovascular disease rely on drugs that reduce serum levels of LDL in the bloodstream. Morerecent studies have found the effects of lowering LDL levels on cardiovascular disease to be somewhat equivocal. Thus, the efficacy of LDL-reducing drugs and therapies continues to be a source of major debate within the medical community.

Lipoprotein(a) ("Lp(a)") binds LDL receptors on the walls of blood vessels. Lp(a) also binds lysine-sepharose, immobilized fibrin and fibrinogen, and the plasminogen receptor on endothelial cells. Additionally, Lp(a) binds other components ofthe arterial wall, including fibrinectin and glycosaminoglycans. High levels of Lp(a) in blood are known to be associated with the incidence of cardiovascular disease, likely due to the cross-linking effects of Lp(a) that has bound to LDL receptors onblood vessel walls.

Some cardiovascular therapies are designed to reduce the binding of Lp(a) by LDL receptors that are present on the interior walls of the arteries and include antioxidants to reduce swelling of the arteries. By way of example, U.S. Pat. No.5,650,418 to Rath et al. (hereinafter "Rath") describes a cardiovascular disease treatment composition which includes lysine or a pharmaceutically acceptable salt thereof, nicotinic acid, and ascorbic acid (i.e., vitamin C). The lysine binds LDLreceptors and, thus, prevents Lp(a) from binding such receptors, thereby reducing the negative effects of Lp(a) on the arteries. Nicotinic acid and ascorbic acid are antioxidants which reduce swelling of the arterial walls, thereby permitting more bloodto flow through the arteries and, to some extent, reducing blood pressure.

In addition to high levels of Lp(a), it is believed that several pathogens, including herpes simplex II virus (HSV-II), may be partially responsible for causing cardiovascular disease. Among other things, it is believed that such pathogens causeswelling of the arterial walls, which results in vasoconstriction. In turn, vasoconstriction restricts the flow rate of blood through the arteries and increases blood pressure.

Also, it is believed that such pathogens may damage the walls of blood vessels, which results in the binding of Lp(a) thereto.

While the composition described in Rath reduces the effects of Lp(a) and reduces some swelling of the arteries, it does not target the causes of such swelling to eliminate the same.

The inventor is not aware of a composition for use in cardiovascular therapy that includes one or more components that target the pathogenic causes of swelling of the arteries and enlist the immune system of a subject (e.g., a mammal, such as ahuman or any other mammal) to reduce or eliminate such pathogenic causes of such inflammation, which may contribute to cardiovascular disease.

SUMMARY OF THE INVENTION

The present invention includes compositions for use in cardiovascular therapy and which may be useful for preventing or treating cardiovascular disease, as well as cardiovascular therapy methods.

Compositions that incorporate teachings of the present invention include, among other things, a component of mammalian, avian, and other immune systems known as "transfer factor." The transfer factor used in the composition may be either anantigen-nonspecific or pathogen-nonspecific transfer factor or one or more transfer factors which have specificity for one or more pathogens or antigens thereof.

Other components that may be included in compositions of the present invention include, but are not limited to, LDL receptor-binding elements, blood flow-enhancing elements, blood cholesterol reducers, fat oxidation prevention elements, andantioxidants. A composition that incorporates teachings of the present invention may include any combination of the foregoing elements and one or more of each such element.

Treatment methods include administration of the composition to a subject either enterally or parenterally, in a known fashion. Due to the presence of transfer factor in the composition, the composition enlists the immune system of a treatedsubject against pathogens that may cause inflammation or lesions that may lead to cardiovascular disease. Other components of such a composition may act to prevent Lp(a) from binding to LDL receptors on the walls of blood vessels, prevent and/or repairdamage to the blood vessels, reduce inflammation, improve blood flow, reduce blood cholesterol levels, and/or prevent fats from oxidizing and, thus, from sticking to the walls of blood vessels.

Other features and advantages of the present invention will become apparent to those of skill in the art through consideration of the ensuing description and the appended claims.

DETAILED DESCRIPTION

Compositions that incorporate teachings of the present invention include transfer factor or other inflammation-reducing or pathogen-reducing components and are useful in cardiovascular therapy and, more particularly, in preventing or evenreducing cardiovascular disease, including atherosclerosis and other disease states that may result therefrom, as well as for improving the overall cardiovascular efficiency of a subject. Cardiovascular therapy methods that employ such compositions arealso within the scope of the present invention.

In addition to transfer factor, an exemplary embodiment of a composition according to the present invention includes an LDL receptor-binding element, a blood flow-enhancing element, a blood cholesterol reducer, and at least one antioxidant.

The transfer factor of a composition according to the present invention may comprise an antigen-nonspecific or pathogen-nonspecific transfer factor, an antigen-specific or pathogen-specific transfer factor, or a combination of nonspecific andspecific transfer factor molecules. If an antigen-specific or pathogen-specific transfer factor is used, the transfer factor may have specificity for pathogenic agents which may cause cardiovascular complications or otherwise affect the circulatorysystem, such as by causing the arterial walls to swell. For example, the composition may include transfer factor which has specificity for one or more of the herpes simplex I and II viruses (respectively, "HSV-I" and "HSV-II"), Chlamydia pneumoniae,cytomegalovirus ("CMV"), Helicobacter pylori, and various oral pathogens.

Also, the transfer factor may comprise a nonmammalian transfer factor, such as egg-derived avian transfer factor or blood-derived transfer factor (e.g., transfer factor from chicken blood) or a mammalian transfer factor, such as colostrum-derivedtransfer factor, spleen-derived transfer factor, or blood-derived transfer factor. U.S. Pat. No. 6,468,534 to Hennen et al., the disclosure of which is hereby incorporated herein in its entirety by this reference, describes egg-derived transfer factoras well as processes for obtaining the same. Colostrum-derived transfer factor and exemplary processes for obtaining the same are described in U.S. Pat. No. 4,816,563 to Wilson et al., the disclosure of which is hereby incorporated herein in itsentirety by this reference. Compositions which include combinations of different types of transfer factor molecules, or transfer factor molecules from different sources, are also within the scope of the present invention.

The transfer factor in a composition incorporating teachings of the present invention enlists the immune system of a subject who receives cardiovascular therapy or is being treated with such a composition against pathogens, including viruses,bacteria, and other pathogenic agents, that may contribute to cardiovascular disease, including atherosclerosis. In enlisting the immune system of a subject in this manner, the transfer factor causes the immune system to reduce inflammation, as is wellknown in the art, and may cause the immune system to reduce or eliminate the number of such pathogens in the body of the subject.

The TEST EXAMPLE that follows provides data that shows the degree to which transfer factor causes an increase in the activity of natural killer cells, which are also referred to in the art as "cytotoxic T-lymphocytes" ("CTLs"), in attackingpathogens. The tests conducted were chromium-51 (radioactive chromium, or 51 Cr) assays. The tests were conducted in vitro on cell cultures, including C. pneumoniae and H. pylori bacterial cells and HSV-1-infected and HSV-2-infected mammalian celllines. In the control, a fixed amount of natural killer cells was introduced into the cellular milieu along with a fixed amount of flour for a period of four hours, then the amount of chromium-51 that had been released was analyzed with a Beckman 2000gamma counter. In a first set of test samples, the same, fixed amount of natural killer cells was introduced into the cellular milieu along with a composition including bovine transfer factor in an amount equal to the amount of flour introduced into thecontrol. A second set of test samples included the fixed amount of natural killer cells, as well as a composition including avian transfer factor in an amount equal to the amounts of flour in the control samples and the bovine transfer factor-containingcomposition in the first set of samples. The results follow:

TABLE-US-00001 Pathogen Additive C. pneumoniae H. pylori HSV-1 HSV-2 chromium-51 counts per minute (cpm): Flour 1323 1121 2017 1262 Bovine TF 2593 2499 2240 2473 percent increase 196% 223% 111% 196% Avian TF 2553 1860 2985 2183 percent increase193% 166% 148% 173%

The data shown in the TEST EXAMPLE indicate that both bovine transfer factor and avian transfer factor increase the activity of natural kill cells, reducing levels of C. pneumoniae, H. pylori, HSV-1 and HSV-2. It follows that the role that eachof these pathogens plays in cardiovascular disease would also be reduced or eliminated by therapy with mammalian transfer factor or nonmammalian transfer factor.

The LDL receptor-binding element may, by way of example only, comprise lysine or a lysine-containing compound, such as magnesium lysinate, lysine hydrochloride, lysine dihydrochloride, lysine orotate, lysine succinate, lysine glutamate, or thelike.

Since LDL receptor-binding elements are capable of occupying the sites on LDL receptors to which Lp(a) and LDL bind, LDL receptor-binding elements are useful for preventing Lp(a) from binding the LDL receptors and, thus, from additionally bindingother components of the arterial walls, which binding is believed to contribute to cardiovascular disease.

Exemplary blood flow-enhancing elements, or vasodilators, that may be included in a composition of the present invention include, without limitation, arginine and arginine-containing compounds, such as magnesium arginate. Other bloodflow-enhancing elements, such as niacinamide, may alternatively or additionally be included in such a composition. By way of example only, the blood flow-enhancing elements may target cardiovascular vessels located around the heart or improve blood flowin a more general fashion (i.e., throughout the body of a treated subject). Combinations of blood flow-enhancing elements that have different blood flow-enhancing characteristics may also be used in a composition that incorporates teachings of thepresent invention.

By increasing the rate at which blood flows through cardiovascular vessels, blood flow-enhancing elements may reduce blood pressure, reduce monocyte adhesion and thereby augment endothelial function, and, as a result, prevent the generation ofand even reduce the occurrence of pathophysiological lesions on the walls of cardiovascular vessels.

Blood cholesterol reducers may also be included in a composition according to the present invention. Examples of useful blood cholesterol reducers include, without limitation, nicotinic acid (which is a form of Niacin, or vitamin B3) or anyother agent which is known to reduce cholesterol levels in blood. Of course, the utility of a blood cholesterol reducer in a composition according to the present invention is that it will reduce the amount of cholesterol, including LDL, in the blood ofa subject, thereby reducing the potential for cholesterol-induced cardiovascular disease.

Antioxidants, including coenzymes, vitamins (e.g., vitamin E, vitamin A, beta-carotene, vitamin C, etc.), and the like are useful for preventing and treating damage to the walls of the arteries, as is well known in the art. In addition, bothmammalian and nonmammalian transfer factors are known to increase the antioxidant and detoxification abilities of a treated subject. Data and specific information regarding these abilities of transfer factor are provided in U.S. Provisional PatentApplication Ser. No. 60/423,965, filed on Nov. 4, 2002, the disclosure of which is hereby incorporated herein in its entirety by this reference.

A composition according to the present invention may also include vitamin B6, which is available as pyridoxine hydrochloride and pyridoxal-5-phosphate. Vitamin B6 deficiency has long been associated with atherosclerosis. It is welldocumented that vitamin B6 is useful for treating atherosclerosis, as well as for reducing blood pressure and for facilitating the removal of toxins from the body of a subject.

Of course, a composition of the present invention which is used to treat or prevent the occurrence of cardiovascular disorders may lack some of the foregoing elements, or it may include additional components that are known or believed to improvethe cardiovascular health of a subject.

The following is an example of a composition that incorporates teachings of the present invention:

TABLE-US-00002 AMOUNT INGREDIENT (per capsule) Transfer factor (Cardio-TF-XF ™) 50 mg Proprietary Blend 144.5 mg Butcher's Broom (root) (22% sterolic heterosides) Ginkgo biloba (leaf) (24% ginkgo flavone glycosides), 6% terpene lactones)Hawthorn (flower and leaf) (1.8% rutin) Garlic (deodorized clove) Coenzyme Q10 Red Rice Yeast Extract Reservatrol (from Polygonum cuspidatum) Ginger Oil Vitamin A (as bete carotene) 2,500 IU Vitamin C (as magnesium dehydroascorbate, ascorbyl 50 mgpalmitate, and ascorbic acid) Vitamin E (as d-alpha tocopherol succinate) 100 IU Niacin (as niacinamide) 5 mg Vitamin B6 (as pyridoxine hydrochloride) 0.5 mg Folate (as folic acid) 100 mcg Vitamin B12 (as cyancobalamin) 2 mcg Magnesium (asmagnesium chloride, magnesium 45 mg dehydroascorbate, magnesium arginate, and magnesium lysinate) Zinc (as zinc arginate) 2.5 mg Selenium (as selenomethionine) 12.5 mcg Copper (as copper glycinate) 0.5 mg Potassium (as potassium citrate) 12.5 TOTAL 310.6mg

The EXEMPLARY COMPOSITION is marketed by 4Life Research, LLC, of Sandy, Utah, as TF CARDIO.RTM.. The above-listed ingredients are contained within each capsule of the EXEMPLARY COMPOSITION.

In the EXEMPLARY COMPOSITION, the transfer factor is at least a part of an inflammation-reducing component or pathogen-reducing component and comprises Cardio-TF XF™, which includes transfer factor specific for HSV-I, HSV-II, Chlamydiapneumoniae, CMV, Helicobacter Pylori, and other pathogens (e.g., those of the oral cavity) that are known to cause lesions and swelling in arterial walls. By enlisting the immune system of a treated subject in resisting such pathogens, the transferfactor component of a composition incorporating teachings of the present invention reduces a cause of inflammation and lesions that are at least partially responsible for many cardiovascular disorders. The transfer factors of Cardio-TF-XF™ are aviantransfer factors which have been derived from the eggs of chickens.

Additionally, transfer factor is known to generally reduce inflammation in a subject, including in blood vessels of the subject, even when pathogens such as those listed above are not present. As is well-known in the art, inflammation, whetherpathogen-induced or not, is known to contribute to cardiovascular disease.

The composition of the EXEMPLARY COMPOSITION also includes an LDL receptor-binding element in the form of magnesium lysinate, a lysine salt. As described previously herein, when a form of lysine binds the LDL receptors on the walls of bloodvessels, including arteries, the level of binding of Lp(a) to the LDL receptors is reduced, thereby reducing the potentially deleterious effects of Lp(a) and, consequently, reducing the incidence of cardiovascular disorders that may be caused by highLp(a) levels.

The magnesium arginate and zinc arginate of the EXEMPLARY COMPOSITION, which are forms of arginine, are known to relax blood vessels and thereby improve blood flow, thus reducing hypertension, or high blood pressure. The niacinamide of theEXEMPLARY COMPOSITION also improves blood flow, although not in as broad a fashion as arginine. Specifically, niacinamide is known to have a flushing affect on peripheral circulation (e.g., in the blood vessels at the surface of the skin). GingkoBiloba is also believed to open blood vessels and, thus, to improve blood flow.

Antioxidants that are included in the EXEMPLARY COMPOSITION include both hydrophilic and hydrophobic antioxidants, although compositions that include only hydrophilic or hydrophobic antioxidants are also within the scope of the invention. Vitamin E and beta-carotene, which are listed as components of the EXEMPLARY COMPOSITION, are examples of hydrophobic antioxidants. Vitamin E and beta-carotene are particularly useful in treating or preventing cardiovascular disease since they may bedissolved in fats, such as LDL cholesterol and Lp(a), and remain therein. Magnesium dehydroascorbic acid and ascorbic acid, both of which are forms of vitamin C, are examples of hydrophilic antioxidants that are included in the EXEMPLARY COMPOSITION. Another antioxidant that is included in the EXEMPLARY COMPOSITION, Coenzyme Q10, or "CoQ10," also acts as an electron-transport carrier.

It is also believed that CoQ10 provides nutrition at the cellular level and that CoQ10 may increase blood flow, thereby reducing high blood pressure, or hypertension. Patients with cardiovascular disorders often exhibit CoQ10deficiency. In view of this knowledge, CoQ10 has long been used in treating the lesions that occur in various cardiovascular disorders, as well as the causes of cardiovascular disorders (e.g., high blood pressure, inflammation, etc.).

Reservatrol, which is an extract of red wine and is also known as the "French Paradox," is known to keep fats from being oxidized and depositing in the arteries. Of course, the inclusion of other fat oxidation prevention elements in acomposition that incorporates teachings of the present invention is also within the scope of the present invention.

Blood cholesterol reducers of the composition described in the EXEMPLARY COMPOSITION include niacinamide, which is also known in the art as "nicotinamide" and is a form of Vitamin B3. Specifically, niacinamide is known to lower LDLcholesterol, Lp(a), triglyceride, and fibrinogen levels while raising high-density lipoprotein (HDL) cholesterol (or "good cholesterol") levels.

The functions and effects (believed, theoretical, or actual) of each of the remaining components of the EXEMPLARY COMPOSITION on the cardiovascular health of a subject are well documented in the art.

Therapeutic methods which include use of a composition according to the present invention or combinations of the components thereof enlist the immune system of a treated subject to attack inflammation-causing pathogens, thereby reducinginflammation that may result in cardiovascular disorders. The immune system of a treated subject is enlisted by administering (e.g., enterally or parenterally) a composition which includes one or more types of transfer factor, as described previouslyherein, to a subject. The manner in which transfer factor initiates activity by various components of a subject's immune system is well known and documented in the art.

A therapy method incorporating teachings of the present invention may also include one or more of the following acts: preventing Lp(a) from binding to LDL receptors on the walls of blood vessels; preventing and/or repairing damage to the bloodvessels (e.g., with an antioxidant or combination of antioxidants); reducing inflammation; improving blood flow; reducing blood cholesterol levels; and preventing fats from oxidizing and, thus, from sticking to the walls of blood vessels.

Examples of the possible benefits of therapy in accordance with teachings of the present invention follow:

Exemplary Benefits

A sixty-four (64) year old male who was suffering from low blood pressure (72 over 52) began taking the EXEMPLARY COMPOSITION, gradually increasing his dosage from two capsules daily to eight capsules per day. In addition, following the initialtwo capsule per day dosage, he began taking three capsules each of TRANSFER FACTOR™ and TRANSFER FACTOR PLUS™, both of which are available from 4Life Research, LLC, of Sandy, Utah, as well as four capsules of PBGS+.RTM., also available from 4LifeResearch, each day. Within six months of initiating therapy, his blood pressure increased to normal levels (110 over 73).

A fifty-one (51) year old female who had been diagnosed with pulmonary fibrosis suffered from a resting heart rate of ninety-nine (99) beats per minute and high blood pressure (156 over 96), despite taking various medications which had beenprescribed for her. Within fourteen days of taking three capsules of TRANSFER FACTOR™ and four capsules of the EXEMPLARY COMPOSITION each day, along with her normal medication, her resting heart rate decreased to sixty-nine (69) beats per minute andher blood pressure returned to normal levels (120 over 78).

A female who had three abnormal electrocardiograms within a three-month period received a normal electrocardiogram within two months of when she began taking four capsules of the EXEMPLARY COMPOSITION each day.

An eighty-nine (89) year old woman suffering from ischemic heart disease and congestive heart failure had symptoms of severe shortness of breath on mild exertion (walking for about five to about ten meters with support) and coughing. Initially,she was given two capsules of the EXEMPLARY COMPOSITION each day. Within three weeks, her breathing had improved somewhat. Further improvements were noted after five weeks of therapy. At two months, walking with support no longer resulted in shortnessof breath. In addition, healthy increases in appetite and weight were noted.

A fifty-four (54) year old male suffering from angina had three coronary arteries that were occluded by about 75% to about 80%. He began therapy with four capsules of the EXEMPLARY COMPOSITION twice daily (i.e., eight capsules per day), alongwith three capsules of TRANSFER FACTOR™ each day. Within four months, the occlusion or blockage of the same three arteries had reduced to about 30% to about 40%.

A forty-nine (49) year old male suffering from Grave's disease and high blood pressure no longer suffered any physical problems within three months of beginning four capsule per day therapy with the EXEMPLARY COMPOSITION.

Another sixty-four (64) year old male who had been diagnosed with cardiomyopathy was suffering symptoms including lack of both energy and stamina and shortness of breath. Almost immediately following the initiation of four capsule per daytherapy with the EXEMPLARY COMPOSITION, these symptoms began to subside. Shortly after increasing his daily dosage to eight capsules per day, the subject no longer had any cardiomyopathy-related symptoms.

A male who had tested at a 7.93 for C-reactive proteins (CRPs), which are indicative of risk for heart attack and measured on a scale of 1.0 to 8.7, began taking, on the day of receiving his CRP results, four capsules of the EXEMPLARY COMPOSITIONand three capsules of TRANSFER FACTOR™ daily. Within four months, his CRP levels were reduced to 1.1.

A thirty-two (32) year old male who takes four capsules of the EXEMPLARY COMPOSITION each day and, prior to taking the EXEMPLARY COMPOSITION, had long maintained a consistent exercise regimen, began recognizing less shortness of breath fromaerobic activity and less joint pain within two months of initiating therapy with the EXEMPLARY COMPOSITION.

A male with chronic pain in his knees no longer had pain within two weeks of taking four capsules of the EXEMPLARY COMPOSITION and three capsules of TRANSFER FACTOR™ each day.

Although the foregoing description contains many specifics, these should not be construed as limiting the scope of the present invention, but merely as providing illustrations of some of the presently preferred embodiments. Similarly, otherembodiments of the invention may be devised which do not depart from the spirit or scope of the present invention. Moreover, features from different embodiments of the invention may be employed in combination. The scope of the invention is, therefore,indicated and limited only by the appended claims and their legal equivalents, rather than by the foregoing description. All additions, deletions, and modifications to the invention, as disclosed herein, which fall within the meaning and scope of theclaims are to be embraced thereby. Other References

* U.S. Appl. No. 60/423,965, filed Nov. 4, 2002, and entitled “Methods and Compositions for Focusing Cell-Mediated Immune Response and Enhancing Efficiency of an Individual's Antioxidant Profile, Detoxification Abilities, and General Cell and Molecular Health.” * PCT International Search Report dated Mar. 19, 2004, Performed for Application PCT/US03.26427. * Williamson et al. 1997. Herbal therapies:The facts and the fiction. Drug Topics. p. 78-87. * Szapary et al. 2000. Alternative Medicine in Cardiovascular Disease: More Questions Than Answers. ACC Current Journal Review. p. 104-108. * Kemper.1999. Ginger (Zingiber officinale). Longwood Herbal Task Force: http://www.mcp.edu/herbal/default.htm. p. 1-18. * Campbell et al. 1998. Chlamydia pneumoniae and Cardiovascular Disease. Emerging Infectious Diseases. vol. 4, No. 4, October-December p. 571-579. * Singh et al. Coenzyme Q in cardiovascular disease. J Assoc Physicians India. Mar. 1998;46(3):299-306; Abstract. * Vercellotti. Microbes, inflammation and atherosclerosis: will old pathology lessons guide new therapies? Trans Am Clin. Climatol. Assoc. 2001, 112:215-222; Abstract. * Kirkpatrick Properties and activities of transfer factor. J. Allergy and Clin. Immunol. 1975, 55(6):411-421; Abstract. * Gordon Cardiovascular Research. Explore, 1999, http://www.explorepub.com/articles/heart—disease.html, p. 3. * Tentolouris et al. L-Arginine in coronary atherosclerosis. Int. J. Cardiol., 2000,75(2-3):123-128. * Focant et al. The Effect of Vitamin E Supplementation of Cow Diets Containing Rapeseed and Linseed on the Prevention of Milk Fat Oxidation. J. Dairy Sci. 1998, 81:1095-1101, Abstract. * Cholesterol-lowering drugs (http://www.americanheart.org/presenter.jhtml?identifier=4510; p. 2).



Inventor

* Hennen, William J.

Assignee

* 4Life Research, LC

Application No. 10646615 filed on 08/22/2003

US Classes: 424/535Milk or colostrum (e.g., butter, whey, etc.)

Examiners Primary: Kim, Taeyoon

Attorney, Agent or Firm

* Durham Jones & Pinegar, IP Law Group

International Classes A61K 35/20 A61K 35/12 A61K 39/00



Abstract

A non-mammalian transfer factor, compositions including the non-mammalian transfer factor, and methods for generating and preparing the non-mammalian transfer factor. The non-mammalian transfer factor may have specificity for one or more antigens. A method of using the non-mammalian transfer factor includes administering either antigen-specific non-mammalian transfer factor or antigen non-specific non-mammalian transfer factor to mammals to treat or prevent pathogenic infections in the mammals. Claims

What is claimed is:

1. A method for obtaining transfer factor, comprising: exposing a non-mammalian source animal to at least one antigenic agent that will cause said non-mammalian source animalto elicit a T-cell mediated immune response; permitting said non-mammalian source animal to elicit a T-cell mediated immune response to said at least one antigenic agent; collecting at least one egg from said non-mammalian source animal following saidT-cell mediated immune response, said at least one egg including transfer factor that transfer cellular immunity to a mammal in vivo and that includes transfer factor molecules having molecular weights of about 4,000 Da to about 5,000 Da.

2. The method of claim 1, wherein said exposing said non-mammalian source animal comprises exposing an avian source animal to said at least one antigenic agent.

3. The method of claim 2, wherein said exposing said avian source animal comprises exposing a hen to said at least one antigenic agent.

4. The method of claim 1, wherein said exposing said non-mammalian source animal to at least one antigenic agent comprises permitting said non-mammalian source animal to be exposed to its natural environment.

5. The method of claim 1, wherein said exposing comprises injecting said non-mammalian source animal with said at least one antigenic agent.

6. The method of claim 1, wherein said exposing is conducted in the presence of an adjuvant.

7. The method of claim 1, wherein said exposing is conducted with substantially no adjuvant.

8. The method of claim 1, wherein said exposing comprises exposing said non-mammalian source animal to Newcastle Virus.

9. The method of claim 1, wherein said exposing comprises exposing said non-mammalian source animal to measles-mumps-rubella vaccine.

10. The method of claim 1, wherein said exposing comprises exposing said non-mammalian source animal to hepatitis B vaccine.

11. The method of claim 1, wherein said exposing comprises exposing said non-mammalian source animal to an antigen of Epstein-Barr Virus.

12. The method of claim 11, wherein said exposing comprises exposing said non-mammalian source animal to a recombinant Epstein-Barr Virus vaccine.

13. The method of claim 1, wherein said exposing comprises exposing said non-mammalian source animal to an antigen of H. pylori.

14. The method of claim 13, wherein said exposing comprises exposing said non-mammalian source animal to a synthetic H. pylori vaccine.

15. The method of claim 1, wherein said exposing comprises exposing said non-mammalian source animal substantially concurrently to a plurality of antigens.

16. The method of claim 1, wherein said exposing comprises exposing said non-mammalian source animal to at least one of a live vaccine, an attenuated vaccine, a killed vaccine, a recombinant antigen, and a natural antigen.

17. The method of claim 1, wherein said exposing comprises exposing said non-mammalian source animal to at least one of a bacterial antigen and a viral antigen.

18. The method of claim 1, wherein said exposing comprises exposing said non-mammalian source animal to an antigen at least based on an antigen of a pathogen from a non-mammalian pathogen source.

19. The method of claim 1, wherein said collecting said at least one egg is effected at least about seven days after said exposing.

20. The method of claim 1, wherein said collecting said at least one egg is effected at least about fourteen days after said exposing.

21. The method of claim 1, further comprising collecting a water soluble fraction of said at least one egg.

22. The method of claim 21, wherein said collecting said water soluble fraction comprises collecting a water soluble fraction of a yolk of said at least one egg.

23. The method of claim 21, further comprising removing substantially all antibodies from said water soluble fraction.

24. A method for obtaining transfer factor specific for a systemic pathogen, comprising: exposing a non-mammalian source animal to at least one antigenic agent for causing said non-mammalian source animal to illicit a T-cell mediated immuneresponse to the systemic pathogen; permitting said non-mammalian source animal to elicit a T-cell mediated immune response to said at least one antigenic agent, said T-cell mediated immune response resulting in generation of transfer factor specific forthe systemic pathogen; and following said T-cell mediated immune response, collecting transfer factor specific for said systemic pathogen, which transfers cellular immunity to a mammal in vivo and includes transfer factor molecules having molecularweights of about 4,000 Da to about 5,000 Da, from at least one egg of said non-mammalian source animal.

25. The method of claim 24, wherein said collecting includes substantially purifying said transfer factor from other proteins or peptides of said at least one egg having molecular weights of greater than about 8,000 Da.

26. The method of claim 24, wherein said exposing comprises exposing said non-mammalian source animal to at least one antigenic agent that causes said non-mammalian source animal to illicit a secondary immune response against at least one ofrubeola virus, rubella virus, mumps virus, hepatitis-B virus, Newcastle Virus, and Epstein-Barr Virus.

27. The method of claim 24, wherein said exposing comprises exposing said non-mammalian source animal to at least one of an MMR vaccine, a Newcastle Virus vaccine, a recombinant Epstein-Barr Virus vaccine, a substantially purified Epstein-BarrVirus antigen, and a recombinant hepatitis B vaccine.

28. The method of claim 1, wherein said collecting includes substantially purifying said transfer factor from other proteins or peptides of said at least one egg having molecular weights of greater than about 8,000 Da.

29. The method of claim 25, wherein substantially purifying transfer factor comprises causing said other proteins or peptides having molecular weights of greater than about 8,000 Da to precipitate from a solution including said transfer factor.

30. The method of claim 28, wherein said substantially purifying transfer factor comprises causing said other proteins or peptides having molecular weights of greater than about 8,000 Da to precipitate from a solution including said transferfactor. Description



BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods for generating antigen-specific transfer factor, compositions including such antigen-specific transfer factor, and uses of these compositions. In particular, the present invention relates to methods for generating antigen-specific transfer factor in an avian host and obtaining the antigen-specific transfer factor from eggs.

2. Background of Related Art

Many deadly pathogens are passed to humans from the animal kingdom. For example, monkeys are the sources of the type I human immunodeficiency virus (HIV-I), which causes acquired immune deficiency syndrome (AIDS) and monkeypox, which is similar to smallpox; ground-dwelling mammals are believed to be the source of the Ebola virus; fruit bats and pigs are the source of the Nipah virus; the Hendra virus comes from horses; the "Hong Kong Flu" originated in chickens; and wild birds, especially ducks, are the sources of many of the deadly influenza viruses. Many diseases also have animal reservoirs. By way of example, mice carry Hanta virus, rats carry the Black Plague, and deer carry Lyme disease.

The Immune System

The immune systems of vertebrates are equipped to recognize and defend the body from invading pathogenic organisms, such as parasites, bacteria, fungi, and viruses. Vertebrate immune systems typically include a cellular component and a noncellular component.

The cellular component of an immune system includes the so-called lymphocytes, or white blood cells, of which there are several types. It is the cellular component of a mature immune system that typically mounts a primary, nonspecific response to invading pathogens, as well as being involved in a secondary, specific response to pathogens.

In the primary, or initial, response to an infection by a pathogen, white blood cells that are known as phagocytes locate and attack the invading pathogens. Typically, a phagocyte will internalize, or "eat" a pathogen, then digest the pathogen. In addition, white blood cells produce and excrete chemicals in response to pathogenic infections that are intended to attack the pathogens or assist in directing the attack on pathogens.

Only if an infection by invading pathogens continues to elude the primary immune response is a specific, secondary immune response to the pathogen needed. As this secondary immune response is typically delayed, it is also known as "delayed-type hypersensitivity". A mammal, on its own, will typically not elicit a secondary immune response to a pathogen until about seven (7) to about fourteen (14) days after becoming infected with the pathogen. The secondary immune response is also referred to as an acquired immunity to specific pathogens. Pathogens have one or more characteristic proteins, which are referred to as "antigens". In a secondary immune response, white blood cells known as B lymphocytes, or "B-cells", and T lymphocytes, or "T-cells", "learn" to recognize one or more of the antigens of a pathogen. The B-cells and T-cells work together to generate proteins called "antibodies", which are specific for one or more certain antigens on a pathogen.

The T-cells are primarily responsible for the secondary, or delayed-type hypersensitivity, immune response to a pathogen or antigenic agent. There are three types of T-cells: T-helper cells, T-suppressor cells, and antigen-specific T-cells, which are also referred to as cytotoxic (meaning "cell-killing") T-lymphocytes ("CTLs"), or T-killer cells. The T-helper and T-suppressor cells, while not specific for certain antigens, perform conditioning functions (e.g., the inflammation that typically accompanies an infection) that assist in the removal of pathogens or antigenic agents from an infected host.

Antibodies, which make up only a part of the noncellular component of an immune system, recognize specific antigens and, thus, are said to be "antigen-specific". The generated antibodies then basically assist the white blood cells in locating and eliminating the pathogen from the body. Typically, once a white blood cell has generated an antibody against a pathogen, the white blood cell and all of its progenitors continue to produce the antibody. After an infection is eliminated, a small number of T-cells and B-cells that correspond to the recognized antigens are retained in a "resting" state. When the corresponding pathogenic or antigenic agents again infect the host, the "resting" T-cells and B-cells activate and, within about forty-eight (48) hours, induce a rapid immune response. By responding in this manner, the immune system mounts a secondary immune response to a pathogen, the immune system is said to have a "memory" for that pathogen.

Mammalian immune systems are also known to produce smaller proteins, known as "transfer factors," as part of a secondary immune response to infecting pathogens. Transfer factors are another noncellular part of a mammalian immune system. Antigen-specific transfer factors are believed to be structurally analogous to antibodies, but on a much smaller molecular scale. Both antigen-specific transfer factors and antibodies include antigen-specific cites and both include highly conserved regions that interact with receptor sites on their respective effector cells. In transfer factor and antibody molecules, a third, "linker", region connects the antigen-specific cites and the highly conserved regions.

The Role of Transfer Factor in the Immune System

Transfer factor is a low molecular weight isolate of lymphocytes. Narrowly, transfer factors may have specificity for single antigens. U.S. Pat. Nos. 5,840,700 and 5,470,835, both of which issued to Kirkpatrick et al. (hereinafter collectively referred to as "the Kirkpatrick Patents"), disclose the isolation of transfer factors that are specific for certain antigens. More broadly, "specific" transfer factors have been generated from cell cultures of monoclonal lymphocytes. Even if these transfer factors are generated against a single pathogen, they have specificity for a variety of antigenic sites of that pathogen. Thus, these transfer factors are said to be "pathogen-specific" rather than antigen-specific. Similarly, transfer factors that are obtained from a host that has been infected with a certain pathogen are pathogen-specific. Although such preparations are often referred to in the art as being "antigen-specific" due to their ability to elicit a secondary immune response when a particular antigen is present, transfer factors having different specificities may also be present. Thus, even the so-called "antigen-specific", pathogen-specific transfer factor preparations may be specific for a variety of antigens.

Additionally, it is believed that antigen-specific and pathogen-specific transfer factors may cause a host to elicit a delayed-type hypersensitivity immune response to pathogens or antigens for which such transfer factor molecules are not specific. Transfer factor "draws" at least the non-specific T-cells, the T-inducer and T-suppressor cells, to an infecting pathogen or antigenic agent to facilitate a secondary, or delayed-type hypersensitivity, immune response to the infecting pathogen or antigenic agent.

Typically, transfer factor includes an isolate of proteins obtained from immunologically active mammalian sources and having molecular weights of less than about 10,000 daltons (D). It is known that transfer factor, when added either in vitro or in vivo to mammalian immune cell systems, improves or normalizes the response of the recipient mammalian immune system.

The immune systems of newborns have typically not developed, or "matured", enough to effectively defend the newborn from invading pathogens. Moreover, prior to birth, many mammals are protected from a wide range of pathogens by their mothers. Thus, many newborn mammals cannot immediately elicit a secondary response to a variety of pathogens. Rather, newborn mammals are typically given secondary immunity to pathogens by their mothers. One way in which mothers are known to boost the immune systems of newborns is by providing the newborn with a set of transfer factors. In mammals, transfer factor is provided by a mother to a newborn in colostrum, which is typically replaced by the mother's milk after a day or two. Transfer factor basically transfers the mother's acquired, specific (i.e., delayed-type hypersensitive) immunity to the newborn. This transferred immunity typically conditions the cells of the newborn's immune system to react against pathogens in an antigen-specific manner, as well as in an antigen- or pathogen-nonspecific fashion, until the newborn's immune system is able on its own to defend the newborn from pathogens. Thus, when transfer factor is present, the immune system of the newborn is conditioned to react to pathogens with a hypersensitive response, such as that which occurs with a typical delayed-type hypersensitivity response. Accordingly, transfer factor is said to "jump start" the responsiveness of immune systems to pathogens.

Much of the research involving transfer factor has been conducted in recent years. Currently, it is believed that transfer factor is a protein with a length of about forty-four (44) amino acids. Transfer factor is believed to have a molecular weight in the range of about 4,000 to about 5,000 Daltons (D), or about 4 kD to about 5 kD. Transfer factor is also believed to include three functional fractions: an inducer fraction; an immune suppressor fraction; and an antigen-specific fraction. Many in the art believe that transfer factor also includes a nucleoside portion, which could be connected to the protein molecule or separate therefrom, that may enhance the ability of transfer factor to cause a mammalian immune system to elicit a secondary immune response. The nucleoside portion may be part of the inducer or suppressor fractions of transfer factor.

The antigen-specific region of the antigen-specific transfer factors is believed to comprise about eight (8) to about twelve (12) amino acids. A second highly-conserved region of about ten (10) amino acids is thought to be a very high-affinity T-cell receptor binding region. The remaining amino acids may serve to link the two active regions or may have additional, as yet undiscovered properties. The antigen-specific region of a transfer factor molecule, which is analogous to the known antigen-specific structure of antibodies, but on a much smaller molecular weight scale, appears to be hyper-variable and is adapted to recognize a characteristic protein on one or more pathogens. The inducer and immune suppressor fractions are believed to impart transfer factor with its ability to condition the various cells of the immune system so that the cells are more fully responsive to the pathogenic stimuli in their environment.

Sources of Noncellular Immune System Components

Conventionally, transfer factor has been obtained from the colostrum of milk cows. While milk cows typically produce large amounts of colostrum and, thus, large amounts of transfer factor over a relatively short period of time, milk cows only produce colostrum for about a day or a day-and-a-half every year. Thus, milk cows are neither a constant source of transfer factor nor an efficient source of transfer factor.

Transfer factor has also been obtained from a wide variety of other mammalian sources. For example, in researching transfer factor, mice have been used as a source for transfer factor. Antigens are typically introduced subcutaneously into mice, which are then sacrificed following a delayed-type hypersensitivity reaction to the antigens. Transfer factor is then obtained from spleen cells of the mice.

While different mechanisms are typically used to generate the production of antibodies, the original source for antibodies may also be mammalian. For example, monoclonal antibodies may be obtained by injecting a mouse, rabbit, or other mammal with an antigen, obtaining antibody-producing cells from the mammal, then fusing the antibody-producing cells with immortalized cells to produce a hybridoma cell line, which will continue to produce the monoclonal antibodies throughout several generations of cells and, thus, for long periods of time.

Antibodies against mammalian pathogens have been obtained from a wide variety of sources, including mice, rabbits, pigs, cows, and other mammals. In addition, the pathogens that cause some human diseases, such as the common cold, are known to originate in birds. As it has become recognized that avian (i.e., bird) immune systems and mammalian immune systems are very similar, some researchers have turned to birds as a source for generating antibodies.

U.S. Pat. No. 5,080,895, issued to Tokoro on Jan. 14, 1992 (hereinafter "the '895 Patent"), discloses a method that includes injecting hens with pathogens that cause intestinal infectious diseases in neonatal mammals. The hens then produce antibodies that are specific for these pathogens, which are present in eggs laid by the hens. The '895 Patent discloses compositions that include these pathogen-specific antibodies and use thereof to treat and prevent intestinal diseases in neonatal piglets and calves. In addition, the '895 Patent assumes that a pathogen-specific transfer factor-like substance is passed from a hen to her eggs. Nonetheless, the '895 Patent does not disclose that such a transfer factor-like substance was in fact present in the eggs, or that an antibody-free composition derived from eggs that were assumed to contain this transfer factor-like substance actually treated or prevented intestinal diseases in neonatal mammals. In fact, the '895 Patent discloses the use of a filter with about 0.45 μm diameter holes to isolate transfer factor from antibodies. As those of skill in the art are aware, however, antibodies, larger molecules, viruses, and even some bacteria will pass through the pores of a 0.45 μm filter. In reality, it is not likely that any individual protein molecules having molecular weights of less than about 12,000 D were separated by such a filter. Based on the pore size of the filter used, however, it is more likely that no individual protein molecules, including antibodies, were removed by the filter.

Avian antibodies that are specific for mammalian pathogens have also been obtained by introducing antigens into eggs.

Treatment of pathogenic infections in mammals with avian antibodies is typically not desirable, however, since the immune systems of mammals are likely to respond negatively to the large avian antibody molecules by eliciting an immune response to the antibodies themselves. Moreover, as mammalian immune systems do not recognize avian antibodies as useful for their abilities to recognize certain pathogens, or the specificities of avian antibodies for antigens of such pathogens, avian antibodies do not even elicit the desired immune responses in mammals.

The inventors are not aware of any art that teaches a method for generating transfer factor in a non-mammalian source, an efficient method for obtaining transfer factor from such a non-mammalian source, such as an avian source, or a method for using such transfer factor in treating or preventing infections by pathogens.

SUMMARY OF THE INVENTION

The present invention includes a method for generating the production of transfer factor in a non-mammalian source and obtaining transfer factor from a non-mammalian source. In addition, compositions including non-mammalian transfer factor are also within the scope of the present invention, as are methods of using these compositions.

The non-mammalian transfer factor generated, obtained, and used in accordance with the present invention may either be antigen non-specific or antigen-specific (i.e., configured to bind or recognize one or more antigens). Unless otherwise indicated, the term "transfer factor", as used herein, includes the previously discussed broad definition, which includes each of the various types of transfer factors, including pathogen-specific, antigen-specific, and transfer factors that are not specific for particular pathogens or antigenic agents. The term "non-specific", when used herein with respect to transfer factors, refers to both transfer factors that are not specific for particular antigens and to mixtures that include transfer factors with different antigen specificities.

Non-specific transfer factor includes transfer factor that the non-mammalian source animal already produces. Individual non-specific transfer factor molecules that are produced by the source animal may have specificity for various antigenic agents, including pathogens, that are present in the source animal's environment. Nonetheless, for purposes of the present invention, transfer factor that is generated merely by a source animal's reaction to its environment is referred to as "non-specific".

On the other hand, antigen-specific transfer factor is generated by exposing a non-mammalian source animal to one or more antigens. The antigens of various types of pathogens, including, but not limited to, bacteria, viruses, fungi, and parasites, have been found by the inventors to induce the production of non-specific transfer factor in non-mammalian sources. Antigen-specific transfer factor has been generated by non-mammalian source animals by both natural antigens (including from live, inactivated, and attenuated sources) and synthetic antigens.

The production of transfer factor in a non-mammalian source may be induced by introducing an antigen characteristic of a certain pathogen into a female non-mammalian source animal. Exemplary types of source animals that may be used include, without limiting the scope of the present invention, birds, reptiles, amphibians, and fish. Preferably, the non-mammalian source animal produces eggs on a frequent basis. Thus, for purposes of the present invention, hens are particularly useful as the non-mammalian source animal. These non-mammalian source animals produce transfer factor, which then appears in the eggs of these source animals. Alternatively, an egg of a non-mammalian source animal may be exposed to the antigenic agent (e.g., by injection of the antigenic agent into the egg) to induce production of transfer factor by the egg itself.

The transfer factor generated by a non-mammalian source animal or by the egg of a non-mammalian source animal may be recovered from the egg and separated from other constituents of the egg, including proteins of larger molecular weight, such as antibodies. Alternatively, transfer factor may be purified from one or more eggs of a non-mammalian source animal.

The non-mammalian transfer factor may then be incorporated into a composition or apparatus for administration to a mammalian or non-mammalian subject or administered directly to the subject. The non-mammalian transfer factor or compositions including the non-mammalian transfer factor may be administered enterally (i.e., orally), or parenterally (i.e., by a non-oral route, such as by injection, through the skin, etc.). Administration of both non-specific and specific non-mammalian transfer factors have been found to initiate an early, specific (i.e., secondary) immune response in mammals to various invading pathogens. Thus, non-mammalian transfer factor has been found to be useful in treating and preventing diseases that may be caused by these various pathogens.

Other features and advantages of the present invention will become apparent to those of skill in the art through consideration of the ensuing description, the accompanying drawings, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, which illustrate exemplary embodiments of the present invention:

FIG. 1 is a schematic representation of an exemplary method for generating non-mammalian transfer factor in a non-mammalian source animal;

FIG. 2 is a schematic representation of an exemplary method for generating non-mammalian transfer factor directly in the eggs of a non-mammalian source animal;

FIG. 3 is a schematic representation of an exemplary method for obtaining non-mammalian transfer factor from eggs; and

FIG. 4 is a schematic representation of an exemplary method for testing for the presence of transfer factor in a solution and for using transfer factor to prevent infection by pathogens or to treat pathogenic infections.

DETAILED DESCRIPTION OF THE INVENTION

As explained previously herein, mammalian mothers pass transfer factor to their newborn children in the colostrum, which is replaced by mother's milk after about a day or two. The transfer factor present in colostrum transfers delayed-type hypersensitivity for certain antigens to the child, thus "jump-starting" the ability of the immune system of the newborn child to respond to certain pathogens, if the child becomes infected with these pathogens.

Over recent years, it has been discovered that avian (i.e., bird) immune systems are very similar to those of mammals. In fact, early studies of the components of immune systems were performed on birds. As a result of these early studies of immune systems, B-cells, one of the types of white blood cells discussed previously herein, was so named due to its origin in the bursa of birds. In addition, it is known that various infectious agents, including some viruses that cause the common cold and influenza A virus, originate in birds and are passed onto humans.

As avian immune systems bear some resemblances to the immune systems of mammals, the inventors believe that transfer factor is also a component of avian immune systems, as well as of the immune systems of other non-mammalian vertebrates. In addition, the inventors believe that although non-mammalian mothers do not provide colostrum to their newborn children, these animals could still transfer immunity to their children by way of transfer factor. In birds and other egg-laying vertebrates, the mother's primary opportunity to provide transfer factor to her children is in the egg-yolk, which supplies the growing embryo with the necessary nutrients during growth. Thus, the inventors have long believed that antigen non-specific and antigen-specific transfer factor could be obtained from eggs.

FIG. 1 schematically illustrates a method for obtaining desired transfer factor from a non-mammalian source 10 of transfer factor, in this case a hen. Non-mammalian source 10 may be exposed to environmental antigenic agents 12a or exposed to specific antigenic agents 12b. Non-mammalian source 10 may be exposed to specific antigenic agents 12b by injection, orally, or otherwise, as known in the art. Non-mammalian source 10 may be exposed to antigenic agents 12b either with or without an adjuvant present. Such exposure to specific antigenic agents 12b may occur once or be repeated. For simplicity, antigenic agents 12a and 12b are also referred to herein as antigenic agents 12 or simply as antigens.

Alternatively, with reference to FIG. 2, an egg 14' of a non-mammalian animal may be directly exposed to one or more antigenic agents 12, such as by injection or otherwise, as known in the art.

With reference to FIG. 3, after non-mammalian source 10 or non-mammalian eggs 14' that were directly exposed to one or more antigenic agents 12 have been given an adequate opportunity to elicit a secondary, or delayed-type hypersensitivity, immune response to antigenic agents 12, eggs 14 are collected. The yolks 16 and whites 18 of eggs 14 are then separated from one another, and various filtration processes are conducted on yolks 16 to obtain a water soluble fraction 20 thereof that includes transfer factor. Larger molecular weight proteins, such as antibodies, may also be removed from water soluble fraction 20 of yolks 16 by known processes, such as by filtering on the basis of molecular weight or by causing these larger molecular weight proteins to precipitate out of solution (e.g., in cold ethyl alcohol), then removing the precipitate 21 from water soluble fraction 20 (e.g., by filtration) to provide a substantially antibody-free, transfer factor-containing solution 22. Alternatively, the yolks 16 and whites 18 need not be separated.

In addition, antigen-specific non-mammalian transfer factor present in water soluble fraction 20 of yolks 16 or in or in solution 22 may be substantially purified from other constituents of water soluble fraction 20 or solution 22 by known techniques, such as by use of the gel permeation and affinity chromatography techniques disclosed in U.S. Pat. Nos. 5,840,700 and 5,470,835, both of which issued to Kirkpatrick et al. (hereinafter collectively referred to as "the Kirkpatrick Patents"), the disclosures of both of which are hereby incorporated by this reference in their entireties. The technique disclosed in the Kirkpatrick Patents is used to isolate biomolecules, such as transfer factor and antibodies, from the other constituents of a solution on the basis of the specificity of these biomolecules for one or more antigens or other specific binding agents. Thus, when the technique disclosed in the Kirkpatrick Patents is used on the antibody- and transfer factor-containing water soluble fraction 20 of egg yolk 16, both transfer factor and antibody may be isolated from the remainder of water soluble fraction 20 with the resulting solution 24 including both antibody and transfer factor. If, on the other hand, the technique disclosed in the Kirkpatrick Patents is conducted on a substantially antibody-free, transfer factor-containing solution 22, the product will be a substantially pure solution 26 of transfer factor specific for one or more antigens. Of course, other methods for obtaining transfer factor from eggs are also within the scope of the present invention, including methods for obtaining transfer factor from various egg preparations, including powdered or freeze-dried whole eggs or egg yolks.

Referring now to FIG. 4, an exemplary method for testing for the presence of non-mammalian transfer factor specific for one or more antigens in a solution, known as a mouse footpad assay, is schematically depicted.

About seven (7) days prior to testing the effectiveness of avian transfer factor in causing mice to elicit a secondary immune response to a particular antigen or pathogen for which the avian transfer factor was specific, a positive control population of six female BALB/c mice is prepared. Each mouse 30 of the positive control population, having ages of about nine (9) weeks to about ten (10) weeks, is anesthetized with isoflurane. About 0.02 ml of a 50/50 (wt/wt) mixture of Freund's adjuvant and the particular antigen 36 against which the avian transfer factor to be tested is specific is administered to each mouse 30 by way of two intramuscular injections, one injection at each side of the base 39 of the tail 38. As these injections are conducted about seven (7) days prior to conducting the mouse footpad assay, the mice of the positive control population are permitted to generate their own secondary, or delayed-type hypersensitivity response to antigen 36.

About twenty-four (24) hours prior to the mouse footpad test, the mice of a first test population, which also includes six female BALB/c mice that are about nine (9) to about ten (10) weeks old (i.e., about the same age as the mice of the positive control population), are also anesthetized with isoflurane. About 0.5 ml of a solution 20, 24 including a preparation containing both avian transfer factor and avian antibody, reconstituted in distilled water, is then administered by subcutaneous injection at the back of the neck 40 of each mouse 30 of the first test population. By comparing the results obtained from these mice with the results obtained from mice of a second test population that had been treated with a substantially antibody-free preparation, the relative contributions of transfer factor and antibody to the swelling could be determined. As antibodies do not elicit a secondary immune response, it was believed prior to conducting the experiments described herein that the measure of the secondary immune response in the first and second test populations of mice would be very similar.

Each mouse of the second test population that includes six female BALB/c mice, having ages of about nine (9) to about ten (10) weeks old (i.e., about the same age as the mice of the positive control and first test populations), are also anesthetized with isoflurane. Each of the six mice 30 is given, by subcutaneous injection in the back of the neck 40, about 0.5 ml of a solution 22, 26 including, reconstituted in distilled water, a lyophilized antigen-specific avian transfer factor preparation with substantially no antibodies.

A negative control population also includes six female BALB/c mice of about nine (9) to about ten (10) weeks in age (i.e., about the same ages as the other three populations of mice).

In order to conduct the mouse footpad assay, the mice of each of the four populations are anesthetized and the distances across each of the largest right hind footpad 32 and the largest left hind footpad 34 of each mouse 30 are measured, such as with a Starrett gauge. Right hind footpad 32 is then subcutaneously injected with an antigen 36-containing solution. Left hind footpad 34, which is used as a control, is injected with about the same volume of a control solution 37, such as a sterile saline diluent, as the volume of solution that is injected into right hind footpad 32.

After a sufficient amount of time (e.g., about sixteen (16) to about twenty-four (24) hours) has elapsed, each mouse 30 is again anesthetized and the distances across right and left hind footpads 32, 34 are again measured. A significant amount of swelling, determined by an increase in the distances across a right hind footpad 32 of mouse 30, is indicative of the occurrence of a delayed-type hypersensitivity reaction in that footpad 32.

Of course, different solutions 24, 26 including transfer factors with specificities for different antigens may be tested on different sets of mice to detect any differences in the abilities of these solutions to transfer delayed-type hypersensitivity immunity to the mice. In addition, the results for each solution may be compared to those obtained from positive control and negative control populations of mice 30. If significant swelling occurs in the right hind footpads 34 of mice 30 to which a substantially antibody-free solution, such as solution 22 or solution 26 of FIG. 3, was administered, the delayed-type hypersensitivity that causes such swelling is attributed to the administered transfer factor.

The following examples are merely illustrative of embodiments of methods for generating, obtaining, and using transfer factor that incorporate teachings of the present invention:

EXAMPLE 1

Transfer factor specific for Newcastle Virus was generated by exposing day-old chicks to a coarse spray of infectious bronchitis/Newcastle virus (IBNC) vaccine, as known in the art, at zero (0) days, forty-two (42) days, and eighty-four (84) days. Eggs laid by these five hens at about one-hundred seventy-five (175) days following the first IBNC vaccine injection were collected.

EXAMPLE 2

The yolks from a first sampling of the antigen specific transfer factor-containing eggs generated in EXAMPLE 1 were separated from the whites, diluted about six (6) to about nine (9) times, by volume, in deionized water (i.e., about one (1) part egg white mixed with about five (5) parts water to about eight (8) parts water) and frozen. The lipid layer from these frozen egg yolks was mechanically separated from the water-soluble fraction of the egg yolks. This water-soluble fraction was then permitted to thaw to a temperature of about 4° C. to about 6° C. and vacuum filtered by use of Whatman qualitative filter paper using a 55 mm diameter porcelain Buchner funnel. The filtrate was then vacuum filtered through a glass microfiber filter, again using a 55 mm diameter Buchner funnel.

A third filtration was then conducted to collect proteins and to remove lipids and lipoproteins from the solution. The third filtration was effected by way of a DURAPORE hydrophilic membrane. The protein-containing fraction, which included both transfer factor and antibody specific for the infectious bronchitis pathogen and Newcastle Virus was collected, frozen, and lyophilized, or freeze-dried, as known in the art.

EXAMPLE 3

The water-soluble fractions of diluted yolk preparations from a second sampling of the eggs collected in EXAMPLE 1 were again mechanically separated from the lipid portions thereof and filtered, as explained previously herein in EXAMPLE 2.

In accordance with the method disclosed in U.S. Pat. No. 4,180,627, which issued to Klesius et al., the disclosure of which is hereby incorporated by this reference in its entirety, an adequate volume of ethyl alcohol (EtOH), or ethanol, was added to the protein-containing fraction to dilute the ethyl alcohol to a concentration of about 60% of the total volume of the alcohol-protein fraction solution. This solution was then cooled to a temperature of about 4 to about 6° C. for a long enough period of time (e.g., overnight, or for about 10-12 hours) for larger molecular weight proteins, including antibodies, present in the solution to precipitate from the solution. Smaller molecular weight proteins (e.g., proteins having molecular weights of about 8,000 D or less), including any transfer factor from the egg yolks, remained in solution.

The larger molecular weight protein-containing precipitate was then removed from the solution by filtering the solution through a Whatman glass microfiber filter in a 55 mm diameter Buchner funnel. CELITE.RTM., a diatomite, or diatomaceous earth, filtration aid available from Celite Corporation of Lompoc, Calif., was used to prevent the precipitate from clogging the filter during filtration of the solution. This substantially precipitate-free solution was then collected, frozen, and lyophilized, as known in the art.

EXAMPLE 4

Each mouse of a test population that included three BALB/c mice, each having an age in the range of about nine (9) to about ten (10) weeks, was tested to determine whether the IBNV-specific avian transfer factor would impart an early secondary, or delayed-type hypersensitivity, immune response to the mice. Each mouse was anesthetized with isoflurane. The distances across the largest footpads of both the left and right hind feet of each mouse were then measured with a Starrett gauge. Each mouse was then given a subcutaneous injection in the back of the neck of about 0.5 ml of a solution that included about 16%, by weight, of the IBNV-specific avian transfer factor reconstituted in distilled water.

After about twenty-four (24) hours, each of the mice was again anesthetized with isoflurane. About 0.01 ml of a sterile saline diluent was then injected into the largest footpad of the left hind foot of each mouse, which footpad served as a control, while the largest footpad of the right hind foot of each mouse was injected with about 0.01 ml of a solution including about 10,000 doses of Newcastle-Bronchitis vaccine reconstituted in about 250 ml of distilled water.

Before another twenty-four (24) hours had elapsed, one of the mice (Mouse #1) died. The two remaining mice were again anesthetized with isoflurane and the largest footpads on their hind feet were again measured. The results follow:

TABLE 1 Newcastle Virus-Test Population Footpad size (μm): Before Sample Injection Final Difference Mouse #1 Left Foot (Control) 1250 Right Foot (Test) 2151 Mouse #2 Left Foot (Control) 2180 2350 50 Right Foot (Test), 2165 2440 85 Mouse #3 Left Foot (Control) 2145 2160 15 Right Foot (Test) 2110 2200 90

The greater increase in size, or swelling, of the right footpad (increases of 85 μm and 90 μm) over that of the left footpad (increases of 50 μm and 15 μm, respectively) indicates that the IBNV-specific avian transfer factor-containing solution induced a delayed-type hypersensitivity reaction in the right feet of Mouse #2 and Mouse #3 within about twenty-four hours following the introduction of the Newcastle-Bronchitis vaccine.

In the remaining examples, substantially the same methods as those disclosed in EXAMPLES 1-3 were used to generate avian transfer factors specific for different types of antigens, including measles, mumps, rubella, Hepatitis B, Epstein-Barr Virus (EBV), and H. pylori.

The effectiveness of each of these various types of antigen-specific avian transfer factors in inducing early secondary, or delayed-type hypersensitivity, immune responses in mammals was then tested by way of mouse footpad assays. Each type of antigen-specific avian transfer factor was tested using four different populations of mice, including a positive control population, a first test population, a second test population, and a negative control population, which were prepared as described previously herein with reference to FIG. 4. The mouse footpad assay for each type of antigen-specific transfer factor was conducted in accordance with the teachings of Petersen E A, Greenberg L E, Manzara T, and Kirkpatrick C H, "Murine transfer factor," I. Description of the model and evidence for specificity, J. Immunol., 126: 2480-84 (1981), the disclosure of which is hereby incorporated by this reference in its entirety.

In each mouse footpad assay, four populations of mice were prepared in the manner described in reference to FIG. 4.

In conducting the various mouse footpad assays on each of a positive control, a first and a second test, and a negative control populations, each mouse was anesthetized with isofluorane, the largest footpad of the left hind footpad of each mouse, which served as a control, was injected with about 0.01 ml of sterile saline diluent, and the largest footpad of the right hind foot of each mouse was injected with about 0.01 ml of a solution including the antigen or pathogen for which the avian transfer factor was specific.

About sixteen (16) to about twenty-four (24) hours following the hind footpad injections, each of the mice of the positive control, test, and negative control populations was again anesthetized with isoflurane and the sizes of the left and right hind footpads of each of the mice were again measured, for example, with a Starrett Gauge.

EXAMPLE 5

Using the same procedures described in EXAMPLES 1-3, avian transfer factor and avian antibodies specific for measles, mumps, and rubella (MMR) vaccine were generated in hens. Each hen received one dose of Merck MMR II vaccine, as described in EXAMPLE 1, at 150 days, 163 days, 190 days, 221 days, and 249 days. Eggs were collected from these hens just after the third innoculation-sometime in the period of about day 192 to about day 223 and prepared as described in EXAMPLE 1. This was done in this EXAMPLE and in the following EXAMPLES to ensure that a high level of transfer factor was present in the eggs. It is believed that transfer factor will be present in eggs about seven (7) days following the first innoculation.

A positive control population of mice was prepared about seven (7) days prior to the beginning of the mouse footpad assay by injecting each mouse of the positive control population with Merck MMR II vaccine, as described previously herein in reference to FIG. 4.

A solution containing both avian antibody and avian transfer factor specific for MMR vaccine was made by reconstituting in distilled water a lyophylized preparation similar to that described in EXAMPLE 2 to a concentration of about 8%, by weight. This transfer factor- and antibody-containing solution was administered to the first test population of mice in the manner described in reference to FIG. 4.

Lyophilized avian transfer factor specific for measles, mumps, and rubella, prepared by a method similar to that described in EXAMPLE 3, was reconstituted in distilled water to a concentration of about 8%, by weight. This reconstituted MMR-specific avian transfer factor was then administered to a second test population of mice in the manner described previously herein in reference to FIG. 4.

About 0.1 ml of a dose of Merck MMR II Vaccine was then administered to the largest footpad of the right hind foot of each mouse of each of positive control, first test, second test, and negative control populations, while substantially the same amount of sterile saline diluent was administered to the largest footpad of the left hind foot of each mouse, as described in reference to FIG. 4.

About sixteen (16) to about twenty-four (24) hours later, the mice were again anesthetized and the sizes of the largest footpads of both hind feet of each mouse measured, as previously described. The results follow:

TABLE 2 MMR Vaccine-First Test Population (Antibody and Transfer Factor Administered) Footpad size (μm): Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2159.00 2235.20 76.20 Right Foot (Test) 2133.60 2387.60 254.00 Mouse #2 Left Foot (Control) 2133.60 2159.00 25.40 Right Foot (Test) 2133.60 2184.40 50.80 Mouse #3 Left Foot (Control) 2159.00 2159.00 0.00 Right Foot (Test) 2159.00 2184.40 25.40 Mouse #4 Left Foot (Control) 2209.80 2235.20 25.40 Right Foot (Test) 2286.00 2311.40 25.40 Mouse #5 Left Foot (Control) 2184.40 2184.40 0.00 Right Foot (Test) 2209.80 2260.60 50.80 Mouse #6 Left Foot (Control) 2260.60 2336.80 76.20 Right Foot (Test) 2235.20 2438.40 203.20

The data for Mouse #6 may have been inaccurate since the scabs from bite marks were present on one or both hind footpads of this mouse at the time the second measurements were taken (i.e., at about sixteen (16) to about twenty-four (24) hours). Nonetheless, with the exception of Mouse #4, each of the remaining mice of the first test population exhibited greater swelling at the time the second footpad measurements were taken in the footpads that were injected with the MMR II vaccine than in the footpads that were injected with the control solution. In Mouse #4, the amount of swelling was about the same in both the left and right footpads.

Overall, as can be seen from the data of TABLE 2, the largest footpads of the right feet of the first test population of mice represented exhibited an average of about 67.73 μm more swelling than the amount of swelling of the largest footpad of the left feet of these mice.

TABLE 3 MMR Vaccine-Second Test Population (Only Transfer Factor Administered) Footpad size (μm): Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2082.80 2133.60 50.80 Right Foot (Test) 2108.20 2235.20 127.00 Mouse #2 Left Foot (Control) 2336.80 2387.60 50.80 Right Foot (Test) 2387.60 2641.60 254.00 Mouse #3 Left Foot (Control) 2184.40 2184.40 0.00 Right Foot (Test) 2184.40 2311.40 127.00 Mouse #4 Left Foot (Control) 2133.60 2133.60 0.00 Right Foot (Test) 2133.60 2133.60 0.00 Mouse #5 Left Foot (Control) 2082.80 2540.00 457.20 Right Foot (Test) 2108.20 2235.20 127.00 Mouse #6 Left Foot (Control) 2260.60 2286.00 25.40 Right Foot (Test) 2286.00 2362.20 76.20

As scabs from bite marks were visible on the footpads of Mouse #2 and Mouse #5 at about twenty-four hours following the injection of antigen and sample, the data form these mice may have been inaccurate. In addition, the largest footpad on the left foot of Mouse #5 was swollen more than three times as much as the corresponding footpad on the left foot of Mouse #5 and several times more than the swelling that occurred in any of the footpads of the other tested mice. Accordingly, the swelling data obtained from Mouse #5 were also omitted as this swelling in the footpad of the left foot was excessive. No increase in swelling in either footpad was measured in Mouse #4. Nonetheless, each of Mouse #1, Mouse #3, and Mouse #6 exhibited greater swelling in the (right) footpad that was injected with the second, substantially antibody-free, transfer factor-containing solution than in the (left) footpad that was injected with the control solution.

Based on the data presented in TABLE 3, on average, the largest footpads on the right feet of Mice ## 1, 3, and 6 were swollen about 91.4 μm more than the largest footpads on the left feet of these mice.

TABLE 4 MMR Vaccine-Positive Control Footpad size (μm): Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2184.40 2235.20 50.80 Right Foot (Test) 2184.40 2260.60 76.20 Mouse #2 Left Foot (Control) 2184.40 2209.80 25.40 Right Foot (Test) 2184.40 2209.80 25.40 Mouse #3 Left Foot (Control) 2006.60 2133.60 127.00 Right Foot (Test) 1981.20 2108.20 127.00 Mouse #4 Left Foot (Control) 2133.60 2184.40 50.80 Right Foot (Test) 2133.60 2260.60 127.00 Mouse #5 Left Foot (Control) 2108.20 2133.60 25.40 Right Foot (Test) 2108.20 2286.00 177.80 Mouse #6 Left Foot (Control) 2082.80 2133.60 50.80 Right Foot (Test) 2057.40 2209.80 152.40

While Mouse #2 and Mouse #3 of the positive control population both exhibited substantially the same amount of swelling in the largest footpads of both the left and right hind feet, each of the other mice had a greater amount of swelling in the largest footpads of their right hind feet and, thus, displayed a secondary immune response to the MMR vaccine that was introduced into the largest footpads of their right hind feet, than the amount of swelling in the largest footpads of the left hind feet of these mice, which were much less swollen.

Based on the data in TABLE 4, it is apparent that the average amount of swelling in the largest footpads of the right hind feet of these mice was about 59.27 μm greater than the swelling of the largest footpads on the left hind feet of these mice.

TABLE 5 MMR Vaccine-Negative Control Footpad size (μm): Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2159.00 2159.00 0.00 Right Foot (Test) 2159.00 2209.80 50.80 Mouse #2 Left Foot (Control) 2159.00 2159.00 0.00 Right Foot (Test) 2108.20 2133.60 25.40 Mouse #3 Left Foot (Control) 2133.60 2133.60 0.00 Right Foot (Test) 2133.60 2133.60 0.00 Mouse #4 Left Foot (Control) 2108.20 2133.60 25.40 Right Foot (Test) 2108.20 2108.20 0.00 Mouse #5 Left Foot (Control) 2057.40 2057.40 0.00 Right Foot (Test) 2032.00 2032.00 0.00 Mouse #6 Left Foot (Control) 2082.80 2133.60 50.80 Right Foot (Test) 2032.00 2082.80 50.80

Two of the mice, Mouse #3 and Mouse #5, of the negative control population exhibited no swelling in the largest footpad of either hind foot. The largest footpads on both hind feet of Mouse #6 were swollen by about the same amount. While the largest footpads on the left hind feet of Mouse #1 and Mouse #4 were not swollen and the footpads on the right hind feet of these two mice were slightly swollen, the largest footpad on the right hind foot of Mouse #4 was not swollen and the largest left hind footpad was only slightly swollen. In fact, the average amount of swelling in the largest footpads of the right hind feet of these mice was only about 8.47 μm greater than the amount of swelling measured in the largest footpads of the left hind feet of the negative control population of mice. Consequently, the data in TABLE 5 indicate that the mice of the negative control population did not elicit a secondary immune response to the MMR vaccine.

Collectively, the data of TABLES 2-5 indicate that a secondary, or delayed-type hypersensitivity, immune response occurred in the majority of mice in each of the first test population, the second test population, and the positive control population, while no such secondary immune response appeared to be present in the negative control population. Accordingly, the data in TABLES 2 and 3 indicate that avian transfer factor specific for MMR vaccine, as well as avian antibody specific for MMR vaccine, are capable of inducing an early secondary immune response in mammals.

EXAMPLE 6

Repeating the procedures described previously herein in EXAMPLES 1-3, avian transfer factor and avian antibodies specific for the Hepatitis B virus were generated by use of a synthetic Hepatitis B antigen vaccine sold under the tradename ENGERIX-B. Each hen received one dose of the Hepatitis B vaccine, as described in EXAMPLE 1, at 150 days, 163 days, 190 days, 221 days, and 249 days. Eggs were collected from these hens sometime in the period of about day 193 and about day 223, as described in EXAMPLE 1 above, and prepared as described in EXAMPLE 1.

A positive control population of mice was prepared about seven (7) days prior to conducting the mouse footpad assay by injecting each mouse of the positive control population with the synthetic Hepatitis B vaccine, ENGERIX-B in the manner described in reference to FIG. 4.

A first solution, which included both avian antibody and avian transfer factor that were specific for Hepatitis B vaccine, was made by reconstituting in distilled water a lyophilized preparation similar to that described in EXAMPLE 2 to a concentration of about 16%, by weight. This transfer factor- and antibody-containing solution was administered to a first test population of mice in the manner described in reference to FIG. 4.

In addition, lyophilized avian transfer factor specific for Hepatitis B vaccine, which was prepared in a similar manner to that described in EXAMPLE 3, was reconstituted in distilled water to a concentration of about 16%, by weight. The reconstituted transfer factor-containing solution was then administered to each of the mice of a second test population, as explained previously herein in reference to FIG. 4.

At the appropriate time, the synthetic Hepatitis B vaccine was administered to the largest footpad of the right foot of each mouse of each of the positive control, first test, second test, and negative control populations, as described previously herein in reference to FIG. 4. The largest footpad of the left foot of each mouse of the four populations was substantially concurrently injected with the same amount of sterile saline diluent, also as described previously herein.

About sixteen (16) to about twenty-four (24) hours later, each of the mice of the four populations was again anesthetized and the sizes of the largest footpads of both hind feet of each mouse were again measured, as described previously herein. The results follow:

TABLE 6 Hepatitis B Vaccine - First Test Population (Antibody and Transfer Factor Administered) Footpad size (μm): Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2032.00 2108.20 76.20 Right Foot (Test) 2032.00 2082.80 50.80 Mouse #2 Left Foot (Control) 2260.60 2362.20 101.60 Right Foot (Test) 2209.80 2336.80 127.00 Mouse #3 Left Foot (Control) 2159.00 2184.40 25.40 Right Foot (Test) 2159.00 2235.20 76.20 Mouse #4 Left Foot (Control) 2108.20 2184.40 76.20 Right Foot (Test) 2108.20 2260.60 152.40 Mouse #5 Left Foot (Control) 1930.40 2032.00 101.60 Right Foot (Test) 1930.40 2108.20 177.80 Mouse #6 Left Foot (Control) 2184.40 2184.40 0.00 Right Foot (Test) 2184.40 2235.20 50.80

Each of the mice of the first test population, with the exception of Mouse #1, exhibited greater swelling in the largest footpad of the right hind foot. On average, the largest footpads of the right hind feet of the mice of the first test population were about 42.17 μm more swollen than the largest footpads of the left hind feet of these mice. Thus, the data of TABLE 6 indicate that the avian transfer factor in the preparation that included transfer factor and antibody specific for the synthetic Hepatitis B vaccine induced an early secondary immune response in each of these mice.

TABLE 7 Hepatitis B Vaccine - Second Test Population (Only Transfer Factor Administered) Footpad size (μm): Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 1981.20 2032.00 50.80 Right Foot (Test) 2006.60 2159.00 152.40 Mouse #2 Left Foot (Control) 1981.20 1981.20 0.00 Right Foot (Test) 1981.20 2006.60 25.40 Mouse #3 Left Foot (Control) 2006.60 2032.00 25.40 Right Foot (Test) 2032.00 2082.80 50.80 Mouse #4 Left Foot (Control) 1955.80 2133.60 177.80 Right Foot (Test) 1981.20 2108.20 127.00 Mouse #5 Left Foot (Control) 1930.40 2006.60 76.20 Right Foot (Test) 1930.40 2057.40 127.00 Mouse #6 Left Foot (Control) 2032.00 2057.40 25.40 Right Foot (Test) 2006.60 2108.20 101.60

On average the largest footpads on the right hind feet of the second test population of mice were about 38.10 μm more swollen than the largest footpads on the left hind feet of these mice. With the exception of Mouse #4, the data of TABLE 7 illustrate that the administration of avian transfer factor specific for Hepatitis B vaccine induced an early secondary, or delayed-type hypersensitivity, immune response in the largest footpad of the right hind foot of each mouse.

TABLE 8 Hepatitis B Vaccine - Positive Control Footpad size (μm): Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2108.20 2133.60 25.40 Right Foot (Test) 2108.20 2159.00 50.80 Mouse #2 Left Foot (Control) 2032.00 2082.80 50.80 Right Foot (Test) 2006.60 2108.20 101.60 Mouse #3 Left Foot (Control) 1854.20 1930.40 76.20 Right Foot (Test) 1879.60 2032.00 152.40 Mouse #4 Left Foot (Control) 2006.60 2108.20 101.60 Right Foot (Test) 2057.40 2209.80 152.40 Mouse #5 Left Foot (Control) 2133.60 2159.00 25.40 Right Foot (Test) 2133.60 2159.00 25.40 Mouse #6 Left Foot (Control) 2006.60 2133.60 127.00 Right Foot (Test) 2006.60 2184.40 177.80

In the positive control population of mice, only Mouse #5 failed to elicit a secondary immune response to the synthetic Hepatitis B vaccine. The largest footpads on the right hind feet of each of the other mice of the positive control population exhibited an average of about 42.33 μm increased swelling over that of the largest footpads on the left hind feet of these mice.

TABLE 9 Hepatitis B Vaccine - Negative Control Footpad size (μm): Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2159.00 2159.00 0.00 Right Foot (Test) 2133.60 2133.60 0.00 Mouse #2 Left Foot (Control) 2057.40 2057.40 0.00 Right Foot (Test) 2082.80 2082.80 0.00 Mouse #3 Left Foot (Control) 2006.60 2032.00 25.40 Right Foot (Test) 1955.80 2032.00 72.60 Mouse #4 Left Foot (Control) 2057.40 2082.80 25.40 Right Foot (Test) 2057.40 2108.20 50.80 Mouse #5 Left Foot (Control) 2133.60 2133.60 0.00 Right Foot (Test) 2133.60 2159.00 25.40 Mouse #6 Left Foot (Control) 2082.80 2133.60 50.80 Right Foot (Test) 2082.80 2133.60 50.80

Three mice of the negative control population exhibited substantially the same amount of swelling in the largest footpads of both the left and right hind feet. Of the remaining three mice, only mouse #3 exhibited a significantly greater amount of swelling in the largest food pad of her right hind foot than in her left hind foot. On average, the difference in swelling between the largest footpads on the right and left hind feet of the mice of the negative control population was only about 16.33 μm.

Collectively, the data presented in TABLES 6-9 indicate the result of EXAMPLE 6 to be that both avian antibody and avian transfer factor specific for synthetic Hepatitis B vaccine cause mammals to elicit an early secondary immune response to the antigen of the synthetic Hepatitis B vaccine, which is also presented by the Hepatitis B virus.

EXAMPLE 7

Again employing substantially the same procedures outlined above in EXAMPLES 1-3, avian transfer factor and avian antibody specific for the H. pylori bacteria were generated in hens. Each of the hens was infected with the H. pylori EIA antigen, in a manner similar to that described in EXAMPLE 1, at day 150, day 163, day 190, day 221, and day 249. Eggs were collected from these hens during the period of about day 193 to about day 223, as described in EXAMPLE 1, and prepared, as described in EXAMPLE 1.

As in the previous EXAMPLES, a positive control population of mice was prepared about seven (7) days prior to conducting the mouse footpad assay by injecting each of the mice of the positive control population with the recombinant, or synthetic, H. pylori EIA antigen, as described in reference to FIG. 4.

A solution including both avian antibody and avian transfer factor specific for the H. pylori EIA antigen was made by reconstituting in distilled water a lyophilized preparation including such avian antibody and avian transfer factor, similar to the preparation described above in EXAMPLE 2, to a concentration of about 16%, by weight. This solution was administered to a first test population of mice, as described previously herein in reference to FIG. 4.

A substantially antibody-free solution including avian transfer factor specific for H. pylori was prepared by reconstituting a lyophilized preparation, obtained in a manner similar to that described in EXAMPLE 3, in distilled water to a concentration of about 16%, by weight. This substantially antibody-free avian transfer factor-containing solution was then administered to each of the mice of a second test population, as described previously herein in reference to FIG. 4.

The largest footpad of the right foot of each mouse of each of the positive control, first test, second test, and negative control populations, was infected with H. pylori EIA antigen, while the same amount of sterile saline diluent was administered to the largest footpad of the left foot of each of these mice in the manner detailed previously herein in reference to FIG. 4.

At the appropriate time, about sixteen (16) to about twenty-four (24) hours following the infection of the largest footpads of the right feet of the mice with H. pylori, the mice were again anesthetized and the sizes of the largest footpads of both hind feet of each mouse was measured, as described previously herein. The results follow:

TABLE 10 H. Pylori - First Test Population (Antibody and Transfer Factor Administered) Footpad size (μm): Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 1955.80 1981.20 25.40 Right Foot (Test) 1930.40 1955.80 25.40 Mouse #2 Left Foot (Control) 2133.60 2133.60 0.00 Right Foot (Test) 2108.20 2260.60 152.40 Mouse #3 Left Foot (Control) 2082.80 2082.80 0.00 Right Foot (Test) 2108.20 2133.60 25.40 Mouse #4 Left Foot (Control) 2082.80 2184.40 101.60 Right Foot (Test) 2082.80 2286.00 203.20 Mouse #5 Left Foot (Control) 2108.20 2133.60 25.40 Right Foot (Test) 2133.60 2133.60 0.00 Mouse #6 Left Foot (Control) 1955.80 2032.00 76.20 Right Foot (Test) 1930.40 2108.20 177.80

The data of TABLE 10 and, particularly those of Mouse #2, Mouse #4, and Mouse #6, indicate that administration of the solution containing both avian antibody and avian transfer factor specific for H. pylori induced an early secondary immune response in the mice of the first test population. While Mouse #1 exhibited substantially equal amounts of swelling in the largest footpads of both her left and right hind feet, Mouse #3 exhibited slightly greater swelling in the largest footpad of her right hind foot than in that of her left hind foot and Mouse #5 exhibited a slightly greater amount of swelling in the largest footpad of her left hind foot than in the largest footpad of her right hind foot. On average, the largest footpads of the right hind feet of the mice of the first test population were about 59.27 μm more swollen than the largest footpads of the left hind feet of these mice.

TABLE 11 H. Pylori - Second Test Population (Only Transfer Factor Administered) Footpad size (μm): Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2235.20 2235.20 0.00 Right Foot (Test) 2184.40 2209.80 25.40 Mouse #2 Left Foot (Control) 2006.60 2006.60 0.00 Right Foot (Test) 2006.60 2032.00 25.40 Mouse #3 Left Foot (Control) 2082.80 2184.40 101.60 Right Foot (Test) 2133.60 2209.80 76.20 Mouse #4 Left Foot (Control) 2133.60 2133.60 0.00 Right Foot (Test) 2133.60 2159.00 25.40 Mouse #5 Left Foot (Control) 2159.00 2184.40 25.40 Right Foot (Test) 2159.00 2235.20 76.20 Mouse #6 Left Foot (Control) 2057.40 2082.80 25.40 Right Foot (Test) 2032.00 2260.60 228.60

The results shown in TABLE 11 were similar to those in TABLE 10. Two of the mice, Mouse #5 and Mouse #6, exhibited much more swelling in the largest footpads of their right hind feet than in the largest footpads of their left hind feet. While the amount of swelling in the largest footpads of the right hind feet of Mouse #1, Mouse #2, and Mouse #4 was greater than that of the largest footpads of the left hind feet of these mice, the difference was only slight. Mouse #3 actually exhibited a slightly greater amount of swelling in the largest footpad of her left hind foot than in the largest footpad of her right hind foot. Nonetheless, as the average swelling in the largest footpads of the right hind feet of these mice is, on average, about 50.80 μm greater than that of the largest footpads on the left hind feet of these mice, the data of TABLE 11 indicate that avian transfer factor specific for H. pylori caused the increased swelling.

TABLE 12 H. Pylori - Positive Control Footpad size (μm): Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2133.60 2133.60 0.00 Right Foot (Test) 2108.20 2184.40 76.20 Mouse #2 Left Foot (Control) 2133.60 2133.60 0.00 Right Foot (Test) 2133.60 2209.80 76.20 Mouse #3 Left Foot (Control) 2032.00 2108.20 76.20 Right Foot (Test) 2082.80 2209.80 127.00 Mouse #4 Left Foot (Control) 1981.20 2082.80 101.60 Right Foot (Test) 1879.60 2133.60 254.00 Mouse #5 Left Foot (Control) 2133.60 2159.00 25.40 Right Foot (Test) 2184.40 2336.80 152.40 Mouse #6 Left Foot (Control) 2133.60 2133.60 0.00 Right Foot (Test) 2082.80 2260.60 177.80

Each of the mice of the positive control population in EXAMPLE 7 elicited at delayed-type hypersensitivity immune response to H. pylori, as indicated by the significant differences in the amount of swelling in the largest footpads of the right hind feet of these mice relative to that in the largest footpads of the left hind feet of these mice. On average, the difference in swelling was about 110.07 μm.

TABLE 13 H. Pylori - Negative Control Footpad size (μm): Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2006.60 2082.80 76.20 Right Foot (Test) 2514.60 2514.60 0.00 Mouse #2 Left Foot (Control) 2032.00 2082.80 50.80 Right Foot (Test) 2032.00 2133.60 101.60 Mouse #3 Left Foot (Control) 2082.80 2108.20 25.40 Right Foot (Test) 2082.80 2108.20 25.40 Mouse #4 Left Foot (Control) 2006.60 2032.00 25.40 Right Foot (Test) 1955.80 2032.00 76.20 Mouse #5 Left Foot (Control) 1930.40 1981.20 50.80 Right Foot (Test) 1955.80 2006.60 50.80 Mouse #6 Left Foot (Control) 2133.60 2159.00 25.40 Right Foot (Test) 2133.60 2159.00 25.40

As indicated by the data of TABLE 13, the amount of swelling in the largest footpads of both the left and right hind feet of Mouse #3, Mouse #5, and Mouse #6, were substantially the same. While the amount of swelling in the largest footpad of the right hind foot of Mouse #2 was greater than the amount of swelling in the largest footpad of the left hind foot of that mouse, the largest footpad of the left hind foot of Mouse #1 was significantly more swollen than the largest footpad of the right hind foot of Mouse #1. The largest footpad of the right hind foot of Mouse #4 was only slightly more swollen than the largest footpad of the left hind foot of Mouse #4. The average difference in swelling of the largest footpads of the right and left hind feet of the mice of the negative control population was only about 4.23 μm.

The data of TABLES 10-13 indicate that avian transfer factor specific for H. pylori facilitates an early secondary immune response in mammals.

EXAMPLE 8

Again, employing substantially the same procedures described previously herein in EXAMPLES 1-3, avian transfer factor and avian antibody specific for the EBNA-1 antigen, a recombinant nuclear antigen of the Epstein-Barr virus (EBV), were generated in hens. Each hen received one dose of EBNA-1, such as described in EXAMPLE 1, at 150 days, 163 days, 190 days, and 249 days. Eggs were collected from these hens during the period of about day 193 to about day 223, as described above in EXAMPLE 1, and prepared as described above in EXAMPLE 1.

A solution with both avian antibody and avian transfer factor specific for EBNA-1 was formed by reconstituting in distilled water a lyophilized preparation similar to that described in EXAMPLE 2. The lyophilized preparation including both avian antibody and avian transfer factor specific for EBNA-1 antigen was diluted to a concentration of about 16%, by weight. This solution was then administered to a first test population of mice in the manner described in reference to FIG. 4.

In addition, a solution containing avian transfer factor specific for EBNA-1, with substantially no avian antibody specific for EBNA-1, was also reconstituted in distilled water to a concentration of about 16%, by weight. This solution was administered to the mice of a second test population in the manner described previously herein in reference to FIG. 4.

A positive control population of mice was prepared by injecting mice with EBNA-1 about seven (7) days before conducting the mouse footpad assay.

Recombinant EBNA-1 antigen was then administered to the largest footpad of the right hind foot of each mouse of each of four populations, including a first test population, a second test population, a positive control population, and a negative control population. Substantially the same amount of sterile saline diluent was administered to the largest footpad of the left hind foot of each mouse. The method of administration was conducted in the same manner as that described previously herein.

About sixteen (16) to about twenty-four (24) hours later, the mice were again anesthetized and the sizes of the largest footpads of both hind feet of each mouse measured, as previously described. The results follow:

TABLE 14 EBV EBNA-1 - First Test Population (Antibody and Transfer Factor Administered) Footpad size (μm): Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2032.00 2057.40 25.40 Right Foot (Test) 2032.00 2057.40 25.40 Mouse #2 Left Foot (Control) 2159.00 2159.00 0.00 Right Foot (Test) 2159.00 2184.40 25.40 Mouse #3 Left Foot (Control) 2159.00 2159.00 0.00 Right Foot (Test) 2133.60 2286.00 152.40 Mouse #4 Left Foot (Control) 2108.20 2108.20 0.00 Right Foot (Test) 2108.20 2209.80 101.60 Mouse #5 Left Foot (Control) 2108.20 2235.20 127.00 Right Foot (Test) 2082.80 2260.60 177.80 Mouse #6 Left Foot (Control) 1981.20 2032.00 50.80 Right Foot (Test) 1981.20 2032.00 50.80

In TABLE 14, it is seen that three of the mice exhibited significantly greater swelling in the largest footpads of their right hind feet than in the largest footpads of their left hind feet. While Mouse #2 also had a greater amount of swelling in the largest footpad of her right hind foot than that in the largest footpad of her left hind foot, the difference was only slight. Two of the mice, Mouse #1 and Mouse #6, had substantially the same amount of swelling in the largest footpads of both their left and right hind feet. Nonetheless, as the amount of swelling in the largest footpads of the right hind feet of the mice of the first test population exceeded that of the largest footpads of the left hind feet of these mice by an average of about 55.03 μm, the data presented in TABLE 14 tend to show that the avian transfer factor in the solution containing both avian antibody and transfer factor specific for EBNA-1 caused the mice of the first test population to elicit an early secondary immune response to the recombinant EBNA-1. As is known in the art, antibodies are passive with respect to secondary immune responses and typically contribute very little to swelling.

TABLE 15 EBV EBNA-1 - Second Test Population (Only Transfer Factor Administered) Footpad size (μm): Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2133.60 2159.00 25.40 Right Foot (Test) 2108.20 2159.00 50.80 Mouse #2 Left Foot (Control) 2006.60 2032.00 25.40 Right Foot (Test) 1955.80 1955.80 0.00 Mouse #3 Left Foot (Control) 2032.00 2133.60 101.60 Right Foot (Test) 2006.60 2159.00 152.40 Mouse #4 Left Foot (Control) 2108.20 2133.60 25.40 Right Foot (Test) 2159.00 2159.00 0.00 Mouse #5 Left Foot (Control) 2184.40 2209.80 25.40 Right Foot (Test) 2159.00 2260.60 101.60 Mouse #6 Left Foot (Control) 2057.40 2108.20 50.80 Right Foot (Test) 2082.80 2133.60 50.80

The mice of the second test population, which were treated with the avian transfer factor-containing solution also exhibited an early secondary immune response to recombinant EBNA-1. This result was particularly evident in Mouse #3 and Mouse #5, which exhibited significantly greater swelling in the largest footpads of their right hind feet than that measured in the largest footpads of their left hind feet. While the amount of swelling in the largest footpad of the right hind foot of Mouse #1 was also greater than the amount of swelling in the largest footpad of the left hind foot of Mouse #1, the difference appears to be slight. Moreover, while Mouse #2 and Mouse #4 displayed a greater amount of swelling in the largest footpads of their left hind feet, the amounts of swelling measured therein were only slightly greater than that measured in the largest footpads of the right hind feet of these mice. On average, the largest footpads of the right hand feet of these mice was about 16.93 μm greater than that measured in the largest footpads of the left hind feet of these mice.

It is believed that transfer factor specific for EBNA-1 may have become unstable when isolated from the corresponding antibody, resulting in the lower measured secondary immune response in the second test population relative to the overall secondary immune response measured in the first test population of mice.

TABLE 16 EBV EBNA-1 - Positive Control Footpad size (μm): Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2209.80 2209.80 0.00 Right Foot (Test) 2235.20 2286.00 50.80 Mouse #2 Left Foot (Control) 2184.40 2184.40 0.00 Right Foot (Test) 2209.80 2260.60 50.80 Mouse #3 Left Foot (Control) 2159.00 2159.00 0.00 Right Foot (Test) 2133.60 2209.80 76.20 Mouse #4 Left Foot (Control) 2159.00 2336.80 177.80 Right Foot (Test) 2133.60 2362.20 228.60 Mouse #5 Left Foot (Control) 2133.60 2133.60 0.00 Right Foot (Test) 2082.80 2260.60 177.80 Mouse #6 Left Foot (Control) 2082.80 2082.80 0.00 Right Foot (Test) 2057.40 2209.80 152.40

As indicated by the greater amounts of swelling in the largest footpads of the right hind feet of each mouse of the positive control population than that of the largest footpads of the left hind feet of these mice, all six of the mice of the positive control population exhibited a delayed-type hypersensitivity immune response to the recombinant EBNA-1 antigen. The measured amount of swelling in the largest footpads of the right hind feet of each of these mice was, on average, about 93.13 μm greater than the measured amount of swelling in the largest footpads of the left hind feet of these mice.

TABLE 17 EBV EBNA-1 - Negative Control Footpad size (μm): Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2133.60 2184.40 50.80 Right Foot (Test) 2082.80 2133.60 50.80 Mouse #2 Left Foot (Control) 2133.60 2133.60 0.00 Right Foot (Test) 2159.00 2184.40 25.40 Mouse #3 Left Foot (Control) 2108.20 2108.20 0.00 Right Foot (Test) 2133.60 2133.60 0.00 Mouse #4 Left Foot (Control) 2082.80 2133.60 50.80 Right Foot (Test) 2159.00 2159.00 0.00 Mouse #5 Left Foot (Control) 2057.40 2082.86 25.40 Right Foot (Test) 1955.80 1981.20 25.40 Mouse #6 Left Foot (Control) 2108.20 2133.60 25.40 Right Foot (Test) 2108.20 2133.60 25.40

In the negative control population, only two of the mice, Mouse #2 and Mouse #4, exhibited different amounts of swelling in the largest footpads of their hind feet. While the amount of swelling in the largest footpad of the right hind foot of Mouse #2 was greater than that exhibited in the largest footpad of the left hind foot, the largest footpad of the left hind foot of Mouse #4 was more swollen than the largest footpad of the right hind foot of Mouse #4. In fact, on average, the largest footpads of the right hind feet of the mice of the negative control population were about 4.23 μm less swollen than the largest footpads of the left hind feet of these mice.

Again, the data of TABLES 14-17 illustrate that avian transfer factor specific for EBNA-1 cause mammals to elicit an early secondary immune response (i.e., within about twenty-four (24) hours as compared to the typical seven (7) to fourteen (14) day time period it takes a mammal to elicit a secondary immune response on its own) to EBNA-1 and viruses and other pathogens that present this antigen.

The foregoing EXAMPLES illustrate that, by way of contrast with the seven (7) to fourteen (14) day time period that it typically takes a mammalian host to elicit a secondary immune response to a pathogen or antigenic agent on its own, when an avian transfer factor incorporating teachings of the present invention has been administered, the mammalian host may elicit a secondary immune response within about twenty-four (24) hours.

The similarities of the differences between the measurements taken at the test and control footpads of each mouse in first and second test groups of each assay indicate that the secondary, or delayed-type hypersensitivity, immune response, was elicited primarily by the transfer factor, not the antibody, which is passive with respect to secondary immune responses and which typically contributes very little to swelling.

It is apparent from EXAMPLES 1-8 and the data generated thereby that avian transfer factor has the ability to generate an early secondary immune response in mammals. As one of skill in the art would readily recognize, avian transfer factor would also generate an early secondary immune response in various types of birds, as well as in reptiles, amphibians, and other non-mammalian species of animals.

As avian transfer factor initiates an early delayed-type hypersensitivity immune reaction in mice, it is reasonable for those of ordinary skill in the art to assume that transfer factor has the same effect in other mammals, including humans.

Although transfer factor was administered to mice in the preceding EXAMPLES by way of injection, it is also within the scope of the present invention to administer avian transfer factor to mammals by other routes. For example, avian transfer factor could be administered orally, by parenteral injection, or by parenteral methods other than injection, such as transdermally, or through the skin, by aerosol via the lungs, or by other methods known in the art. Oral administration of avian transfer factor to mammals is supported by the fact that mammalian mothers supply transfer factor to their newborn children by way of colostrum, which the newborns ingest orally. Transfer factor survives the conditions of both the stomach and the small intestine, where transfer factor is absorbed into the bloodstream of the mammalian newborn. Thus, transfer factor is known to survive the intestinal tracts of mammals. The ability of transfer factor to withstand the conditions of the digestive tracts of mammals was demonstrated in Kirkpatrick C H, "Activities and characteristics of transfer factors," Biotherapy, 9: 13-16 (1996), the disclosure of which is hereby incorporated by this reference in its entirety.

Although the foregoing description contains many specifics, these should not be construed as limiting the scope of the present invention, but merely as providing illustrations of some of the presently preferred embodiments. Similarly, other embodiments of the invention may be devised which do not depart from the spirit or scope of the present invention. Features from different embodiments may be employed in combination. The scope of the invention is, therefore, indicated and limited only by the appended claims and their legal equivalents, rather than by the foregoing description. All additions, deletions and modifications to the invention as disclosed herein which fall within the meaning and scope of the claims are to be embraced thereby.

* * * * * Other References

* Fudenberg, H. H., et al., "Transfer Factor 1993: New Frontiers," Progress in Drug Research, vol. 42 (1994), pp. 309-318 * Qureshi, M.A., et al., "Understanding Immunology in Disease Development and Control," Poultry Science, vol. 77 (1998), pp. 1126-1129 * Sharma, J.M., "The Structure and Function of the Avian Immune System," Acta Veterinaria Hungarica, vol. 45(3) (1997), pp. 229-238 * Xlth International Congress on Transfer Factor, Universidad Autonoma de Nuevo Leon, Mar. 1999 * Egcel™ and BioChoice™, Overview for Health Care Professionals, DCV, Apr. 1999, pp. 1-4 * Millipore Sterile Membrane Filters, http://www.millipore.com. (2000) * Celite Filter Media; RH 1010, Funnel, Buchner Type; http://www.celtic-eng.com, http://www.glassfilter.com, http://www.worldminerals.com. (2000) * Fabio, Anthony di, "Scope of Protection Immune Milk." pp. 1-8, The Arthritis Trust, Dedicated to Eradicating Rheumatoid Disease, from the Earth, 2000 * Klesius et al., "Adoptive transfoer of Delayed Hypersensitivity and Protective Immunity to Elmeria Tenalta with Chicken-Derived Transfer Factor,"pp. 1333-1337, Poultry Science, vol. 63, 1984 * Glambrone et al., "Adoptive Transfer of Delayed Wattle Reactivity in Chickens wiht a Dialyzable Leukocyte Extract Containing Transfer Factor," pp. 767-771, Poultry Science, vol. 62, 198



Inventors

* Hennen, William J. * Lisonbee, David T.

Assignee

* 4Life Research, LC

Application No. 667147 filed on 09/21/2000

US Classes: 424/157.1, Derived from, or present in, food product (e.g., milk, colostrum, whey, eggs, etc.)424/130.1, IMMUNOGLOBULIN, ANTISERUM, ANTIBODY, OR ANTIBODY FRAGMENT, EXCEPT CONJUGATE OR COMPLEX OF THE SAME WITH NONIMMUNOGLOBULIN MATERIAL424/184.1, ANTIGEN, EPITOPE, OR OTHER IMMUNOSPECIFIC IMMUNOEFFECTOR (E.G., IMMUNOSPECIFIC VACCINE, IMMUNOSPECIFIC STIMULATOR OF CELL-MEDIATED IMMUNITY, IMMUNOSPECIFIC TOLEROGEN, IMMUNOSPECIFIC IMMUNOSUPPRESSOR, ETC.)424/201.1, Combination of viral and bacterial antigens (e.g., multivalent viral and bacterial vaccine, etc.)424/204.1, Virus or component thereof424/227.1, Hepatitis B virus (e.g., hepatitis B surface antigen (HBsAg), pre-S region, hepatitis B core antigen (HBcAg), hepatitis B e-antigen, Dane particle, etc.)435/41, MICRO-ORGANISM, TISSUE CELL CULTURE OR ENZYME USING PROCESS TO SYNTHESIZE A DESIRED CHEMICAL COMPOUND OR COMPOSITION530/300, PEPTIDES OF 3 TO 100 AMINO ACID RESIDUES530/350PROTEINS, I.E., MORE THAN 100 AMINO ACID RESIDUES

Examiners Primary: Park, Hankyel T. Assistant: Brown, Stacy S.

Attorney, Agent or Firm

* Traskbritt

International Classes A61K 039/395 A61K 039/00 A61K 039/12 C12P 001/00 C07K 001/00



Abstract

A composition for eliciting a T-cell mediated immune response in a subject includes transfer factor from at least two different types of source animals. For example, the composition may include mammalian transfer factor and nonmammalian transfer factor. An example of the composition includes a combination of a colostrum-derived product, which includes the mammalian transfer factor, and an egg-derived product, which includes the nonmammalian transfer factor. Additionally, the egg-derived product may be substantially free of fat. Methods for forming the composition and eliciting T-cell mediated immune responses in subjects that have been treated with the composition are also disclosed. Claims



What is claimed:

1. A method for reducing the cleaning frequency of processing equipment used for capsulating an egg-derived product, comprising:

combining a colostrum-derived product with an egg-derived product before or during introduction of the egg-derived product into the capsulation equipment.

2. The method of claim 1, wherein said combining comprises combining about equal weights of said colostrum-derived product and the egg-derived product.

3. The method of claim 1, wherein said combining comprises combining said colostrum-derived product in a greater amount, by weight, than the egg-derived product with the egg-derived product.

4. The method of claim 1, wherein said combining comprises combining said colostrum-derived product, in a lesser amount, by weight, than the egg-derived product with the egg-derived product.

5. The method of claim 1, further comprising:

defatting the egg-derived product.

6. The method of claim 1, further comprising:

combining at least one vitamin with at least one of the egg-derived product and said colostrum-derived product.

7. The method of claim 1, wherein said combining comprises combining said colostrum-derived product and the egg-derived product with at least one of said colostrum-derived product and the egg-derived product including transfer factor.

8. A method for reducing the cleaning frequency of equipment used for processing an egg-derived product, comprising:

combining a colostrum-derived product with an egg-derived product before or during introduction of the egg-derived product into the equipment.

9. The method of claim 8, wherein said combining comprises combining about equal weights of said colostrum-derived product and the egg-derived product.

10. The method of claim 8, wherein said combining comprises combining said colostrum-derived product in a greater amount, by weight, than the egg-derived product with the egg-derived product.

11. The method of claim 8, wherein said combining comprises combining said colostrum-derived product, in a lesser amount, by weight, than the egg-derived product with the egg-derived product.

12. The method of claim 8, further comprising:

defatting the egg-derived product.

13. The method of claim 8, further comprising:

combining at least one vitamin with at least one of the egg-derived product and said colostrum-derived product.

14. The method of claim 8, wherein said combining comprises combining said colostrum-derived product and the egg-derived product with at least one of said colostrum-derived product and the egg-derived product including transfer factor. Description



BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to compositions which include transfer factor and, more specifically, to compositions which include transfer factor from different types of source animals. The present invention also relates to methods for making compositions that include different types of transfer factor and to methods for eliciting or enhancing a T-cell mediated immune response by the immune system of a subject.

2. Background of Related Art

Many, deadly pathogens are passed to humans from the animal kingdom. For example, monkeys are the sources of the type I human immunodeficiency virus (HIV-I), which causes acquired immune deficiency syndrome (AIDS) and monkeypox, which is similar to smallpox; ground-dwelling mammals are believed to be the source of the Ebola virus; fruit bats and pigs are the source of the Nipah virus; the Hendra virus comes from horses; the virus responsible for the "Hong Kong Flu" originated in chickens; and wild birds, especially ducks, are the sources of many of the deadly influenza viruses. Many diseases also have animal reservoirs. By way of example, mice carry Hanta virus, rats carry the Black Plague, and deer carry Lyme disease.

The Immune System

The immune systems of vertebrates are equipped to recognize and defend the body from invading pathogenic organisms, such as parasites, bacteria, fungi, and viruses. Vertebrate immune systems typically include a cellular component and a noncellular component.

The cellular component of an immune system includes the so-called "lymphocytes," or white blood cells, of which there are several types. It is the cellular component of a mature immune system that typically mounts a primary, non-specific response to invading pathogens, as well as being involved in a secondary, specific response to pathogens.

In the primary, or initial, response to an infection by a pathogen, white blood cells that are known as phagocytes locate and attack the invading pathogens. Typically, a phagocyte will internalize, or "eat" a pathogen, then digest the pathogen. In addition, white blood cells produce and excrete chemicals in response to pathogenic infections that are intended to attack the pathogens or assist in directing the attack on pathogens.

Only if an infection by invading pathogens continues to elude the primary immune response is a specific, secondary immune response to the pathogen needed. As this secondary immune response is typically delayed, it is also known as "delayed-type hypersensitivity." A mammal, on its own, will typically not elicit a secondary immune response to a pathogen until about seven (7) to about fourteen (14) days after becoming infected with the pathogen. The secondary immune response is also referred to as an acquired immunity to specific pathogens. Pathogens have one or more characteristic proteins, which are referred to as "antigens." In a secondary immune response, white blood cells known as B lymphocytes, or "B-cells," and T lymphocytes, or "T-cells," "learn" to recognize one or more of the antigens of a pathogen. The B-cells and T-cells work together to generate proteins called "antibodies," which are specific for (e.g., configured to bind to or otherwise "recognize") one or more certain antigens on a pathogen.

The T-cells are primarily responsible for the secondary, or delayed-type hypersensitivity, immune response to a pathogen or antigenic agent. There are three types of T-cells: T-helper cells, T-suppressor cells, and antigen-specific T-cells, which are also referred to as cytotoxic (meaning "cell-killing") T-lymphocytes (CTLs), or T-killer cells or natural killer (NK) cells. The T-helper and T-suppressor cells, while not specific for certain antigens, perform conditioning functions (e.g., the inflammation that typically accompanies an infection) that assist in the removal of pathogens or antigenic agents from an infected host.

Antibodies, which make up only a part of the noncellular component of an immune system, recognize specific antigens and, thus, are said to be "antigen-specific." The generated antibodies then basically assist the white blood cells in locating and eliminating the pathogen from the body. Typically, once a white blood cell has generated an antibody against a pathogen, the white blood cell and all of its progenitors continue to produce the antibody. After an infection is eliminated, a small number of T-cells and B-cells that correspond to the recognized antigens are retained in a "resting" state. When the corresponding pathogenic or antigenic agents again infect the host, the "resting" T-cells and B-cells activate and, within about forty-eight (48) hours, induce a rapid immune response. By responding in this manner, the immune system mounts a secondary immune response to a pathogen, the immune system is said to have a "memory" for that pathogen.

Mammalian immune systems are also known to produce smaller proteins, known as "transfer factors," as part of a secondary immune response to infecting pathogens. Transfer factors are another noncellular part of a mammalian immune system. Antigen-specific transfer factors are believed to be structurally analogous to antibodies, but on a much smaller molecular scale. Both antigen-specific transfer factors and antibodies include antigen-specific sites. In addition, both transfer factors and antibodies include highly conserved regions that interact with receptor sites on their respective effector cells. In transfer factor and antibody molecules, a third, "linker," region connects the antigen-specific sites and the highly conserved regions.

The Role of Transfer Factor in the Immune System

Transfer factor is a low molecular weight isolate of lymphocytes. Narrowly, transfer factors may have specificity for single antigens. U.S. Pat. Nos. 5,840,700 and 5,470,835, both of which issued to Kirkpatrick et al. (hereinafter collectively referred to as "the Kirkpatrick Patents"), disclose the isolation of transfer factors that are specific for certain antigens. More broadly, "specific" transfer factors have been generated from cell cultures of monoclonal lymphocytes. Even if these transfer factors are generated against a single pathogen, they have specificity for a variety of antigenic sites of that pathogen. Thus, these transfer factors are said to be "pathogen-specific" rather than antigen-specific. Similarly, transfer factors that are obtained from a host that has been infected with a certain pathogen are pathogen-specific. Although such preparations are often referred to in the art as being "antigen-specific" due to their ability to elicit a secondary immune response when a particular antigen is present, transfer factors having different specificities may also be present in such preparations. Thus, even the so-called "antigen-specific," pathogen-specific transfer factor preparations may be specific for a variety of antigens.

Additionally, it is believed that antigen-specific and pathogen-specific transfer factors may cause a host to elicit a delayed-type hypersensitivity immune response to pathogens or antigens for which such transfer factor molecules are not specific. Transfer factor "draws" at least the non-specific T-cells, the T-inducer and the T-suppressor cells, to an infecting pathogen or antigenic agent to facilitate a secondary, or delayed-type hypersensitivity, immune response to the infecting pathogen or antigenic agent.

Typically, transfer factor includes an isolate of proteins having molecular weights of less than about 10,000 daltons (D) that have been obtained from immunologically active mammalian sources. It is known that transfer factor, when added either in vitro or in vivo to mammalian immune cell systems, improves or normalizes the response of the recipient mammalian immune system.

The immune systems of newborns have typically not developed, or "matured," enough to effectively defend the newborn from invading pathogens. Moreover, prior to birth, many mammals are protected from a wide range of pathogens by their mothers. Thus, many newborn mammals cannot immediately elicit a secondary response to a variety of pathogens. Rather, newborn mammals are typically given secondary immunity to pathogens by their mothers. One way in which mothers are known to boost the immune systems of newborns is by providing the newborn with a set of transfer factors. In mammals, transfer factor is provided by a mother to a newborn in colostrum, which is typically replaced by the mother's milk after a day or two. Transfer factor basically transfers the mother's acquired, specific (i.e., delayed-type hypersensitive) immunity to the newborn. This transferred immunity typically conditions the cells of the newborn's immune system to react against pathogens in an antigen-specific manner, as well as in an antigen- or pathogen-nonspecific fashion, until the newborn's immune system is able on its own to defend the newborn from pathogens. Thus, when transfer factor is present, the immune system of the newborn is conditioned to react to pathogens with a hypersensitive response, such as that which occurs with a typical delayed-type hypersensitivity response. Accordingly, transfer factor is said to "jump start" the responsiveness of immune systems to pathogens.

Much of the research involving transfer factor has been conducted in recent years. Currently, it is believed that transfer factor is a protein with a length of about forty-four (44) amino acids. Transfer factor typically has a molecular weight in the range of about 3,000 to about 5,000 Daltons (Da ), or about 3 kDa to about 5 kDa, but it may be possible for transfer factor molecules to have molecular weights outside of this range. Transfer factor is also believed to include three functional fractions, each of which may include different types of transfer factor molecules: an inducer fraction; an immune suppressor fraction; and an antigen-specific fraction. Many in the art believe that transfer factor also includes a nucleoside portion, which could be connected to the protein molecule or separate therefrom, that may enhance the ability of transfer factor to cause a mammalian immune system to elicit a secondary immune response. The nucleoside portion may be part of the inducer or suppressor fractions of transfer factor.

The antigen-specific region of the antigen-specific transfer factors is believed to comprise about eight (8) to about twelve (12) amino acids. A second highly-conserved region of about ten (10) amino acids is thought to be a very high-affinity T-cell receptor binding region. The remaining amino acids may serve to link the two active regions or may have additional, as yet undiscovered properties. The antigen-specific region of a transfer factor molecule, which is analogous to the known antigen-specific structure of antibodies, but on a much smaller molecular weight scale, appears to be hyper-variable and is adapted to recognize a characteristic protein on one or more pathogens. The inducer and immune suppressor fractions are believed to impart transfer factor with its ability to condition the various cells of the immune system so that the cells are more fully responsive to the pathogenic stimuli in their environment.

Sources of Noncellular Immune System Components

Conventionally, transfer factor has been obtained from the colostrum of milk cows, such as by the method described in U.S. Pat. No. 4,816,563 to Wilson et al. (hereinafter "Wilson"). While milk cows typically produce large amounts of colostrum and, thus, large amounts of transfer factor over a relatively short period of time, milk cows only produce colostrum for about a day or a day-and-a-half every year. Thus, milk cows are neither a constant source of transfer factor nor an efficient source of transfer factor.

Transfer factor has also been obtained from a wide variety of other mammalian sources. For example, in researching transfer factor, mice have been used as a source for transfer factor. Antigens are typically introduced subcutaneously into mice, which are then sacrificed following a delayed-type hypersensitivity reaction to the antigens. Transfer factor is then obtained from spleen cells of the mice.

While different mechanisms are typically used to generate the production of antibodies, the original source for antibodies may also be mammalian. For example, monoclonal antibodies may be obtained by injecting a mouse, a rabbit, or another mammal with an antigen, obtaining antibody-producing cells from the mammal, then fusing the antibody-producing cells with immortalized cells to produce a hybridoma cell line, which will continue to produce the monoclonal antibodies throughout several generations of cells and, thus, for long periods of time.

Antibodies against mammalian pathogens have been obtained from a wide variety of sources, including mice, rabbits, pigs, cows, and other mammals. In addition, the pathogens that cause some human diseases, such as the common cold, are known to originate in birds. As it has become recognized that avian (i.e., bird) immune systems and mammalian immune systems are very similar, some researchers have turned to birds as a source for generating antibodies.

Avian antibodies that are specific for pathogens that infect mammals, or "mammalian pathogens," have been obtained by introducing antigens into eggs. Alternatively, antibodies may be present in eggs following exposure of the source animal to antigens, including antigens of mammalian pathogens. U.S. Pat. No. 5,080,895, issued to Tokoro on Jan. 14, 1992 (hereinafter "the '895 patent"), discloses a method that includes injecting hens with pathogens that cause intestinal infectious diseases in neonatal mammals. The hens then produce antibodies that are specific for these pathogens, which are present in eggs laid by the hens. The '895 patent discloses compositions that include these pathogen-specific antibodies and use thereof to treat and prevent intestinal diseases in neonatal piglets and calves. Treatment of pathogenic infections in mammals with avian antibodies may have undesirable results, however, since the immune systems of mammals may respond negatively to the large avian antibody molecules by eliciting an immune response to the antibodies themselves. Moreover, as mammalian immune systems do not recognize avian antibodies as useful for their abilities to recognize certain pathogens, or the specificities of avian antibodies for antigens of such pathogens, avian antibodies often do not elicit the desired immune responses in mammals.

It is also known that transfer factor may be obtained from eggs. U.S. Pat. No. 6,468,534 to Hennen et al. (hereinafter "Hennen") describes a process by which female chickens (i.e., hens) are exposed to one or more antigens, which results in the elicitation of an immune response, including a secondary immune response, by the chickens. As a result of the secondary immune response, transfer factor molecules are present in the eggs of the chicken. The eggs may then be processed to provide a product in which the transfer factor is present. Such a product may take the form of a freeze-dried, or lyophilized, egg powder, and may include all or part of the egg. The egg powder may then be incorporated directly into gelatin capsules or mixed with other substances, then introduced into gelatin capsules.

FIG. 2 schematically depicts capsulation equipment of a type that is currently useful for capsulating egg-derived avian transfer factor in the form of an egg powder. Capsulation equipment 20 includes a composition supply hopper 24, a feed station 28, and an auger 26 in communication between each composition supply hopper 24 and feed station 28. Auger 26 transports the whole egg powder from composition supply hopper 24 to feed station 28.

When auger 26 operates, it is heated to a temperature which exceeds the relatively low melting point of cholesterol, from egg yolk, in the whole egg powder. The warmed cholesterol is sticky, coating auger 26, the conduit in communication therewith, and feed station 28, thereby decreasing the efficiency with which capsulation equipment 20 operates. Consequently, capsulation equipment must be disassembled and cleaned periodically, which may take a considerable amount of time (e.g., up to about 8 hours), resulting in a significant decrease in the productivity of capsulation equipment 20 and, thus, the number of capsules that may be formed therewith. Thus, processing of whole egg powder to obtain a transfer factor-containing product is somewhat undesirable.

Additionally, compositions which are derived from products (e.g., eggs or colostrum) from a single source animal typically only include transfer factor molecules which have specificity to antigens to which the source animal has been exposed. The consequence of such limited exposure may be that the effectiveness of such transfer factor-containing compositions in preventing or treating certain types of infections or conditions is also limited.

Accordingly, there is a need for a composition which is useful for causing an immune system of a treated subject to elicit an immune response to a broader array of pathogens, as well as for a method for improving the efficiency and productivity with which capsulation and other composition-forming equipment operates.

SUMMARY OF THE INVENTION

The present invention includes a composition for eliciting a T-cell mediated immune response in a subject. The composition includes transfer factor from at least two different types of source animals. The term "type," as used herein with respect to source animals, describes the source animals from which transfer factor may be obtained and refers to source animals from different classes (e.g. mammals, birds, reptiles, amphibians, insects, etc.). The term "type," as used herein, also refers to source animals from different subclasses, orders (e.g., artiodactyls, primates, carnivores, etc.), families (bovine, hominids, felines, etc.), subfamilies, genuses (e.g., cattle, humans, domestic cats, etc.), and even species and subspecies. Use of the term "type" herein with respect to transfer factor denotes the type of source animal from which the transfer factor was obtained.

An exemplary embodiment of the composition includes transfer factor from both mammalian and nonmammalian source animals, which types of transfer factor are also referred to herein as "mammalian transfer factor" and "nonmammalian transfer factor," respectively. By way of nonlimiting example, the mammalian transfer factor may be included in the composition as colostrum or a fraction or extract thereof, which are collectively referred to herein as "colostrum-derived products," or otherwise, as known in the art (e.g., as a leukocyte (white blood cell) extract, as a splenic ("from the spleen") extract, etc.). Also by way of example, the nonmammalian transfer factor of the exemplary composition may be obtained from an egg or a fraction or extract thereof, which are also referred to herein as "egg-derived products."

When a composition of the present invention includes a colostrum-derived product and an egg-derived product, both products may be included in the mixture in amounts (e.g., by weight, by volume, etc., of the total mixture) that are about equal, or more of one of the colostrum-derived product and the egg-derived product than the other.

In another aspect, the present invention includes a method for capsulating an egg-derived product which includes transfer factor. The inventive capsulation method includes mixing a substantially fat-free component, such as a colostrum-derived product, which may or may not include transfer factor, with the egg-derived product before or while the egg-derived product is being introduced into capsulation equipment.

Additionally, the present invention includes a method for reducing the cleaning frequency of capsulation equipment used for capsulating an egg-derived product. That method includes mixing a less fatty or substantially fat-free substance, such as a colostrum-derived product, with the egg-derived product before or during introduction of the egg-derived product into the capsulation equipment.

The present invention also includes methods for treating a subject. Treatment methods that incorporate teachings of the present invention include administration of a composition according to the present invention to a subject. As the composition includes transfer factor, administration of the composition to the subject will cause the subject's immune system to elicit a T-cell mediated immune response or will enhance a T-cell mediated immune response by the subject's immune system which is already underway.

Other features and advantages of the present invention will become apparent to those of ordinary skill in the art through consideration of the ensuing description, the accompanying drawings, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, which depict exemplary embodiments of various aspects of the present invention:

FIG. 1 depicts an example of the manner in which a composition that incorporates teachings of the present invention may be embodied; and

FIG. 2 is a schematic representation of capsulation equipment that may be used to introduce a powdered embodiment of the composition of the present invention into gelatin capsules.

DETAILED DESCRIPTION

An exemplary embodiment of composition that incorporates teachings of the present invention includes transfer factor from at least two different types of source animals. By way of nonlimiting example, a composition according to the present invention may include mammalian transfer factor and nonmammalian transfer factor.

The different types of transfer factor of the inventive composition may be obtained from any suitable source. For example, mammalian transfer factor may be obtained from colostrum, as described in Wilson, the disclosure of which is hereby incorporated herein in its entirety by this reference, or otherwise, as known in the art (e.g., a leukocyte (white blood cell) extract, a splenic (i.e., "from the spleen") extract, etc.). An exemplary source for nonmammalian transfer factor is an egg of an animal, such as a chicken, as described in Hennen, the disclosure of which is hereby incorporated herein in its entirety by this reference. Thus, a composition according to the present invention may include a first component which comprises a colostrum-derived product, as well as a second component that comprises an egg-derived product.

As compositions that incorporate teachings of the present invention include transfer factor from different types of source animals, they may include transfer molecules with a broader array of antigen-specificity or pathogen-specificity than conventional transfer factor-containing compositions. Thus, a composition according to the present invention is capable of enlisting the immune system of a treated animal to elicit a T-cell mediated immune response against a broader array of pathogens than those against which conventional transfer factor-containing compositions are effective. This is because different types of animals may be exposed to different types of antigens or pathogens, such as by vaccination, the animals' environments, or the like.

As an example, a composition which includes transfer factor-containing components from both cows and chickens will include transfer factor molecules which are specific to antigens or pathogens to which cows are exposed, as well as transfer factor molecules that have specificity for antigens or pathogens to which chickens are exposed. As both cows and chickens may be exposed to antigens or pathogens to which the other is not exposed, such a composition may include transfer factor molecules with antigen or pathogen specificities that would not be present in a composition that includes only transfer factor from cows (e.g., by way of a colostrum-derived product) or transfer factor from chickens (e.g., through an egg-derived product).

A composition of the present invention may include about the same amounts, measured in terms of weight or volume, of a colostrum-derived product and an egg-derived product (i.e., about 50% colostrum-derived product and about 50% egg-derived product). Alternatively, a composition that incorporates teachings of the present invention may include more colostrum-derived product (e.g., about 85% or 60%, by combined weight of the colostrum-derived product and egg-derived product) than egg-derived product (about 15% or 40%, by weight). As another alternative, the inventive composition may include more egg-derived product (e.g., about 60% or 85%, by weight) than colostrum-derived product (e.g., about 40% or 15% by weight). As another example, a composition that incorporates teachings of the present invention may include about one percent, by weight, of one of a colostrum-derived product and an egg-derived product and about 99%, by weight, of the other of the colostrum-derived product and the egg-derived product. Although specific amounts of colostrum-derived product and egg-derived product have been provided, any combination thereof is within the scope of the present invention.

In addition to including a source of transfer factor (e.g., a colostrum-derived product, an egg-derived product, etc.), a composition that incorporates teachings of the present invention may include one or more other ingredients, including, but not limited to, vitamins, minerals, proteins, natural products (e.g., herbs, mushrooms, roots, etc., or extracts thereof, and the like. Additional ingredients may be useful for providing further advantages to subjects to which the composition is administered, or may enhance the ability of the transfer factor in the composition to elicit or enhance a secondary, or delayed-type hypersensitivity, immune response.

As shown in FIG. 1, without limiting the scope of the present invention, a composition 10 according to the present invention may take the form of a powdered or particulate substance, which includes the multiple types of transfer factor (not shown). In order to ensure that an appropriate and precise dosage of composition 10 is administered to a subject (not shown), composition 10 may be contained within a gelatin capsule 12 of a type which is well-known and readily available to those in the art. The result is the illustrated capsule 14. Alternatively, a composition according to the present invention may be embodied as tablet, a so-called "caplet," an unencapsulated powder, a liquid, a gel, or in any other pharmaceutically acceptable form. Suitable processes for placing the inventive composition into any such form are readily apparent to those of skill in the art.

In an exemplary embodiment of a method for making or forming a composition according to the present invention, a first type of transfer factor may be combined with a second type of transfer factor. Additionally, one or more other types of transfer factor may be combined with the first and second types of transfer factor. The different types of transfer factor that are combined may be substantially purified transfer factor, components or "products" that include transfer factor, or any combination thereof.

Turning again to FIG. 2, a process for forming composition-filled capsules 14, such as that shown in FIG. 1, is provided merely as an example for a method for making a composition that incorporates teachings of the present invention. As illustrated, the composition 10 is made and composition-filled capsules 14 are formed using standard capsulation equipment 20 of a type known in the industry, such as the SF-135 capsule-filling machine available from CapPlus Technologies of Phoenix, Ariz.

In addition to one or more composition supply hoppers 24, an auger 26 associated with each composition supply hopper 24, and a feed station 28 with which each auger 26 and the conduit 27 within which auger 26 is contained communicates, capsulation equipment 20 includes one or more capsule hoppers 30, as well as a pneumatic feed system 32 for transporting capsule bodies 12a and/or caps 12b to feed station 28.

As the capsulation equipment will introduce the mixture into capsules, which may be swallowed by a subject, it is currently preferred that the substantially fat-free component and the egg-derived product be introduced into the capsulation equipment in powdered form. The substantially fat-free component dilutes the amount, or concentration, of fat (e.g., from egg yolk) present in the mixture relative to the concentration of fat which is present in the egg-derived product. Accordingly, the relative amounts of the substantially fat-free product and the egg-derived product may be tailored to provide a fat concentration that will minimize clogging of the capsulation equipment.

Continuing with the example of a composition 10 which includes a colostrum-derived product 10a as the substantially fat-free component and an egg-derived product 10b, colostrum-derived product 10a and egg-derived product 10b may be introduced simultaneously into a single composition supply hopper 24 of capsulation equipment 20. For example, colostrum-derived product 10a and egg-derived product 10b may be mixed upon introduction thereof into composition supply hopper 24, as shown, or premixed. By introducing a substance which has a lower fat content than egg-derived product 10b into composition supply hopper 24 along with egg-derived product 10b, the fat content (e.g., concentration) of the resulting mixture is less than that of egg-derived product 10b, reducing or eliminating the likelihood that composition supply hopper 24, auger 26, conduit 27, feed station 28, or any other component of capsulation equipment 20 will be coated with cholesterol or fat.

Following introduction of a predetermined amount of composition 10 into capsule bodies 12a at feed station 28, the filled capsule bodies 12a are transported to a capsule closing station 34, where capsule caps 12b are assembled therewith to fully contain composition 10 within capsule 12.

Again, a composition-filled capsule 14 is only one example of the manner in which a composition that incorporates teachings of the present invention may be embodied. The inventive composition may also take other forms, such as tablets, caplets, loose powder, liquid, gel, liquid-filled or gel-filled capsules, or any other pharmaceutically acceptable form known in the art, each of which may be made by known processes.

The composition of the present invention may be administered to a subject (e.g., a mammal, such as a human, a dog, or a cat, a bird, a reptile, a fish, etc.) by any suitable process (e.g., enterally, parenterally, etc.), depending, of course, upon the form thereof. For example, virtually any form of the composition (e.g., a capsule, tablet, caplet, powder, liquid, gel, etc.) may be administered orally (i.e., through the mouth of the subject), provided that the composition includes a pharmaceutically acceptable carrier of a type known in the art that will prevent degradation or destruction of transfer factor molecules by the conditions that persist in the digestive tract of the subject without substantially interfering with the efficacy of the transfer factor molecules included in the composition.

The dosage of composition or transfer factor within the composition that is administered to the subject may depend on a variety of factors, including, without limitation, the subject's weight, the health of the subject, or conditions (e.g., pathogens) to which the subject has been exposed.

Administration of the composition to the subject may cause the immune system of the subject to elicit a T-cell mediated immune response against one or more antigens or pathogens. Thus, the composition may be administered to a subject to treat a disease state that the subject is experiencing, to prevent the subject from exhibiting a disease state caused by a particular pathogen, or to merely enhance the overall health of the subject's immune system and abilities to fight off infecting or invading pathogens.

The following EXAMPLES illustrate the enhanced ability of a composition which includes transfer factor from multiple types of source animals to cause an immune system of a treated subject to elicit a T-cell mediated immune response to various types of pathogens, in the form of target cells. The target cells included bacteria (e.g., C. pneumoniae and H. pylori) and viruses (e.g., herpes simplex virus-1 (HSV-1) and herpes simplex virus-2 (HSV-2)) in the form of virally infected cells, as well as to cancerous, or malignant, cells (e.g., K562 erythroleukemic cells).

The in vitro technique that was used to make these determinations was the so-called "chromium-51 release assay," which includes measurement of the amount of radioactive chromium-51 (Cr-51) released by cells that have been attacked by NK cells. The radioactivity measurement may be obtained, for example, with a Beckman 2000 Gamma Counter, which is available from Beckman Coulter, Inc., of Fullerton, Calif.

In the EXAMPLES, a fixed amount (5 micrograms per milliliter of nutrient media and cellular milieu) of a powdered composition was provided in the nutrient media and cellular milieu, along with a substantially fixed amount of NK cells. Examples of the powdered compositions that were used include bleached wheat flour, Transfer Factor™ (TF), available from 4Life Research, LLC, of Sandy, Utah, Transfer Factor Plus™ (TFP), also available from 4Life Research, avian transfer factor available in a lyophilized (ie., freeze-dried) whole egg powder, and mixtures of TF and TFP (both the formula marketed in the United States and that marketed internationally) with avian transfer factor in a ratio of about 85% TF or TFP (ie., bovine transfer factor), by weight, to about 15% avian transfer factor, by weight. The powdered composition, nutrient media, NK cells, and target cells were mixed and incubated for four hours prior to measuring the radioactive atoms that were released by disruption of the target cells by the NK cells. Each exemplary reaction was conducted in triplicate, with the results of the three reactions having been averaged.

The following TABLE includes data of the counts per minute obtained with each combination of target cells and powdered composition, as well as the effectiveness of each powdered composition in eliciting an NK cell-mediated immune response against the target cells relative to the NK cell-mediated immune response relative to (measured in percent increase) the same types and concentrations of target cells in the presence of bleached wheat flour.

TABLE Target Cells Composition C. Pneu H. Pyl K562 HSV-1 HSV-2 Flour 1323/ 1121/ 1267/ 2017/ 1262/ TF 2593/ 2499/ 2445/ 2240/ 2473/ % increase 196% 223% 193% 110% 196% TFP 3386/ 2701/ 3243/ 2944/ 1956/ % increase 256% 241% 256% 146% 155% 100% 2553/ 1860/ 2483/ 2985/ 2183/ Avian TF % increase 193% 166% 196% 148% 173% Bov-Av TF 14,857/ 11,434/ 6,639/ 17,910/ 10,626/ % increase 1123% 1020% 524% 888% 842% Bov-Av 61,956/ 55,432/ 40,075/ 80,498/ 46,933/ TFP US % increase 4683% 4944% 3163% 3991% 3719% Bov-Av 57,471/ 47,855/ 36,401/ 73,660/ 42,693/ TFP Intl % increase 4344% 4267% 2873% 3652% 3383%

The results that are set forth in the TABLE show that administration of a composition of the present invention to a subject will likely increase the subject's secondary, or delayed-type hypersensitivity, immune response, as effected by NK cells, against one or more pathogens to a degree which far exceeds the NK cell activity initiated by both colostrum-derived transfer factor and egg-derived transfer factor alone. In fact, the results show that a composition that incorporates teachings of the present invention may result in facilitation of the activity of NK cells with an unexpected degree of synergy.

Although the foregoing description contains many specifics, these should not be construed as limiting the scope of the present invention, but merely as providing illustrations of some of the presently preferred embodiments. Similarly, other embodiments may be devised without departing from the spirit or scope of the present invention. Features from different embodiments may be employed in combination. The scope of the invention is, therefore, indicated and limited only by the appended claims and their legal equivalents rather than by the foregoing description. All additions, deletion and modifications to the invention as disclosed herein which fall within the meaning and scope of the claims are to be embraced thereby.



Inventors

* Lisonbee, David * Hennen, William J. * Daugherty, F. Joseph

Assignee

* 4Life Research, LC

Application No. 10663353 filed on 09/15/2003

US Classes: 424/535, Milk or colostrum (e.g., butter, whey, etc.)424/581Egg enclosed in shell or a part thereof (e.g., eggshell, egg yolk, etc.)

Examiners Primary: Witz, Jean C.

Attorney, Agent or Firm

* TraskBritt, PC

Foreign Patent References

* 914831 EP 05/01/1999 * 930316 EP 07/01/1999

International Class A61K035/20



Abstract text

A composition for eliciting a T-cell mediated immune response in a subject includes transfer factor from at least two different types of source animals. For example, the composition may include mammalian transfer factor and nonmammalian transfer factor. An example of the composition includes a combination of a colostrum-derived product, which includes the mammalian transfer factor, and an egg-derived product, which includes the nonmammalian transfer factor. Additionally, the egg-derived product may be substantially free of fat. Methods for forming the composition and eliciting T-cell mediated immune responses in subjects that have been treated with the composition are also disclosed. Claims

1. A composition for eliciting a cell-mediated immune response in a treated subject, comprising: an active component consisting essentially of: mammalian transfer factor; and non-mammalian transfer factor; and at least one additional component.

2. The composition of claim 1, wherein the at least one additional component comprises at least a portion of a source from which at least one of the mammalian transfer factor and the non-mammalian transfer factor was derived.

3. The composition of claim 1, wherein the at least one additional component is substantially free of fat.

4. The composition of claim 3, wherein the at least partially purified extract is substantially free of at least one of casein, cells, cell debris, antibodies, and allergenic agents.

5. The composition of claim 1, wherein the mammalian transfer factor comprises bovine transfer factor.

6. The composition of claim 5, wherein the bovine transfer factor comprises colostrum-derived transfer factor.

7. The composition of claim 1, wherein the non-mammalian transfer factor comprises avian transfer factor.

8. The composition of claim 7, wherein the avian transfer factor comprises egg-derived transfer factor.

9. The composition of claim 1, wherein the non-mammalian transfer factor comprises egg-derived transfer factor.

10. The composition of claim 1, further comprising: a supplemental component.

11. The composition of claim 10, wherein the supplemental component includes at least one of vitamins, minerals, and at least one natural supplement.

12. The composition of claim 1, wherein the at least one additional component comprises a filler.

13. The composition of claim 1, wherein the mammalian transfer factor is configured to elicit a broad cell-mediated immune response and the non-mammalian transfer factor is configured to elicit a focused cell-mediated immune response.

14. The composition of claim 1, wherein respective amounts of the mammalian transfer factor and the non-mammalian transfer factor are tailored to synergistically elicit the cell-mediated immune response.

15. A composition for eliciting a cell-mediated immune response in a treated subject, comprising: a first quantity of mammalian transfer factor; and a second quantity of non-mammalian transfer factor, the first and second quantities being tailored to synergistically elicit the cell-mediated immune response.

16. The composition of claim 15, further comprising: at least a portion of a source from which at least one of the first and second quantities of transfer factor was derived.

17. The composition of claim 16, wherein at least the portion of the source is substantially free of fat.

18. The composition of claim 17, wherein at least the portion of the source is substantially free of at least one of casein, cells, cell debris, antibodies, and allergenic agents.

19. The composition of claim 15, wherein the mammalian transfer factor comprises bovine transfer factor.

20. The composition of claim 19, wherein the bovine transfer factor comprises colostrum-derived transfer factor.

21. The composition of claim 15, wherein the non-mammalian transfer factor comprises avian transfer factor.

22. The composition of claim 21, wherein the avian transfer factor comprises egg-derived transfer factor.

23. The composition of claim 1, wherein the non-mammalian transfer factor comprises egg-derived transfer factor.

24. The composition of claim 15, further comprising: a supplemental component.

25. The composition of claim 24, wherein the supplemental component includes at least one of vitamins, minerals, and at least one natural supplement.

26. The composition of claim 15, further comprising: a filler.

27. The composition of claim 15, wherein the mammalian transfer factor is configured to elicit a broad cell-mediated immune response and the non-mammalian transfer factor is configured to elicit a focused cell-mediated immune response.

28. A cell-free composition for eliciting a cell mediated immune response in a treated subject, comprising: mammalian transfer factor; and non-mammalian transfer factor.

29. The cell-free composition of claim 28, further comprising: at least a portion of a source from which at least one of the mammalian transfer factor and the non-mammalian transfer factor was derived.

30. The cell-free composition of claim 29, wherein at least the portion is substantially free of fat.

31. The cell-free composition of claim 30, wherein at least the portion extract is substantially free of at least one of casein, cell debris, antibodies, and allergenic agents.

32. The cell-free composition of claim 28, wherein the mammalian transfer factor comprises bovine transfer factor.

33. The cell-free composition of claim 32, wherein the bovine transfer factor comprises colostrum-derived transfer factor.

34. The cell-free composition of claim 28, wherein the non-mammalian transfer factor comprises avian transfer factor.

35. The cell-free composition of claim 34, wherein the avian transfer factor comprises egg-derived transfer factor.

36. The cell-free composition of claim 28, wherein the non-mammalian transfer factor comprises egg-derived transfer factor.

37. The cell-free composition of claim 28, further comprising: a supplemental component.

38. The cell-free composition of claim 37, wherein the supplemental component includes at least one of vitamins, minerals, and at least one natural supplement.

39. The cell-free composition of claim 28, further comprising: a filler.

40. The cell-free composition of claim 28, wherein the mammalian transfer factor is configured to elicit a broad cell-mediated immune response and the non-mammalian transfer factor is configured to elicit a focused cell-mediated immune response.

41. A composition for eliciting a cell-mediated immune response in a treated subject, comprising mammalian source component including mammalian transfer factor and non-mammalian source component including non-mammalian transfer factor, at least one of the mammalian source component and the non-mammalian source component being substantially antibody-free.

42. The composition of claim 41, further comprising: at least a portion of a source from which at least one of the mammalian transfer factor and the non-mammalian transfer factor was derived.

43. The composition of claim 42, wherein at least the portion is substantially free of fat.

44. The composition of claim 43, wherein at least the portion is also substantially free of at least one of casein, cell debris, and allergenic agents.

45. The composition of claim 41, wherein the mammalian transfer factor comprises bovine transfer factor.

46. The composition of claim 45, wherein the bovine transfer factor comprises colostrum-derived transfer factor.

47. The composition of claim 41, wherein the non-mammalian transfer factor comprises avian transfer factor.

48. The composition of claim 47, wherein the avian transfer factor comprises egg-derived transfer factor.

49. The composition of claim 41, wherein the non-mammalian transfer factor comprises egg-derived transfer factor.

50. The composition of claim 41, further comprising: a supplemental component.

51. The composition of claim 50, wherein the supplemental component includes at least one of vitamins, minerals, and at least one natural supplement.

52. The composition of claim 41, further comprising: a filler.

53. The composition of claim 41, wherein the mammalian transfer factor is configured to elicit a broad cell-mediated immune response and the non-mammalian transfer factor is configured to elicit a focused cell-mediated immune response.

54. A composition for eliciting a cell mediated immune response in a treated subject, comprising: a first extract including mammalian transfer factor; and a second extract comprising non-mammalian transfer factor, at least one of the first extract and the second extract being at least partially purified.

55. The composition of claim 54, wherein at least one of the first extract and the second extract comprises an extract from which at least some components having molecular weights of greater than about 160,000 daltons have been removed.

56. A composition for eliciting a cell mediated immune response in a treated subject, comprising: a focused component including transfer factor from a first source, the transfer factor from the first source being targeted toward at least one specific antigen; and an unfocused component including transfer factor from a second source, the transfer factor from the second source being configured to provide protection against a broad array of antigens.

57. The composition of claim 56, wherein the broad array of antigens includes antigens to which an animal from which the second source was derived is exposed in its natural environment.

58. A composition for eliciting a cell-mediated immune response in a treated subject, comprising mammalian source component including mammalian transfer factor and non-mammalian source component including non-mammalian transfer factor, at least one of the mammalian source component and the non-mammalian source component being substantially antibody-free. Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of PCT international patent application PCT/US04/30307, filed Sep. 15, 2004, which claims the benefit of the filing date of U.S. patent application Ser. No. 10/663,353, filed Sep. 15, 2003, now U.S. Pat. No. 6,866,868, issued Mar. 15, 2005, the disclosures of both of which are hereby incorporated herein, in their entireties, by this reference.

FIELD OF THE INVENTION

[0002] The present invention relates generally to compositions which include transfer factor and, more specifically, to compositions which include transfer factor from different types of source animals. The present invention also relates to methods for making compositions that include different types of transfer factor and to methods for eliciting or enhancing a T-cell mediated immune response by the immune system of a subject.

BACKGROUND OF RELATED ART

[0003] Many deadly pathogens are passed to humans from the animal kingdom. For example, monkeys are the sources of the type I human immunodeficiency virus (HIV-I), which causes acquired immune deficiency syndrome (AIDS) and monkeypox, which is similar to smallpox; ground-dwelling mammals are believed to be the source of the Ebola virus; fruit bats and pigs are the source of the Nipah virus; the Hendra virus comes from horses; the virus responsible for the "Hong Kong Flu" originated in chickens; and wild birds, especially ducks, are the sources of many of the deadly influenza viruses. Many diseases also have animal reservoirs. By way of example, mice carry Hanta virus, rats carry the Black Plague, and deer carry Lyme disease.

The Immune System

[0004] The immune systems of vertebrates are equipped to recognize and defend the body from invading pathogenic organisms, such as parasites, bacteria, fungi, and viruses. Vertebrate immune systems typically include a cellular component and a noncellular component.

[0005] The cellular component of an immune system includes the so-called "lymphocytes," or white blood cells, of which there are several types. It is the cellular component of a mature immune system that typically mounts a primary, nonspecific response to invading pathogens, as well as being involved in a secondary, specific response to pathogens.

[0006] In the primary, or initial, response to an infection by a pathogen, white blood cells that are known as phagocytes locate and attack the invading pathogens. Typically, a phagocyte will internalize, or "eat" a pathogen, then digest the pathogen. In addition, white blood cells produce and excrete chemicals in response to pathogenic infections that are intended to attack the pathogens or assist in directing the attack on pathogens.

[0007] Only if an infection by invading pathogens continues to elude the primary immune response is a specific, secondary immune response to the pathogen needed. As this secondary immune response is typically delayed, it is also known as "delayed-type hypersensitivity." A mammal, on its own, will typically not elicit a secondary immune response to a pathogen until about seven (7) to about fourteen (14) days after becoming infected with the pathogen. The secondary immune response is also referred to as an acquired immunity to specific pathogens. Pathogens have one or more characteristic proteins, which are referred to as "antigens." In a secondary immune response, white blood cells known as B lymphocytes, or "B-cells," and T lymphocytes, or "T-cells," "learn" to recognize one or more of the antigens of a pathogen. The B-cells and T-cells work together to generate proteins called "antibodies," which are specific for (e.g., configured to bind to or otherwise "recognize") one or more certain antigens on a pathogen.

[0008] The T-cells are primarily responsible for the secondary, or delayed-type hypersensitivity, immune response to a pathogen or antigenic agent. There are three types of T-cells: T-helper cells, T-suppressor cells, and antigen-specific T-cells, which are also referred to as cytotoxic (meaning "cell-killing") T-lymphocytes (CTLs), or T-killer cells or natural killer (NK) cells. The T-helper and T-suppressor cells, while not specific for certain antigens, perform conditioning functions (e.g., the inflammation that typically accompanies an infection) that assist in the removal of pathogens or antigenic agents from an infected host. The NK cells, which comprise about ten to about fifteen percent of circulating lymphocytes, are important mediators of both natural and adaptive immunity.

[0009] Antibodies, which make up only a part of the noncellular component of an immune system, recognize specific antigens and, thus, are said to be "antigen-specific." The generated antibodies then basically assist the white blood cells in locating and eliminating the pathogen from the body. Typically, once a white blood cell has generated an antibody against a pathogen, the white blood cell and all of its progenitors continue to produce the antibody. After an infection is eliminated, a small number of T-cells and B-cells that correspond to the recognized antigens are retained in a "resting" state. When the corresponding pathogenic or antigenic agents again infect the host, the "resting" T-cells and B-cells activate and, within about forty-eight (48) hours, induce a rapid immune response. By responding in this manner, the immune system mounts a secondary immune response to a pathogen, the immune system is said to have a "memory" for that pathogen.

[0010] Mammalian immune systems are also known to produce smaller proteins, known as "transfer factors," as part of a secondary immune response to infecting pathogens. Transfer factors are another noncellular part of a mammalian immune system. Antigen-specific transfer factors are believed to be structurally analogous to antibodies, but on a much smaller molecular scale. Both antigen-specific transfer factors and antibodies include antigen-specific sites. In addition, both transfer factors and antibodies include highly conserved regions that interact with receptor sites on their respective effector cells. In transfer factor and antibody molecules, a third, "linker," region connects the antigen-specific sites and the highly conserved regions.

The Role of Transfer Factor in the Immune System

[0011] Transfer factor is a low molecular weight isolate of lymphocytes. Narrowly, transfer factors may have specificity for single antigens. U.S. Pat. Nos. 5,840,700 and 5,470,835, both of which issued to Kirkpatrick et al. (hereinafter collectively referred to as "the Kirkpatrick Patents"), disclose the isolation of transfer factors that are specific for certain antigens. More broadly, "specific" transfer factors have been generated from cell cultures of monoclonal lymphocytes. Even if these transfer factors are generated against a single pathogen, they have specificity for a variety of antigenic sites of that pathogen. Thus, these transfer factors are said to be "pathogen-specific" rather than antigen-specific. Similarly, transfer factors that are obtained from a host that has been infected with a certain pathogen are pathogen-specific. Although such preparations are often referred to in the art as being "antigen-specific" due to their ability to elicit a secondary immune response when a particular antigen is present, transfer factors having different specificities may also be present in such preparations. Thus, even the so-called "antigen-specific," pathogen-specific transfer factor preparations may be specific for a variety of antigens.

[0012] Additionally, it is believed that antigen-specific and pathogen-specific transfer factors may cause a host to elicit a delayed-type hypersensitivity immune response to pathogens or antigens for which such transfer factor molecules are not specific. Transfer factor "draws" at least the non-specific T-cells, the T-inducer and T-suppressor cells, to an infecting pathogen or antigenic agent to facilitate a secondary, or delayed-type hypersensitivity, immune response to the infecting pathogen or antigenic agent.

[0013] Typically, transfer factor includes an isolate of proteins having molecular weights of less than about 10,000 daltons (D) that have been obtained from immunologically active mammalian sources. It is known that transfer factor, when added either in vitro or in vivo to mammalian immune cell systems, improves or normalizes the response of the recipient mammalian immune system.

[0014] The immune systems of newborns have typically not developed, or "matured," enough to effectively defend the newborn from invading pathogens. Moreover, prior to birth, many mammals are protected from a wide range of pathogens by their mothers. Thus, many newborn mammals cannot immediately elicit a secondary response to a variety of pathogens. Rather, newborn mammals are typically given secondary immunity to pathogens by their mothers. One way in which mothers are known to boost the immune systems of newborns is by providing the newborn with a set of transfer factors. In mammals, transfer factor is provided by a mother to a newborn in colostrum, which is typically replaced by the mother's milk after a day or two. Transfer factor basically transfers the mother's acquired, specific (i.e., delayed-type hypersensitive) immunity to the newborn. This transferred immunity typically conditions the cells of the newborn's immune system to react against pathogens in an antigen-specific manner, as well as in an antigen- or pathogen-nonspecific fashion, until the newborn's immune system is able on its own to defend the newborn from pathogens. Thus, when transfer factor is present, the immune system of the newborn is conditioned to react to pathogens with a hypersensitive response, such as that which occurs with a typical delayed-type hypersensitivity response. Accordingly, transfer factor is said to "jump start" the responsiveness of immune systems to pathogens.

[0015] Much of the research involving transfer factor has been conducted in recent years. Currently, it is believed that transfer factor is a protein with a length of about forty-four (44) amino acids. Transfer factor typically has a molecular weight in the range of about 3,000 to about 5,000 Daltons (Da), or about 3 kDa to about 5 kDa, but it may be possible for transfer factor molecules to have molecular weights outside of this range. Transfer factor is also believed to include three functional fractions, each of which may include different types of transfer factor molecules: an inducer fraction; an immune suppressor fraction; and an antigen-specific fraction. Many in the art believe that transfer factor also includes a nucleoside portion, which could be connected to the protein molecule or separate therefrom, that may enhance the ability of transfer factor to cause a mammalian immune system to elicit a secondary immune response. The nucleoside portion may be part of the inducer or suppressor fractions of transfer factor.

[0016] The antigen-specific region of the antigen-specific transfer factors is believed to comprise about eight (8) to about twelve (12) amino acids. A second highly-conserved region of about ten (10) amino acids is thought to be a very high-affinity T-cell receptor binding region. The remaining amino acids may serve to link the two active regions or may have additional, as yet undiscovered properties. The antigen-specific region of a transfer factor molecule, which is analogous to the known antigen-specific structure of an antibody, but on a much smaller molecular weight scale, appears to be hyper-variable and is adapted to recognize a characteristic protein on one or more pathogens. The inducer and immune suppressor fractions are believed to impart transfer factor with its ability to condition the various cells of the immune system so that the cells are more fully responsive to the pathogenic stimuli in their environment.

Sources of Noncellular Immune System Components

[0017] Conventionally, transfer factor has been obtained from the colostrum of milk cows, such as by the method described in U.S. Pat. No. 4,816,563 to Wilson et al. (hereinafter "Wilson"). While milk cows typically produce large amounts of colostrum and, thus, large amounts of transfer factor over a relatively short period of time, milk cows only produce colostrum for about a day or a day-and-a-half every year. Thus, milk cows are neither a constant source of transfer factor nor an efficient source of transfer factor.

[0018] Transfer factor has also been obtained from a wide variety of other mammalian sources. For example, in researching transfer factor, mice have been used as a source for transfer factor. Antigens are typically introduced subcutaneously into mice, which are then sacrificed following a delayed-type hypersensitivity reaction to the antigens. Transfer factor is then obtained from spleen cells of the mice.

[0019] While different mechanisms are typically used to generate the production of antibodies, the original source for antibodies may also be mammalian. For example, monoclonal antibodies may be obtained by injecting a mouse, a rabbit, or another mammal with an antigen, obtaining antibody-producing cells from the mammal, then fusing the antibody-producing cells with immortalized cells to produce a hybridoma cell line, which will continue to produce the monoclonal antibodies throughout several generations of cells and, thus, for long periods of time.

[0020] Antibodies against mammalian pathogens have been obtained from a wide variety of sources, including mice, rabbits, pigs, cows, and other mammals. In addition, the pathogens that cause some human diseases, such as the common cold, are known to originate in birds. As it has become recognized that avian (i.e., bird) immune systems and mammalian immune systems are very similar, some researchers have turned to birds as a source for generating antibodies.

[0021] Avian antibodies that are specific for pathogens that infect mammals, or "mammalian pathogens," have been obtained by introducing antigens into eggs. Alternatively, antibodies may be present in eggs following exposure of the source animal to antigens, including antigens of mammalian pathogens. U.S. Pat. No. 5,080,895, issued to Tokoro on Jan. 14, 1992 (hereinafter "the '895 Patent"), discloses a method that includes injecting hens with pathogens that cause intestinal infectious diseases in neonatal mammals. The hens then produce antibodies that are specific for these pathogens, which are present in eggs laid by the hens. The '895 Patent discloses compositions that include these pathogen-specific antibodies and use thereof to treat and prevent intestinal diseases in neonatal piglets and calves. Treatment of pathogenic infections in mammals with avian antibodies may have undesirable results, however, since the immune systems of mammals may respond negatively to the large avian antibody molecules by eliciting an immune response to the antibodies themselves. Moreover, as mammalian immune systems do not recognize avian antibodies as useful for their abilities to recognize certain pathogens, or the specificities of avian antibodies for antigens of such pathogens, avian antibodies often do not elicit the desired immune responses in mammals.

[0022] It is also known that transfer factor may be obtained from eggs. U.S. Pat. No. 6,468,534 to Hennen et al. (hereinafter "Hennen") describes a process by which female chickens (i.e., hens) are exposed to one or more antigens, which results in the elicitation of an immune response, including a secondary immune response, by the chickens. As a result of the secondary immune response, transfer factor molecules are present in the eggs of the chicken. The eggs may then be processed to provide a product in which the transfer factor is present. Such a product may take the form of a spray dried or freeze dried, or lyophilized, egg powder, and may include all or part of the egg. The egg powder may then be incorporated directly into gelatin capsules or mixed with other substances the introduced into gelatin capsules.

[0023] FIG. 2 schematically depicts capsulation equipment of a type that is currently useful for capsulating egg-derived avian transfer factor in the form of an egg powder. Capsulation equipment 20 includes a composition supply hopper 24, a feed station 28, and an auger 26 in communication between each composition supply hopper 24 and feed station 28. Auger 26 transports the whole egg powder from composition supply hopper 24 to feed station 28.

[0024] When auger 26 operates, it is heated to a temperature which exceeds the relatively low melting point of cholesterol, from egg yolk, in the egg powder. The warmed cholesterol is sticky, coating auger 26, the conduit in communication therewith, and feed station 28, thereby decreasing the efficiency with which capsulation equipment 20 operates. Consequently, capsulation equipment must be disassembled and cleaned periodically, which may take a considerable amount of time (e.g., up to about 8 hours), resulting in a significant decrease in the productivity of capsulation equipment 20 and, thus, the number of capsules that may be formed therewith. Thus, processing of whole egg powder to obtain a transfer factor-containing product is somewhat undesirable.

[0025] Additionally, compositions which are derived from products (e.g., eggs or colostrum) from a single source animal typically only include transfer factor molecules which have specificity to antigens to which the source animal has been exposed. The consequence of such limited exposure may be that the effectiveness of such transfer factor-containing compositions in preventing or treating certain types of infections or conditions is also limited.

[0026] Accordingly, there is a need for a composition which is useful for causing an immune system of a treated subject to elicit an immune response to a broader array of pathogens, as well as for a method for improving the efficiency and productivity with which capsulation and other composition-forming equipment operates.

SUMMARY OF THE INVENTION

[0027] The present invention includes compositions for eliciting a T-cell mediated immune responses in subjects. The composition includes an active component with transfer factor from at least two different types of source animals. The term "type," as used herein with respect to source animals, describes the source animals from which transfer factor may be obtained and refers to source animals from different classes (e.g., mammals, birds, reptiles, amphibians, insects, etc.). The term "type," as used herein, also refers to source animals from different subclasses, orders (e.g., artiodactyls, primates, carnivores, etc.), families (bovine, hominids, felines, etc.), subfamilies, genuses (e.g., cattle, humans, domestic cats, etc.), and even species and subspecies. Use of the term "type" herein with respect to transfer factor denotes the type of source animal from which the transfer factor was obtained.

[0028] An exemplary embodiment of the active component of such a composition includes transfer factor from both mammalian and nonmammalian source animals, which types of transfer factor are also referred to herein as "mammalian transfer factor" and "nonmammalian transfer factor," respectively. By way of nonlimiting example, the mammalian transfer factor may be included in the composition as colostrum or a fraction or extract thereof, which are collectively referred to herein as "colostrum-derived products," or otherwise, as known in the art (e.g., as a cellular extract, such as a leukocyte (white blood cell) extract, a splenic ("from the spleen") extract, or the like, etc.). Also by way of example, the nonmammalian transfer factor of the exemplary composition may be obtained from an egg or a fraction or extract thereof, which are also referred to herein as "egg-derived products." It has been discovered that when different types of transfer factors are combined and administered to a treated animal (e.g., a mammal), some synergy occurs.

[0029] When a composition of the present invention includes a colostrum-derived product and an egg-derived product, both products may be included in the mixture in amounts (e.g., by weight, by volume, etc., of the total mixture) that are about equal, or more of one of the colostrum-derived product and the egg-derived product than the other. Experimental results show that transfer factor from source animals that have highly dependent young, such as cows, induces a relatively quick secondary immune response, with anergy (i.e., a lack of sensitivity by white blood cells to the transfer factor molecules) setting in relatively quickly.

[0030] The different types of transfer factor of the active component may be selected or provided in amounts that are tailored to cause a treated subject to synergistically elicit a T-cell mediated immune response. For example, transfer factor from source animals that have independent young, such as chickens or other "gallinaceous" birds, does not induce as quick a secondary immune response, but does provide for a more sustained secondary immune response. Accordingly, the relative concentrations of colostrum-derived transfer product and egg-derived product may be tailored to elicit a secondary immune response that occurs or is sustained for a particular period of time. As another example of such synergism, transfer factor from one source may facilitate elicitation of a cell-mediated immune response against a corresponding set of pathogens or other antigenic agents, while transfer factor from another source may cause a treated subject to elicit a cell-mediated response against another set of pathogens or other antigenic agents. As a further example, one set of pathogens against which transfer factor from one source (e.g., from a source animal that has been exposed to a broad array of pathogens or other antigenic agents) is most effective may cause a subject to elicit a broad, or unfocused immune response, while transfer factor from another source (e.g., a source from a source animal that has been exposed to a limited number (e.g., only one or a few) pathogens or other antigenic agents) may cause a subject to elicit a narrow, focused immune response.

[0031] An active component of such a composition may consist essentially of the two or more types of transfer factor (including dialysate or another at least partially purified fraction having an upper-end molecular weight cutoff of about 10,000 Da), or include additional components.

[0032] Additional components may include a variety of different things, such as a portion of a source (e.g., egg, colostrum, cells, etc.) from which the transfer factor was derived, a supplement, beneficial microorganisms, and the like.

[0033] If a portion, or extract, of a source of transfer factor is included in a composition according to the present invention, the extract may be purified or at least partially to remove one more components therefrom. By way of nonlimiting example, proteins (e.g., antibodies and other proteins having molecular weights of about 160,000 Da or more), fat, casein, cells, or cell debris may be substantially removed from the extract and, thus, the extract, or even the composition, may be substantially free of these components. Allergenic components, including, but not limited to, some of the components listing immediately above, may also be separated from the transfer factor from at least one source. Of course, there is no requirement that any components be substantially removed from non-transfer factor portions of one or more sources, or that the non-transfer factor portions of one or more sources otherwise be purified.

[0034] Supplements are also referred to herein as "supplemental components." A supplement that may be included in a composition of the present invention include, without limitation, one or more vitamins, minerals, proteins, or natural products (e.g., herbs, mushrooms, roots, etc.) or extracts thereof. Polysaccharides are believed to provide further synergy in the effectiveness of a composition of the present invention in eliciting secondary immune responses in treated animals. Exemplary polysaccharides are available in the form of beta-glucans and mushroom extracts (which, of course, include other components).

[0035] While a composition according to the present invention may also or alternatively include one or more beneficial microorganisms, compositions that incorporate teachings of the present invention may also lack microorganisms and, thus, be microorganism-free or cell-free.

[0036] According to another aspect, the present invention includes methods for forming compositions that include two or more types of transfer factor. One or more of transfer factor (e.g., colostrum, eggs, cells, tissues, etc.) may be processed to obtain and, optionally, at least partially purify transfer factor. Such processing may also be used to obtain, extract, or at least partially purify other components from the one or more sources. For example, processes such as those disclosed in Wilson and Hennen, may be employed. If desired, other components may be included in the composition.

[0037] In another aspect, the present invention includes a method for processing or manufacturing an egg-derived product which includes transfer factor. The inventive method of processing or manufacture includes mixing a substantially fat-free component, such as a colostrum-derived product, which may or may not include transfer factor, with the egg-derived product before or while the egg-derived product is being introduced into manufacturing or other processing equipment. Capsulation is one example of a processing or manufacturing method in which such techniques may be employed.

[0038] Additionally, the present invention includes a method for reducing the cleaning frequency of manufacturing or other processing equipment, such as capsulation equipment, used for processing an egg-derived product. That method includes mixing a less fatty or substantially fat free substance, such as a colostrum-derived product, with the egg-derived product before or during introduction of the egg-derived product into the processing equipment.

[0039] The present invention also includes methods for treating a subject. Treatment methods that incorporate teachings of the present invention include administration of a composition according to the present invention to a subject. As the composition includes transfer factor, administration of the composition to the subject will cause the subject's immune system to elicit a T-cell mediated immune response or will enhance a T-cell mediated immune response by the subject's immune system which is already underway.

[0040] Other features and advantages of the present invention will become apparent to those of ordinary skill in the art through consideration of the ensuing description, the accompanying drawings, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In the drawings, which depict exemplary embodiments of various aspects of the present invention:

[0042] FIG. 1 depicts an example of the manner in which a composition that incorporates teachings of the present invention may be embodied;

[0043] FIG. 2 is a schematic representation of capsulation equipment that may be used to introduce a powdered embodiment of the composition of the present invention into gelatin capsules; and

[0044] FIG. 3 schematically illustrates an exemplary test protocol that was conducted to determine the efficacy of various aspects of the present invention.

DETAILED DESCRIPTION

[0045] An exemplary embodiment of composition that incorporates teachings of the present invention includes transfer factor from at least two different types of source animals. By way of nonlimiting example, a composition according to the present invention may include mammalian transfer factor and nonmammalian transfer factor.

[0046] The different types of transfer factor of the inventive composition may be obtained from any suitable source. For example, mammalian transfer factor may be obtained from colostrum, as described in Wilson or otherwise as known in the art (e.g., a leukocyte (white blood cell) extract, a splenic (i.e., "from the spleen") extract, etc.). An exemplary source for nonmammalian transfer factor is an egg of an animal, such as a chicken, as described in Hennen. Thus, a composition according to the present invention may include a first component which comprises colostrum or a fraction or extract thereof, which are collectively referred to herein as a "colostrum-derived product," as well as a second component that comprises egg or a fraction or extract thereof, which are also referred to herein as an "egg-derived products."

[0047] As compositions that incorporate teachings of the present invention include transfer factor from different types of source animals, they may include transfer molecules with a broader array of antigen-specificity or pathogen-specificity than conventional transfer factor-containing compositions. Thus, a composition according to the present invention is capable of enlisting the immune system of a treated animal to elicit a T-cell mediated immune response against a broader array of pathogens than those against which conventional transfer factor-containing compositions are effective. This is because different types of animals may be exposed to different types of antigens or pathogens, such as by vaccination, the animals' environments, or the like. Moreover, it is known that some conditions in certain animals are caused by multiple infections, even further expanding the specificity of a composition according to the present invention. For example, one or more pathogens may adversely affect (e.g., suppress or monopolize) the host's immune system, while one or more other pathogens may be permitted to cause a disease state in the host. As another example, some disease states are caused by a combination of pathogens.

[0048] As an example, a composition which includes transfer factor-containing components from both cows and chickens will include transfer factor molecules which are specific to antigens or pathogens to which cows are exposed, as well as transfer factor molecules that have specificity for antigens or pathogens to which chickens are exposed. As both cows and chickens may be exposed to antigens or pathogens to which the other is not exposed, such a composition may include transfer factor molecules with antigen or pathogen specificities that would not be present in a composition that includes only transfer factor from cows (e.g., by way of a colostrum-derived product) or transfer factor from chickens (e.g., through an egg-derived product).

[0049] A composition of the present invention may include about the same amounts, measured in terms of weight or volume, of a colostrum-derived product and an egg-derived product (i.e., about 50% colostrum-derived product and about 50% egg-derived product). Alternatively, a composition that incorporates teachings of the present invention may include about more colostrum-derived product (e.g., about 85% or 60%, by combined weight of the colostrum-derived product and egg-derived product) than egg-derived product (about 15% or 40%, by weight). As another alternative, the inventive composition may include more egg-derived product (e.g., about 60% or 85%, by weight) than colostrum-derived product (e.g., about 40% or 15% by weight). As another example, a composition that incorporates teachings of the present invention may include about one percent, by weight, of one of a colostrum-derived product and an egg-derived product and about 99%, by weight, of the other of the colostrum-derived product and the egg-derived product. Although specific amounts of colostrum-derived product and egg-derived product have been provided, any combination thereof is within the scope of the present invention.

[0050] In addition to including a source of transfer factor (e.g., a colostrum-derived product, an egg-derived product, etc.) a composition that incorporates teachings of the present invention may include one or more other ingredients, including, but not limited to, vitamins, minerals, proteins, natural products (e.g., herbs, mushrooms, roots, etc., or extracts thereof), and the like. Additional ingredients may be useful for providing further advantages to subjects to which the composition is administered, or may enhance the ability of the transfer factor in the composition to elicit or enhance a secondary, or delayed-type hypersensitivity, immune response.

[0051] As shown in FIG. 1, without limiting the scope of the present invention, a composition 10 according to the present invention may take the form of a powdered or particulate substance, which includes the multiple types of transfer factor (not shown). In order to ensure that an appropriate and precise dosage of composition 10 is administered to a subject (not shown), composition 10 may be contained within a gelatin capsule 12 of a type which is well-known and readily available to those in the art. The result is the illustrated capsule 14. Alternatively, a composition according to the present invention may be embodied as tablet, a so-called "caplet," an unencapsulated powder, a liquid, a gel, or in any other pharmaceutically acceptable form. Suitable processes for placing the inventive composition into any such form are readily apparent to those of skill in the art.

[0052] In an exemplary embodiment of a method for making or forming a composition according to the present invention, a first type of transfer factor may be combined with a second type of transfer factor. Additionally, one or more other types of transfer factor may be combined with the first and second types of transfer factor. The different types of transfer factor that are combined may be substantially purified transfer factor, components or "products" that include transfer factor, or any combination thereof.

[0053] Turning again to FIG. 2, a process for forming composition-filled capsules 14, such as that shown in FIG. 1, is provided merely as an example for a method for making a composition that incorporates teachings of the present invention. As illustrated, the composition 10 is made and composition-filled capsules 14 are formed using standard capsulation equipment 20 of a type known in the industry, such as the SF-135 capsule filling machine available from CapPlus Technologies of Phoenix, Ariz.

[0054] In addition to one or more composition supply hoppers 24, an auger 26 associated with each composition supply hopper 24, and a feed station 28 with which each auger 26 and the conduit 27 within which auger 26 is contained communicates, capsulation equipment 20 includes one or more capsule hoppers 30, as well as a pneumatic feed system 32 for transporting capsule bodies 12a and/or caps 12b to feed station 28.

[0055] As the capsulation equipment will introduce the mixture into capsules, which may be swallowed by a subject, it is currently preferred that the substantially fat-free component and the egg-derived product be introduced into the capsulation equipment in powdered form. The substantially fat-free component dilutes the amount, or concentration, of fat (e.g., from egg yolk) present in the mixture relative to the concentration of fat which is present in the egg-derived product. Accordingly, the relative amounts of substantially-fat free product and the egg-derived product may be tailored to provide a fat concentration that will minimize clogging of the capsulation equipment.

[0056] Continuing with the example of a composition 10 which includes a colostrum-derived product 10a as the substantially fat-free component and an egg-derived product 10b, colostrum-derived product 10a and egg-derived product 10b may be introduced simultaneously into a single composition supply hopper 24 of capsulation equipment 20. For example, colostrum-derived product 10a and egg-derived product 10b may be mixed upon introduction thereof into composition supply hopper 24, as shown, or premixed. By introducing a substance which has a lower fat content than egg-derived product 10b into composition supply hopper 24 along with egg-derived product 10b, the fat content (e.g., concentration) of the resulting mixture is less than that of egg-derived product 10b, reducing or eliminating the likelihood that composition supply hopper 24, auger 26, conduit 27, feed station 28, or any other component of capsulation equipment 20 will be coated with cholesterol or fat.

[0057] Following introduction of a predetermined amount of composition 10 into capsule bodies 12a at feed station, the filled capsule bodies 12a are transported to a capsule closing station 34, where capsule caps 12b are assembled therewith to fully contain composition 10 within capsule 12.

[0058] Again, a composition-filled capsule 14 is only one example of the manner in which a composition that incorporates teachings of the present invention may be embodied. The inventive composition may also take other forms, such as tablets, caplets, loose powder, liquid, gel, liquid-filled or gel-filled capsules, any other pharmaceutically acceptable form known in the art, each of which may be made by known processes.

[0059] The composition of the present invention may be administered to a subject (e.g., a mammal, such as a human, a dog, or a cat, a bird, a reptile, a fish, etc.) by any suitable process (e.g., enterally, parentarlly, etc.), depending, of course, upon the form thereof. For example, virtually any form of the composition (e.g., a capsule, tablet, caplet, powder, liquid, gel, etc.) may be administered orally (i.e., through the mouth of the subject), provided that the composition includes a pharmaceutically acceptable carrier of a type known in the art that will prevent degradation or destruction of transfer factor molecules by the conditions that persist in the digestive tract of the subject without substantially interfering with the efficacy of the transfer factor molecules included in the composition.

[0060] The dosage of composition or transfer factor within the composition that is administered to the subject may depend on a variety of factors, including, without limitation, the subject's weight, the health of the subject, or conditions (e.g., pathogens) to which the subject has been exposed.

[0061] Administration of the composition to the subject may cause the immune system of the subject to elicit a T-cell mediated immune response against one or more antigens or pathogens. Thus, the composition may be administered to a subject to treat a disease state that the subject is experiencing, to prevent the subject from exhibiting a disease state caused by a particular pathogen, or to merely enhance the overall health of the subject's immune system and abilities to fight off infecting or invading pathogens.

[0062] The following EXAMPLES illustrate the enhanced ability of a composition which includes transfer factors from multiple types of source animals to cause an immune system of a treated subject to elicit a T-cell mediated immune response to various types of pathogens, in the form of target cells. The ratios used in the EXAMPLES are based on the weigh of the material (e.g., egg powder, colostrum powder) used in a particular test sample.

EXAMPLE 1

[0063] In EXAMPLE 1, a preliminary test, the target cells included bacteria (e.g., C. pneumoniae and H. pylori) and viruses (e.g., herpes simplex virus-1 (HSV-1) and herpes simplex virus-2 (HSV-2)) in the form of virally infected cells, as well as to cancerous, or malignant, cells (e.g., K562 erythroleukemic cells).

[0064] The in vitro technique that was used to make these determinations was the so-called "chromium-5 1 release assay," which includes measurement of the amount of radioactive chromium-51 (Cr-51) released by cells that have been attacked by NK cells. The radioactivity measurement may be obtained, for example, with a Beckman 2000 Gamma Counter, which is available from Beckman Coulter, Inc., of Fullerton, Calif.

[0065] In EXAMPLE 1, which was a preliminary test, a fixed amount (5 micrograms per milliliter of nutrient media and cellular milieu) of a powdered composition was provided in the nutrient media and cellular milieu, along with a substantially fixed amount of NK cells. Examples of the powdered compositions that were used include bleached wheat flour, Transfer Factor™ (TF), available from 4Life Research, LLC, of Sandy, Utah, Transfer Factor Plus™ (TFP or TF ), also available from 4Life Research, avian transfer factor available in a lyophilized (i.e., freeze-dried) whole egg powder, and mixtures of TF and TFP (both the formula marketed in the United States and that marketed internationally) with avian transfer factor in a ratio of about 85% TF or TFP (i.e., bovine transfer factor), by weight, to about 15% avian transfer factor, by weight. The powdered composition, nutrient media, NK cells, and target cells were mixed and incubated for four hours prior to measuring the radioactive atoms that were released by disruption of the target cells by the NK cells. Each exemplary reaction was conducted in triplicate, with the results of the three reactions having been averaged.

[0066] In addition to including one or more types of transfer factor, TFP includes a variety of other components, including maitake and shiitake Mushrooms, cordyceps, inositol hexaphosphate, beta glucans, beta sitosterol, and olive leaf extract. Maitake and shiitake mushrooms are known to be good sources for polysaccharides and to promote T-cell function. Cordyceps are also rich in polysaccharides. Beta glucans, another class of polysaccharides, is also known to be an important immune cell stimulator.

[0067] The following TABLE includes data of the counts per minute obtained with each combination of target cells and powdered composition, as well as the effectiveness of each powdered composition in eliciting an NK cell-mediated immune response against the target cells relative to the NK cell-mediated immune response relative to (measured in percent increase) the same types and concentrations of target cells in the presence of bleached wheat flour.

EXAMPLE 1

[0068] TABLE-US-00001 TABLE 1 Target Cells Composition C. Pneu H. Pyl K562 HSV-1 HSV-2 Spontaneous 1,256/ 1,875/ 1,620/ 974/ 1,476/ Flour 1,323/ 1,121/ 1,267/ 2,017/ 1,262/ Average 1,290/ 1,498/ 1,444/ 1,496/ 1,365/ TF 2,593/ 2,499/ 2,445/ 2,240/ 2,473/ % increase over flour 96% 123% 93% 11% 96% % increase over average 101% 67% 69% 50% 81% TFP 3,386/ 2,701/ 3,243/ 2,944/ 1,956/ % increase over flour 156% 141% 156% 46% 55% % increase over average 163% 80% 125% 97% 43% Bov-Av TF 14,857/ 11,434/ 6,639/ 17,910/ 10,626/ % increase over flour 1023% 920% 424% 788% 742% % increase over average 1052% 663% 360% 1098% 679% Bov-Av 6,196/ 5,543/ 4,008/ 8,050/ 4,693/ TFP US % increase over flour 458% 485% 306% 389% 362% % increase over average 380% 270% 178% 438% 244% Bov-Av 5,747/ 4,786/ 3,640/ 7,366/ 4,269/ TFP Intl % increase over flour 424% 417% 277% 355% 328% % increase over average 346% 219% 152% 393% 213% 100% 2,553/ 1,860/ 2,483/ 2,985/ 2,183/ Avian TF % increase over flour 93% 66% 96% 48% 73% % increase over average 98% 24% 72% 100% 60%

[0069] Notably, the formulations denoted "TFP" include only about half (0.466667) of the transfer factor as that present in the formulations denoted "TF." Accordingly, one of ordinary skill in the art would expect the data that corresponds to cytotoxicity induced by the products identified as "Bov-Av TFP US" and "Bov-Av TFP Intl" to be somewhat less than the cytotoxicity induced by the product identified as Bov-Av TF. Instead, these numbers were much higher. In fact, it appears that the data that corresponds to "Bov-Av TFP US" and "Bov-Av TFP Intl" is about ten times too high. Accordingly, appropriate corrections have been made to TABLE 1. Additionally, further testing has been conducted, as is evident from the ensuing EXAMPLES, to evaluate and verify the abilities of combinations of different types of transfer factor to elicit T-cell responses in treated animals.

[0070] The preliminary results that are set forth in TABLE 1 show that administration of a composition of the present invention to a subject will likely increase the subject's secondary, or delayed-type hypersensitivity, immune response, as effected by NK cells, against one or more pathogens to a degree which far exceeds the NK cell activity initiated by both colostrum-derived transfer factor and egg-derived transfer factor alone. In fact, the results show that a composition that incorporates teachings of the present invention may result in facilitation of the activity of NK cells with an unexpected degree of synergy.

[0071] In view of these results, further experimentation was conducted to determine the efficacy of a broader range of aspects of the present invention.

EXAMPLE 2

[0072] The effects of various transfer factor compositions, including compositions that incorporate teachings of the present invention, on the activity of lymphocytes in attacking cancer cells was evaluated. FIG. 3 schematically represents the protocol for the evaluation. Blood from healthy donors was obtained, at reference 40. Mononuclear cells, including natural killer cells, were separated from other constituents of the blood, at reference character 42, by standard phycol-urographin methodology, employing a density gradient p=i 0.077 g/cm3. The isolated mononuclear cells, or "effector cells," at a dilution of about 60,000 cells/100 μl of culture medium, were then introduced in 100 μl aliquots into the wells of a 96-well microtitre plate, such as that available from Coming Incorporated of Coming, N.Y., under the trade name COSTAR.RTM., as shown at reference character 44.

[0073] Thereafter, transfer factor-containing test samples, or "additives," as noted in TABLES 2 through 5 below, were introduced into each well, with resulting concentrations of transfer factor in the test samples being 1 mg/ml, 0.1 mg/ml, 0.01 mg/ml, 0.001 mg/ml, 0.0001 mg/ml, and 0.00001 mg/ml, as is also shown at reference character 44. A control including no transfer factor product was also employed. The microtitre plates were then placed in a CO2-incubator with conditions of 5% CO2 atmosphere, 100% humidity, and a temperature of 37° C., and incubated for periods of 24 hours and 48 hours. Each study variant was conducted in triplicate. After incubation, about 30,000 K-562 tumor cells (i.e., erythroblastotic human leukemia), or "target cells," were introduced into each well, as illustrated at reference character 46, providing a ratio of effector cells-to-target cells of about 2:1. The effector and target cells were then incubated for periods of 18 hours and 24 hours in the CO2 incubator, under the same conditions listed above.

[0074] Thereafter, at reference character 48, the MTT method of defining the viability of cellular cultures, which employs a soluble yellow bromide, 3-(4,5-dimethylthiasol-2-il)-2,5-tetrazol (MTT), was used to determine the number of K-562 tumor cells that were killed in each well. In such a test, live cells reduce the MTT to insoluble purple-blue intracellular crystals of MTT-formazan (MTT-f). Nonviable dead cells are not capable of reducing the MTT to MTT-f. Thus, the optical properties of the resulting solution may be evaluated to provide an indication of the affect of various transfer factor-containing products on the ability of the effector cells to kill the K-562 tumor cells. More specifically, the intensity of MTT transformation into MTT-f reflects the general level of the studied cells' dehydrogenase activity and is modulated by the activity of conjugated fermentation systems; e.g., respiratory chain of electrons transmission, etc.

[0075] The MTT solution used in this EXAMPLE was prepared in 5 mg/ml of Henks' saline solution, as known in the art. Equal volume aliquots of the MTT solution were introduced into the wells of the microtitre plates, and the plates were incubated in a CO2 incubator, under the same conditions noted above, for a period of about three to about four hours. The microtitre plates were then centrifuged at about 1,500 rpm for about 5 minutes, the supernatant was removed, and 150 μl aliquots of dimethylsulfoxide (DMSO) were introduced into the wells.

[0076] The microtitre plates were then permitted to sit at room temperature for a period of thirty minutes, allowing formazan crystals to completely dissolve. Thereafter, a multiwell spectrophotometer (LABSYSTEMS MultiScan MSS 340, available from Cambridge Scientific Products of Cambridge, Mass.) was used to evaluate each well of each microtitre plate at a wavelength of 540 nm.

[0077] As shown at reference character 50, the optical density (OD) measurements that were obtained with the spectrophotometer were then used to calculate the cytotoxic index (%) (CI (%)) of each well. The CI (%) calculation was performed according to the standard formula: Cl (%)=[I-(Oe t-ODe)/ODt]*100,

[0078] where ODe t is the OD in experimental series, ODe is the OD in wells including only effector cells, and ODt is the OD in the wells including only target cells. TABLE-US-00002 TABLE 2 CI (%) at 24 Hours Additive 1 mg/ml 10-1 mg/ml 10-2 mg/ml 10-3 mg/ml 10-4 mg/ml 10-5 mg/ml TF (bovine) 35 17 29 18 18 15 TF 13.5 20.3 35 28.5 10 20.3 (international formulation) TF (85:15, 13.3 10.6 29 30 21.6 76 bovine:avian) TF (70:30, 80 47 24 12 30 26.3 bovine:avian) TF (avian) 16 37 47 47 16.1 34.3 None 18 18 18 18 18 18 (spontaneous cell death) (. -.6%)

[0079] TABLE-US-00003 TABLE 3 % Increase in CI (over spontaneous CI) at 24 Hours Additive 1 mg/ml 10-1 mg/ml 10-2 mg/ml 10-3 mg/ml 10-4 mg/ml 10-5 mg/ml TF (bovine) 94 -6 61 0 0 -17 TF -25 13 94 58 -44 13 (international formulation) TF (85:15, -26 -41 61 67 20 322 bovine:avian) TF (70:30, 344 161 33 -33 67 46 bovine:avian) TF (avian) -11 106 161 161 -11 91 None 0 0 0 0 0 0 (spontaneous cell death) (. -.6%)

[0080] TABLE-US-00004 TABLE 4 CI (%) at 48 Hours Additive 1 mg/ml 10-1 mg/ml 10-2 mg/ml 10-3 mg/ml 10-4 mg/ml 10-5 mg/ml TF (bovine) 19.3 50 54.7 15.3 40.7 11.3 TF 23.3 12 17 42 48 62 (international formulation) TF (85:15) 48 82.7 96.7 69.4 54 91 TF (70:30) 97 94 99 90 96 91 TF (avian) 68 49 45 35 58 70 None 18 18 18 18 18 18 (spontaneous cell death) (. -.6%)

[0081] TABLE-US-00005 TABLE 5 % Increase in CI (over spontaneous CI) at 48 Hours Additive 1 mg/ml 10-1 mg/ml 10-2 mg/ml 10-3 mg/ml 10-4 mg/ml 10-5 mg/ml TF (bovine) 7 178 204 -15 126 -37 TF 29 -33 -6 133 167 244 (international formulation) TF (85:15, 167 359 437 286 200 406 bovine:avian) TF (70:30, 439 422 450 400 433 406 bovine:avian) TF (avian) 278 172 150 94 222 289 None 0 0 0 0 0 0 (spontaneous cell death) (. -.6%)

[0082] The data provided in TABLES 2 through 5 confirms that the majority of test samples (i.e., transfer factor-containing compositions) stimulated increased (relative to spontaneous tumor cell death) antitumor and cytotoxic activity of healthy donors' lymphocytes against K-562 tumor cells.

[0083] The greatest stimulating effect appears in the 48 hour results, with the most effective range of stimulating concentrations being from about 0.1 mg/ml to about 0.0001 mg/ml. The test samples that included both colostrum-derived transfer factor and egg-derived transfer factor again appear to be the most effective in the given conditions of the experiment, lysing as many as 80-98% of the K-562 tumor cells.

[0084] Additionally the results of TABLE 5 indicate that combinations of different types of transfer factor, particularly the 85:15 ratio of TF to egg-derived transfer factor, may be more effective that other courses of therapy for eliminating undesirable cells and pathogens from the body of a treated animal. More specifically, inasmuch as the inventors are aware, in equivalent testing, the best results that could be achieved with interleukin-2 treatment have been 76% cytotoxicity of K-562 tumor cells with a 24 hour incubation (which amounts to a 322% increase over spontaneous deaths of such cells)and an 88% cytotoxicity of K-562 tumor cells with a 48 hour incubation (which amounts to a 389% increase over spontaneous deaths of such cells).

EXAMPLE 3

[0085] Another confirmatory test was conducted to verify the above-stated results and to evaluate the effects of a greater variety of compositions of the present invention on inducing NK and other mononuclear cells to kill K-562 tumor cells. The same protocol described in EXAMPLE 2 was employed in the tests of EXAMPLE 3.

[0086] The results of 24 and 48 hour incubation periods for a variety of compositions formulations, each including egg powder and bovine colostrum powder, are listed in TABLES 6 through 9. TABLE-US-00006 TABLE 6 CI (%) at 24 Hours 1 10-1 10-2 Bovine:Avian mg/ml mg/ml mg/ml 10-3 mg/ml 10-4 mg/ml 85:15 45 29 67.5 28 50 50:50 67.5 23 66 63.5 22.5 30:70 64.6 68.8 39.1 45.6 44 15:85 55.2 28 20.1 20 18.8 None 18 18 18 18 18 (spontaneous cell death) (. -.6%)

[0087] TABLE-US-00007 TABLE 7 % Increase in CI (over spontaneous CI) at 24 Hours 1 10-1 10-2 Bovine:Avian mg/ml mg/ml mg/ml 10-3 mg/ml 10-4 mg/ml 85:15 150 61 275 56 178 50:50 275 28 267 253 25 30:70 259 282 117 153 144 15:85 207 56 12 11 4 None 0 0 0 0 0 (spontaneous cell death) (. -.6%)

[0088] TABLE-US-00008 TABLE 8 CI (%) at 48 Hours 1 10-1 10-2 Bovine:Avian mg/ml mg/ml mg/ml 10-3 mg/ml 10-4 mg/ml 85:15 46 60 69 67 64 50:50 69 74 74 63 49 30:70 75 83 67 63 45 15:85 77 69 51 42 40 None 18 18 18 18 18 (spontaneous cell death) (. -.6%)

[0089] TABLE-US-00009 TABLE 9 % Increase in CI (over spontaneous CI) at 48 Hours 1 10-1 10-2 Bovine:Avian mg/ml mg/ml mg/ml 10-3 mg/ml 10-4 mg/ml 85:15 156 233 283 272 256 50:50 283 311 311 250 172 30:70 317 361 272 250 150 15:85 328 283 183 133 122 None 0 0 0 0 0 (spontaneous cell death) (. -.6%)

[0090] For the sake of comparison, a whole colostrum sample and a processed transfer factor sample including 100% bovine transfer factor sample (an no avian transfer factor), each including 0.01 mg/ml of transfer factor, were evaluated. At 24 hours, the whole colostrum sample demonstrated a 22% increase in lysis over spontaneous lysis, while the 100% bovine transfer factor sample was responsible for a 103% increase in lysis over spontaneous cell lysis. At 48 hours, the increases in cell lysis were 26% and 203%, respectively.

[0091] The data of TABLES 6 through 9, particularly of TABLES 6 and 8, shows that when more colostrum-derived transfer factor is present in a composition according to the present invention (e.g., 85:15), the initial (24 hour test) response may be greater than the response generated by compositions that include less colostrum-derived transfer factor, but does not increase significantly over time (48 hour test).

[0092] Compositions (e.g., 50:50 and 30:70) that include more egg-derived transfer factor may provide comparable short term results (24 hour test), but provide much better long term (48 hour test) results.

[0093] These results support the theory that combining different types of transfer factors provides a synergistic effect. They also indicate that the proportions of different types of transfer factor in a composition may be tailored to provide a desired result.

EXAMPLE 4

[0094] TABLE-US-00010 TABLE 10 CI (%) 1 mg/ml 10-1 mg/ml 10-2 mg/ml 10-3 mg/ml 10-4 mg/ml 10-5 mg/ml 24 hrs. TF (85:15) 13.3 10.6 29 30 21.6 76 (colostrum:egg) 85:15 45 29 67.5 28 50 (colostrum:egg) 48 hrs. TF (85:15) 48 82.7 96.7 69.4 54 91 (colostrum:egg) 85:15 46 60 69 67 64 (colostrum:egg)

[0095] EXAMPLE 4 compares data obtained in EXAMPLES 2 and 3 above to illustrate that the inclusion of additional components, primarily polysaccharides, in TFP improves the efficiency with which a composition that incorporates teachings of the present invention induces NK and other mononuclear blood cells to kill K-562 tumor cells and, thus, elicits a secondary immune response.

[0096] Notably, in the 48 hour test,where polysaccharides were included, cytotoxicity was greater at all dilutions above 0.0001 mg/ml than in comparable compositions that lacked polysaccharides. Thus, polysaccharides are believed to either increase the synergism with which the two or more types of transfer factors act or to provide additional synergism in the elicitation of a secondary immune response.

[0097] While the foregoing EXAMPLES and accompanying data demonstrate the effectiveness of compositions that include transfer factor and, in particular, compositions that include two or more different types of transfer factor, in eliciting a T-cell (e.g., NK cell) mediated immune response, transfer factor is also believed to affect the immune system of a treated subject in a number of other ways. For example, and not to limit the scope of the present invention, transfer factor may provide the biochemical benefits disclosed in U.S. patent application Ser. No. 11/122,430, filed May 4, 2005, the disclosure of which is hereby incorporated herein, in its entirety, by this reference. As the benefits of transfer factor are not limited to elicitation of T-cell mediated immune responses, synergy in the biochemical effects of transfer factor may also be recognized when two or more types of transfer factor are combined.

[0098] Although the foregoing description contains many specifics, these should not be construed as limiting the scope of the present invention, but merely as providing illustrations of some of the presently preferred embodiments. Similarly, other embodiments may be devised without departing from the spirit or scope of the present invention. Features from different embodiments may be employed in combination. The scope of the invention is, therefore, indicated and limited only by the appended claims and their legal equivalents rather than by the foregoing description. All additions, deletions and modifications to the invention as disclosed herein which fall within the meaning and scope of the claims are to be embraced thereby.



Inventors

* Lisonbee, David * Hennen, William J. * Daugherty, F. Joseph

US Class

Attorney, Agent or Firm

* TRASK BRITT

International Class A61K 39/00



Abstract text

A composition for eliciting a T-cell mediated immune response in a subject includes transfer factor from at least two different types of source animals. For example, the composition may include mammalian transfer factor and nonmammalian transfer factor. An example of the composition includes a combination of a colostrum-derived product, which includes the mammalian transfer factor, and an egg-derived product, which includes the nonmammalian transfer factor. Additionally, the egg-derived product may be substantially free of fat. Methods for forming the composition and eliciting T-cell mediated immune responses in subjects that have been treated with the composition are also disclosed. Claims

1-38. (Cancelled)

39. A method for reducing the cleaning frequency of processing equipment used for capsulating an egg-derived product, comprising: combining a colostrum-derived product with an egg-derived product before or during introduction of the egg-derived product into the capsulation equipment.

40. The method of claim 39, wherein said combining comprises combining about equal weights of said colostrum-derived product and the egg-derived product.

41. The method of claim 39, wherein said combining comprises combining said colostrum-derived product in a greater amount, by weight, than the egg-derived product with the egg-derived product.

42. The method of claim 39, wherein said combining comprises combining said colostrum-derived product, in a lesser amount, by weight, than the egg-derived product with the egg-derived product.

43. The method of claim 39, further comprising: defatting the egg-derived product.

44. The method of claim 39, further comprising: combining at least one vitamin with at least one of the egg-derived product and said colostrum-derived product.

45. The method of claim 39, wherein said combining comprises combining said colostrum-derived product and the egg-derived product with at least one of said colostrum-derived product and the egg-derived product including transfer factor.

46. A method for reducing the cleaning frequency of equipment used for processing an egg-derived product, comprising: combining a colostrum-derived product with an egg-derived product before or during introduction of the egg-derived product into the equipment.

47. The method of claim 46, wherein said combining comprises combining about equal weights of said colostrum-derived product and the egg-derived product.

48. The method of claim 46, wherein said combining comprises combining said colostrum-derived product in a greater amount, by weight, than the egg-derived product with the egg-derived product.

49. The method of claim 46, wherein said combining comprises combining said colostrum-derived product, in a lesser amount, by weight, than the egg-derived product with the egg-derived product.

50. The method of claim 46, further comprising: defatting the egg-derived product.

51. The method of claim 46, further comprising: combining at least one vitamin with at least one of the egg-derived product and said colostrum-derived product.

52. The method of claim 46, wherein said combining comprises combining said colostrum-derived product and the egg-derived product with at least one of said colostrum-derived product and the egg-derived product including transfer factor. Description

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates generally to compositions which include transfer factor and, more specifically, to compositions which include transfer factor from different types of source animals. The present invention also relates to methods for making compositions that include different types of transfer factor and to methods for eliciting or enhancing a T-cell mediated immune response by the immune system of a subject.

[0003] 2. Background of Related Art

[0004] Many deadly pathogens are passed to humans from the animal kingdom. For example, monkeys are the sources of the type I human immunodeficiency virus (HIV-I), which causes acquired immune deficiency syndrome (AIDS) and monkeypox, which is similar to smallpox; ground-dwelling mammals are believed to be the source of the Ebola virus; fruit bats and pigs are the source of the Nipah virus; the Hendra virus comes from horses; the virus responsible for the "Hong Kong Flu" originated in chickens; and wild birds, especially ducks, are the sources of many of the deadly influenza viruses. Many diseases also have animal reservoirs. By way of example, mice carry Hanta virus, rats carry the Black Plague, and deer carry Lyme disease.

The Immune System

[0005] The immune systems of vertebrates are equipped to recognize and defend the body from invading pathogenic organisms, such as parasites, bacteria, fungi, and viruses. Vertebrate immune systems typically include a cellular component and a noncellular component.

[0006] The cellular component of an immune system includes the so-called "lymphocytes," or white blood cells, of which there are several types. It is the cellular component of a mature immune system that typically mounts a primary, nonspecific response to invading pathogens, as well as being involved in a secondary, specific response to pathogens.

[0007] In the primary, or initial, response to an infection by a pathogen, white blood cells that are known as phagocytes locate and attack the invading pathogens. Typically, a phagocyte will internalize, or "eat" a pathogen, then digest the pathogen. In addition, white blood cells produce and excrete chemicals in response to pathogenic infections that are intended to attack the pathogens or assist in directing the attack on pathogens.

[0008] Only if an infection by invading pathogens continues to elude the primary immune response is a specific, secondary immune response to the pathogen needed. As this secondary immune response is typically delayed, it is also known as "delayed-type hypersensitivity." A mammal, on its own, will typically not elicit a secondary immune response to a pathogen until about seven (7) to about fourteen (14) days after becoming infected with the pathogen. The secondary immune response is also referred to as an acquired immunity to specific pathogens. Pathogens have one or more characteristic proteins, which are referred to as "antigens." In a secondary immune response, white blood cells known as B lymphocytes, or "B-cells," and T lymphocytes, or "T-cells," "learn" to recognize one or more of the antigens of a pathogen. The B-cells and T-cells work together to generate proteins called "antibodies," which are specific for (e.g., configured to bind to or otherwise "recognize") one or more certain antigens on a pathogen.

[0009] The T-cells are primarily responsible for the secondary, or delayed-type hypersensitivity, immune response to a pathogen or antigenic agent. There are three types of T-cells: T-helper cells, T-suppressor cells, and antigen-specific T-cells, which are also referred to as cytotoxic (meaning "cell-killing") T-lymphocytes (CTLs), or T-killer cells or natural killer (NK) cells. The T-helper and T-suppressor cells, while not specific for certain antigens, perform conditioning functions (e.g., the inflammation that typically accompanies an infection) that assist in the removal of pathogens or antigenic agents from an infected host.

[0010] Antibodies, which make up only a part of the noncellular component of an immune system, recognize specific antigens and, thus, are said to be "antigen-specific." The generated antibodies then basically assist the white blood cells in locating and eliminating the pathogen from the body. Typically, once a white blood cell has generated an antibody against a pathogen, the white blood cell and all of its progenitors continue to produce the antibody. After an infection is eliminated, a small number of T-cells and B-cells that correspond to the recognized antigens are retained in a "resting" state. When the corresponding pathogenic or antigenic agents again infect the host, the "resting" T-cells and B-cells activate and, within about forty-eight (48) hours, induce a rapid immune response. By responding in this manner, the immune system mounts a secondary immune response to a pathogen, the immune system is said to have a "memory" for that pathogen.

[0011] Mammalian immune systems are also known to produce smaller proteins, known as "transfer factors," as part of a secondary immune response to infecting pathogens. Transfer factors are another noncellular part of a mammalian immune system. Antigen-specific transfer factors are believed to be structurally analogous to antibodies, but on a much smaller molecular scale. Both antigen-specific transfer factors and antibodies include antigen-specific sites. In addition, both transfer factors and antibodies include highly conserved regions that interact with receptor sites on their respective effector cells. In transfer factor and antibody molecules, a third, "linker," region connects the antigen-specific sites and the highly conserved regions.

The Role of Transfer Factor in the Immune System

[0012] Transfer factor is a low molecular weight isolate of lymphocytes. Narrowly, transfer factors may have specificity for single antigens. U.S. Pat. Nos. 5,840,700 and 5,470,835, both of which issued to Kirkpatrick et al. (hereinafter collectively referred to as "the Kirkpatrick Patents"), disclose the isolation of transfer factors that are specific for certain antigens. More broadly, "specific" transfer factors have been generated from cell cultures of monoclonal lymphocytes. Even if these transfer factors are generated against a single pathogen, they have specificity for a variety of antigenic sites of that pathogen. Thus, these transfer factors are said to be "pathogen-specific" rather than antigen-specific. Similarly, transfer factors that are obtained from a host that has been infected with a certain pathogen are pathogen-specific. Although such preparations are often referred to in the art as being "antigen-specific" due to their ability to elicit a secondary immune response when a particular antigen is present, transfer factors having different specificities may also be present in such preparations. Thus, even the so-called "antigen-specific," pathogen-specific transfer factor preparations may be specific for a variety of antigens.

[0013] Additionally, it is believed that antigen-specific and pathogen-specific transfer factors may cause a host to elicit a delayed-type hypersensitivity immune response to pathogens or antigens for which such transfer factor molecules are not specific. Transfer factor "draws" at least the non-specific T-cells, the T-inducer and T-suppressor cells, to an infecting pathogen or antigenic agent to facilitate a secondary, or delayed-type hypersensitivity, immune response to the infecting pathogen or antigenic agent.

[0014] Typically, transfer factor includes an isolate of proteins having molecular weights of less than about 10,000 daltons (D) that have been obtained from immunologically active mammalian sources. It is known that transfer factor, when added either in vitro or in vivo to mammalian immune cell systems, improves or normalizes the response of the recipient mammalian immune system.

[0015] The immune systems of newborns have typically not developed, or "matured," enough to effectively defend the newborn from invading pathogens. Moreover, prior to birth, many mammals are protected from a wide range of pathogens by their mothers. Thus, many newborn mammals cannot immediately elicit a secondary response to a variety of pathogens. Rather, newborn mammals are typically given secondary immunity to pathogens by their mothers. One way in which mothers are known to boost the immune systems of newborns is by providing the newborn with a set of transfer factors. In mammals, transfer factor is provided by a mother to a newborn in colostrum, which is typically replaced by the mother's milk after a day or two. Transfer factor basically transfers the mother's acquired, specific (i.e., delayed-type hypersensitive) immunity to the newborn. This transferred immunity typically conditions the cells of the newborn's immune system to react against pathogens in an antigen-specific manner, as well as in an antigen- or pathogen-nonspecific fashion, until the newborn's immune system is able on its own to defend the newborn from pathogens. Thus, when transfer factor is present, the immune system of the newborn is conditioned to react to pathogens with a hypersensitive response, such as that which occurs with a typical delayed-type hypersensitivity response. Accordingly, transfer factor is said to "jump start" the responsiveness of immune systems to pathogens.

[0016] Much of the research involving transfer factor has been conducted in recent years. Currently, it is believed that transfer factor is a protein with a length of about forty-four (44) amino acids. Transfer factor typically has a molecular weight in the range of about 3,000 to about 5,000 Daltons (Da), or about 3 kDa to about 5 kDa, but it may be possible for transfer factor molecules to have molecular weights outside of this range. Transfer factor is also believed to include three functional fractions, each of which may include different types of transfer factor molecules: an inducer fraction; an immune suppressor fraction; and an antigen-specific fraction. Many in the art believe that transfer factor also includes a nucleoside portion, which could be connected to the protein molecule or separate therefrom, that may enhance the ability of transfer factor to cause a mammalian immune system to elicit a secondary immune response. The nucleoside portion may be part of the inducer or suppressor fractions of transfer factor.

[0017] The antigen-specific region of the antigen-specific transfer factors is believed to comprise about eight (8) to about twelve (12) amino acids. A second highly-conserved region of about ten (10) amino acids is thought to be a very high-affinity T-cell receptor binding region. The remaining amino acids may serve to link the two active regions or may have additional, as yet undiscovered properties. The antigen-specific region of a transfer factor molecule, which is analogous to the known antigen-specific structure of antibodies, but on a much smaller molecular weight scale, appears to be hyper-variable and is adapted to recognize a characteristic protein on one or more pathogens. The inducer and immune suppressor fractions are believed to impart transfer factor with its ability to condition the various cells of the immune system so that the cells are more fully responsive to the pathogenic stimuli in their environment.

Sources of Noncellular Immune System Components

[0018] Conventionally, transfer factor has been obtained from the colostrum of milk cows, such as by the method described in U.S. Pat. No. 4,816,563 to Wilson et al. (hereinafter "Wilson"). While milk cows typically produce large amounts of colostrum and, thus, large amounts of transfer factor over a relatively short period of time, milk cows only produce colostrum for about a day or a day-and-a-half every year. Thus, milk cows are neither a constant source of transfer factor nor an efficient source of transfer factor.

[0019] Transfer factor has also been obtained from a wide variety of other mammalian sources. For example, in researching transfer factor, mice have been used as a source for transfer factor. Antigens are typically introduced subcutaneously into mice, which are then sacrificed following a delayed-type hypersensitivity reaction to the antigens. Transfer factor is then obtained from spleen cells of the mice.

[0020] While different mechanisms are typically used to generate the production of antibodies, the original source for antibodies may also be mammalian. For example, monoclonal antibodies may be obtained by injecting a mouse, a rabbit, or another mammal with an antigen, obtaining antibody-producing cells from the mammal, then fusing the antibody-producing cells with immortalized cells to produce a hybridoma cell line, which will continue to produce the monoclonal antibodies throughout several generations of cells and, thus, for long periods of time.

[0021] Antibodies against mammalian pathogens have been obtained from a wide variety of sources, including mice, rabbits, pigs, cows, and other mammals. In addition, the pathogens that cause some human diseases, such as the common cold, are known to originate in birds. As it has become recognized that avian (i.e., bird) immune systems and mammalian immune systems are very similar, some researchers have turned to birds as a source for generating antibodies.

[0022] Avian antibodies that are specific for pathogens that infect mammals, or "mammalian pathogens," have been obtained by introducing antigens into eggs. Alternatively, antibodies may be present in eggs following exposure of the source animal to antigens, including antigens of mammalian pathogens. U.S. Pat. No. 5,080,895, issued to Tokoro on Jan. 14, 1992 (hereinafter "the '895 Patent"), discloses a method that includes injecting hens with pathogens that cause intestinal infectious diseases in neonatal mammals. The hens then produce antibodies that are specific for these pathogens, which are present in eggs laid by the hens. The '895 Patent discloses compositions that include these pathogen-specific antibodies and use thereof to treat and prevent intestinal diseases in neonatal piglets and calves. Treatment of pathogenic infections in mammals with avian antibodies may have undesirable results, however, since the immune systems of mammals may respond negatively to the large avian antibody molecules by eliciting an immune response to the antibodies themselves. Moreover, as mammalian immune systems do not recognize avian antibodies as useful for their abilities to recognize certain pathogens, or the specificities of avian antibodies for antigens of such pathogens, avian antibodies often do not elicit the desired immune responses in mammals.

[0023] It is also known that transfer factor may be obtained from eggs. U.S. Pat. No. 6,468,534 to Hennen et al. (hereinafter "Hennen") describes a process by which female chickens (i.e., hens) are exposed to one or more antigens, which results in the elicitation of an immune response, including a secondary immune response, by the chickens. As a result of the secondary immune response, transfer factor molecules are present in the eggs of the chicken. The eggs may then be processed to provide a product in which the transfer factor is present. Such a product may take the form of a freeze dried, or lyophilized, egg powder, and may include all or part of the egg. The egg powder may then be incorporated directly into gelatin capsules or mixed with other substances the introduced into gelatin capsules.

[0024] FIG. 2 schematically depicts capsulation equipment of a type that is currently useful for capsulating egg-derived avian transfer factor in the form of an egg powder. Capsulation equipment 20 includes a composition supply hopper 24, a feed station 28, and an auger 26 in communication between each composition supply hopper 24 and feed station 28. Auger 26 transports the whole egg powder from composition supply hopper 24 to feed station 28.

[0025] When auger 26 operates, it is heated to a temperature which exceeds the relatively low melting point of cholesterol, from egg yolk, in the whole egg powder. The warmed cholesterol is sticky, coating auger 26, the conduit in communication therewith, and feed station 28, thereby decreasing the efficiency with which capsulation equipment 20 operates. Consequently, capsulation equipment must be disassembled and cleaned periodically, which may take a considerable amount of time (e.g., up to about 8 hours), resulting in a significant decrease in the productivity of capsulation equipment 20 and, thus, the number of capsules that may be formed therewith. Thus, processing of whole egg powder to obtain a transfer factor-containing product is somewhat undesirable.

[0026] Additionally, compositions which are derived from products (e.g., eggs or colostrum) from a single source animal typically only include transfer factor molecules which have specificity to antigens to which the source animal has been exposed. The consequence of such limited exposure may be that the effectiveness of such transfer factor-containing compositions in preventing or treating certain types of infections or conditions is also limited.

[0027] Accordingly, there is a need for a composition which is useful for causing an immune system of a treated subject to elicit an immune response to a broader array of pathogens, as well as for a method for improving the efficiency and productivity with which capsulation and other composition-forming equipment operates.

SUMMARY OF THE INVENTION

[0028] The present invention includes a composition for eliciting a T-cell mediated immune response in a subject. The composition includes transfer factor from at least two different types of source animals. The term "type," as used herein with respect to source animals, describes the source animals from which transfer factor may be obtained and refers to source animals from different classes (e.g., mammals, birds, reptiles, amphibians, insects, etc.). The term "type," as used herein, also refers to source animals from different subclasses, orders (e.g., artiodactyls, primates, carnivores, etc.), families (bovine, hominids, felines, etc.), subfamilies, genuses (e.g., cattle, humans, domestic cats, etc.), and even species and subspecies. Use of the term "type" herein with respect to transfer factor denotes the type of source animal from which the transfer factor was obtained.

[0029] An exemplary embodiment of the composition includes transfer factor from both mammalian and nonmammalian source animals, which types of transfer factor are also referred to herein as "mammalian transfer factor" and "nonmammalian transfer factor," respectively. By way of nonlimiting example, the mammalian transfer factor may be included in the composition as colostrum or a fraction or extract thereof, which are collectively referred to herein as "colostrum-derived products," or otherwise, as known in the art (e.g., as a leukocyte (white blood cell) extract, as a splenic ("from the spleen") extract, etc.). Also by way of example, the nonmammalian transfer factor of the exemplary composition may be obtained from an egg or a fraction or extract thereof, which are also referred to herein as "egg-derived products."

[0030] When a composition of the present invention includes a colostrum-derived product and an egg-derived product, both products may be included in the mixture in amounts (e.g., by weight, by volume, etc., of the total mixture) that are about equal, or more of one of the colostrum-derived product and the egg-derived product than the other.

[0031] In another aspect, the present invention includes a method for capsulating an egg-derived product which includes transfer factor. The inventive capsulation method include mixing a substantially fat-free component, such as a colostrum-derived product, which may or may not include transfer factor, with the egg-derived product before or while the egg-derived product is being introduced into capsulation equipment.

[0032] Additionally, the present invention includes a method for reducing the cleaning frequency of capsulation equipment used for capsulating an egg-derived product. That method includes mixing a less fatty or substantially fat free substance, such as a colostrums-derived product, with the egg-derived product before or during introduction of the egg-derived product into the capsulation equipment.

[0033] The present invention also includes methods for treating a subject. Treatment methods that incorporate teachings of the present invention include administration of a composition according to the present invention to a subject. As the composition includes transfer factor, administration of the composition to the subject will cause the subject's immune system to elicit a T-cell mediated immune response or will enhance a T-cell mediated immune response by the subject's immune system which is already underway.

[0034] Other features and advantages of the present invention will become apparent to those of ordinary skill in the art through consideration of the ensuing description, the accompanying drawings, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In the drawings, which depict exemplary embodiments of various aspects of the present invention:

[0036] FIG. 1 depicts an example of the manner in which a composition that incorporates teachings of the present invention may be embodied; and

[0037] FIG. 2 is a schematic representation of capsulation equipment that may be used to introduce a powdered embodiment of the composition of the present invention into gelatin capsules.

DETAILED DESCRIPTION

[0038] An exemplary embodiment of composition that incorporates teachings of the present invention includes transfer factor from at least two different types of source animals. By way of nonlimiting example, a composition according to the present invention may include mammalian transfer factor and nonmammalian transfer factor.

[0039] The different types of transfer factor of the inventive composition may be obtained from any suitable source. For example, mammalian transfer factor may be obtained from colostrum, as described in Wilson, the disclosure of which is hereby incorporated herein in its entirety by this reference, or otherwise, as known in the art (e.g., a leukocyte (white blood cell) extract, a splenic (i.e., "from the spleen") extract, etc.). An exemplary source for nonmammalian transfer factor is an egg of an animal, such as a chicken, as described in Hennen, the disclosure of which is hereby incorporated herein in its entirety by this reference. Thus, a composition according to the present invention may include a first component which comprises colostrum or a fraction or extract thereof, which are collectively referred to herein as a "colostrum-derived product," as well as a second component that comprises egg or a fraction or extract thereof, which are also referred to herein as an "egg-derived products."

[0040] As compositions that incorporate teachings of the present invention include transfer factor from different types of source animals, they may include transfer molecules with a broader array of antigen-specificity or pathogen-specificity than conventional transfer factor-containing compositions. Thus, a composition according to the present invention is capable of enlisting the immune system of a treated animal to elicit a T-cell mediated immune response against a broader array of pathogens than those against which conventional transfer factor-containing compositions are effective. This is because different types of animals may be exposed to different types of antigens or pathogens, such as by vaccination, the animals' environments, or the like.

[0041] As an example, a composition which includes transfer factor-containing components from both cows and chickens will include transfer factor molecules which are specific to antigens or pathogens to which cows are exposed, as well as transfer factor molecules that have specificity for antigens or pathogens to which chickens are exposed. As both cows and chickens may be exposed to antigens or pathogens to which the other is not exposed, such a composition may include transfer factor molecules with antigen or pathogen specificities that would not be present in a composition that includes only transfer factor from cows (e.g., by way of a colostrum-derived product) or transfer factor from chickens (e.g., through an egg-derived product).

[0042] A composition of the present invention may include about the same amounts, measured in terms of weight or volume, of a colostrum-derived product and an egg-derived product (i.e., about 50% colostrum-derived product and about 50% egg-derived product). Alternatively, a composition that incorporates teachings of the present invention may include about more colostrum-derived product (e.g., about 85% or 60%, by combined weight of the colostrum-derived product and egg-derived product) than egg-derived product (about 15% or 40%, by weight). As another alternative, the inventive composition may include more egg-derived product (e.g., about 60% or 85%, by weight) than colostrum-derived product (e.g., about 40% or 15% by weight). As another example, a composition that incorporates teachings of the present invention may include about one percent, by weight, of one of a colostrum-derived product and an egg-derived product and about 99%, by weight, of the other of the colostrum-derived product and the egg-derived product. Although specific amounts of colostrum-derived product and egg-derived product have been provided, any combination thereof is within the scope of the present invention.

[0043] In addition to including a source of transfer factor (e.g., a colostrum-derived product, an egg-derived product, etc.) a composition that incorporates teachings of the present invention may include one or more other ingredients, including, but not limited to, vitamins, minerals, proteins, natural products (e.g., herbs, mushrooms, roots, etc., or extracts thereof), and the like. Additional ingredients may be useful for providing further advantages to subjects to which the composition is administered, or may enhance the ability of the transfer factor in the composition to elicit or enhance a secondary, or delayed-type hypersensitivity, immune response.

[0044] As shown in FIG. 1, without limiting the scope of the present invention, a composition 10 according to the present invention may take the form of a powdered or particulate substance, which includes the multiple types of transfer factor (not shown). In order to ensure that an appropriate and precise dosage of composition 10 is administered to a subject (not shown), composition 10 may be contained within a gelatin capsule 12 of a type which is well-known and readily available to those in the art. The result is the illustrated capsule 14. Alternatively, a composition according to the present invention may be embodied as tablet, a so-called "caplet," an unencapsulated powder, a liquid, a gel, or in any other pharmaceutically acceptable form. Suitable processes for placing the inventive composition into any such form are readily apparent to those of skill in the art.

[0045] In an exemplary embodiment of a method for making or forming a composition according to the present invention, a first type of transfer factor may be combined with a second type of transfer factor. Additionally, one or more other types of transfer factor may be combined with the first and second types of transfer factor. The different types of transfer factor that are combined may be substantially purified transfer factor, components or "products" that include transfer factor, or any combination thereof.

[0046] Turning again to FIG. 2, a process for forming composition-filled capsules 14, such as that shown in FIG. 1, is provided merely as an example for a method for making a composition that incorporates teachings of the present invention. As illustrated, the composition 10 is made and composition-filled capsules 14 are formed using standard capsulation equipment 20 of a type known in the industry, such as the SF-135 capsule filling machine available from CapPlus Technologies of Phoenix, Ariz.

[0047] In addition to one or more composition supply hoppers 24, an auger 26 associated with each composition supply hopper 24, and a feed station 28 with which each auger 26 and the conduit 27 within which auger 26 is contained communicates, capsulation equipment 20 includes one or more capsule hoppers 30, as well as a pneumatic feed system 32 for transporting capsule bodies 12a and/or caps 12b to feed station 28.

[0048] As the capsulation equipment will introduce the mixture into capsules, which may be swallowed by a subject, it is currently preferred that the substantially fat-free component and the egg-derived product be introduced into the capsulation equipment in powdered form. The substantially fat-free component dilutes the amount, or concentration, of fat (e.g., from egg yolk) present in the mixture relative to the concentration of fat which is present in the egg-derived product. Accordingly, the relative amounts of substantially-fat free product and the egg-derived product may be tailored to provide a fat concentration that will minimize clogging of the capsulation equipment.

[0049] Continuing with the example of a composition 10 which includes a colostrum-derived product 10a as the substantially fat-free component and an egg-derived product 10b, colostrum-derived product 10a and egg-derived product 10b may be introduced simultaneously into a single composition supply hopper 24 of capsulation equipment 20. For example, colostrum-derived product 10a and egg-derived product 10b may be mixed upon introduction thereof into composition supply hopper 24, as shown, or premixed. By introducing a substance which has a lower fat content than egg-derived product 10b into composition supply hopper 24 along with egg-derived product 10b, the fat content (e.g., concentration) of the resulting mixture is less than that of egg-derived product 10b, reducing or eliminating the likelihood that composition supply hopper 24, auger 26, conduit 27, feed station 28, or any other component of capsulation equipment 20 will be coated with cholesterol or fat.

[0050] Following introduction of a predetermined amount of composition 10 into capsule bodies 12a at feed station, the filled capsule bodies 12a are transported to a capsule closing station 34, where capsule caps 12b are assembled therewith to fully contain composition 10 within capsule 12.

[0051] Again, a composition-filled capsule 14 is only one example of the manner in which a composition that incorporates teachings of the present invention may be embodied. The inventive composition may also take other forms, such as tablets, caplets, loose powder, liquid, gel, liquid-filled or gel-filled capsules, any other pharmaceutically acceptable form known in the art, each of which may be made by known processes.

[0052] The composition of the present invention may be administered to a subject (e.g., a mammal, such as a human, a dog, or a cat, a bird, a reptile, a fish, etc.) by any suitable process (e.g., enterally, parentarlly, etc.), depending, of course, upon the form thereof. For example, virtually any form of the composition (e.g., a capsule, tablet, caplet, powder, liquid, gel, etc.) may be administered orally (i.e., through the mouth of the subject), provided that the composition includes a pharmaceutically acceptable carrier of a type known in the art that will prevent degradation or destruction of transfer factor molecules by the conditions that persist in the digestive tract of the subject without substantially interfering with the efficacy of the transfer factor molecules included in the composition.

[0053] The dosage of composition or transfer factor within the composition that is administered to the subject may depend on a variety of factors, including, without limitation, the subject's weight, the health of the subject, or conditions (e.g., pathogens) to which the subject has been exposed.

[0054] Administration of the composition to the subject may cause the immune system of the subject to elicit a T-cell mediated immune response against one or more antigens or pathogens. Thus, the composition may be administered to a subject to treat a disease state that the subject is experiencing, to prevent the subject from exhibiting a disease state caused by a particular pathogen, or to merely enhance the overall health of the subject's immune system and abilities to fight off infecting or invading pathogens.

[0055] The following EXAMPLES illustrate the enhanced ability of a composition which includes transfer factor from multiple types of source animals to cause an immune system of a treated subject to elicit a T-cell mediated immune response to various types of pathogens, in the form of target cells. The target cells included bacteria (e.g., C. pneumoniae and H. pylori) and viruses (e.g., herpes simplex virus-1 (HSV-1) and herpes simplex virus-2 (HSV-2)) in the form of virally infected cells, as well as to cancerous, or malignant, cells (e.g., K562 erythroleukemic cells).

[0056] The in vitro technique that was used to make these determinations was the so-called "chromium-51 release assay," which includes measurement of the amount of radioactive chromium-51 (Cr-51) released by cells that have been attacked by NK cells. The radioactivity measurement may be obtained, for example, with a Beckman 2000 Gamma Counter, which is available from Beckman Coulter, Inc., of Fullerton, Calif.

[0057] In the EXAMPLES, a fixed amount (5 micrograms per milliliter of nutrient media and cellular milieu) of a powdered composition was provided in the nutrient media and cellular milieu, along with a substantially fixed amount of NK cells. Examples of the powdered compositions that were used include bleached wheat flour, Transfer Factor™ (TF), available from 4Life Research, LLC, of Sandy, Utah, Transfer Factor Plus™ (TFP), also available from 4Life Research, avian transfer factor available in a lyophilized (i.e., freeze-dried) whole egg powder, and mixtures of TF and TFP (both the formula marketed in the United States and that marketed internationally) with avian transfer factor in a ratio of about 85% TF or TFP (i.e., bovine transfer factor), by weight, to about 15% avian transfer factor, by weight. The powdered composition, nutrient media, NK cells, and target cells were mixed and incubated for four hours prior to measuring the radioactive atoms that were released by disruption of the target cells by the NK cells. Each exemplary reaction was conducted in triplicate, with the results of the three reactions having been averaged.

[0058] The following TABLE includes data of the counts per minute obtained with each combination of target cells and powdered composition, as well as the effectiveness of each powdered composition in eliciting an NK cell-mediated immune response against the target cells relative to the NK cell-mediated immune response relative to (measured in percent increase) the same types and concentrations of target cells in the presence of bleached wheat flour.

1 TABLE Target Cells Composition C. Pneu H. Pyl K562 HSV-1 HSV-2 Flour 1323/ 1121/ 1267/ 2017/ 1262/ TF 2593/ 2499/ 2445/ 2240/ 2473/ % increase 196% 223% 193% 110% 196% TFP 3386/ 2701/ 3243/ 2944/ 1956/ % increase 256% 241% 256% 146% 155% 100% Avian TF 2553/ 1860/ 2483/ 2985/ 2183/ % increase 193% 166% 196% 148% 173% Bov-Av TF 14,857/ 11,434/ 6,639/ 17,910/ 10,626/ % increase 1123% 1020% 524% 888% 842% Bov-Av TFP US 61,956/ 55,432/ 40,075/ 80,498/ 46,933/ % increase 4683% 4944% 3163% 3991% 3719% Bov-Av TFP Intl 57,471/ 47,855/ 36,401/ 73,660/ 42,693/ % increase 4344% 4267% 2873% 3652% 3383%

[0059] The results that are set forth in the TABLE show that administration of a composition of the present invention to a subject will likely increase the subject's secondary, or delayed-type hypersensitivity, immune response, as effected by NK cells, against one or more pathogens to a degree which far exceeds the NK cell activity initiated by both colostrum-derived transfer factor and egg-derived transfer factor alone. In fact, the results show that a composition that incorporates teachings of the present invention may result in facilitation of the activity of NK cells with an unexpected degree of synergy.

[0060] Although the foregoing description contains many specifics, these should not be construed as limiting the scope of the present invention, but merely as providing illustrations of some of the presently preferred embodiments. Similarly, other embodiments may be devised without departing from the spirit or scope of the present invention. Features from different embodiments may be employed in combination. The scope of the invention is, therefore, indicated and limited only by the appended claims and their legal equivalents rather than by the foregoing description. All additions, deletions and modifications to the invention as disclosed herein which fall within the meaning and scope of the claims are to be embraced thereby.



Inventors

* Lisonbee, David * Hennen, William J. * Daugherty, F. Joseph

US Classes 424/535, Milk or colostrum (e.g., butter, whey, etc.)426/605Egg containing, e.g., mayonnaise, etc.

Attorney, Agent or Firm

* TRASK BRITT

International Class 07 A61K035/20 A23L001/24

Issued Patent Number: 6866868



Abstract text

Compositions are provided comprising transfer factor alone or combined with an antibody. The antibody may be contained in an antibody fraction. The transfer factor and/or the antibody or antibody fraction may be lyophilized. Also provided are formulations further comprising glucans, as well as additional optional components. Also provided are methods for making the compositions and formulations, as well as kits containing the compositions. Methods of preventing and/or treating a condition in a subject using the compositions and/or formulations are also provided. Such conditions may include malignant and benign tumors. Claims

1. A composition comprising transfer factor and an antibody, each independently derived from a source selected from the group consisting of ova, colostrum, blood, egg, and combinations thereof.

2. The composition of claim 1 wherein the transfer factor is lyophilized.

3. The composition of claim 1 wherein the antibody is lyophilized.

4. The composition of claim 1 wherein the source is bovine or avian.

5. The composition of claim 1 wherein the antibody is contained in an antibody fraction.

6. The composition of claim 5 wherein the antibody fraction is lyophilized.

7. The composition of claim 5 wherein the antibody fraction comprises antibodies at about 1% to about 99% per weight of the fraction.

8. The composition of claim 5 wherein the antibody fraction comprises antibodies at about 50% per weight of the fraction.

9. The composition of claim 1 wherein the transfer factor is encapsulated by a hydrophobic or lipid coating.

10. The composition of claim 1 wherein the antibody is encapsulated by a hydrophobic or lipid coating.

11. The composition of claim 5 wherein the antibody fraction is encapsulated by a hydrophobic or lipid coating.

12. The composition of claim 1 wherein both of the transfer factor and the antibody are encapsulated by a hydrophobic or lipid coating.

13. The composition of claim 5 wherein both of the transfer factor and the antibody fraction are encapsulated by a hydrophobic or lipid coating.

14. The composition of claim 5 wherein the antibody fraction is present from about 1% to about 99% by weight of the composition.

15. The composition of claim 5 wherein the antibody fraction is present at about 20% by weight of the composition.

16. The composition of claim 1 wherein the transfer factor is present from about 1% to about 99% by weight of the composition.

17. The composition of claim 1 wherein the transfer factor is present at about 80% by weight of the composition.

18. A composition comprising transfer factor, wherein the transfer factor is lyophilized.

19. A formulation comprising the composition of claim 1, 5, or 18 and a glucan.

20. The formulation of claim 19 wherein the glucan is hydrolyzed.

21. The formulation of claim 19 wherein the glucan is a fungal glucan.

22. The formulation of claim 21 wherein the fungus is a hybrid fungus.

23. The formulation of claim 19 wherein the glucan is supplied as a whole fungus organism.

24. The formulation of claim 23 wherein the fungus comprises a Cordyceps strain.

25. The formulation of claim 24 wherein the Cordyceps is Cordyceps sinensis.

26. The formulation of claim 23 wherein the glucan is present from about 10 mg to about 18 gm of whole organism per ounce.

27. The formulation of claim 23 wherein the glucan is present from about 100 mg to about 5 gm of whole organism per ounce.

28. The formulation of claim 19 wherein the glucan is encapsulated by a hydrophobic or lipid coating.

29. A formulation comprising the composition of claim 1, 5, or 18 wherein the formulation is encapsulated by a hydrophobic or lipid coating.

30. The formulation of claim 19 wherein the formulation is encapsulated by a hydrophobic or lipid coating.

31. The formulation of claim 19 further comprising a member selected from the group consisting of essential fats, lactic acid-producing bacteria, inositol hexaphosphate, olive leaf extract, aloe extract, β-sitosterol, yeast extract, montmorillonite, amino acids, methylsulfonylmethane, choline bitartrate, di-potassium phosphate, potassium chloride, magnesium sulfate, calcium pantothenate, vitamin E, vitamin C, vitamin A, vitamin D3, vitamin B1, vitamin B2, vitamin B12, zinc, and mixtures thereof.

32. The formulation of claim 19 further comprising a member selected from the group consisting of glucosamines, chondroitins, boswella, tumeric, super oxide dismutase, and mixtures thereof.

33. The composition of claim 1 or 18 wherein the transfer factor is a targeted transfer factor.

34. The composition of claim 33 wherein the targeted transfer factor is targeted to an organism selected from the group consisting of Herpes Simplex Virus 1, Herpes Simplex Virus 2, H. Pylori, Camphobactor, Chlamydia, Bovine Rhinotracheitis Virus, Parainfluenza, Respiratory Syncytial Virus Vaccine, modified live virus, Campylobacter Fetus, Leptospira Canicola, Grippotyphosa, Hardjo, Leterohaemorrhagiae, Pomona Bacterin, Bovine Rota-Coronaviras, Escherichia Coli Bacterin, Clostridium Chauvoei, Septicum, Haemolyticum, Novy, Sordellii, Perfringens Types C & D, Bacterin, Toxoid, Haemophilus Somnus, Pasteurella Haemolytica, Multocida Bacterin, and combinations thereof.

35. A kit comprising at least a first container comprising a composition comprising transfer factor, wherein said transfer factor is lyophilized.

36. A kit comprising at least a first container and a composition comprising transfer factor and an antibody.

37. The kit of claim 36 wherein the antibody is contained in an antibody fraction.

38. The kit of claim 36 wherein the transfer factor is lyophilized.

39. The kit of claim 36 wherein the antibody is lyophilized.

40. The kit of claim 37 wherein the antibody fraction is lyophilized.

41. The kit of claim 36 further comprising a second container comprising a reconstitution solution.

42. The kit of claim 36 further comprising instructions for reconstitution of one or more lyophilized components of the kit.

43. The kit of claim 36 further comprising instructions for administering the components to a subject.

44. A method of making a composition comprising the steps of:A) Fractionating colostrum to obtain a first fraction having a molecular weight of about 10,000 Daltons or less;B) Fractionating colostrum to obtain a second fraction having a molecular weight of about 10,000 to about 150,000 Daltons; andC) Combining a first amount of the first fraction with a second amount of said second fraction to form the composition.

45. The method of claim 44 wherein the first amount contributes about 80% of the composition by weight, and the second amount contributes about 20% of the composition by weight.

46. The method of claim 44 additionally comprising the step of lyophilizing the first fraction.

47. The method of claim 44 additionally comprising the step of lyophilizing the second fraction.

48. The method of claim 44 additionally comprising the step of C) lyophilizing the composition.

49. A method of making a formulation comprising the steps of:A) Fractionating colostrum to obtain a first fraction having a molecular weight of about 10,000 Daltons or less;B) Fractionating colostrum to obtain a second fraction having a molecular weight of about 10,000 to about 150,000 Daltons;C) Combining a first amount of the first fraction with a second amount of said second fraction to form a composition;D) Combining the composition with a member selected from the group consisting of essential fats, lactic acid-producing bacteria, inositol hexaphosphate, olive leaf extract, aloe extract, β-sitosterol, yeast extract, montmorillonite, amino acids, methylsulfonylmethane, choline bitartrate, di-potassium phosphate, potassium chloride, magnesium sulfate, calcium pantothenate, vitamin E, vitamin C, vitamin A, vitamin D3, vitamin B1, vitamin B2, vitamin B12, zinc, and mixtures thereof.

50. A method of making a formulation comprising the steps of:A) Fractionating colostrum to obtain a first fraction having a molecular weight of about 10,000 Daltons or less;B) Fractionating colostrum to obtain a second fraction having a molecular weight of about 10,000 to about 150,000 Daltons;C) Combining a first amount of the first fraction with a second amount of said second fraction to form a composition; andD) Combining the composition with a member selected from the group consisting of glucosamines, chondroitins, boswella, turmeric, super oxide dismutase, and mixtures thereof.

51. A method of reducing tumor size in a subject in need thereof comprising administering to the subject a composition comprising transfer factor and an antibody or antibody fraction.

52. A method of reducing tumor size in a subject in need thereof comprising administering to the subject a composition comprising lyophilized transfer factor.

53. The method of claim 51 or 52 wherein the tumor size is reduced by at least about 50%.

54. The method of claim 51 or 52 wherein the tumor size is reduced by at least about 90%.

55. The method of claim 51 or 52 wherein the subject is avian or a mammal.

56. The method of claim 55 wherein the mammal is a human.

57. The method of claim 51 or 52 wherein the tumor is a malignant tumor.

58. The method of claim 57, wherein the malignant tumor is selected from the group consisting of a carcinoma, a melanoma, a sarcoma, a bone tumor, a mast cell tumor, a keratoma, a lymphoma, a histiocytoma, leukemia, and combinations thereof.

59. The method of claim 58, wherein the lymphoma is selected from the group consisting of Hodgkin's lymphoma and non-Hodgkin's lymphoma.

60. The method of claim 58 wherein the sarcoma is selected from the group consisting of a melanosarcoma, an osteosarcoma, and a hemangiosarcoma.

61. The method of claim 60, wherein the melanosarcoma is an amelanotic melanosarcoma.

62. The method of claim 58, wherein the carcinoma is an adenocarcinoma.

63. The method of claim 58, wherein the melanoma is an oral melanoma.

64. The method of claim 60, wherein the hemangiosarcoma is a splenic hemangiosarcoma.

65. A method of treating at least one condition in a subject comprising administering to the subject a composition comprising lyophilized transfer factor, wherein the condition is selected from the group consisting of hyperthyroidism, lymphopenia, Cushing's disease, Addison's disease, weight loss, hair loss, fatigue, and anorexia.

66. A method for the treatment of at least one condition in a subject comprising administering to the subject a composition comprising a transfer factor and an antibody or antibody fraction, wherein the condition is selected from the group consisting of hyperthyroidism, lymphopenia, Cushing's disease, Addison's disease, weight loss, hair loss, fatigue, and anorexia.

67. A method of reducing morbidity in a subject comprising the step of administering to the subject a composition comprising transfer factor and an antibody or antibody fraction.

68. A method of reducing morbidity in a subject comprising the step of administering to the subject a composition comprising lyophilized transfer factor.

69. A method of increasing feed conversion in a subject comprising the step of administering to the subject a composition comprising transfer factor and an antibody or antibody fraction.

70. A method of increasing feed conversion in a subject comprising the step of administering to the subject a composition comprising lyophilized transfer factor. Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application claims benefit of U.S. Provisional Application No. 60/814,777, filed Jun. 14, 2006, and also claims benefit of U.S. Provisional Application No. 60/834,739, filed Jul. 31, 2006, the disclosures of which are hereby incorporated by reference herein, in their entireties.

FIELD OF THE INVENTION

[0002]This invention relates generally to compositions comprising transfer factor, particularly lyophilized transfer factor; and to compositions comprising transfer factor in combination with an antibody. Such compositions are useful in the prevention and/or treatment of certain conditions, including benign and malignant tumors.

BACKGROUND OF THE INVENTION

[0003]Transfer factors, which are produced by leucocytes and lymphocytes, are small water soluble polypeptides of between about 44 amino acids that stimulate or transfer cell mediated immunity from one individual to another and across species but do not create an allergic response. Since transfer factors are smaller than antibodies, they do not transfer antibody mediated responses nor do they induce antibody production. The properties, characteristics and processes for obtaining transfer factor or transfer factors are discussed in U.S. Pat. Nos. 4,816,563; 5,080,895; 5,840,700, 5,883,224 and 6,468,534, the contents of which are hereby incorporated by reference into the present application.

[0004]Transfer factor has been described as an effective therapeutic for Herpes simplex virus (Viza, et al.), a treatment for acne blemishes, U.S. Pat. No. 4,435,384 and as a treatment against C. albicans (Khan et al.). Transfer factor has also been used to treat intestinal cryptosporidiosis in recipients treated with specific transfer factor (McMeeking, et al.). Still, et al. also showed that chicken pox infections were prevented by pretreatment of children treated with transfer factor from individuals that had chicken pox or who in other words had been sensitized to the varicella antigen. The antigen specific transfer factors are the most well studied and have been demonstrated to be able to convey the antigen recognition ability of the experienced donor to the naive recipient. It may be assumed that the individual or animal that is the source of the transfer factor has been sensitized to the antigen of interest. However, transfer factor as found in commercial bovine colostrum extract coming from a pool of animals (e.g., cows) contains the acquired immunity from all of the pool and therefore provides a type of generalized adoptive transfer of immunity. Transfer factors or transfer factor can be obtained from a dialyzable extract of the lyzed cells or from an extract of extracellular fluid containing transfer factor. Common sources of transfer factors are colostrums and ova. It is common practice to refer to preparations that contain transfer factor by the name of the active component (i.e., transfer factor or TF). Transfer factor extract containing transfer factors is also herein referred to as transfer factor. Transfer factor from bovine colostrum extract is defined as defatted water soluble material from colostrum that will pass through a nominal 10,000 molecular weight filter. The colostral derived transfer factor has been prepared with activity against various organisms including infectious bovine rhinotracheitis virus. One of the specific effects of transfer factor is a significantly increased natural killer (NK) cell activity. Natural killer cells provide protection against viruses as part of the innate immune defense system.

[0005]Although transfer factor is a polypeptide, it has been reported that it is surprising stable in the gastrointestinal tract. For example, Kirkpatrick compared oral versus parental administration of transfer factor in clinical studies. Kirkpatrick, Biotherapy, 9:13-16, 1996. He concluded that the results refute any arguments that the acidic or enzymatic environment of the gastrointestinal tract would prevent oral therapy using transfer factors.

[0006]When attempts were made to sequence TF, it was reported that an N-terminal end of the transfer factor peptide is resistant to sequential Edman degradation. Kirkpatrick, Molecular Medicine, 6(4):332-341 (2000).

[0007]Accordingly, transfer factor was believed to be stable in the gastrointestinal tract and rumen. However, it has since been shown that transfer factor is not as stable as once believed. It appears to be particularly unstable in the digestive tract of ruminants.

[0008]Transfer factors have been used successfully in compositions for treating animal diseases and syndromes including those in ruminants. See, for example U.S. Pat. No. 6,962,718.

[0009]The present invention relates to compositions and formulations containing transfer factor, as well as methods of making the same and methods of treatment and/or prevention of conditions using the same. Other U.S. patents and U.S. patent applications relate to the present invention, including without limitation, U.S. Pat. Nos. 6,506,413 and 6,962,718, U.S. Patent Provisional Application Nos. 60/573,113, 60/649,363, 60/701,860, and 60/814,777, U.S. Patent Application Publication Nos. 2006/0029585 A1, 2006/0073197 A1, and 2007/0128253 A1, all of which are incorporated herein by reference. Also related are PCT publications WO/2002/087599 and WO/2005/112891, incorporated herein by reference.

BRIEF SUMMARY OF THE INVENTION

[0010]In certain aspects, the invention relates to compositions comprising lyophilized transfer factor. In other aspects, the invention relates to compositions comprising transfer factor in combination with an antibody. In certain aspects, the antibody is contained in an antibody fraction. In certain embodiments, the transfer factor and/or the antibody or antibody fraction with which it is combined may be lyophilized.

[0011]In further aspects, compositions and formulations of the invention may further comprise additional components. In certain preferred embodiments, the compositions and formulations comprising transfer factor and/or antibody or antibody fraction may additionally comprise glucans.

[0012]Additional aspects of the invention relate to compositions and formulations comprising encapsulated components; including, but not limited to, encapsulated transfer factor and/or encapsulated antibody or antibody fraction.

[0013]Additional aspects of the present invention are directed to methods of making compositions and formulations according to the invention.

[0014]Further aspects of the present invention are directed to methods of treating and/or preventing certain conditions comprising administering an effective amount of a composition and/or formulation comprising transfer factor and/or antibody or antibody fraction.

[0015]Both the foregoing general description and the following detailed description are exemplary and explanatory, and are intended to provide further explanation of the invention as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0016]FIG. 1 shows the results of an assay for lymphocyte stimulation following application of a formulation containing lyophilized transfer factor.

[0017]FIG. 2 shows the results of an assay for lymphocyte stimulation following application of a formulation containing glucans.

DETAILED DESCRIPTION OF THE INVENTION

[0018]In certain embodiments, the present invention is directed to compositions comprising transfer factor and an antibody. In certain embodiments, the antibody is contained within an antibody fraction.

[0019]In other embodiments, the invention is directed to compositions comprising transfer factor that is lyophilized. In certain embodiments, lyophilized transfer factor may be combined with antibody or antibody fraction. In certain preferred embodiments of the invention, compositions are provided comprising lyophilized transfer factor and lyophilized antibody. In certain embodiments, the antibody may be in an antibody fraction that is lyophilized.

[0020]Other aspects of the invention are directed to formulations further comprising components, including, but not limited to, nutraceutical ingredients, in addition to lyophilized transfer factor. In other aspects of the invention, there are provided formulations comprising additional components in addition to transfer factor and antibody or antibody fraction.

[0021]Transfer Factor

[0022]According to particular embodiments of the invention, compositions and formulations are provided comprising transfer factor. According to certain embodiments of the invention, various forms of transfer factor may be used. They include, without limitation, excreted transfer factor released from transfer factor containing cells such as lymphocytes, leukocytes, and ova, and collected from extracellular fluids such as colostrums and blood. Another form includes preexcreted transfer factor found within the cell or on the cell surface. In certain embodiments of the invention, substantially purified transfer factor originating from leukocytes, colostrum, or ova and having a molecular weight of less than 10,000 daltons and a specific activity of at least 5000 units per absorbance unit at 214 nanometers, may also be used. The transfer factor used in the Examples of this invention and referred to in the following Tables and further referred to in the rest of the detailed description is generally extracted from colostrum collected from a general pool of lactating cows; although, in some cases, it is derived from eggs. Though bovine colostral derived transfer factor was generally used to develop the formulations of this invention, it is well known to anyone skilled in the art that other kinds and sources of transfer factor could be used.

[0023]Alternative sources of transfer factor include, but are not limited to, avian transfer factor, ova transfer factor, and transfer factor isolated from colostrum collected from non-bovine animals such as goats, pigs, horses and humans. In addition, combinations of transfer factors from any number of sources may be used in the formulations of the instant invention. Transfer factor may also be derived from recombinant cells that are genetically engineered to express one or more transfer factors or by clonal expansion of leukocytes.

[0024]Isolation of Transfer Factor and Antibody Fraction

[0025]In certain embodiments of the invention, transfer factor may be obtained from colostrum. In a preferred embodiment, transfer factor is obtained from bovine colostrum. The colostrum is fractionated, removing most of the curds and whey, to produce the transfer factor and antibody fractions. The fraction having a molecular weight of approximately 10,000 daltons (Da) or below is designated as transfer factor. The fraction obtained that is approximately 10,000 to 150,000 Da is designated the antibody fraction, also known as the antibody-colostrum fraction. In certain embodiments, the antibody fraction comprises antibodies from about 1% to about 99%, about 5% to about 95%, about 10% to about 90%, about 15% to about 85%, about 20% to about 80%, about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, about 45% to about 55%, or about 50% by weight, the remainder comprising other colostrum components.

[0026]According to certain embodiments of the invention, transfer factor, as used in the formulations described in the Tables, particularly when not defined as obtained from an avian source, may be further defined as defatted water soluble material from bovine colostrum that will pass through a nominal 10,000 molecular weight filter.

[0027]In other embodiments, the transfer factor is obtained from an avian source. In one embodiment, chickens are given a feed mixture containing excrement from an animal, including without limitation, a human, a fish, a goat, a llama, an alpaca, a pig, a sheep, a cow, and a horse. The excrement will contain a large variety of pathogens and upon administration of a feed to an animal, it will develop transfer factor and/or antibodies to such pathogens. Avian transfer factor can then be obtained from the eggs produced by the above-treated chickens. In certain embodiments of the invention, transfer factor may be found in whole egg yolks. As a non-limiting example, the transfer factor of avian source (which is believed to also contain antibodies) listed in the formulation of Table 7 is supplied as powdered whole egg yolks.

[0028]Alternative kinds of transfer factor include, but are not limited to, targeted transfer factors. Target transfer factors include transfer factor collected from sources which have been exposed to (1) one or more viral or otherwise infectious organisms; (2) one or more antigens that produce an immune response; or (3) a combination of organisms and antigens. The term antigen is defined herein is anything that will initiate the cell mediated immune response. Examples of such viral or other infectious organisms include Herpes Simplex Virus 1, Herpes Simplex Virus 2, H. Pylori, Camphobactor and Chlamydia, Bovine Rhinotracheitis Virus, Parainfluenza, Respiratory Syncytial Virus Vaccine, modified live virus, Campylobacter Fetus, Leptospira Canicola, Grippotyphosa, Hardjo, Leterohaemorrhagiae, Pomona Bacterin, Bovine Rota-Coronavirus, Escherichia Coli Bacterin, Clostridium Chauvoei, Septicum, Haemolyticum, Novy, Sordellii, Perfringens Types C & D, Bacterin, Toxoid, Haemophilus Somnus, Pasteurella Haemolytica, Multocida Bacterin. However, one of skill in the art would readily recognize that a wide variety of other viral and otherwise infectious organisms can find use in the instant invention. Examples include those set forth in Appendix I and Appendix II.

[0029]Antibodies

[0030]In another aspect of the present invention, the formulations and compositions of the present invention include an antibody. The antibody may be present in an antibody fraction. In certain embodiments, the antibody or the antibody fraction is present in a composition also comprising transfer factor. The antibody or antibody fraction may be present at about 1% to about 99% of a composition having the transfer factor. In other embodiments, the antibody or antibody fraction is present from about 5% to about 95%, about 10% to about 90%, about 15% to about 85%, about 20% to about 80%, about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, about 45% to about 55%, or about 50% of a composition having the transfer factor. In other embodiments, the antibody or antibody fraction is present at about 15% to about 25%, about 17.5% to about 22.5%, or about 20% of a composition having a transfer factor. In some embodiments, the antibody is provided as a lyophilized antibody or antibody fraction.

[0031]Lyophilization

[0032]The present invention also provides compositions, formulations, and kits containing the same, that have one or more lyophilized component(s). Lyophilization or "freeze-drying" is a process well known to those of ordinary skill in the art. For example, some techniques of lyophilization are described in Akers, Michael J., Chapter 41 in Remington The Science and Practice of Pharmacy, 828 (David B. Troy ed., Lippincott Williams & Wilkins 2006), which is incorporated herein by reference. In certain embodiments, formulations and/or compositions of the present invention may include lyophilized transfer factor. In certain embodiments, transfer factor, which may be lyophilized, may be combined with antibody or an antibody fraction, which may, in certain embodiments, be lyophilized. In other embodiments, additional components of formulations and compositions of the invention may be lyophilized, including, without limitation, other peptides and proteins.

[0033]In certain embodiments, the transfer factor is present in a composition also comprising antibody or antibody fraction. In certain embodiments, the transfer factor is present from 1% to about 99% by weight in a composition also comprising antibody or antibody fraction. In other embodiments, the transfer factor is present from about 5% to about 95%, about 10% to about 90%, about 15% to about 85%, about 20% to about 80%, about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, about 45% to about 55%, or about 50%, all by weight of a composition also comprising antibody or antibody fraction. In certain embodiments, transfer factor is present in a composition from about 70% to about 90%, about 75% to about 85%, or about 77.5 to about 82.5% by weight of a composition comprising antibody or antibody fraction. In certain preferred embodiments, the transfer factor is present in a composition at approximately 80% by weight of a composition also comprising antibody or antibody fraction.

Formulations

[0034]In certain embodiments of the invention, transfer factor is provided in a formulation that further comprises one or more additional ingredients. In some embodiments, transfer factor and an antibody or antibody fraction is provided in a formulation that further comprises one or more additional ingredients. In certain embodiments, the transfer factor may be lyophilized. In certain embodiments, the antibody or antibody fraction may be lyophilized.

[0035]In a preferred embodiment, transfer factor is present in the formulation in the amount of about 10 mg to about 12 gm/oz, more preferably about 100 mg to about 6 gm/oz and most preferably about 10 mg to about 3 gm/oz. In certain preferred embodiments, such a formulation comprising transfer factor is provided to an animal in an amount of about 1 oz per 1000 lb of animal.

[0036]In certain embodiments of the invention, formulations are provided which comprise glucans. Glucans may be derived from any suitable source, including, but not limited to, fungi, oats, and yeast. Preferably, glucans are present in or derived from fungi.

[0037]In certain embodiments, the glucans which may be included in the formulations are present in whole fungi.

[0038]In certain preferred embodiments, glucans are present in or derived from Cordyceps, more preferably, Cordyceps sinensis.

[0039]In certain embodiments, glucans are derived from hybrid strains of fungi. In a preferred embodiment the hybrid glucans used in the invention are present in, or derived from, hybrid strains of Cordyceps and in particular Cordyceps sinensis.

[0040]One technique to induce the hybridization of Cordyceps involves plating two different strains or species on a single agar plate which has been inoculated with rattlesnake venom as described in, for example, U.S. Patent Application Publication No. 2006/0073197, published Apr. 6, 2006, and U.S. Patent Application Publication No. 2007/0128253, published Jun. 7, 2007, each of which is incorporated herein by reference. In a preferred embodiment, the hybrid strain producing the hybrid glucans that may be used in compositions and formulations of the invention is Cordyceps sinensis Alohaensis, which is available from Pacific Myco Products, Santa Cruz, Calif.

[0041]There are a number of different Cordyceps sinensis strains and due to their variable asexual mycelial growth forms they have been considered to be different species by many taxonomists. A non-exhaustive list of strains includes: Paecilomyces hepiali Chen, Cephalsporim sinensis, Paecilomyces sinensis Cn80-2, Scydalilum sp., Hirstutella sinenis, Mortierella hepiali, Chen Lu, Topycladium sinensis, Scytalidium hepiali, G. L. Li. Preferred embodiments of the instant invention make use of hybrid glucans from hybrids of one or more of these different strains, however, the invention may alternatively preferentially include glucans from non-hybridized strains. Alternative embodiments utilize the whole hybrid Cordyceps, e.g., Cordyceps sinensis Alohaensis. Hybrid glucans may also include those obtained by crossing sources of feed, e.g., oats, etc.

[0042]When glucans are used, the formulation preferably contains about 10 mg to about 18 gm of whole organism/oz, more preferably about 100 mg to about 10 gm of whole organism/oz and most preferably about 100 mg to about 5 gm of whole organism/oz.

[0043]Equivalent amounts of purified or partially purified glucan as well as the nucleosides associated therewith (e.g., Cordycepin (3'deoxyadenosine), adenosine and N6-(2 hydroxyethyl)-adenosine) may also be used.

[0044]In certain embodiments, compositions and formulations comprising transfer factor may be combined with minerals, antioxidants, amino acids, and other neutraceuticals.

[0045]The use of nutraceuticals to treat vitamin and mineral deficiencies is well known. However, the use of nutraceuticals, such as vitamins, minerals and other nutritional components to prevent and treat diseases other than those caused by the deficiency of those nutraceuticals, though still controversial, is receiving more consideration from both laymen and physicians. The following is a non-limiting list of nutraceuticals and some of their generally acknowledged nutritional and health benefits. Any of these may be included in formulations comprising transfer factor, including lyophilized transfer factor and/or transfer factor in combination with an antibody or an antibody fraction.

[0046]Vitamin A--is important in preventing eye epithelial disorders; deficiency results in night blindness

[0047]Vitamin B2--is essential to human nutrition relating to the oxidation of carbohydrates and amino acids

[0048]Mixed tocopherols--are antioxidants

[0049]Choline Chloride--is a member of the vitamin B complex and a dietetic factor for furnishing free methyl groups for transmethylation.

[0050]Vitamin B6--functions in the formation and breakdown of amino acids and is involved in the synthesis of serotonin and norepinephrine. However, exact dietary requirements are uncertain

[0051]Vitamin B12--is an antipernicious-anemia factor essential for normal hemopoiesis.

[0052]Vitamin E--is an antioxidant that protects against free radicals.

[0053]Vitamin K--is essential for the formation of prothrombin

[0054]Biotin--functions in metobolic processes leading to the formation of fats and utilization of carbon dioxide

[0055]Folic Acid--a growth factor involved in the formation of nucleic acids and necessary for the formation of heme

[0056]Niacin--a component of the Vitamin B complex, a deficiency results in pellagra

[0057]Vitamin D3--is important in the absorption of calcium

[0058]Pantothenic Acid--is considered essential for growth and well being of animals; deficiency results in growth retardation, skin lesions and graying of hair

[0059]Thiamine--is necessary in diet of all animals except ruminants; used to prevent beriberi and important in carbohydrate metabolism

[0060]Lysine--is an essential amino acid

[0061]Methionine--is a sulfur containing essential amino acid

[0062]Arginine--is an amino acid important in the synthesis of urea (principal form in which mammals excrete)

[0063]Soy--is a source of proteins

[0064]Methyl Sulfonyl Methane--is a form of organic sulfur involved in cell membrane permeability

[0065]Zinc--is an essential mineral for growth; deficiency creates susceptibility to various pathogens

[0066]Omega 3-, 6-, and 9-Fatty Acids--are essential fatty acids and polyunsaturated fats; a deficiency results in hypertension and high blood pressure; they are believed to improve immune function

[0067]Yeast--(e.g., brewers, bakers, etc.) contains beta glucans which appear to increase production and/or activation of natural killer cells

[0068]Calcium--is required for bone development

[0069]Phosphorus--is required for bone development

[0070]Selenium--a deficiency results in heart muscle disease

[0071]Iron--is required for formation of hemoglobin; deficiency results in anemia

[0072]Magnesium--is an element required for growth in all living organisms

[0073]Manganese--is an element required for growth in all living organisms

[0074]Copper--is an element required for growth in plants, animals and most microorganisms

[0075]Iodine--is an element necessary for the synthesis of hormone production by the thyroid gland

[0076]Cobalt--is a trace element essential in the nutrition of ruminants (cattle, sheep) and in the maturation of human red blood cells in the form of Vitamin B.sub. 12

[0077]Molybdenum--is a trace element believed to be necessary in animal diets but its function in the minimal levels have not been established

[0078]Lactic Acid Generating Bacteria--are a digestive aid and growth inhibitor of harmful bacteria

[0079]Chrondroitin--is a component of connective tissue which may relieve joint pain and arthritis.

[0080]Glucosamine--is a component of micropolysaccharides and glycoprotein which may be helpful in arthritis.

[0081]Di-methyl glycine--is a methylated amino acid found in all cells and an antioxidant.

[0082]Montmorillonite--is collodial clay containing trace elements which are considered by some to be important for well being and to compensate for elements no longer in foods because of depleted soils (the components are shown below in Table 1)

[0083]Super oxide dismutase (SOD)--is an antioxidant enzyme present in the mammalian body. It converts super oxide free radicals to the less active peroxide. It stimulates hair growth and is believed to protect cells against ultraviolet-B irradiation and to protect the heart.

[0084]Boswellia--is an herb Boswellia serrata. Boswellic acids, the biologically active ingredients of the gum resin of this herb, are considered to have anti-inflammatory and anti-arthritic actions.

[0085]Octocosonol--is derived from wheat germ oil and provides 17% more residual energy before fatigue.

[0086]In certain preferred embodiments of the invention, formulations useful for the prevention and/or treatment of conditions in a subject, such as, for example, a mammal, may include one or more of the following: lyophilized transfer factor (mammalian) in combination with mammalian antibody-colostrum fraction, avian antibodies or antibody fraction (may, in certain embodiments, be obtained from whole egg yolk), glucans, preferably hybrid glucans, essential fats, lactic acid producing bacteria, Vitamin C, zinc, 1p6 (Inositol hexaphosphate), ace mannins, olive leaf extract, phytosterols, montmorillinite, amino acids, Methyl Sulfonyl Methane, and choline bitartrate, as well as additional vitamins and minerals.

[0087]In other preferred embodiments of the invention, formulations may additionally comprise one or more of glucosamines, chondroitins, Boswella, tumeric, and super oxide dismutase. In certain embodiments, adding one or more of these ingredients may make the formulation particularly effective in treating cancer, as pain reduction and cutting inflammation appear to be a large factor in cancer remission, as well as getting the animal to eat.

[0088]Table 1 sets forth typical components of Montmorillonite.

[0089]Tables 2-6 set forth transfer factor formulations that have been used to treat various animals and pathologies. In each case, the transfer factor is not lyophilized as set forth herein. However, the transfer factor in each of these formulations can be readily lyophilized prior to admixture with the other components of the formulation. In certain embodiments, transfer factor may be added to these formulations along with antibody or an antibody fraction. In such embodiments, the transfer factor and/or the antibody or antibody fraction may be lyophilized.

[0090]Table 2, shows a breakdown of a formulation of transfer factor, nutraceuticals and carriers useful for treating a number of conditions, including, without limitation, Cushing syndrome, Cushings disease, adenomas, onchocerciasis, hypothyroidism or EPM. In Table 2 and all the other tables references to "lb" (pounds) means pounds of body weight.

[0091]Columns 2, 3 and 4 of Tables 2-6 show the approximate high, low and preferred amounts, respectively, of the formulation components, in amounts per body weight, to be given to an animal in a single dosage. The formulations in Tables 3 and 4 are very similar to the formulation of Table 2 but they are preferably used for dogs and cats, respectively. The formulation represented in Table 2 is designed preferably for livestock. The 5 ounces of the formula listed in column 5 is designed to be given to a 1000 pound animal but that will vary and could be given to a 500 pound animal in some cases. The average horse is around 1000 pounds. The 28.3 gm dosage in Table 3 is calculated for a dog weighing about 100-200 pounds but that dosage may also be given to a 15 pound dog. The 2.2 gm formula in Table 4 is for a cat weighing around 15 pounds. However, since these formulas are comprised of nutraceuticals and transfer factor, one skilled in the art will recognize that the ranges are not certain and as critical as the ranges for allopathic drugs.

[0092]Further, the formulations in Tables 2-4 are designed to treat preferably chronic diseases, the formulation in Table 5 is designed for treatment preferably of acute diseases and the formulation in Table 6 is useful for both acute and chronic diseases. All the formulations may be given in megadoses to achieve an acute response.

[0093]In certain embodiments, the invention provides compositions in which a transfer factor and/or antibody or antibody fraction is "encapsulated." The encapsulation protects the transfer factor and/or antibody or antibody fraction from inactivation in the gastrointestinal tract. Such encapsulation is important especially in the case of ruminants where digestion within the rumen has been found to be problematic. Enhanced bioavailability has been demonstrated when a transfer factor is encapsulated and administered to ruminants. In preferred embodiments, the transfer factor and/or antibody or antibody fraction is encapsulated by mixing with a hydrophobic substance or a lipid to form a coating around the transfer factor and/or the antibody or antibody fraction. In certain aspects, the composition may contain encapsulated glucans. Other optional components of compositions and formulations of the invention may be encapsulated, such as, without limitation, β-sitosterol, inositol hexaphosphate, olive leaf extract, aloe extract, vitamin C, and glucans, including, but not limited to, glucans obtained from fungi as described herein. The transfer factor and/or antibody or antibody fraction can be individually encapsulated or encapsulated as a mixture. Alternatively, the entire formulation can be encapsulated. The encapsulated transfer factor and/or encapsulated antibody formulation can be produced in a variety of ways. In a preferred embodiment, each of the transfer factor and/or antibody or antibody fraction in the formulation is encapsulated as described in U.S. Pat. Nos. 5,190,775, 6,013,286 and U.S. Application 2003/0129295, each of which is incorporated herein by reference in their entirety.

[0094]In certain embodiments, glucans of the formulation may be encapsulated, preferably with a hydrophobic or lipid coating. It is preferred that the amount of hydrophobic or lipid coating be between about 25% and 150 wt % of the glucan, about 50-150 wt %, or about 75-125 wt % with an equal weight being most preferred.

[0095]Table 7 provides an encapsulated transfer factor formulation for treating pathologies. This transfer factor formulation includes at least encapsulated transfer factor derived from both bovine and avian sources, and/or one or more of hybrid glucans. It is preferred that the glucan portion of this formulation also be encapsulated. Other components include zinc proteinate, targeted avian transfer factors, β-sitosterol, inositol hexaphosphate (IP6), olive leaf extract, aloe extract powder, probiotics, B. subtlis, B. longum, B. thermophilium, L. acidophilus, E. faecium, and S. cerevisia. In a preferred embodiment, all of the foregoing are included in this transfer factor formulation.

[0096]In another preferred embodiment, a formulation is provided according to Table 7, but with the following modifications. The component listed as "Transfer factor (mammal source)" is substituted with a composition containing 80% bovine colostrum transfer factor as described herein, combined with 20% bovine colostrum antibody fraction as described herein (both weight percents of the composition). The mammalian transfer factor and the colostrum antibody fraction are both lyophilized. In addition, the component listed as "Transfer factor (avian source)" is present in the formulation in an amount of 3000.0 mg/oz. This component is supplied as powdered whole egg yolk that was obtained from hyperimmunized chickens, i.e., chickens that had been exposed to pathogens prior to laying the eggs which serve as a source of transfer factor. The avian transfer factor may be obtained from commercial sources such as, for example, 4Life.RTM. Research; Labelle, Inc., Bellingham, Wash.; Troue; and Ghen Corporation, Japan.

[0097]The transfer factor may be encapsulated with a hydrophobic or lipid coating that is preferably between about 25% and about 150 wt % of the transfer factor, about 50-150 wt % and about 75-125 wt %, with an equal weight being most preferred.

[0098]In additional embodiments of the invention, additional components may be used in the formulation. For example, IP6, β-sitosterol, olive leaf extract, aloe extract matter and/or vitamin C may be used. In preferred embodiments, IP6 is present at between 10 mg and 3 gm/oz, or one preferably between 100 mg and 2 gm/oz, and most preferably between 100 mg and 1 gm/oz. The β-sitosterol is preferable in the amount of between 10 mg and 3 gm/oz, or preferably between 100 mg and 2 gm/oz, and most preferably between 100 mg and 1 gm/oz. Olive leaf extract is preferably present in the amount of 2 mg to 2 gm/oz, more preferably between 5 mg and 1 gm/oz, and most preferably between 5 mg and 500 gm/oz. Aloe extract is preferably present at between 2 mg and 1000 mg, more preferably between 5 and 500 mg/oz, and most preferably between 5 and 250 mg/oz. Vitamin C may be present at between 10 mg/oz and 10 gm/oz, or preferably between 100 mg and 8 gm/oz, and most preferably between 100 mg and 5 gm/oz.

[0099]The amount of transfer factor and/or antibody or antibody fraction used in the formulation or the amount of formulation administered will vary depending upon the severity of the clinical manifestations presented. In addition, the amount of transfer factor administered to a recipient will vary depending upon the species from the transfer factor is derived as compared to the species of the recipient. It has been observed that transfer factor derived from bovine species administered to cattle is more efficacious than transfer factor from another species such as avian species. Accordingly, when the source of the transfer factor and recipient are different species, it is preferred that the amount of transfer factor be increased.

[0100]Administration of a formulation of a transfer factor with zinc and at least one essential fatty acid is expected to result in at least a partially effective treatment of Cushings syndrome, Cushings disease, adenomas and other benign tumors, onchocerciasis, hypothyroidism or EPM. The treatment is more effective as other nutraceuticals listed in Table 2 are added. The dosage is in milligrams per pound unless otherwise stated. The amounts of the components present in a 5 ounce transfer factor formulation containing the other preferred nutraceuticals is shown in column 5 of Table 2.

[0101]Transfer factor at a dosage of about 0.75 mg/lb transfer factor in combination with about 0.49 mg/lb zinc and 20.57 mg/lb of canola oil, safflower oil or flax oil, sources of essential fatty acids (i.e., 3, 6, 9 omega fatty acids), given once daily to an animal suffering from Cushings syndrome, Cushings disease, adenomas or other benign tumors, onchocerciasis, hypothyroidism or equine protozoal myelytis should result in approximately a 30% to 50% reduction in the size of the benign tumors and/or the symptoms of these listed diseases. All of these components should of course be pharmaceutically acceptable to the animal receiving them.

[0102]A combination of Vitamin C at about 2.16 mg/lb and 2.29 mg/lb of yeast in combination with the above listed transfer factor and other fatty acid nutraceuticals should result in approximately a 40% to 50% reduction in the size of benign tumors and/or symptoms of the above listed diseases.

[0103]It is preferred in formulations of the invention that the metal nutraceuticals are proteinated because these forms are easier for the animal to digest and also because the proteinate forms are more stable to pH. The nutraceutical components in the formulations in Tables 2-7 are the active components for treating the various described diseases and syndromes. The fillers and carriers are included to make the formulations more palatable to the animal and also to help preserve the mixture. These include silicon dioxide, maltodextrin, soy and peanut flour, peanut oil, dextrose, whey, spices and flavorings. Mixed tocopherols and choline chloride are nutraceuticals but the effective results described herein can still be achieved by deleting these two components from the formulations.

[0104]Previous use of non-encapsulated transfer factor in ruminants, e.g., cows, produced significant beneficial results. See, e.g. U.S. Patent Publication 2003/0077254, published Apr. 24, 2003 incorporated herein by reference in its entirety. Subsequently, it was discovered that transfer factor was not stable by oral administration in a stressed population of cattle. After discovering that transfer factor is inactivated in vitro in the presence of rumen fluid and flora, it was determined that prior success with transfer factor in ruminants was due to the presence of the esophageal groove. When not stressed, the esophageal groove provides partial bypass of the rumen. However, in a stressed population the esophageal groove closes and shunts the transfer factor formulation into the rumen. It was discovered that encapsulating transfer factor and/or glucans with a hydrophobic substance or a lipid to form an encapsulated formulation is sufficient to provide substantial by-pass of (e.g., 85%) of the rumen even in a stressed population.

[0105]A variety of other methods for rumen by-pass are known. In one embodiment, the encapsulated or non-encapsulated formulation is directly injected (subcutaneously, intramuscularly, or intravenously) to by-pass not only the rumen but also the entire digestive system. Similarly, intravaginal, intrarectal or other direct administration to mucus membranes, such as the eye subconjunctival, by-pass the digestive system and the rumen in particular. Alternatively, the formulation can be mixed with various solvents which allow for direct skin absorption. Furthermore, methods are known in the art to stimulate opening of the esophageal groove in various ruminants and such opening allows for immediate passage of an orally administered formulation to the gastrointestinal tract, by-passing the rumen.

[0106]Preferred embodiments for human consumption include, but are not limited to incorporation of transfer factor formulations in processed foods such as cereals, snacks, chips, or bars. Preferred embodiments for animal consumption include, but are not limited to, transfer factor formulations admixed in feed pellets, salt licks, molasses licks or other processed feed products.

[0107]In certain embodiments, the transfer factor formulations find use in increasing food conversion efficiency. Food conversion efficiency is the rate at which an organism can convert food to body mass, and is also known in the cattle industry as feed conversion efficiency. Transfer factor compositions and formulations have been successfully used to increase the body weight of cattle at an enhanced rate as compared to non-treated cattle, even in situations where the treated cattle are diseased. Accordingly, the compositions and formulations are not limited to prophylaxis and treatment of pathologies, but find use in other aspects of overall organismal health and development. In certain embodiments, methods of improving feed conversion in a subject comprise the administration to the subject of compositions and formulations comprising lyophilized transfer factor. In certain embodiments, methods of improving feed conversion in a subject comprise the administration of compositions and formulations to a subject comprising transfer factor in combination with an antibody or antibody fraction.

[0108]The transfer factor formulations of the present invention include pharmaceutical compositions suitable for administration. In a preferred embodiment, the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. "Pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.

[0109]The pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers such as sodium acetate; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol. Additives are well known in the art, and are used in a variety of formulations.

[0110]In a further embodiment, the pharmaceutical compositions may be added in a micellular formulation; see U.S. Pat. No. 5,833,948, hereby expressly incorporated by reference in its entirety.

[0111]In one aspect, the components of the compositions and pharmaceutical formulations of the present invention may have an effect upon administration individually, such as for example the reduction of a tumor, but upon administration in one or more combinations, have an effect that is synergistic. By "synergistic" is meant an enhancement of the effect of one or more combined components in a more than additive fashion relative to the effect of each component when used alone.

Method of Detecting Assaying Activity of Active Agents

[0112]According to certain aspects of the invention, quality control methods are employed to assess whether the compositions and/or formulations of the present invention stimulate lymphocyte activation. Lymphocyte function in response to antigens or mitogens may be measured by several techniques known to those skilled in the art. For example, it is known in the art that upon an appropriate stimuli, certain T lymphocytes are activated and expand their population. The expansion of this subset of lymphocytes reactive to the particular stimuli are characterized by various cellular events in the expanding cells. The events include without limitation, increased synthesis of ATP, NADP, and Proliferating Cell Nuclear Antigen (PCNA). Such intracellular components may be used to correlate the activation of the T lymphocytes as described, for example, in U.S. Pat. No. 6,630,316 (the '316 patent) to Wier, which is incorporated herein by reference. In certain embodiments of the present invention, compositions and/or formulations as described herein may be assayed for lymphocyte activation by methods, such as those described in the '316 patent.

[0113]In certain embodiments, the present invention provides methods involving administration of compositions and/or formulations according to the invention to a subject. As used herein, the term "subject" is used to mean an animal, including, without limitation, an avian or a mammal. Mammalian subjects include, without limitation, primates, bovines, porcines, ovines, equines, and carnivores, including, but not limited to, felines and canines. The mammal may be a human.

[0114]Combinations of pharmaceutical compositions may be administered. Moreover, the compositions may be administered in combination with other therapeutics.

[0115]A daily dosage of 141 mg per pound of body weight of any of the formulations in column 5 of Tables 2, 3 or 4, for 14 days has been successful in treating feline pneumonitis, feline leukemia, feline autoimmune dysfunction, feline flea bit dermatitis, feline hyperthyroidism, feline viral infection, feline ulcerations, feline bacterial infection, canine flea bite dermatitis, canine Cushings disease, malignant tumors, canine autoimmune dysfunction, canine viral and bacterial infection. These treatments for the most part have resulted in complete cures. The use of lyophilized transfer factor in these formulations is expected to produce the same or better results. In other embodiments, transfer factor in combination with antibody or antibody fraction may be used in these formulations and is expected to produce the same or better results.

[0116]Administering a formulation comprising all of the nutraceuticals in Table 2 at the preferred dosage to an animal with benign tumors resulted in about a 60% reduction in the size of the benign tumors and about a 90% reduction in the symptoms exhibited by the animal suffering the above listed diseases and syndromes. The use of lyphilized transfer factor in these formulation is expected to produce the same or better results. In other embodiments, transfer factor in combination with antibody or antibody fraction may be used in these formulations and is expected to produce the same or better results.

[0117]Administration of all of the nutraceuticals in Table 2 at the low dosage in column 3 of those tables results in about a 7% to 100% reduction in the size of the tumors and/or a 30% to 100% reduction in the symptoms exhibited by the animal suffering from those diseases or syndromes. The use of lyophilized transfer factor in these formulations is expected to produce the same or better results. In other embodiments, transfer factor in combination with antibody or antibody fraction may be used in these formulations and is expected to produce the same or better results.

[0118]The stress formulation in Table 5 is also used to treat numerous animal diseases and syndromes and as stated previously, mainly their acute stages. This formulation is also water soluble so it can be given in the animal's drinking water. A mixture of about 0.75 mg/lb transfer factor and about 1.42 mg/lb lactobacillus acidophilus 109 colony forming units (CFU) given twice daily will result in at least a 30% reduction in clinical symptoms resulting from strangles, dust cough, hypothyroidism and lymphopenia. The same dosage given to young calves will also reduce morbidity by about 30%. The addition of ionic salts or chelates of calcium, magnesium sodium and potassium twice daily in amounts approximating those in column 4 of Table 5 to the above amounts of transfer factor and lactic acid generating bacterial results in a 40% reduction in clinical symptoms of the above mentioned diseases. The addition of about 0.482 mg/lb of citric acid to the above formulation results in about a 45% reduction in the symptoms of the above mentioned diseases. Further addition of Vitamins A, B2, B6, B 12, C and E, and thiamine results in a 50% reduction in the symptoms of these diseases. The stress formulations given once or twice a day in the dosage presented in column 4 of Table 5 will cure or at least treat and reduce the symptoms of autoimmune dust cough, diarrhea from viral etiology, abscessation, in strangles, snotty nose in strangles, acute viremia in swine, scratches in the horse, hypersensitivity from scratches and onchoceriasis, PURRS, BRD, calf dysentery, coliform infections, Rhodococcus infections, Clostidium infections, circo virus in birds, and pnemonitis in cats. A combination of transfer factor and lactic acid producing bacteria or this combination further combined with yeast as shown in Table 5 will also treat these diseases but to a lesser extent. The use of lyophilized transfer factor is expected to produce the same or better results. In other embodiments, transfer factor in combination with antibody or antibody fraction may be used in these formulations and is expected to produce the same or better results.

[0119]The stress formulation as shown in Table 5 given once or twice daily will also increase the weight gain and feed efficiency of livestock. The weight gain will increase by at least 8%. A combination of transfer factor and lactic acid producing bacteria or this combination further combined with yeast as shown in Table 5 will also increase weight gain but to a lesser extent. The use of lyophilized transfer factor is expected to produce the same or better results. In other embodiments, transfer factor in combination with antibody or antibody fraction may be used in these formulations and is expected to produce the same or better results.

[0120]In a preferred embodiment, 2 gm of encapsulated hybrid glucan containing 1 gm of hybrid glucan is used.

[0121]Table 6 shows a breakdown of a performance formulation of transfer factor and nutraceuticals for treating and curing numerous diseases such as arthritis, laminitis, inflammation and malignant tumors. These diseases may also be treated with a combination of transfer factor and super oxide dismutase; transfer factor and glucosamine salts; transfer factor, glucosamine salts and super oxide dismutase; transfer factor, glucosamine salts, super oxide dismutase and glycine; transfer factor, glucosamine salts, super oxide dismutase, glycine and methyl sulfonyl methane; transfer factor, glucosamine salts, super oxide dismutase, glycine, methyl sulfonyl methane and octocosonol or transfer factor, glucosamine salts, super oxide dismutase, glycine, methyl sulfonyl methane, octocosonol and montmorillinite.

[0122]Table 7 shows a formula containing transfer factor and glucan both hybridized and non-hybridized.

[0123]Any of the aforementioned formulations may include lyophilized components, such as, for example, lyophilized transfer factor and/or lyophilized antibody or antibody fraction. Any of the aforementioned formulations may include an antibody or antibody fraction along with transfer factor.

TABLE-US-00001 TABLE 1 Montmorillonite Components Average Nutrient Content Per Ounce (1 Tablespoon = ~0.36 oz.) (mg) Silicon 6933 Tungsten 0.218 Aluminum Silica 2505 Vanadium 0.215 Sodium Chloride 1320 Ruthenium 0.210 Potassium 1293 Baron 0.189 Protein 1116 Bromine 0.140 Calcium 1104 Cobalt 0.129 Sulfur 431 Selenium 0.110 Iron 431 Syprosium 0.107 Magnesium 224 Fluorine 0.102 Chlorine 164 Scandium 0.0997 Titanium 61.9 Samarium 0.0943 Carbon 48.2 Nobelium 0.0754 Sodium 37.2 Copper 0.0593 Barium 10.5 Praseodymium 0.0539 Phosphate 8.62 Erbium 0.0539 Strontium 6.46 Hafnium 0.0539 Cesium 4.93 Ytterbium 0.0377 Manganese 4.04 Lithium 0.0377 Thorium 2.69 Yttrium 0.0323 Uranium 2.69 Holmium 0.0296 Arsenic 1.97 Cadmium 0.0296 Chromium 1.89 Palladium 0.0189 Molybdenum 1.64 Terbium 0.0161 Nickel 1.62 Thulium 0.0161 Iodine 1.28 Gold 0.0161 Lead 1.17 Tantalum 0.0135 Cerium 1.08 Iridium 0.0135 Rubidium 0.983 Lutetium 0.0108 Antimony 0.781 Europium 0.0108 Gallium 0.673 Rhodium 0.0108 Germanium 0.673 Tin 0.0108 Neodymium 0.539 Silver 0.00808 Zinc 0.539 Indium 0.00808 Lanthanum 0.486 Oxygen 0.00539 Bismuth 0.385 Mercury 0.00269 Zirconium 0.269 Tellurium 0.00269 Rhenium 0.269 Beryllium 0.00269 Thallium 0.269

TABLE-US-00002 TABLE 2 Premix Formulation (Amounts in mg/lb of body weight unless otherwise stated) Dosage: mg/5 oz. Component High Low Preferred of formula l-Arginine 0.5 0.005 0.05 50.00 *Lacto yeast (4.9% of blend) 69.51 0.6951 6.91 6951.88 Montmorillinite 1 gm/lb 0.24118 2.4118 2411.88 Canola oil (14.5% mix) 1.5 gm/lb 2.05 20.571 20571.88 Safflower oil (14.5% mix) 1.5 gm/lb 2.05 20.57 20571.88 Flax seed oil (55% Alpha Linolenic 1.5 gm/lb 2.05 20.571 1418.75 Acid) (1.0% mix) Phosphorous (Monosodium 15.750 gm 0.0525 5.08 5080.00 phosphate) 12% Calcium carbonate 8.5% 13.68 gm 0.0485 4.88 4880.00 (38% calcium) Methyl sulfonyl methane 20 0.02 2 2000.00 Transfer factor 50.00 0.05 0.75 750.00 Vitamin C (ascorbic acid) 21.62 0.2162 2.162 2162.50 d-Biotin (Vitamin H 2%) 9.73 0.000973 0.00973 10.00 Vitamin D3 29.16 IU 0.7298 IU 7.298 IU 7298.38 IU Vitamin B12 0.092 0.000092 0.00092 0.92 Folic Acid 1 0.001006 0.01006 10.06 Niacinimide 12 0.012157 0.12157 121.57 Pantothenic acid (d-Calcium 0.324 0.0108 0.108 108.00 Pantothenate) 91.6% Vitamin B6 (Pyridine Hcl) 82.3%) 1.158 0.001158 0.01158 11.58 Vitamin A (Retinol Palmitate) 650M 600 IU 4.02 IU 40.212 IU 40232.50 IU IU/g feed grade Vitamin B2 0.0554 0.002776 0.02776 27.76 Thiamine (Mononitrate) 83% 3.09 0.00308 0.0308 30.80 Vitamin E 72.9 IU 0.0729 IU 0.729 IU 729.42 IU Vitamin K 1 0.0007 0.007 7.00 Cobalt (Proteinate) 5% 0.00043 0.000043 0.00043 0.43 Copper (Proteinate) 10% 0.56 0.0112 0.112 112.00 Iodine (Potassiumiodide) 98% 0.005 0.000053 0.00053 0.53 Iron (Proteinate) 15% 3.31 0.0331 0.331 331.16 Magnesium (Oxide) 58% 10 0.04 0.4 400.00 Manganese (Proeinate) 15% 1.65 0.04 0.4 332.10 Molybdenum (Sodium Molybdate 0.05 0.001 0.01 10.00 Dihydrate) 39% Selenium (Sodium Selenite) 44.8% 0.00162 0.000081 0.00081 1.00 Zinc (Proteinate) 15% 50 0.04987 0.4987 498.72 l-Lysine (Mono HCl) 8.41 0.0841 0.841 841.57 d,l-Methionine 11.03 0.1103 1.103 1103.86 Mixed Tocopherols 300.00 Choline Chloride 2434.00 Sipernat 50 (Silicon dioxide) 12768.75 Lodex-5 (maltodextrin) 7519.38 Soy flour (17.5% mix) 24828.13 Sweet whey 996.00 BF70 spice 146.00 Dextrose powder 750.00 *Lactic acid generating bacteria is two-thirds of component and yeast is one-third; lactic acid generating bacteria is 500,000,000 CFU/gm, yeast (e.g., "Saccharamyces") 250,000,000 CFU/gm

TABLE-US-00003 TABLE 3 Canine Premix Formulation (Amounts in mg/lb of body weight unless otherwise stated) Dosage: mg/oz Component High Low Preferred of formula l-Arginine 0.5 0.005 0.05 10.00 *Lacto yeast (4.9% of blend) 69.51 0.6951 6.91 1390.38 Montmorillinite 1 gm/lb 0.24118 2.4118 482.20 Canola oil (14.5% mix) 1.5 gm/lb 2.05 20.571 3887.00 Safflower oil (14.5% mix) 1.5 gm/lb 2.05 20.57 3887.00 Flax seed oil (55% Alpha 1.5 gm/lb 2.05 20.571 240.00 Linolenic Acid) (1.0% mix) Phosphorous (Monosodium 15.750 gm 0.0525 5.08 1010.00 phosphate) 12% Calcium carbonate 8.5% 13.68 gm 0.0485 4.88 977.00 (38% calcium) Methyl sulfonyl methane 20 0.02 2 400.00 Transfer factor 50.00 0.05 2.50 500.00 Vitamin C (ascorbic acid) 21.62 0.2162 2.162 432.50 d-Biotin (Vitamin H 2%) 9.73 0.000973 0.00973 2.00 Vitamin D3 29.16 IU 0.7298 IU 7.298 IU 1459.68 IU Vitamin B12 0.092 0.000092 0.00092 0.18 Folic Acid 1 0.001006 0.01006 2.16 Niacinimide 12 0.012157 0.12157 24.31 Pantothenic acid (d-Calcium 0.324 0.0108 0.108 21.60 Pantothenate) 91.6% Vitamin B6 (Pyridine Hcl) 82.3%) 1.158 0.001158 0.01158 2.32 Vitamin A (Retinol Palmitate) 600 IU 4.02 IU 40.212 IU 8046.50 IU 650 M IU/g feed grade Vitamin B2 0.0554 0.002776 0.02776 5.55 Thiamine (Mononitrate) 83% 3.09 0.00308 0.0308 0.16 Vitamin E 72.9 IU 0.0729 IU 0.729 IU 145.88 IU Vitamin K 1 0.0007 0.007 1.40 Cobalt (Proteinate) 5% 0.00043 0.000043 0.00043 0.086 Copper (Proteinate) 10% 0.56 0.0112 0.112 22.40 Iodine (Potassiumiodide) 98% 0.005 0.000053 0.00053 0.106 Iron (Proteinate) 15% 3.31 0.0331 0.331 66.23 Magnesium (Oxide) 58% 10 0.04 0.4 80.00 Manganese (Proeinate) 15% 1.65 0.04 0.4 66.42 Molybdenum (Sodium Molybdate 0.05 0.001 0.01 2.00 Dihydrate) 39% Selenium (Sodium Selenite) 0.00162 0.000081 0.00081 0.20 44.8% Zinc (Proteinate) 15% 50 0.04987 0.4987 99.74 l-Lysine (Mono HCl) 8.41 0.0841 0.841 176.91 d,l-Methionine 11.03 0.1103 1.103 220.77 Mixed Tocopherols 60.00 Choline Chloride 486.80 Sipernat 50 (Silicon dioxide) 2553.35 Lodex-5 (maltodextrin) 1508.87 Peanut oil 496.56 Soy flour (17.5% mix) 4965.02 Peanut flour 4965.02 Sweet whey 400.00 BF70 spice 29.20 Dextrose powder 500.00 *Lactic acid generating bacteria is two-thirds of component and yeast is one-third; lactic acid generating bacteria is 500,000,000 CFU/gm, yeast (e.g., "Saccharamyces") 250,000,000 CFU/gm

TABLE-US-00004 TABLE 4 Feline Premix Formulation (Amounts in mg/lb of body weight unless otherwise stated) Dosage: mg/2.2 gm Component High Low Preferred of formula l-Arginine 0.5 0.005 0.05 0.78 *Lacto yeast (4.9% of blend) 69.51 0.6951 6.91 108.42 Montmorillinite 1 gm/lb 0.24118 2.4118 37.00 Canola oil (14.5% mix) 1.5 gm/lb 2.05 20.571 323.25 Safflower oil (14.5% mix) 1.5 gm/lb 2.05 20.57 323.25 Flax seed oil (55% Alpha 1.5 gm/lb 2.05 20.571 22.13 Linolenic Acid) (1.0% mix) Phosphorous (Monosodium 15.750 gm 0.0525 5.08 78.70 phosphate) 12% Calcium carbonate 8.5% 13.68 gm 0.0485 4.88 75.69 (38% calcium) Methyl sulfonyl methane 20 0.02 2 31.20 Transfer factor 50.00 0.05 16.00 250.00 Vitamin C (ascorbic acid) 21.62 0.2162 2.162 33.73 d-Biotin (Vitamin H 2%) 9.73 0.000973 0.00973 0.156 Vitamin D3 29.16 IU 0.7298 IU 7.298 IU 113.90 IU Vitamin B12 0.092 0.000092 0.00092 0.014 Folic Acid 1 0.001006 0.01006 0.168 Niacinimide 12 0.012157 0.12157 1.90 Pantothenic acid (d-Calcium 0.324 0.0108 0.108 1.68 Pantothenate) 91.6% Vitamin B6 (Pyridine Hcl) 82.3%) 1.158 0.001158 0.01158 0.18 Vitamin A (Retinol Palmitate) 600 IU 4.02 IU 40.212 IU 627.60 IU 650 M IU/g feed grade Vitamin B2 0.0554 0.002776 0.02776 0.43 Thiamine (Mononitrate) 83% 3.09 0.00308 0.0308 0.48 Vitamin E 72.9 IU 0.0729 IU 0.729 IU 11.38 IU Vitamin K 1 0.0007 0.007 0.11 Cobalt (Proteinate) 5% 0.00043 0.000043 0.00043 0.006 Copper (Proteinate) 10% 0.56 0.0112 0.112 1.75 Iodine (Potassiumiodide) 98% 0.005 0.000053 0.00053 0.008 Iron (Proteinate) 15% 3.31 0.0331 0.331 5.17 Magnesium (Oxide) 58% 10 0.04 0.4 6.24 Manganese (Proeinate) 15% 1.65 0.04 0.4 5.18 Molybdenum (Sodium Molybdate 0.05 0.001 0.01 0.156 Dihydrate) 39% Selenium (Sodium Selenite) 0.00162 0.000081 0.00081 0.156 44.8% Zinc (Proteinate) 15% 50 0.04987 0.4987 7.78 l-Lysine (Mono HCl) 8.41 0.0841 0.841 13.80 d,l-Methionine 11.03 0.1103 1.103 17.22 Mixed Tocopherols 4.68 Choline Chloride 38.0 Sipernat 50 (Silicon dioxide) 199.06 Lodex-5 (maltodextrin) 117.30 Sweet whey 155.37 BF70 spice 2.28 Dextrose powder 250.00 Glucosamine HCl 100.00 Pernaconniculus-Chondroitin 200.00 *Lactic acid generating bacteria is two-thirds of component and yeast is one-third; lactic acid generating bacteria is 500,000,000 CFU/gm, yeast (e.g., "Saccharamyces") 250,000,000 CFU/gm

TABLE-US-00005 TABLE 5 Stress Formula (Amounts in mg/lb of body weight unless otherwise stated) Dosage: mg/ounce Component High Low Preferred of formula Calcium 1.80 0.09 0.028 28.00 Pantothenate Vitamin C 20.00 0.056 0.017 17.00 (ascorbic acid) Vitamin B12 13.00 0.13 0.198 198.59 Vitamin A 600.00 IU 0.10 IU 0.014 14.00 Vitamin B2 1.20 0.065 0.018 18.00 Thiamine 16.00 0.0308 0.017 17.00 Vitamin E 72.9 IU 0.729 IU 0.012 12.48 Magnesium Sulfate 10.00 0.113 0.113 113.00 *Lactobacillus 10.00 0.467 1.418 1418.00 acidophilus Sodium 166.00 0.236 2.368 2368.00 Chloride Dipotassium 116.00 5.85 1.773 1773.00 phosphate Citric acid 31.00 1.59 0.482 482.00 Yeast 180.00 0.1957 0.283 283.00 (hydrolyzed) Glycine 0.142 0.0142 0.142 141.80 Potassium 18.00 0.93 0.283 283.00 chloride Vitamin D3 29.00 0.729 0.002 1.56 Dextrose 40.00 2.00 21.38 21375.00 Artificial flavor 0.028 0.0028 28.548 28.30 Transfer Factor 50.00 0.05 0.75 750.00 Sipernat 0.05 56.70 (silicon dioxide) *109 colony forming units (CFU)/gm

TABLE-US-00006 TABLE 6 Performance Formula (Amounts in mg/lb of body weight unless otherwise stated) Dosage: mg/oz. Component High* Low* Average* of formula Super oxide dismutase 60.0 0.6 6.0 6000.0 Glucosamine salts 65.0 0.65 6.5 6500.0 Transfer factor1 (horses, cows) 15.0 0.15 1.5 1500.0 Transfer factor1 (goats) 10.0 0.10 1.0 3000.0 Transfer factor1 (dogs, cats) 50.0 0.5 5.0 14000.0 Pernaconniculus-Chondroitin 16.5 0.165 1.65 1650.0 (mucopolysaccharides) Boswellic acids 30 0.3 3.0 3000.0 Di-methyl glycine 27.0 0.27 2.7 2700.0 Methyl sulfonyl methane 27.0 0.27 2.7 2700.0 Octocosonol 2.0 0.004 0.04 400.0 Montmorillinite 30.0 0.3 3.0 3000.0 *These amounts are calculated for livestock animals weighing about 450 to 1,000 pounds, goats weighing about 150 pounds, and dogs and cats weighing from about 8 to about 15 pounds. 1The amount of transfer factor may vary for different species but the amounts for the other components remain the same for each species.

TABLE-US-00007 TABLE 7 Livestock Stress Rumen By-Pass (Amounts in mg/lb of body weight unless otherwise stated) Dosage: mg/oz. (unless otherwise Component noted) of formula Stabilized1 Transfer factor (mammal source) 3500.0 Transfer factor (avian source) 1000.0 β-sitosterol (90% phytosterols) 300.0 Inositol hexaphosphate 350.0 Olive leaf extracts 35.0 Aloe extract powder (200:1) 17.0 Hybridized and non-hybridized 4000.0 Glucans (from Hybridized Cordycepts sinensis, Agaricus blazeii, Miatake, Shitake, Coriolis, Inonotus, Obliquus, and Poris cocos mushrooms) Vitamin C 2000.0 Non-Stabilized Vitamin A 4434 IU/oz Vitamin D3 1140 IU/oz Vitamin E 500 IU/oz Vitamin B1 12.77 Vitamin B2 12.77 Vitamin B12 1.5 Di-potassium phosphate 1.5 g/oz Potassium chloride 207 Magnesium sulfate 83 Calcium pantothenate 23 Ascorbic acid 23 Lactic acid bacteria 2.5 × 106 CFU/oz Yeast (S. cerivisiea) 15.0 × 106 CFU/oz Zinc proteinate 10 *These amounts are calculated for livestock animals weighing about 450 to 1,000 pounds, goats weighing about 150 pounds, and dogs and cats weighing from about 8 to about 15 pounds. 1Stabilized active ingredients are included in a formulation of 50% soybean oil and 50% active ingredient.

Kits

[0124]In one aspect, the present invention provides kits suitable for treatment of an subject, for example, an animal. The kits may further include instructions for use. Instructions may be included as a separate insert and/or as part of the packaging or container, such as a label affixed to a container or as a writing or other communication integrated as part of a container. The instructions may inform the user of methods of administration of the compositions and formulations contained therein, precautions, expected results, warnings concerning improper use, and the like.

[0125]In one embodiment, the kit includes a first container having a composition or a formulation that includes a transfer factor. In addition to the transfer factor, the formulations and compositions of the present invention may also include an antibody or antibody fraction. In certain embodiments, the antibody or antibody fraction may be present at about 5% to about 35% of the formulation or composition. In other embodiments, the antibody or antibody fraction is present at about 10% to about 30%, from about 15% to about 25%, from about 17.5% to about 22.5%, or about 20%. The first container of the present invention may contain compositions or formulations that have either (1) transfer factor or (2) transfer factor and antibody or antibody fraction in a lyophilized form.

[0126]In another aspect, the kits provide a first container having a composition or formulation of the present invention that is encapsulated by a hydrophobic or lipid coating. In another embodiment, the hydrophobic or lipid coating may include essential fats and/or plant oils. The plant oil may be soybean oil.

[0127]In another aspect, the kit may include a composition or formulation containing a glucan, including without limitation a hybrid glucan, a hydrolyzed glucan, and a hydrolyzed hybrid glucan, as described herein. The glucan may be derived from a fungus. The fungus may be a whole fungus. As described herein, the glucan may be derived from a Cordyceps strain, including without limitation the Cordyceps sinensis strain. It may also be a hybrid glucan, as described herein. In one embodiment, the glucan is encapsulated by a hydrophobic or lipid coating as described herein. The coating may include an essential fat and/or a plant oil. The plant oil may be soybean oil. The glucan may be hydrolyzed as described herein. In some embodiments, the glucan is provided in the first container, which already contains the transfer factor. However, the glucan may also be provided in a second container separate from the first container. The composition or formulation having the glucan may include a lyophilized glucan.

[0128]The present invention provides kits having a first container with a transfer factor and a second container with a glucan. The transfer factor may be lyophilized. If the transfer factor and/or the glucan is lyophilized, the kit may further comprise a third container having a reconstitution solution for reconstitution of the lyophilisate(s). The reconstitution solution may be any solution suitable for reconstituting a lyophilistate known to those skilled in the art. For example, water or any suitable solvent may be used. In addition, the kit may contain instructions for the reconstitution of the lyophilisates in the reconstitution solution.

[0129]In another aspect, the kit includes instructions for the administration of the formulations and/or compositions included in the containers described herein. The kits as described herein may further comprise suitable packaging of the respective compositions, instructions, and/or other optional components. In one embodiment, kits of the present invention may further contain components useful in the application of the compositions and formulations described herein.

[0130]In one aspect, the kit includes instructions for the prevention and/or treatment of a condition in a subject. The conditions suitable for treatment are described herein, including without limitation, benign and malignant tumors. The present invention includes methods, compositions, and pharmaceutical formulations for the prevention and/or treatment of conditions and/or diseases in a subject, including a mammal. Included are methods, compositions and formulations suitable for animals, including mammals, including humans.

Compositions, Pharmaceutical Formulations and Methods of Treatment

[0131]In certain embodiments, the present invention provides methods of treating a subject with a condition by administering a composition or a pharmaceutical formulation, as described herein. In one embodiment, the pharmaceutical formulation is prepared from a kit described herein. The compositions may contain one or more of the following components, in any combination: transfer factor, lyophilized transfer factor, an antibody or antibody fraction, a lyophilized antibody or antibody fraction, and a glucan. For example, the composition or pharmaceutical formulation may comprise (i) a transfer factor, (ii) a transfer factor and an antibody or antibody fraction (iii) a transfer factor, an antibody or antibody fraction and a glucan, (iv) a lyophilized transfer factor and an antibody or antibody fraction (v) a lyophilized transfer factor and a lyophilized antibody or antibody fraction, (vi) a lyophilized transfer factor, a lyophilized antibody or antibody fraction, and a glucan. In certain embodiments, the compositions and formulations prepared from a kit may optionally include additional components. In one embodiment, the transfer factor of any of these combinations may be lyophilized. In other embodiments, the antibody or antibody fraction of a composition or pharmaceutical formulation described herein may be lyophilized.

[0132]In another aspect, the compositions and pharmaceutical formulations provided by the present invention comprise glucans from a particular strains of fungus. In one embodiment, a glucan is derived from one or more strains of fungus. By "fungus" herein is meant a fungus other than yeast unless otherwise noted. The present invention contemplates the derivation of one or more glucans, hybrid glucans, hydrolyzed glucans, and hydrolyzed hybrid glucans from one of the following strains: Cordyceps sinensis, Agaricus blazeii, Miatake, Shiake, Coriolis, Inonotus, Obliquus, and Poris cocos. In another embodiment, glucans are derived from more than one strain. U.S. Patent Application Publication No. 2006/0073197 A1, which is incorporated herein by reference in its entirety, also relates to the glucan-containing compositions and pharmaceutical formulations of the present invention.

[0133]In one embodiment, the compositions and pharmaceutical formulations of the present invention include one or more of the following: lyophilized transfer factor, a hybrid glucan, a hydrolyzed hybrid glucan, and an antibody or antibody fraction. Glucans may be derived from different sources, including various species of fungus. For example, a composition or formulation of the present invention may contain hydrolyzed glucans derived from Cordyceps sinensis, Agaricus blazei, Grifola frondosa, Ganoderma lucidum, Lentinula edodes, and/or Coriolus versicolor. In one embodiment, the glucans may be hybrid or non-hybrid glucans derived from Cordyceps sinensis.

[0134]In another embodiment, the compositions and pharmaceutical formulations containing various combinations of transfer factor and antibody or antibody fraction may further comprise one or more ingredients of the performance formula according to Table 6 above.

[0135]The conditions suitable for treatment using these compositions, pharmaceutical formulations and by these methods include, without limitation, a malignant tumor, a benign tumor, hyperthyroidism, lymphopenia, Cushing's disease, Addison's disease, weight loss, hair loss, fatigue, and anorexia.

[0136]In one embodiment, the malignant tumor may be a carcinoma, including without limitation, a squamous cell carcinoma, a transitional cell carcinoma, an adenocarcinoma or a thyroid carcinoma. Tumors may be found in any organ or tissue of the body. The primary tumor or original tumor is the place where the cancer begins but it can spread or metastasize and form metastatic tumors in other parts of the body. Carcinomas are tumors that begins in the skin or in tissues that line or cover internal organs. In some cases, the primary cancer site may not be known and the tumor is called a carcinoma of unknown primary origin. Subjects with such tumors may have a cell type called adenocarcinoma, which refers to cancer that begins in the cells in glandular structures in the lining or covering of certain organs in the body. Common primary sites for adenocarcinomas include without limitation the lung, pancreas, breast, prostate, stomach, liver, and colon.

[0137]In another embodiment, the malignant tumor may be a sarcoma, including without limitation, a fibrosarcoma, a chondrosarcoma, a lymphosarcoma, a melanosarcoma, an osteocsarcoma, or a hemangiosarcoma. In one embodiment, the melanosarcoma may be an amelanotic melanosarcoma. In one other embodiment, the hemangiosarcoma is splenic. A sarcoma includes cancers of the bone, bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue.

[0138]In one embodiment, the malignant tumor may be a mast cell tumor. Mast cell tumors may be graded as grade 1, grade 2, grade 3, or grade 4. Grading of mast cell tumors is typically performed by a pathologist during a biopsy. The grade assigned indicates the malignant characteristic of the cells, where grade 1 is benign, grade 3 is malignant, grade 2 is between 1 and 3, and grade 4 is a rapidly growing malignant tumor with metastasis. Mast cell tumors are frequently found in canines.

[0139]In one embodiment, the malignant tumor is a histiocytoma, which may originate from a Langerhans cell found in the skin. Such cells are part of the immune system and process and present external antigens to other cells in the immune system. Histiocytomas are frequently found in canines, including without limitation Labrador retrievers, Stafforshire terriers, Boxers, and Daschunds.

[0140]In some embodiments, the malignant tumors suitable for treatment also include, without limitation, a melanoma, a bone tumor, a keratoma, a lipoma, plepharitis eye tumors, dermal tumors, and leukemia. The lymphoma may be a Hodgkin's lymphoma or a non-Hodgkin's lymphoma.

[0141]In other embodiments, the condition suitable for treatment by the methods, compositions, and pharmaceutical formulations of the present invention is a benign tumor or an adenoma, including without limitation meibomian gland adenomas, a glandular adenoma of the skin, and perinanal adenomas. In one other embodiment, the malignant tumor is a granulosa tumor of the ovary.

[0142]In another embodiment, the condition suitable for treatment by the methods, compositions, and pharmaceutical formulations of the present invention is a fungal infection, including without limitation blastomycosis and coccidiomycosis. Infection may occur by inhalation. Blastomycosis is caused by the fungus Blastomyces dermatitidis. Coccidiomycosis is caused by spores from the fungus, Coccidiodes immitis. Fungal infections may occur in subjects with compromised immune systems, including without limitation people with HIV and organ transplant recipients.

[0143]In one aspect of the present invention, the methods, compositions, and pharmaceutical formulations provide a way to reduce tumor size in a subject. In one embodiment of the invention, there is provided a method of reducing tumor size in a subject in need thereof comprising administering to the subject a composition comprising transfer factor and an antibody or antibody fraction. In another embodiment, there is provided a method of reducing tumor size in a subject in need thereof comprising administering to the subject a composition comprising lyophilized transfer factor. In certain embodiments, the tumor size may be reduced by at least about 50%, or at least about 90%.

[0144]By "reduce" or "reduction" and any grammatical equivalents, is meant a cytotoxic effect on tumor cells and/or a cytostatic effect on tumor cells, as well as the regression of a tumor, such that its size is reduced. In one embodiment, tumors are reduced from about 1% to about 100%, about 5% to about 95%, about 10% to about 90%, about 15% to about 85%, about 20% to about 80%, about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, about 45% to about 55%, and about 50%. In other embodiments, the tumor reduced may be a malignant or a benign tumor. Some embodiments provide a reduction of particular types of tumors. For example, a malignant tumor may be reduced from about 20% to about 40% and a benign tumor may be reduced from about 80% to about 100%. In addition, particular types of malignant tumors may be reduced. For example, an amelanotic melanosarcoma may be reduced by about 80%, a lytic bone tumor may be reduced by about 100%, a keratoma may be reduced by about 100%, a mast cell tumor may be reduced from about 50% to about 80%, an osteosarcoma may be reduced by about 80%, and a melanoma may be reduced from about 50% to about 80%. In other embodiments, the reduction may be transient. For example, a lymphoma may be transiently reduced from about 50% to about 80% and a hemangiosarcoma may be transiently reduced for about one year from about 50% to about 80%.

[0145]The following examples serve to more fully describe the manner of using the above-described invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes.

EXAMPLE 1

[0146]The compositions and/or formulations of the present invention may be assayed for lymphocyte stimulation activity using procedures known in the art, such as the ImmunKnow™ Immune Cell Function Assay provided by Cylex, Inc. and discussed herein (See Wier U.S. Pat. No. 6,630,316). Dilutions of the compositions and/or formulations if the present invention may be prepared as a stock solution having a concentration of 1 mg/ml and stored at 4° C. Dilutions of the stock solution can be prepared for analysis. For example, dilutions were prepared at 0.04, 0.2, 0.4, 2.0, 4.0, 10, 20, and 100 μg/mL, as well as 1 mg/mL, for a number of samples. Each diluted sample was added to a 96 well assay plate, followed by the addition of diluted whole blood to each assay well containing the diluted sample. The plates were incubated at 37° C. for one hour and then a sample diluent of Phytohemagglutinin-L (PHA) was added to each well. The plates were then incubated at 37° C. for 15-18 hours, after which magnetic beads coated with mouse monoclonal anti-human CD4 (Dynabeads.RTM.* CD4) were added. Following a 15 second agitation on a plate shaker, the plates were incubated at room temperature (18-28° C.) for 15 minutes. This step was repeated and a final agitation step on a plate shaker for 15-3-seconds was performed. The magnetic beads were then separated from the sample, followed by a washing step to remove any residual unbound cells. A lysis reagent was added to each well. A luminescent reagent was added and the amount of ATP present in each cell lysate was assessed. The results shown in FIGS. 1-2 are in terms of a stimulation index. The samples analyzed include a composition comprising a lyophilized transfer factor (FIG. 1) and a composition comprising hydrolyzed glucans and glucans (Immune-Assist as provided by Aloha Medicinals and containing Agaricus blazei, Cordyceps sinesis hybrid, Lentinula edodes, Grifola frondosa, Ganoderma lucidum, and Coriolus versicolor) (FIG. 2). Results were also obtained for a composition comprising a lyophilized transfer factor with a 20% antibody fraction, spray-dried.

EXAMPLE 2

[0147]A calf study was performed using the stress formula disclosed in Table 5 above but containing lyophilized transfer factor. A 1-2% death loss was observed. When the same formula containing a spray-dried lyophilized transfer factor was administered, the death loss increased to 37.5%, that is, six out of sixteen animals died after being treated three times.

EXAMPLE 3

[0148]A cow study was performed using the stress formula disclosed in Table 5 above but containing lyophilized transfer factor. An 11.7% death loss was observed. A similar study performed thereafter resulted in a 6.5% death loss. Another study was performed using the stress formula from Table 5 above but containing spray-dried transfer factor (4-Life) and a 35% death loss was observed (48 out of 137 died).

TABLE-US-00008 Head treated dead % January 2006 130 34 26% On lyophilized transfer factors February 2006 137 16 12% On lyophilized transfer factors March 2006 124 8 6% On lyophilized transfer factor April 2006 (Ran out of lyophilized 137 48 35% transfer factors and switched to spray dried product) May 5, 2006 (Animals given 20 1 5% lyophilized transfer factors)

[0149]Their problems subsided to 3% death loss with use of lyophilized transfer factors.

COMPARATIVE EXAMPLE

[0150]Spray dried transfer factor product was tested on 16 calves out of 100 head shipment. Sixteen calves got spray dried transfer factor on days one, two, and twelve. Results:

[0151]three died

[0152]13 head treated twice, none doing well in feed efficiency and poor hair coat

EXAMPLE 4

[0153]baby calves two to three days old.

[0154]15 head on lyophilized transfer factor product

a) treated one with mild scours,b) all calves sucking on own day 3c) calves on full feed Day 7

[0155]15 head treated with spray dried transfer factor product

a) five treated calvesb) calves sucking on day 7c) calves on full feed day 14

[0156]15 head of Controls no transfer factor.

a) treated all 15 calves over two times eachb) calves all sucking on day 10 to 14c) calves still not on full feed day 24

EXAMPLE 5

[0157]1500 head of dairy calves in Oakdale California was treated with spray dried transfer factor product since November, 2002, averaging about 5 to 8% death loss. When switched to lyophilized transfer factor product dropped the death loss dropped to about 2.8%.

[0158]The above Examples 2-5 demonstrate reduced mortality and morbidity in calves treated with lyophilized transfer factor in comparison with spray dried transfer factor and transfer factor-free controls.

[0159]It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present inventions without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modification and variations of the inventions provided they come within the scope of the appended claims and their equivalents.

[0160]The terms and expressions which have been employed are used as terms of descriptions and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope on this invention.

[0161]In addition, where features or aspects of the invention are described in terms of Markush group or other grouping of alternatives, those skilled in the art will recognized that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

[0162]Unless indicated to the contrary, all numerical ranges described herein include all combinations and subcombinations of ranges and specific integers encompassed therein. Such ranges are also within the scope of the described invention.

[0163]The disclosures of each patent, patent application and publication cited or described in this document are hereby incorporated herein by reference, in their entirety.

TABLE-US-00009 APPENDIX 1 HUMAN AND BOVINE PATHOGENS: POTENTIONAL CROSS REACTIVITY Human Pathogen or Disease Commonality Bovine Pathogen BACTERIA Travelers Disease (E. coli) very Toxigenic E. coli very Campylobacter jejuni Bloody diarrhea/hemolytic uremia increasing E. coli 0157:H7 Verotoxic Salmonellosis/Typhoid Fever common Salmonella thyphimurium, Salmonella typhosa dublin Diarrhea, from food or water very Campylobacter jejuni Clostridial Infection (non-tetanus) common Clostridia (many species) C. dificil Mycobacterium Infections Mycobacterium species johnei, Crohn's Disease common common in Jersey cattle Staphylococcal super infections common Staph. aureus Streptococcal infections common Streptococcus Endocarditis common Beta Strep. Superinfection increasing S. pyogenes S. pyogenes increasing Enterococci common Enterococci (most spp. & VRE) Hospital/VRE strains serious common Helicobacter pylon (ulcers) common Bovine/Porcine association VIRUS Influenza common Influenza virus Pneumonia Resp. Syncytial Virus common Bovine Resp. Sync. Virus Papilloma, Condylomaya common Bovine Papilloma Virus Virus Diarrhea common Bovine Virus Diarrhea Rotavirus Rotavirus Coronavirus Cytomegalovirus common Bovine CMV and IBR Herpes Infections common Bovine Rhinotracheitis HIV (Retrovirus) common Bovine Immune Deficiency Virus Rhinovirus (common cold) very Bovine Rhinovirus YEAST, FUNGI and PROTOZOA Candidiasis common Candida exp. common Cryptosporidiosis very Calf diarrhea, C. parvum Giardiasis common Calf diarrhea, G. lamblia OTHER Mycoplasma pneumonia, arthritis common Bvn. Mycopl. Pneumonia

TABLE-US-00010 APPENDIX 2 HUMAN AND AVIAN PATHOGENS: POTENTIAL CROSS REACTIVITY Common- Human Pathogen or Disease ality Avian Pathogen BACTERIA Travelers Diarrhea (E. coli) very Toxigenic E. coli very Campylobacter jejuni Bloody diarrhea/hemolytic increasing E. coli 0157:H7 verotoxic uremia Diarrhea O1, O2, O47, others Salmonellosis very Salmonella sp. Diarrhea, from food and water very Campylobacter jejuni Clostridial Infection common Clostridia sp. Pasteurellosis very Pasteurella multocida Pneumonia common Haemophilus gallinarium common Mycoplasma gallispeticum common Chlamydia pneumona Systemic infection common Erysipeloxthrix insidiosa Diarrhea, systemic infection very Lisreria monocytogenes VIRUS Chicken pox very Fowl pox Influenza very Influenza virus Infectious bronchitis common Infectious Bronchitis Adult Leukemia virus rare Marek's disease virus (ATLV-1) Pneumonia common Paramyxovirus Herpetic infections common Herpes simplex virus FUNGAL Pneumonia, systemic disease very Aspergillus sp. Diarrhea, systemic disease very Aspergillus sp. Diarrhea, thrush, vaginitis very Candida albicans Systemic disease very Histoplasma capsulatum Systemic disease very Coccidia PARASITES Trichomoniasis very Trichomonas Diarrhea very Giardia



Inventor

* Ramaekers, Joseph C.

US Class 424/130.1IMMUNOGLOBULIN, ANTISERUM, ANTIBODY, OR ANTIBODY FRAGMENT, EXCEPT CONJUGATE OR COMPLEX OF THE SAME WITH NONIMMUNOGLOBULIN MATERIAL

Attorney, Agent or Firm

* FOX ROTHSCHILD LLP;PRINCETON PIKE CORPORATE CENTER

International Classes A61K 39/395 A61P 3/00



Abstract text

Compositions and formulations are provided comprising a growth factor fraction and at least one glucan. Also provided are methods for making the compositions and formulations. Methods of promoting growth in an animal using the compositions and/or formulations are also provided. Claims

1. A composition comprising a growth factor fraction and at least one glucan.

2. The composition of claim 1 wherein the growth factor fraction is lyophilized.

3. The composition of claim 1 further comprising an antibody.

4. The composition of claim 3 wherein the antibody is contained in an antibody fraction.

5. The composition of claim 3 wherein the antibody is lyophilized.

6. The composition of claim 1 further comprising a transfer factor.

7. The composition of claim 6 wherein the transfer factor is lyophilized.

8. The composition of claim 1 wherein the growth factor fraction is encapsulated by a hydrophobic or lipid coating.

9. The composition of claim 1 wherein the glucan is encapsulated by a hydrophobic or lipid coating.

10. The composition of claim 3 wherein the antibody is encapsulated by a hydrophobic or lipid coating.

11. Composition of claim 1 wherein the composition is encapsulated by a hydrophobic or lipid coating.

12. The composition of claim 1 wherein the growth factor fraction is obtained from colostrum.

13. The composition of claim 3 wherein the antibody is derived from an avian or a mammalian source.

14. The composition of claim 6 wherein the transfer factor is derived from an avian or mammalian source.

15. The composition of claim 1 wherein the glucan is derived from a member selected from the group consisting of Cordyceps sinensis, Agaricus Blazeii, Coriolus Poira Cocos, Inontus obliquus, Maitake, Shiitake, and mixtures thereof.

16. The composition of claim 16 wherein the Cordyceps sinensis is a hybrid.

17. The composition of claim 1 further comprising a member selected from the group consisting of inositol hexaphosphate, olive leaf extract, mannans, and phytosterols.

18. The composition of claim 17 wherein the mannans are obtained from Aloe vera leaf.

19. The composition of claim 1 further comprising a member selected from the group consisting of dipotassium phosphate, potassium chloride, magnesium sulfate, calcium pantothenate, vitamin E, vitamin C, vitamin A, vitamin D3, vitamin B1, vitamin B2, vitamin B 12, and mixtures thereof.

20. A method of making a composition for promoting animal growth comprising the steps of:A) Fractionating colostrum to obtain a fraction having a molecular weight of about 10,000 Daltons or greater; andB) Mixing the fraction with at least one glucan.

21. A method of making a formulation for promoting animal growth comprising the steps of:A) Fractionating colostrum to obtain a fraction having a molecular weight of about 10,000 Daltons or greater;B) Mixing the fraction with at least one glucan to form a composition; andC) Combining the composition with a member selected from the group consisting of dipotassium phosphate, potassium chloride, magnesium sulfate, calcium pantothenate, vitamin E, vitamin C, vitamin A, vitamin D3, vitamin B1, vitamin B2, vitamin B 12, and mixtures thereof.

22. A method of promoting growth in an animal comprising administering to the animal a composition comprising a growth factor fraction and at least one glucan.

23. The method of claim 22 wherein the animal is a calf.

24. A method of increasing feed conversion in an animal comprising the step of administering to the animal a composition comprising a growth factor fraction and at least one glucan.

25. The method of claim 24 wherein the animal is a calf. Description

FIELD OF THE INVENTION

[0001]This invention relates generally to compositions and formulations comprising a growth factor fraction and at least one glucan. Such formulations are useful in providing health benefits to animals, including promoting growth.

BACKGROUND OF THE INVENTION

[0002]The present invention relates to compositions and formulations comprising a growth factor fraction, as well as methods of making the same and methods of administration using the same. Other U.S. patents and U.S. patent applications relate to the present invention, including without limitation, U.S. Pat. Nos. 6,506,413 and 6,962,718, U.S. Patent Provisional Application Nos. 60/573,113, 60/649,363, 60/701,860, and 60/814,777, U.S. patent application Ser. No. 11/762,727, U.S. Patent Application Publication Nos. 2006/0029585 A1, 2006/0073197 A1, and 2007/0128253 A1, all of which are incorporated herein by reference. Also related are PCT publications WO/2002/087599 and WO/2005/112891, incorporated herein by reference.

BRIEF SUMMARY OF THE INVENTION

[0003]In certain aspects, the invention relates to compositions comprising a growth factor fraction. Preferably, the growth factor fraction is combined with at least one glucan. In certain embodiments, the growth factor fraction may be combined with an antibody. In certain aspects, the antibody is contained in an antibody fraction. In certain embodiments, the growth factor fraction and/or the antibody or antibody fraction with which it is combined may be lyophilized.

[0004]In further aspects, compositions and formulations of the invention may further comprise additional components. In certain embodiments, the compositions and formulations comprising a growth factor fraction may additionally comprise transfer factor. In certain embodiments, the transfer factor may be lyophilized.

[0005]Additional aspects of the invention relate to compositions and formulations comprising a growth factor fraction that is encapsulated by a hydrophobic or lipid coating. Additional components of the compositions may also be encapsulated, including, but not limited to, encapsulated glucans, and encapsulated antibody or antibody fraction, encapsulated transfer factor, and combinations thereof.

[0006]Additional aspects of the present invention are directed to methods of making compositions and formulations comprising a growth factor fraction.

[0007]Further aspects of the present invention are directed to methods of promoting growth in an animal by administering an effective amount of a composition and/or formulation comprising a growth factor fraction.

[0008]Both the foregoing general description and the following detailed description are exemplary and explanatory, and are intended to provide further explanation of the invention as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0009]In certain embodiments, the present invention is directed to compositions and formulations comprising a growth factor fraction. In preferred embodiments, the growth factor fraction may be obtained from colostrum. Preferably, compositions comprising growth factor fraction further comprise at least one glucan.

[0010]In certain embodiments, the growth factor fraction is combined with an antibody. In particular embodiments, the antibody is contained within an antibody fraction.

[0011]In certain embodiments, growth factor fraction may be combined with transfer factor. In certain embodiments, the transfer factor may be obtained from an avian and/or a mammalian source.

[0012]Other aspects of the invention are directed to formulations further comprising components, including, but not limited to, nutraceutical ingredients, in addition to the growth factor fraction. In other aspects of the invention, there are provided formulations comprising additional components in addition to growth factor fraction and glucan.

[0013]In certain embodiments of the invention, a fraction containing a growth factor or growth factors may be obtained from colostrum. In a preferred embodiment, this growth factor fraction is obtained from bovine colostrum. The colostrum may be fractionated; the fraction comprising material having a molecular weight of approximately 10,000 daltons (Da) and above is designated as the growth factor fraction. The growth factor fraction may include high molecular weight proteins. The fraction obtained that is approximately 10,000 to approximately 150,000 Da is designated the antibody fraction, also known as the antibody-colostrum fraction. The fraction comprising material having a molecular weight of approximately 10,000 Da and below is designated as transfer factor.

[0014]In other embodiments, the transfer factor and/or antibody is obtained from an avian source. One such source is chickens that are inoculated with or exposed to one or more pathogens. Inoculation with pathogens may be accomplished by any effective means. In one embodiment, chickens are given a feed mixture containing excrement from an animal, including without limitation, a human, a fish, a goat, a llama, an alpaca, a pig, a sheep, a cow, and a horse. The excrement will contain a large variety of pathogens and upon administration of a feed to an animal, it will develop transfer factor and/or antibodies to such pathogens. Avian transfer factor and avian antibodies can then be obtained from the eggs produced by the above-treated chickens. In certain embodiments of the invention, avian transfer factor and avian antibodies may be found in whole egg yolks. As a non-limiting example, the antibodies of avian source are supplied as powdered whole egg yolks (which is believed to also contain transfer factor).

[0015]Additionally, transfer factor and antibodies may be derived from any suitable source, as described, for example, in U.S. Pat. Nos. 4,816,563; 5,080,895; 5,840,700; 5,883,224; and 6,468,534; and U.S. patent application Ser. No. 11/762,727, the contents of which are hereby incorporated by reference herein.

[0016]Lyophilization

[0017]The present invention also provides compositions and formulations that have one or more lyophilized component(s). Lyophilization or "freeze-drying" is a process well known to those of ordinary skill in the art. For example, some techniques of lyophilization are described in Akers, Michael J., Chapter 41 in Remington The Science and Practice of Pharmacy, 828 (David B. Troy ed., Lippincott Williams & Wilkins 2006), which is incorporated herein by reference. In certain embodiments, formulations and/or compositions of the present invention may include lyophilized growth factor fraction. In certain embodiments, growth factor fraction, which may be lyophilized, may be combined with antibody or an antibody fraction, which may, in certain embodiments, be lyophilized. In other embodiments, transfer factor, which may be combined with growth factor fraction, may be lyophilized. In other embodiments, additional components of formulations and compositions of the invention may be lyophilized, including, without limitation, other peptides and proteins.

Formulations

[0018]In certain embodiments of the invention, growth factor fraction is provided in a formulation that further comprises one or more additional ingredients. In some embodiments, growth factor fraction and at least one glucan is provided in a formulation that further comprises one or more additional ingredients. In certain embodiments, the growth factor fraction may be lyophilized. In certain embodiments, the optional antibody or antibody fraction may be lyophilized.

[0019]In a preferred embodiment, growth factor fraction is present in the formulation in the amount of about 10 mg to about 12 gm/oz, more preferably about 100 mg to about 6 gm/oz and most preferably about 10 mg to about 3 gm/oz. In certain preferred embodiments, such a formulation comprising transfer factor is provided to an animal in an amount of about 1 oz per 1000 lb of animal.

[0020]In certain embodiments of the invention, formulations are provided which comprise glucans. Glucans may be derived from any suitable source, including, but not limited to, fungi, oats, and yeast. Preferably, glucans are present in or derived from fungi. In certain embodiments, the glucans which may be included in the formulations are present in whole fungi. In certain preferred embodiments, glucans are present in or derived from Cordyceps, more preferably, Cordyceps sinensis.

[0021]In certain embodiments, glucans are derived from hybrid strains of fungi. In a preferred embodiment the hybrid glucans used in the invention are present in, or derived from, hybrid strains of Cordyceps and in particular Cordyceps sinensis. One technique to induce the hybridization of Cordyceps involves plating two different strains or species on a single agar plate which has been inoculated with rattlesnake venom as described in, for example, U.S. Patent Application Publication No. 2006/0073197, published Apr. 6, 2006, and U.S. Patent Application Publication No. 2007/0128253, published Jun. 7, 2007, each of which is incorporated herein by reference. In a preferred embodiment, the hybrid strain producing the hybrid glucans that may be used in compositions and formulations of the invention is Cordyceps sinensis Alohaensis, which is available from Pacific Myco Products, Santa Cruz, Calif.

[0022]In addition to Cordycep sinensis hybrids, suitable sources of glucans may include, but are not limited to, Agaricus Blazeii, Coriolus Poira Cocos, Inonotus Obliquus, Maitake Mushroom, Shiitake Mushroom, and combinations thereof.

[0023]When glucans are used, the formulation preferably contains about 10 mg to about 18 gm of whole organism/oz, more preferably about 100 mg to about 10 gm of whole organism/oz and most preferably about 100 mg to about 5 gm of whole organism/oz.

[0024]Equivalent amounts of purified or partially purified glucan as well as the nucleosides associated therewith (e.g., Cordycepin (3'deoxyadenosine), adenosine and N6-(2 hydroxyethyl)-adenosine) may also be used.

[0025]In certain embodiments, compositions and formulations comprising growth factor fraction may be combined with minerals, antioxidants, amino acids, and other nutraceuticals.

[0026]In certain preferred embodiments of the invention, formulations that may be administered to a subject, may, in addition to growth factor fraction and one or more glucans, include one or more of the following: mammalian antibody-colostrum fraction, avian antibodies or antibody fraction (which may, in certain embodiments, be obtained from whole egg yolk), and transfer factor, which, in preferred form, may be derived from mammalian or avian source.

[0027]In certain preferred embodiments, compositions may further comprise one or more of inositol hexaphosphate (Ip6), mannans, olive leaf extract, and phytosterols. In certain preferred embodiments, mannans are derived from Aloe vera. In certain preferred embodiments, phytosterols may be derived from soya bean.

[0028]In certain embodiments, compositions may further comprise one or more of lactic acid producing bacteria, ascorbic acid, Vitamin A, Vitamin D3, Vitamin E, Vitamin B1, Vitamin B2, Vitamin B 12, dipotassium phosphate, potassium chloride, magnesium sulfate, and calcium pantothenate.

[0029]In certain embodiments, the invention provides compositions and formulations in which one or more components are encapsulated. Encapsulation may be achieved by mixing the component to be encapsulated with a hydrophobic substance or a lipid to form a coating around the component. Encapsulation may protect labile components from inactivation in the gastrointestinal tract. Such encapsulation may be important especially in the case of ruminants where digestion within the rumen has been found to interfere with the administration of certain labile factors. Enhanced bioavailability has been demonstrated, for example, when a transfer factor is encapsulated and administered to ruminants.

[0030]Previous use of non-encapsulated transfer factor in ruminants, e.g., cows, produced significant beneficial results. See, e.g. U.S. Patent Publication 2003/0077254, published Apr. 24, 2003 incorporated herein by reference in its entirety. Subsequently, it was discovered that transfer factor was not stable by oral administration in a stressed population of cattle. After discovering that transfer factor is inactivated in vitro in the presence of rumen fluid and flora, it was determined that prior success with transfer factor in ruminants was due to the presence of the esophageal groove. When not stressed, the esophageal groove provides partial bypass of the rumen. However, in a stressed population the esophageal groove closes and shunts the transfer factor formulation into the rumen. It was discovered that encapsulating transfer factor and/or glucans with a hydrophobic substance or a lipid to form an encapsulated formulation is sufficient to provide substantial by-pass of (e.g., 85%) of the rumen even in a stressed population.

[0031]While not seeking to be bound by any theory or theories, it is believed that encapsulation of growth factor fraction may increase its bioavailability upon administration to fermenting animals, such as adult ruminants.

[0032]In preferred embodiments, the growth factor fraction is encapsulated by mixing with a hydrophobic substance or a lipid to form a coating around the growth factor(s). In additional embodiments, one or more additional components such as antibody, antibody fraction, transfer factor and/or glucans may be encapsulated. Other optional components of compositions and formulations of the invention may be encapsulated, such as, without limitation, inositol hexaphosphate, olive leaf extract, mannans, phytosterol, vitamin C and mixtures thereof. The growth factor fraction, antibody or antibody fraction, and/or transfer factor may each be individually encapsulated or encapsulated as a mixture. Alternatively, the entire formulation can be encapsulated. The encapsulated component(s) and/or formulation can be produced in a variety of ways. In a preferred embodiment, each of the growth factor fraction, glucans, antibody or antibody fraction, and/or transfer factor in the formulation may be encapsulated as described in U.S. Pat. Nos. 5,190,775, 6,013,286 and U.S. Application 2003/0129295, each of which is incorporated herein by reference in its entirety.

[0033]In certain embodiments, glucans of the formulation may be encapsulated, preferably with a hydrophobic or lipid coating. It is preferred that the amount of hydrophobic or lipid coating be between about 25% and 150 wt/% of the glucan, about 50-150 wt %, or about 75-125 wt/%, with an equal weight being most preferred.

[0034]The formulation described in Example 2 provides an exemplary encapsulated growth factor formulation. In certain embodiments, the growth factor formulation includes encapsulated growth factor fraction and one or more glucans. In certain preferred embodiments, the formulation may further comprise encapsulated antibodies and/or encapsulated transfer factor. It is preferred that the glucan portion of this formulation also be encapsulated. In preferred form, additional components include one or more of the following that has been encapsulated: inositol hexaphosphate (IP6), phytosterol, olive leaf extract, mannans, and Vitamin C. Additional optional components may include one or more of Vitamin A, Vitamin D3, Vitamin E, Vitamin B1, Vitamin B2, Vitamin B 12, dipotassium phosphate, potassium chloride, magnesium sulfate, calcium pantothenate, and lactic acid producing bacteria. In a preferred embodiment, all of the foregoing are included in a growth factor formulation.

[0035]The growth factor fraction may be encapsulated with a hydrophobic or lipid coating that is preferably between about 25% and about 150 wt/% of the growth factor fraction, about 50-150 wt/% and about 75-125 wt/%, with an equal weight of growth factor fraction and hydrophobic or lipid coating being most preferred.

[0036]A variety of other methods for rumen by-pass are known. In one embodiment, the encapsulated or non-encapsulated formulation is directly injected (subcutaneously, intramuscularly, or intravenously) to by-pass not only the rumen but also the entire digestive system. Similarly, intravaginal, intrarectal or other direct administration to mucus membranes, such as the eye subconjunctival, by-pass the digestive system and the rumen in particular. Alternatively, the formulation can be mixed with various solvents which allow for direct skin absorption. Furthermore, methods are known in the art to stimulate opening of the esophageal groove in various ruminants and such opening allows for immediate passage of an orally administered formulation to the gastrointestinal tract, by-passing the rumen.

[0037]In additional embodiments of the invention, additional components may be used in the formulation. For example, IP6, olive leaf extract, aloe extract and/or vitamin C may be used. In preferred embodiments, IP6 is present at between 10 mg and 3 gm/oz, or one preferably between 100 mg and 2 gm/oz, and most preferably between 100 mg and 1 gm/oz. Olive leaf extract is preferably present in the amount of 2 mg to 2 gm/oz, more preferably between 5 mg and 1 gm/oz, and most preferably between 5 mg and 500 gm/oz. Aloe extract is preferably present at between 2 mg and 1000 mg, more preferably between 5 and 500 mg/oz, and most preferably between 5 and 250 mg/oz. Vitamin C may be present at between 10 mg/oz and 10 gm/oz, or preferably between 100 mg and 8 gm/oz, and most preferably between 100 mg and 5 gm/oz.

Methods of Administration

[0038]In certain embodiments, the present invention provides methods involving administration of compositions and/or formulations according to the invention to a subject. As used herein, the "subject" is preferably an animal, including, but not limited to, a calf.

[0039]In certain embodiments, the present invention provides methods of administering to a subject a composition or a pharmaceutical formulation, as described herein. Embodiments of the invention are directed to use of compositions and formulations comprising growth factor fraction as described herein for promoting growth in an animal.

[0040]In certain embodiments, the growth factor formulations find use in increasing food conversion efficiency. Food conversion efficiency is the rate at which an organism can convert food to body mass, and is also known in the cattle industry as feed conversion efficiency. The compositions and formulations described herein find use in a number of aspects of overall organismal health and development. In certain embodiments, methods of improving feed conversion in an animal subject comprises the administration to the subject of compositions and formulations comprising growth factor fraction.

[0041]The growth factor formulations of the present invention include pharmaceutical compositions suitable for administration. In a preferred embodiment, the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. "Pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.

[0042]The pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers such as sodium acetate; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol. Additives are well known in the art, and are used in a variety of formulations.

[0043]In a further embodiment, the pharmaceutical compositions may be added in a micellular formulation; see U.S. Pat. No. 5,833,948, hereby expressly incorporated by reference in its entirety.

[0044]In one aspect, the components of the compositions and pharmaceutical formulations of the present invention may have an effect upon administration individually, but upon administration in one or more combinations, have an effect that is synergistic. By "synergistic" is meant an enhancement of the effect of one or more combined components in a more than additive fashion relative to the effect of each component when used alone.

[0045]Combinations of pharmaceutical compositions may be administered. Moreover, the compositions may be administered in combination with other therapeutics.

[0046]The following examples serve to more fully describe the manner of using the above-described invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes.

EXAMPLE 1

Effects of a Formulation Containing Growth Factor Fraction on Sale Barn Stressed Calves

[0047]The following study was performed on Holstein calves obtained at 1 to 3 days of age. The calves were sale barn stressed; bought from an order buyer who collects calves, usually all bull calves to be castrated, from a large number of dairies. When the order buyer's truck is full, usually holding several hundred head, the animals are taken to a farm such as the one associated with this study. All of the calves used in this study were from one truck load. They arrive at the feeding facility one to three days old, highly stressed. They were placed in hutches individually, the operation having over 500 hutches. The animals were fed initially with milk replacer supplied in a nipple bucket. Since some calves do not take readily to the nipple bucket, the number of days to sucking has been noted. After several days, feed was placed in a trough for the animals. Since animals that start eating full feed there generally have less trouble with disease, time to full feed has been noted. The animals continued feeding from the milk nipple bucket for approximately 60 days. Fresh water was supplied daily in a separate bucket at all times.

[0048]After about 60 days the animals were transferred to growing pens on full feed, and weaned off milk altogether. Calves are usually vaccinated, wormed, and castrated in the first five to ten days at the calf growing facility.

[0049]Forty-eight (48) calves were divided into three groups. One group of 16 calves (Group 1) was given Formulation 1 comprising growth factor fraction. The formulation was administered by dissolving it in the milk replacer. One ounce of Formulation 1 was administered to the animals on each of Day 1, Day 2 and Day 12.

Formulation 1

TABLE-US-00001 [0050]Amounts are given per ounce of Formulation Freeze dried (lyophilized) growth factor fraction 2000 mg (Available from Sterling Technology, Inc., Brookings, SD) Avian antibodies against: 3000 mg E. Coli, Rota and corona virus, Salmonella (Avian antibodies supplied as whole egg yolk from chickens immunized with the indicated pathogens. Available from Labelle, Inc., Bellingham, WA) Poly R-Glucans derived from: Cordyceps sinensis hybrid 1000 mg Agaricus Blezeii Coriolus Poira Cocos Inontus Obliquus Maitake Shiitake (Available from Aloha Medicinals Inc., Carson City, NV) Vitamin A 12150 IU Vitamin D3 3125 IU Vitamin E 400 IU Vitamin B1 35 mg Vitamin B2 35 mg Vitamin B 12 4 mg Dipotassium Phosphate 4000 mg Potassium Chloride 566 mg Magnesium Sulfate 226 mg Calcium Pantothenate 62 g Ascorbic Acid 62 mg Lactic Acid Bacteria 2.5 Billion CFU

[0051]Group 2 consisted of 16 calves to which the following Comparative Formulation was administered by dissolving it in the milk replacer on Day 1, Day 2, and Day 12. Amounts are given per ounce of formulation. This formulation is available from 4Life.RTM. Research.

TABLE-US-00002 Transfer factor 1450 mg (Proprietary mixture from avian and mammalian sources) Proprietary Blend: 1000 mg Ip6 (Inositol hexaphosphate) Soya bean extract (Phytosterols) Olive leaf extract (Olea europea) Mannans (from Aloe vera leaf) Poly-R ™ Proprietary Blend: 1000 mg Cordyceps sinensis hybrid Agaricus Blazeii Extract Coriolus Poira Cocos Inonotus Obliquus Maitake Mushroom Shiitake Mushroom Vitamin A 12,150 IU Vitamin D3 3125 IU Vitamin E 400 IU Vitamin B1 35 mg Vitamin B2 35 mg Vitamin B12 4 mg Dipotassium Phosphate 4000 mg Potassium Chloride 566 mg Magnesium Sulfate 226 mg Calcium Pantothenate 62 mg Ascorbic Acid 62 mg Lactic Acid Bacteria 2.8 Billion CFU

[0052]Group 3 (Controls) consisted of 16 calves that were not administered either of the above formulations.

Results

[0053]For Group 1 consisting of 16 baby Holstein calves that received one ounce of Formulation 1 on days 1-2-12: [0054]two animals required treatments with 1500 IU of Penicillin each; both responded to the treatment [0055]Mortality=0 [0056]animals were purchased in August of 2005, sold in January of 2007 (after 403 days) [0057]Finish weight--animals sold at a weight of 1340 to 1450 pounds; average finish weight approximately 1420 pounds [0058]All sold at USDA Grade ChoiceBehavior All calves sucking by day 3 and on full feed by day 7.Condition: alert, active clearly good flesh gaining weight.Group 2 consisting of 16 Holstein calves bought at the same time, which received the Comparative Formulation: [0059]required treatment of 5 calves with 1500 IU of Penicillin; all responded to this treatment [0060]Mortality=0 [0061]animals purchased in August of 2005, sold March of 2007, (time to finish was 463 days) [0062]animals sold at weight of approximately 1405-1470 pounds, average finish weight of approximately 1430 pounds. [0063]All sold at USDA Grade ChoiceBehavior All calves sucking by day 7 and on full feed by day 14.Condition: good flesh gaining weight.Group 3 (controls) consisting of 16 Holstein calves purchased at the same time as Groups 1 and 2, to which neither of the above formulations was administered. [0064]All calves in this group were treated over three times each with Nuflor, A180, Banamine, and Micotil. A total of 52 treatments was required. [0065]Mortality=0 [0066]Sold in May at approximately 1290 to 1370 pounds, average finish weight of approximately 1320 pounds. [0067]Time to sale (finish) was 484 days [0068]All sold at USDA Grade Select (lower grade than Choice)Behavior Calves all sucking on day 10 to 14, still not on full feed by day 24.Not as alert, or playful as the other groups.Condition Showed poor body condition, including peaked backs and pelvis

[0069]The above example demonstrates the beneficial effects, including those relating to growth and to reduced morbidity, achieved in calves administered a formulation comprising a growth factor fraction.

EXAMPLE 2

[0070]Below is described an exemplary formulation, in which certain components are encapsulated, that may prove beneficial for mature ruminants; including, for example, calves older than about 45 to 60 days (the age by which rumination generally begins).

TABLE-US-00003 Freeze dried (lyophilized) growth factor 4000 mg fraction (encapsulated) Avian antibodies (encapsulated) 480 mg Transfer factor (encapsulated) 1120 mg Poly R (encapsulated) 2000 mg Proprietary blend (encapsulated) 300 mg Phytosterol olive leaf mannans Ip6 Vitamin C (encapsulated) 2000 mg Vitamin A 12150 IU Vitamin D3 3125 IU Vitamin E 400 IU Vitamin B1 35 mg Vitamin B2 35 mg Vitamin B 12 4 mg Dipotassium Phosphate 4000 mg Potassium Chloride 566 mg Magnesium Sulfate 226 mg Calcium Pantothenate 62 g Lactic Acid Bacteria 2.5 Billion CFU

[0071]It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present inventions without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modification and variations of the inventions provided they come within the scope of the appended claims and their equivalents.

[0072]The terms and expressions which have been employed are used as terms of descriptions and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope on this invention.

[0073]In addition, where features or aspects of the invention are described in terms of Markush group or other grouping of alternatives, those skilled in the art will recognized that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

[0074]Unless indicated to the contrary, all numerical ranges described herein include all combinations and subcombinations of ranges and specific integers encompassed therein. Such ranges are also within the scope of the described invention.

[0075]The disclosures of each patent, patent application and publication cited or described in this document are hereby incorporated herein by reference, in their entirety.



Inventor

* Ramaekers, Joseph C.

Assignee

* RAMAEKERS NUTRITION, INC.

US Classes 424/130.1, IMMUNOGLOBULIN, ANTISERUM, ANTIBODY, OR ANTIBODY FRAGMENT, EXCEPT CONJUGATE OR COMPLEX OF THE SAME WITH NONIMMUNOGLOBULIN MATERIAL514/2, Peptide containing (e.g., protein, peptones, fibrinogen, etc.) DOAI424/725, PLANT MATERIAL OR PLANT EXTRACT OF UNDETERMINED CONSTITUTION AS ACTIVE INGREDIENT (E.G., HERBAL REMEDY, HERBAL EXTRACT, POWDER, OIL, ETC.)424/601, Phosphorus or phosphorus compound424/663, Chloride424/682Aluminum, calcium or magnesium element, or compound containing

Attorney, Agent or Firm

* FOX ROTHSCHILD LLP;PRINCETON PIKE CORPORATE CENTER

International Classes A61K 39/395 A61K 38/02 A61K 36/00 A61K 33/42 A61K 33/14 A61K 33/06 A61P 3/00



Abstract text

Compositions that include extracts from sources of immune modulators that include nanofraction immune modulator molecules (i.e., molecules having molecular weights of about 3,000 Da and less) are disclosed. These compositions may also include other immune modulators, such as transfer factor. Administration of compositions with extracts that include nanofraction immune modulator molecules modulates the cell-mediated immunity (e.g., down-regulates undesired T cell activity) of a subject to which such compositions are administered. When administered with transfer factor, the combination of nanofraction immune modulator molecules and transfer factor down-regulates undesired T cell activity while increasing, or up-regulating, T cell activity against pathogens and other undesirable entities, such as cancer cells and other aberrant or mutated cells. Assays and assay techniques for evaluating the immune modulation capabilities of various substances are also disclosed. Claims

1. A composition for improving immune function, comprising: an immune modulating component, including: a first part including a first extract of a source of immune modulators having an upper molecular weight cutoff of about 10,000 Da, the food product extract including transfer factor and nanofraction immune modulating molecules having molecular weights of about 3,000 Da or less; and a second part including a second extract of a source of immune modulators having an upper molecular weight cutoff of about 3,000 Da, the extract including more of the nanofraction immune modulating molecules having molecular weights of about 3,000 Da or less.

2. The composition of claim 1, wherein the extract comprises an extract of bovine colostrum.

3. The composition of claim 1, wherein the extract further comprises an extract of chicken eggs.

4. The composition of claim 3, wherein the second extract comprises another extract of bovine colostrum.

5. The composition of claim 4, wherein the another extract of bovine colostrum comprises at least about two percent of the weight of the immune modulating component.

6. The composition of claim 5, wherein: the extract of bovine colostrum comprises about 68% of the weight of the immune modulating component; and the extract of chicken eggs comprises about 30% of the weight of the immune modulating component.

7. A composition for improving immune function, comprising: an immune modulating component consisting essentially of: a powdered extract of bovine colostrum having an upper molecular weight cutoff of about 10,000 Da; a powdered extract of bovine colostrum having an upper molecular weight cutoff of about 3,000 Da; and powdered egg yolk.

8. The composition of claim 7, wherein the powdered extract of bovine colostrum having an upper molecular weight cutoff of about 3,000 Da comprises at least about two percent of the weight of the immune modulating component.

9. The composition of claim 8, wherein: the powdered extract of bovine colostrum having an upper molecular weight cutoff of about 10,000 Da comprises about 68% of the weight of the immune modulating component; the powdered extract of bovine colostrum having an upper molecular weight cutoff of about 10,000 Da comprises about 2% of the weight of the immune modulating component; and the powdered a whole egg yolk comprises about 30% of the weight of the immune modulating component.

10. A composition for improving immune function, comprising: an immune modulating component consisting essentially of nanofraction molecules having molecular weights of at most about 3,000 Da.

11. The composition of claim 10, wherein the nanofraction molecules are obtained from at least one of bovine colostrum and chicken eggs.

12. A method for modulating an immune system of a subject, comprising: administering a composition with an extract from a source of immune modulators that consists essentially of nanofraction immune modulator molecules having molecular weights of at most about 3,000 Da to the subject.

13. The method of claim 12, wherein administering consists essentially of administering a composition that consists essentially of the extract.

14. The method of claim 12, wherein administering includes administering to the subject a composition additionally including an extract of a source of immune modulators that includes immune modulator molecules having molecular weights of up to about 10,000 Da.

15. The method of claim 14, wherein administering comprises administering a composition including at least about two percent, by weight, of the extract that consists essentially of immune modulator molecules having molecular weights of up to about 3,000 Da.

16. The method of claim 15, wherein administering comprises administering a composition including: an immune modulating component consisting of: about 2%, by weight, of a dietary supplement comprising an extract of bovine colostrum and consisting essentially of immune modulator molecules having molecular weights of up to about 3,000 Da; about 68%, by weight, of a dietary supplement comprising an extract of bovine colostrum and consisting essentially of immune modulator molecules having molecular weights of up to about 10,000 Da; and about 30%, by weight, of a dietary supplement comprising chicken egg yolk.

17. The method of claim 16, wherein administering increases activity of at least one of T memory cells, T helper cells, and natural killer cells against pathogens, cancer cells, and aberrant or mutated cells.

18. The method of claim 17, wherein administering decreases undesired cell-mediated immune activity.

19. An assay method for determining the immune modulating ability of at least one molecule, comprising: evaluating a non-stimulated activity of at least one type of immune cell; exposing the at least one type of immune cell to a stimulator molecule; evaluating a stimulated activity of the at least one type of immune cell; exposing the at least one type of immune cell to a potential immune modulator; and evaluating an effect of the potential immune modulator on the stimulated activity of the at least one type of immune cell.

20. The assay method of claim 19, wherein evaluating the activity of the at least one type of immune cell comprises evaluating the activity of at least one type of T-cell.

21. The assay method of claim 20, wherein evaluating the activity of the at least one type of T-cell comprises evaluating the activity of at least one of a CD3 cell and a CD4 cell.

22. The assay method of claim 19, wherein exposing the at least one type of immune cell to a stimulator molecule comprises exposing the at least one type of immune cell to a mitogen; and evaluating the stimulated activity comprises evaluating the increase in activity of the at least one type of immune cell following exposure to the mitogen.

23. The assay method of claim 19, further comprising: exposing the at least one type of immune cell to a stimulator molecule comprises exposing the at least one type of immune cell to an antigen; and evaluating the stimulated activity comprises evaluating the increase in activity of the at least one type of immune cell following exposure to the antigen.

24. The assay method of claim 19, wherein the at least one type of immune cell comprises at least one of a healthy immune cell, an immune cell from an immunologically compromised source, or an immune cell from a source that is recovering or has recently recovered from an infection. Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 60/848,348, filed Sep. 29, 2006, the disclosure of which is hereby incorporated herein, in its entirety, by this reference.

FIELD OF INVENTION

[0002] The present invention relates to molecules that modulate (e.g., elicit, enhance, suppress undesirable activity, etc.) cell-mediated immunity in a subject, including methods for generating and obtaining such molecules, preparations and compositions that include such molecules, methods for evaluating the effectiveness of such molecules, and methods of use. More specifically, the present invention relates to small molecules, which are referred to herein as "nanofraction" molecules that modulate cell-mediated immunity.

BACKGROUND OF RELATED ART

[0003] The ability of antibodies to provide and transfer immunity is well known and widely researched, as are the characteristics of antibodies and the mechanisms by which antibodies are produced.

[0004] Not so well known or so widely researched are the roles transfer factors, which includes a family of molecules having molecular weights of between 3,500 Da and 7,500 Da, play in modulating cellular, or T-cell-mediated, immunity. Over time, the understanding that those of skill in the pertinent art have about the characteristics of transfer factors and their roles in an organism's immune system has improved and continues to improve.

[0005] While further research continues to shed light on the characteristics and functions of a wide variety of immune system components, there may be a large number of poorly understood, or even overlooked molecules that may have an impact on the manner in which immunity is developed, maintained, conveyed, and transferred, as well as on the effects of immunity on longevity.

SUMMARY OF THE INVENTION

[0006] The effectiveness of various molecules in modulating cell-mediated immunity has recently been characterized in a quantifiable manner. Molecules that may directly or indirectly modulate cell-mediated immunity are known in the art as "immune modulators." One class of immune modulators includes small, or low molecular weight, nanofraction (e.g., up to 3,000 Da, up to 3,500 Da, 250 Da to 2,000 Da, 2,000 Da to 4,000 Da) molecules that elicit, enhance, suppress, or otherwise modulate a cell-mediated immune response. Due to the relatively small sizes, or molecular weights, of such immune modulators, they are referred to herein as "nanofraction" immune modulators and as "nanofraction" molecules.

[0007] Nanofraction immune modulators may be obtained from a variety of different types of source animals. Examples of source animals include, but are not limited to, mammals (e.g., cows) and birds (e.g., chickens). Without limiting the scope of the present invention, nanofraction immune modulators may be obtained from colostrum, or even milk, produced by a mammal. As another non-limiting example, nanofraction immune modulators may be acquired from eggs produced by birds or any other type of animal. Colostrum, eggs, and other sources of nanofraction molecules are collectively referred to herein as "nanofraction sources."

[0008] The natural production of nanofraction immune modulators by a source animal may be enhanced by exposing the source animal to a greater amount, or concentration, of one or more antigens than the amount(s) of such antigen(s) to which the source animal would normally be exposed. For example, if a particular type of source animal, or even a specific source animal, would, in its typical environment, normally be exposed to a certain amount or concentration of a given antigen, the source animal's production of immune modulators, including nanofraction molecules, may be enhanced by exposing the source animal to an even greater amount (e.g., concentration) of that antigen (e.g., by vaccinating the source animal, by placing the source animal into an environment where a greater amount or concentration of that antigen is present, etc.). As another example, if a particular type of source animal, or even a specific source animal, were typically vaccinated with a given antigen, the source animal's production of one or more nanofraction immune modulators could be enhanced by increasing the exposure of the source animal to an antigen (e.g., by exposing the source animal to an increased concentration of the antigen, a more effective or more virulent form of the antigen, etc.), although the nanofraction molecules are not themselves believed to be antigen specific.

[0009] Known processes may be used to partially, substantially, or completely purify nanofraction immune modulators from other molecules present in the nanofraction source animal from which they are obtained and, optionally, to concentrate the nanofraction immune modulators. Such processes include, without limitation, mechanical separation, phase separation (e.g., separation of aqueous and non-aqueous components from one another), precipitation, centrifugation, filtration (including microfiltration, with a molecule weight cutoff (MWCO) in the range of about 12,000 Da down to about 4,000 Da, and nanofiltration, with an MWCO of less than about 4,000 Da), dialysis, chromatographic, and electrophoretic purification processes. Such processes may be effected individually or in any combination to produce a preparation in which one or more types of immune modulators are present.

[0010] In one aspect, the present invention includes preparations of at least partially purified, substantially purified (e.g., to a degree accepted by those in the pertinent art), and completely purified immune modulators. Additionally, the present invention includes compositions that include nanofraction molecules. In addition to nanofraction molecules, such compositions may include other components that are useful in supporting or modulating the immune system of a subject (e.g., transfer factor, antibodies, etc.), as well as components that may benefit the subject in other ways.

[0011] Methods that include use or administration of nanofraction molecules or compositions including the same, alone or with other immune modulators, are also within the scope of the present invention. Methods of use include the administration of one or more types of immune modulators (e.g., in raw, partially purified, substantially purified, or completely purified form, in a preparation, in a composition, etc.) to a subject (e.g., a human or any type of animal that is believed to benefit from the immune modulation provided by nanofraction molecules). The immune modulators are administered to a subject in an amount that increases the level (e.g., concentration) of a particular, administered type of immune modulator in the body of the subject to an above-normal amount for the subject. Without limiting the scope of this aspect of the present invention, a subject may receive an amount of one or more immune modulators that is clinically effective for causing the immune system of the subject to elicit a cell-mediated immune response or an amount that effectively enhances a cell-mediated immune response by the subject.

[0012] In addition, tests and testing methods that evaluate the effectiveness of immune modulators are within the scope of the present invention. As an example, a T-cell immune function assay may be used to evaluate the ability of a potential immune modulator to modulate the activity of (e.g., production of adenosine tri-phosphate (ATP) by) one or more types of cells that participate in cell-mediated immunity, either alone or in conjunction with other molecules (e.g., antigens, mitogens (which induce mitosis, or cell replication, etc.).

[0013] Other features and advantages will become apparent to those of skill in the art through consideration of the ensuing description and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In the drawings, FIGS. 1 through 4 are graphic illustrations of the results of various tests performed on compositions that incorporate teachings of the present invention.

DETAILED DESCRIPTION

[0015] It has recently been discovered that small molecules in a variety of molecular weight ranges are useful in modulating the activity of immune cells. The following EXAMPLES set forth the acts that were performed to reach these conclusions.

EXAMPLE 1

[0016] Using known processes, including phase separation, precipitation, filtration, microfiltration or nanofiltration, and dialysis, a variety of molecular weight fractions were prepared from both bovine colostrum and chicken eggs. The molecular weight fractions that were obtained from bovine colostrum were: 250 Da to 2,000 Da, 2,000 Da to 4,000 Da, 4,000 Da to 8,000 Da (which includes transfer factor, and was included for the sake of comparison), and 8,000 Da to 12,000 Da. Similarly, 2,000 Da to 4,000 Da, 4,000 Da to 8,000 Da (which includes transfer factor, and was included for the sake of comparison), and 8,000 Da to 12,000 Da molecular weight fractions were prepared from chicken egg yolks. The molecular weight fractions were then dried to powder form (e.g., by spray drying, freeze drying, etc.).

[0017] Various assays were then conducted using these preparations to evaluate the effects of molecules in each fraction to modulate the activity of cells that convey cellular immunity (e.g., CD4 T helper cells). Specifically, assays of the type disclosed in U.S. Pat. Nos. 5,773,232 and 6,630,316 and in U.S. Patent Application Publication 2005/0260563, the entire disclosure of each of which is hereby incorporated herein, in its entirety, by this reference, were modified and used to evaluate the activities of different molecular weight fractions from EXAMPLE 1 in a variety of conditions. The aforementioned assays are used to evaluate the production of adenosine tri-phosphate (ATP) by immune cells (e.g., CD4 T helper cells, CD3 cells (which includes all T-cells), etc.). The amount of ATP produced by the cells may be measured in a manner known in the art (e.g., by use of the so-called "Luciferin reaction" with a lumenometer).

EXAMPLE 2

[0018] A first series of assays was conducted using the white blood cells of healthy individuals that include, or express, so-called "CD4" glycoproteins on their surfaces using the ImmuKnow™ assay produced by Cylex Incorporated of Columbia, Md. These white blood cells are also referred to as "CD4 " cells due to their expression of the CD4 glycoproteins. Expression of the CD4 glycoprotein distinguishes so-called "T helper" cells from other types of white blood cells, including other T cells.

[0019] The components of the ImmuKnow™ assay test kit were: a standard 96-well "Assay Plate," including removable eight well strips; a "Sample Diluent," which includes growth medium and a preservative; a "Stimulant," which includes phytohemagglutinin-L (PHA-L) (a substance from beans (e.g., red kidney beans) that is known to nonspecifically stimulate mitosis (a process in which a cell grows and splits into two new cells) and, thus, the preparatory production of adenosine tri-phosphate (ATP) in white blood cells (i.e., a "mitogen")), introduced into the remaining four "stimulated" wells of the eight well strip. diluted in growth medium and a preservative; "Dynabeads.RTM.*CD4," which are magnetic beads coated with mouse monoclonal anti-human CD4 antibodies and are carried by a buffered saline solution with bovine serum albumin (BSA) and preservative; a "Wash Buffer," which includes a buffered saline solution with BSA; a "Lysis Reagent," which includes a hypotonic basic solution with detergent, a "Calibrator Panel" with ATP concentrations of 0, 1, 10, 100, and 1,000 ng/ml; a "Luminescence Reagent" including luciferin and luciferase in a buffered solution, which reacts with ATP to create light in an amount indicative of the amount of ATP to which the Luminescence Reagent has been exposed; and a "Measurement Plate" with 96 wells that have opaque boundaries (i.e., walls and bases).

[0020] One eight well strip of the 96 well Assay Plate, which may be referred to as a "control strip," was used to provide controls, including four "nonstimulated" (NS) control wells and four "stimulated" control wells.

[0021] Another eight well strip of a 96 well Assay Plate, which may be referred to as a "test strip," was used for each sample to be tested. Four of the wells of each strip were designated as "nonstimulated" wells, while the other four wells of each strip were "stimulated" wells. Fifty microliters (50 μl) of Sample Diluent was introduced into each of the four "nonstimulated" wells of the control strip, while 25 μl of Sample Diluent was introduced into each of the "nonstimulated" wells of each test strip. Twenty-five microliters (25 μl) of Stimulant was introduced into each of the four "stimulated" wells of the control strip and into each of the four "stimulated" wells of each test strip.

[0022] In addition to the Sample Diluent or Stimulant, a 25 μl sample of one of the molecular weight fractions identified in EXAMPLE 1 was introduced into each of the eight wells of each test strip. More specifically, each of the molecular weight fractions of EXAMPLE 1 was reconstituted in Sample Diluent and diluted with a volume of Sample Diluent to provide three different concentrations that would ultimately, upon addition of a 25 μl sample to a well, respectively amount to the addition of 10 μg, 100 μg, and 1,000 μg of the dried powder to the well.

[0023] A 1:3 (blood:"Sample Diluent") dilution of a blood sample, which had been gently agitated to uniformly distribute its constituents, including white blood cells, was prepared. Seventy-five microliters (75 μl) of diluted blood was added to each well of each strip. The contents of the wells were then mixed (e.g., by placing the plate on a shaker plate for about 30 seconds), then incubated at a temperature of 37° C. in a 5% CO2 environment for about 18 hours.

[0024] Once incubation was complete, the contents of the wells were again mixed (e.g., by placing the plate on a shaker plate for about three minutes). Thereafter, the Dynabeads.RTM. solution was mixed to homogenously suspend the Dynabeads.RTM. within the liquid by which they were carried (e.g., with a vortex). As noted above, the Dynabeads.RTM. in this example include magnetic beads coated with mouse monoclonal anti-human CD4 antibodies. Fifty microliters (50 μl) of the Dynabead.RTM.-carrying solution was added to each well of each strip.

[0025] The contents of the wells of each strip were again mixed (e.g., on a shaker plate for about 15 seconds), then allowed to set, or incubate, at room temperature for a duration of about 15 minutes. The process of mixing and incubation was then repeated. The mouse anti-human CD4 antibodies on the Dynabeads.RTM. bind only to white blood cells exhibiting the CD4 glycoprotein. During this incubation, CD4 white blood cells, which include T helper cells, were immobilized by, or bound to, the mouse monoclonal anti-human CD4 antibodies on the Dynabeads.RTM..

[0026] Following incubation, the contents of each well were mixed again (e.g. for about 15 seconds to about 30 seconds on a shaker plate) to resuspend the Dynabeads.RTM.. The contents of each well were then introduced into a magnetic field (e.g., by placing each eight well strip in a magnet tray available from Cylex), in accordance with the protocol set forth in the instructions that accompany the ImmuKnow™ assay. When subjected to the magnetic field, the Dynabeads.RTM. are pulled to one side of each well in which they are present. The remaining contents of the well may then be removed (e.g., aspirated with a pipette, etc.) and the beads and T helper cells washed one or more times (e.g., three times, each with 200 μl Wash Buffer) to substantially purify the same.

[0027] Two hundred microliters (200 μl) of Lysis Reagent was then added to each well. Following removal of the contents of each well from the magnetic field, the contents of each well (i.e., the Dynabeads.RTM., cells attached thereto, and the Lysis Reagent) were mixed (e.g., for about five minutes on a plate shaker). The Lysis Reagent disrupted the membranes of the CD4 cells that were immobilized by antibody on the Dynabeads.RTM. Among other things, ATP was released from the lysed cells.

[0028] Once cell lysis was complete, the contents of each well were again subjected to a magnetic field, pulling the Dynabeads.RTM. within each well to one side of the well. A 50 μl sample was then transferred from each well to a corresponding well of the Measurement Plate. In addition to transferring samples, several wells of the 96 well Measurement Plate were reserved for 50 μl samples of the various ATP concentrations of the Calibrator Panel solutions.

[0029] One hundred fifty microliters (150 μl) of the Luminescence Reagent was then added to each well of the Measurement Plate that included either a test sample or a sample of Calibrator Panel solution. The luminescence from each well was then measured. The measured luminescence from each well provides an indication of the amount of ATP present in that well. The amount of ATP present in each well is, in turn, indicative of an amount of metabolic activity within the cells (i.e., CD4 cells) from which the contents of each well of the Measurement Plate. In the samples that were derived from cells that were nonspecifically stimulated with PHA, a relatively high level of ATP is expected to be present. The addition of an immune modulator (e.g., from one of the fractions identified in EXAMPLE 1) would increase or decrease, or modulate, the metabolic activity in CD4 cells that was nonspecifically stimulated by PHA.

[0030] The results of such testing are set forth in the following table, with the illustrated numbers, representing the mean (average) amount of ATP produced by the white blood cells of each subset: TABLE-US-00001 TABLE 1 Con- 10 μg 100 μg 1000 μg Sample trol per well per well per well 250 Da to Non-Stimulated 14 52 50 37 2,000 Da (NS) Colostrum fraction Stimulated with 388 336 253 127 PHA % reduction in 13.4 34.8 67.3 PHA stimulation 2,000 Da to NS 14 52 62 42 4,000 Da Colostrum fraction Stimulated with 388 388 377 339 PHA % reduction in 0 2.8 12.6 PHA stimulation 4,000 Da to NS 14 69 50 46 8,000 Da (includes TF) Colostrum fraction Stimulated with 388 378 250 207 PHA % reduction in 2.6 35.6 46.6 PHA stimulation 8,000 Da to NS 14 49 45 39 12,000 Da Colostrum fraction Stimulated with 388 337 237 181 PHA % reduction in 13.1 38.9 53.4 PHA stimulation 2,000 Da to NS 14 49 33 44 4,000 Da Egg fraction Stimulated with 388 228 161 148 PHA % reduction in 41.2 58.5 61.9 PHA stimulation 4,000 Da to NS 14 54 47 44 8,000 Da (includes TF) Egg fraction Stimulated with 388 354 230 158 PHA % reduction in 8.8 40.7 59.3 PHA stimulation

[0031] These data show that in the non-simulated tests, where cells were not exposed to PHA, each of the 4,000 Da to 8,000 Da molecular weight fractions (from colostrum and egg), both of which are known to contain transfer factor, stimulated additional metabolic activity in the CD4 white blood cells. These data confirm the ability of transfer factor to up-regulate cell-mediated immunity.

[0032] Conversely, the 4,000 Da to 8,000 Da colostrum and egg fractions down-regulated the nonspecific ability of PHA to stimulate metabolic activity in CD4 cells. Since PEA is an artificial, nonspecific stimulant, the down-regulation of its activity by transfer factor, which participates in cell-mediated immunity, is not surprising. It is believed, and previous research has shown, that transfer factor helps balance, and even focus, immune activity by T-cells (e.g., by helping the cells "remember" their primary purpose, by reducing autoimmunity and associated disorders, while improving activity against undesirable entities, such as infection of a subject's body by microorganisms (bacteria, viruses, etc.), etc.). The down-regulation of PHA-stimulated activity by T-cells appears to confirm this role of transfer factor in cell-mediated immunity.

[0033] Similar results were seen in a number of other molecular weight fractions that do not include transfer factor, including the 250 Da to 2,000 Da colostrum fraction (both up-regulation and down-regulation), the 2,000 Da to 4,000 Da colostrum faction (up-regulation), and the 2,000 Da to 4,000 Da egg fraction (up-regulation and down-regulation). The 8,000 Da to 10,000 Da colostrum fraction also caused up-regulation of activity in CD4 white blood cells that were not stimulated with PHA and down-regulation of PHA stimulation of metabolic activity in CD4 white blood cells.

[0034] These data establish that immune modulators other than transfer factor are present in at least the 250 Da to 2,000 Da colostrum fraction, the 2,000 Da to 4,000 Da colostrum fraction, and the 2,000 Da to 4,000 Da egg fraction. The immune modulation capabilities of the "nanofraction molecules" present in each of these fractions has been at least partially confirmed by the experiment that is set forth in EXAMPLE 3.

EXAMPLE 3

[0035] A second series of assays of activity induced in a healthy individual's white blood cells exhibiting the CD3 glycoprotein (i.e., CD3 cells), which are known to include all T-cells, including so-called "T memory" cells. Specifically, Cylex's T-Cell Memory™ assay was used. The protocol of Cylex's T-Cell Memory™ assay is very similar to that set forth in EXAMPLE 2, with the following exceptions: 25 μl of the Stimulant, which included Concanavalin A (ConA) instead of PHA, was only introduced into the "stimulated" wells of the control strip, while 25 μl of a 1:10 dilution of cytomegalovirus (CMV) vaccine was added to the "stimulated" wells of each test strip (for a final, per well dilution of 1:50); mouse anti-human CD3 antibodies were immobilized to the surfaces of magnetic beads (per instructions accompanying the T-Cell Memory™ test kit) to prepare the Dynabeads.RTM.; and the Dynabeads.RTM.; were added to the blood samples, Sample Diluent, Stimulant (if any), and sample fraction (if any) before the initial incubation.

[0036] In the T-Cell Memory™ assay, antigen is used in place of a mitogen (e.g., PHA) so that the ability of the T memory cells to recognize a particular antigen may be evaluated. Notably, the intensity of the light emitted from each well of the Measurement Plate is less, as T memory cells make up only a portion of the cells that have been bound to antibody molecules of the Dynabeads.RTM..

[0037] The results of these assays are set forth in the following table, with the illustrated numbers representing the mean (average) amount of ATP produced by the white blood cells for each sample (and amount) tested: TABLE-US-00002 TABLE 2 Con- Sample trol 10 μg 100 μg 1000 μg 250 Da to Non-Stimulated 12 14 12 17 2,000 Da (NS) Colostrum fraction Stimulated with 316 468 338 345 CMV % increase in 48.1 7.0 9.2 CMV stimulation 2,000 Da to NS 12 15 12 15 4,000 Da Colostrum fraction Stimulated with 316 501 503 440 CMV % increase in 58.5 59.1 39.2 CMV stimulation 4,000 Da to NS 12 22 16 19 8,000 Da (includes TF) Colostrum fraction Stimulated with 316 473 476 475 CMV % increase in 49.7 50.6 50.3 CMV stimulation 8,000 Da to NS 12 14 18 26 12,000 Da Colostrum fraction Stimulated with 316 453 404 370 CMV % increase in 43.4 27.8 17.1 CMV stimulation 2,000 Da to NS 12 26 61 108 4,000 Da Egg fraction Stimulated with 316 305 349 350 CMV % increase in -3.5 10.4 10.8 CMV stimulation 4,000 Da to NS 12 34 39 108 8,000 Da (includes TF) Egg fraction Stimulated with 316 310 280 381 CMV % increase in -1.9 -11.4 20.6 CMV stimulation

[0038] The data obtained from the testing conducted in this EXAMPLE 3 demonstrate that, in the presence of an antigen (i.e., a specific stimulant, as opposed to the nonspecificity of a mitogen, such as Conk or PHA), the three non-transfer factor-containing colostral fractions enhance the activity of the tested T memory cells to CMV to a degree that is comparable to (the 10 μg samples of the 250 Da to 2,000 Da and 8,000 Da to 12,000 Da colostrum fractions) or exceeds (the 10 μg and 100 μg samples of the 2,000 Da to 4,000 Da colostrum fraction) the ability of the comparably sized samples of the 4,000 Da to 8,000 Da, transfer factor-containing colostrum fraction to enhance the activity of the tested cells when exposed to CMV.

EXAMPLE 4

[0039] Another set of assays was conducted to determine whether or not either the nanofraction immune modulator molecules (i.e., the immune modulators of the 2,000 Da to 4,000 Da colostrum fraction) or the transfer-factor containing fraction (i.e., the 4,000 Da to 8,000 Da colostrum fraction) could modulate (e.g., enhance) the immune memory of a subject who had recently been exposed to a high dose of a particular antigen. Specifically, a blood sample was obtained from an individual who had been exposed to an influenza virus, which causes a systemic infection, and suffered from influenza symptoms for four weeks.

[0040] The assay was conducted in the manner described in EXAMPLE 3, using the Cylex T-Cell Memory™ assay in accordance with the instructions provided with that assay and set forth in EXAMPLE 3, except a influenza antigen, in the form of 1:25 dilution of a the influenza vaccine manufactured by Aventis Pasteur of Paris, France, for the 2006-2007 flu season (for a final, per well dilution of 1:125), was used in place of the CMV vaccine of EXAMPLE 3.

[0041] The results from that assay are set forth in the following table: TABLE-US-00003 TABLE 3 Sample Control 10 μg 100 μg 1000 μg 2,000 Da to NS 4 10 3 4 4,000 Da Colostrum fraction Stimulated with 827 1003 906 936 influenza antigen ConA 694 % increase in 21.3 9.6 13.2 influenza antigen stimulation 4,000 Da to NS 4 36 24 11 8,000 Da (includes TF) Colostrum fraction Stimulated with 827 989 997 830 influenza antigen ConA 694 % increase in 19.6 20.6 0.4 influenza antigen stimulation

[0042] The results shown in TABLE 3 (which are also shown graphically in FIG. 1) indicate that, when T memory cells of a subject who has recently been exposed to a particular antigen are exposed to that antigen, particularly in the presence of nanofraction molecules or transfer factor, activity of the CD3 memory T-cells increases significantly. In fact, relatively small amounts of nanofraction molecules and of transfer factor caused an increase of about 20% in T memory cell activity. In fact, it appears that the immune modulators of the 2,000 Da to 4,000 Da fraction are about as effective as the transfer factor and any other molecules present in the 4,000 Da to 8,000 Da fraction in modulating the activity of the tested cells.

[0043] The results from EXAMPLES 1-4 indicate that immune modulators having molecular weights in the 250 Da to 2,000 Da, 2,000 Da to 4,000 Da, and 8,000 Da to 12,000 Da ranges are effective in modulating immune activity of various types of T-cells. Thus, by administering such immune modulators or preparations or compositions including the immune modulators to a subject, the subject's cell-mediated immunity may be modulated.

[0044] Based on these results, a process was developed for producing various dietary supplements (e.g., from (bovine) colostrum, (chicken) egg, etc.) that include molecules of a predetermined MWCO. For example, and not by way of limitation, a liquid preparation of a source of nanofraction immune modulators, from which at least macroscopic particles (e.g., colostrum/milk solids, egg shells and membranes, etc.) (e.g., by phase separation, filtration processes, etc.) have been removed may be forced through a filter with pores that are sized to provide the predetermined upper MWCO. As a nonlimiting example, a filter that provides a molecular weight cutoff of about 3,000 Da may be used. Alternatively, dialysis processes, which include use of dialysis membranes having pores that provide the desired MWCO, may be used. The use of such processes provides a "nanofraction" from which larger molecules, including transfer factor, antibodies, and a variety of other molecules having molecular weights of greater than about 3,000 Da, are excluded. (e.g., colostrum, chicken and various powdered compositions were produced. The filtrate (i.e., the portion of the liquid that has passed through the filter) may then be further processed by known techniques (e.g., freeze drying, spray drying, evaporation to form a more concentrated liquid, incorporation into a gel, etc.). The resulting "nanofraction product" may then be used alone or incorporated into other compositions.

[0045] It is believed that by including nanofraction molecules, even in very small amounts, in preparations that also include transfer factor (and which may also include baseline levels (i.e., those levels already present in the source (e.g., colostrum, egg, etc.) from which transfer factor is obtained), the resulting compositions will down-regulate undesired activity by T-cells (e.g., autoimmunity and associated disorders, etc.), while improving, or up-regulating, desired T-cell activity. The nanofraction-and-transfer factor compositions that are set forth in TABLES 4 and 5 were developed. TABLE-US-00004 TABLE 4 TRANSFER FACTOR TRI-FACTOR Relative Amount Ingredient (by weight) Bovine Colostrum fraction, upper MWCO 10,000 Da 68% (spray dried) Bovine Colostrum fraction, upper MWCO 3,000 Da 2% (nanofraction) (spray dried) Chicken Egg Yolk (spray dried) 30%

[0046] The composition of TABLE 4 may also be referred to as an "immune modulating component." Such an "immune modulating component" may consist essentially of a combination of sources of immune modulators (including sources of nanofraction immune modulators) or extracts of immune modulator sources, such as those listed in TABLE 4, or it may include other ingredients.

[0047] Likewise, a composition that incorporates teachings of the present invention may consist essentially of an "immune modulating component," such as that disclosed in TABLE 4, or it may include other ingredients, as set forth in TABLE 5. TABLE-US-00005 TABLE 5 TRANSFER FACTOR PLUS .RTM. TRI-FACTOR Amount (per serving, serving size = one Ingredient capsule) Transfer Factor Tri-Factor 150 mg Zinc (as monomethionine) 5 mg Cordyvant ™ Proprietary Polysaccharide Complex 440 mg IP-6 (Inositol hexaphosphate) Soya bean Extract (phytosterols) Cordyceps sinensis (7% cordyceptic acids) Beta-Glucan (from baker's yeast) (Saacharomyces cerevisiae) Beta-Glucan (from Oat) (Avena sativa) Agaricus blazeii Extract Mannans (from Aloe vera) (leaf) Olive Leaf Extract (Olea europaea) Maitake Mushroom (Grifola frondosa) (whole plant) Shiitake Mushroom (Lentinus edodes) (whole plant) (5:1 extract)

[0048] A composition according to the present invention may be embodied as a liquid (e.g., into the Riovida.RTM. drink available from 4Life Research, LLC, of Sandy, Utah), a powder (which may include additional ingredients to provide a desirable flavor, dissolution properties, and the like), a tablet (which additionally includes other ingredients, such as binders (e.g., starch) and the like, a gel (in which gelatin or other ingredients may be added), or in any other suitable form. It should be understood that, for purposes of this disclosure, the additional ingredients that are used to manufacture a such embodiments of a composition of the present invention may, for purposes, of this disclosure, merely be considered to be optional and nonessential to the composition, unless otherwise required by an appended claim.

EXAMPLE 5

[0049] Blood was collected from an individual who had been suffering from shingles (varicella zoster virus (VZV) infection) symptoms for about four weeks. The blood was then assayed using the ImmuKnow™ assay in the manner described in EXAMPLE 2, with the sample fractions of EXAMPLE 2 having been replaced with the following: (a) a control that included no immune modulators; (b) a colostrum fraction having a MWCO of about 3,000 Da that had been spray dried; (c) 4Life Transfer Factor.RTM. XF, which is currently available from 4Life Research and includes a bovine colostrum extract with an upper MWCO of about 10,000 Da, was added; (d) the composition in TABLE 4, which is labeled as "TF Tri-Factor"; and (e) the composition of TABLE 5, which is labeled as "TFPlus Tri-Factor." Each of (b) through (e) was reconstituted in the Sample Diluent that accompanied the ImmuKnow™ assay, and diluted to a concentration that resulted in a final, per-well concentration of 1 mg/ml once blood samples and all other liquids had been added to each well.

[0050] The results from those assays are set forth in the following table: TABLE-US-00006 TABLE 6 TF Nano- Tri- TFPlus Control fraction TF XF Factor Tri-Factor Non-Stimulated (NS) 26 35 77 47 19 Stimulated with PHA 220 234 150 140 27

[0051] These results also appear in the graph of FIG. 2.

[0052] It is reiterated that these results were obtained at a point in time (about four weeks following initial infection; i.e., during convalescence) where, in the absence of stimulation, T helper (CD4 ) cell activity is expected to decrease, although a large number of T helper cells remain in the subject's blood. T helper cell activity was stimulated only slightly, in the absence of the nonspecific stimulant PHA, by the nanofraction, TF XF and TF Tri-Factor compositions, and does not appear to have been stimulated by TFPlus Tri-Factor. The nonspecific stimulation of T helper cells by PHA was, however, reduced significantly by the TF XF and TF Tri-Factor compositions, and to an even larger extent by the TFPlus Tri-Factor composition, as might be expected from the results of EXAMPLE 2, as set forth in TABLE 1.

EXAMPLE 6

[0053] At an earlier point in time (about one week following the onset of shingles symptoms), it would be expected that T memory cells, although not present in the subject's blood in large concentrations due to the localized nature of the VZV infections that cause shingles (i.e., relatively low blood titers of VZV), would have already recognize the VZV infection and be readily stimulated by the presence of VZV antigen. Accordingly, a T-Cell Memory™ assay was conducted to determine the effects of the nanofraction, TF XF, TF Tri-Factor, and TFPlus Tri-Factor on T memory cells from blood from the same patient as that tested in EXAMPLE 5. The protocol set forth in EXAMPLE 3 was followed, with the following exceptions: VZV vaccine, which had been diluted 1:10, was used in place of CMV vaccine (for a final, per well dilution of 1:50); and the sample fractions of EXAMPLE 3 were replaced with the control and immune modulators used in EXAMPLE 5, with each immune modulator having been diluted to a final, per-well concentration of 100 μg/ml.

[0054] The following table sets forth the results of the assay: TABLE-US-00007 TABLE 7 TF Nano- Tri- TFPlus Control fraction TF XF Factor Tri-Factor Non-Stimulated (NS) 1 2 13 27 51 Stimulated with VZV 1 5 30 32 69 Stimulated with ConA 288

[0055] This data is also depicted graphically in FIG. 3.

[0056] As expected, transfer factor, which is present in TF XF, stimulated activity by the T memory cells. The addition of a small amount of extra nanofraction molecules to the transfer factor significantly increased the activity of T memory cells, both with and without additional VZV stimulation. Thus, the results of EXAMPLES 5 and 6 confirm that the addition of extra nanofraction molecules to preparations that also include transfer factor, even in very small amounts, will down-regulate undesired activity by T-cells (e.g., autoimmunity and associated disorders, etc.), while improving, or up-regulating, desired T-cell activity.

EXAMPLE 7

[0057] In another study, which was conducted to determine the abilities of various compositions, including the compositions including transfer factor and extra nanofraction molecules set forth in TABLES 4 and 5, to stimulate natural killer (NK) cell activity against the human erythroblast leukemia cell line K-562, which is sensitive to NK cells, were evaluated. Accordingly, the NK cells are also referred to herein as "effector cells," while the K-562 cells are also referred to herein as "tumor cells" and as "target cells." Specifically, MTT Assay technology was used, in which 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which is yellow, is reduced to formazan, which is purple, by reductase enzymes in the active mitochondria of living cells. Just prior to analysis of cytotoxicity, a solution (e.g., dimethyl sulfoxide, sodium dodecyl sulfate (SDS) in dilute hydrochloric acid (HCl), etc.) that will dissolve formazan is added to each well. Spectrophotometry, in which the amount of light of a certain wavelength (e.g., a wavelength in the range of about 500 nm to about 600 nm) absorbed by the solution in each well is measured, is then used to determine the numbers of living cells in the assayed wells, relative to the number of living cells in one or more control wells into which no test compositions were added.

[0058] The compositions that were evaluated included Transfer Factor Advanced™, available from 4Life Research, LLC; Transfer Factor Plus Advanced™, also available from 4Life; Transfer Factor Tri-Factor, which includes both transfer factor and an elevated amount of nanofraction molecules; Transfer Factor Plus Tri-Factor, which includes transfer factor, an elevated amount of nanofraction molecules, and other ingredients that are believed to enhance immune system activity; nanofraction molecules from bovine colostrum; nanofraction molecules from chicken eggs; and interleukin-2 (IL-2), available under the trade name Proleukin from Chiron of the Netherlands, which is known to enlist NK cells against cancer cells.

[0059] Blood was obtained from five healthy donors. Known processes were used to isolate white blood cells from other constituents of the blood. Known density gradient centrifugation techniques (e.g., using a Histopaque.RTM. density gradient available from Sigma-Aldrich Corporation of St. Louis, Mo.) were used to isolate mononuclear cells, including NK cells, from other types of white blood cells. The mononuclear cells were introduced into RPMI 1640 growth medium with 10% fetal calf serum (FCS). Equal volumes of this mixture, including a concentration of about 60,000 white blood cells in 100 μl of culture medium, were introduced into different wells of a standard 96-well plate. Reconstituted samples of the transfer factor and/or nanofraction molecule-containing compositions identified above, each having a concentration of 0.100 mg powder to 1 ml sterile, deionized water, were each added to three different wells containing the white blood cells and growth medium, for a total of eighteen wells. In addition, 1,000 IU/ml IL-2 were introduced into three positive control wells containing the mononuclear cell-growth medium mixture. Three negative control wells included only the mononuclear cell-growth medium mixture, with no immune modulators. Three effector cell-only negative control wells also included only the mononuclear cell-growth medium mixture, while three target cell-only negative control wells included only 100 μl of growth medium.

[0060] The mononuclear cells were incubated with their respective immune modulation compositions (except for the three negative control wells) in the presence of 5% CO2 at a temperature of 37° C. and 100% humidity for 48 hours.

[0061] Following incubation, about 30,000 K-562 cells were introduced into each well, except for the three effector cell-only negative control wells, that contained mononuclear cells and growth medium. The 96-well plate and the mixtures in its wells were again incubated in the presence of 5% CO2 at a temperature of 37° C. and 100% humidity for 48 hours.

[0062] The MTT solution, having a concentration of 5 mg MTT/ml Henk's saline solution, was prepared in accordance with known, standard techniques. Twenty microliters (20 μl) of the MTT solution was introduced into each mononuclear cell-growth medium-tumor cell-containing well of the 96-well plate. The plate and the contents of its wells were again incubated in the presence of 5% CO2 at a temperature of 37° C. and 100% humidity, this time for about four hours.

[0063] Following this final incubation, the 96-well plate was centrifuged at about 1,500 rpm for about five minutes. Thereafter, the supernatant (liquid) was removed from each well, and 150 μl of dimethylsulfoxide (DMSO) was introduced into to each mononuclear cell and tumor cell-containing well. A spectrophotometer was then used to measure the optical density, at a wavelength of 540 nm, of each cell-containing well. The measured optical density was then used to determine the cytotoxic index (%) (CI (%)) of the NK cells, as activated by each tested substance, using the following formula: CI (%)=[1-(ODe t-ODe)/ODt]×100,

[0064] where ODe t is optical density of each test well corresponding to a tested composition, including the IL-2 of the positive control, ODe is the average optical density of the three effector cell-only negative control wells, and ODt is the average optical density of the three target cell-only negative control wells. The CI(%) represents the percentage of target cells that have been killed by NK cells in each well that also contained a tested immune modulation composition. The results are presented in the following table: TABLE-US-00008 TABLE 8 Immune Modulation Composition CI (%) Relative Activity Transfer Factor Advanced 43.1 55 Transfer Factor Plus Advanced 38.5 49 Transfer Factor Tri-Factor 60.3 77 Transfer Factor Plus .RTM. Tri-Factor 57.9 74 Nanofraction molecules, colostrum 77.9 100 Nanofraction molecules, egg 68.7 88 IL-2 77.0 84

[0065] These data, which are also depicted in the chart of FIG. 4, show that compositions including nanofraction molecules, particularly those from bovine colostrum, are about as effective as or more effective than IL-2 at eliciting NK cell activity against K-562 tumor cells, while compositions that include transfer factor from colostrum and eggs and nanofraction molecules from colostrum (i.e., the Transfer Factor Tri-Factor and the Transfer Factor Plus.RTM. Tri-Factor) activate NK cells more effectively than compositions that lack nanofraction molecules.

[0066] By adding as little as 2% more nanofraction molecules, by weight, to a composition that includes transfer factor, the nano fraction molecules may boost action by nonspecific components (e.g., NK cells) of the cell-mediated portion of a subject's immune system, complementing the ability of transfer factor to elicit activity by antigen-specific components of the cell-mediated portion of the subject's immune system.

[0067] When considered together, the results of EXAMPLES 5 through 7 demonstrate that transfer factors regulate and prime T helper cells, which enable a subject's immune system to respond more quickly and efficiently to pathogens and other undesired entities. In addition, EXAMPLES 5 through 7 illustrate that transfer factor may enhance the activity of T memory cells.

[0068] EXAMPLES 5 through 7 also show that the addition of extra nanofraction immune modulator molecules to compositions that include transfer factor may fortify and enhance the immune modulation (e.g., of T helper cells, T memory cells, and NK cells) of transfer factor and of existing compositions that include transfer factor.

[0069] A method of modulating the cell-mediated immunity of a subject includes administering (e.g., enterally, parenterally, etc.) a composition including nanofraction molecules to the subject. The nanofraction molecules may be administered alone, or as part of a composition that consists essentially of nanofraction molecules, or they may be administered with a composition (e.g., a composition such as that set forth in TABLE 4 or TABLE 5) that includes transfer factor. Administration may occur on a regular basis in an effort to maintain an overall balance in the subject's cell-mediated immunity, or it may be effected in response to an infection, an autoimmune disorder, tissue transplant, or another event that affects (activates or suppresses) the cell-mediated immunity of the subject.

[0070] Administration of compositions that include nanofraction immune modulator molecules in accordance with teachings of the present invention is believed to modulate cell-mediated immune activity based on physiological need. For example, undesired cell-mediated immune activity (e.g., autoimmunity, etc.) may be reduced. As another example, the ability of T cells to remove undesirable pathogens, as well as other undesirable entities, such as cancer cells and other aberrant or mutated cells, from the body of a subject (e.g., by activating T helper (CD4 ) cells, which in turn activate natural killer (NK) cells, by increasing antigen-specific immunity by enabling T memory cells, etc.) may also be focused and enhanced, particularly when transfer factor is administered with an additional amount of nanofraction immune modulator molecules.

[0071] Although the foregoing description contains many specifics, these should not be construed as limiting the scope of the present invention, but merely as providing illustrations of some of the presently preferred embodiments. Similarly, other embodiments of the invention may be devised which do not depart from the spirit or scope of the present invention. Features from different embodiments may be employed in combination. The scope of the invention is, therefore, indicated and limited only by the appended claims and their legal equivalents, rather than by the foregoing description. All additions, deletions and modifications to the invention as disclosed herein which fall within the meaning and scope of the claims are to be embraced thereby.



Inventors

* Lisonbee, David A. * McCausland, Calvin W. * Bennett, Richard H. * Vaughan, Brent M. * Lefler, Shane M.

US Classes 424/535, Milk or colostrum (e.g., butter, whey, etc.)424/581, Egg enclosed in shell or a part thereof (e.g., eggshell, egg yolk, etc.)435/29Involving viable micro-organism

Attorney, Agent or Firm

* TRASK BRITT

International Classes A61K 35/20 A61K 35/54 A61P 37/04 C12Q 1/02



Abstract text

A nutriceutical food product includes a solid matrix and a liquid combined into a gel. The nutriceutical food product may include an immune modulator, such as transfer factor and/or a nanofraction immune modulator. A fruit component may be included in the nutriceutical food product. The fruit component may include at least one oligoproanthocyanidin-containing fruit, such as acai. Claims

1. A nutriceutical food product, comprising:a solid gel matrix; anda liquid component dispersed throughout the solid gel matrix, the liquid component including an immune modulator.

2. The nutriceutical food product of claim 1, wherein the solid gel matrix comprises xanthan and guar gum.

3. The nutriceutical food product of claim 2, wherein the solid gel matrix further comprises pectin.

4. The nutriceutical food product of claim 3, wherein the pectin comprises pectin from at least one oligoproanthocyanidin-containing fruit.

5. The nutriceutical food product of claim 4, wherein the liquid component includes an extract of the at least one oligoproanthocyanidin-containing fruit.

6. The nutriceutical food product of claim 5, wherein the at least one oligoproanthocyanidin-containing fruit comprises at least one of acai, elderberry, grape, and pomegranate.

7. The nutriceutical food product of claim 6, further comprising:a fruit component comprising at least an extract of one or more of acai, elderberry, grape, and pomegranate.

8. The nutriceutical food product of claim 1, wherein the immune modulator comprises transfer factor.

9. The nutriceutical food product of claim 8, wherein the immune modulator further comprises a nanofraction immune modulator.

10. The nutriceutical food product of claim 1, wherein the immune modulator comprises a nanofraction immune modulator.

11. A nutriceutical food product, comprising:a solid gel matrix; anda liquid component dispersed throughout the solid gel matrix,at least one of the solid gel matrix and the liquid component comprising acai or an extract of acai.

12. The nutriceutical food product of claim 11, wherein the solid gel matrix comprises xanthan and guar gum.

13. The nutriceutical food product of claim 12, wherein the solid gel matrix further comprises pectin.

14. The nutriceutical food product of claim 13, wherein the pectin comprises pectin from the acai.

15. The nutriceutical food product of claim 11, wherein the liquid component includes an immune modulator and a surfactant.

16. The nutriceutical food product of claim 11, wherein the immune modulator comprises transfer factor.

17. The nutriceutical food product of claim 16, wherein the immune modulator further comprises a nanofraction immune modulator.

18. The nutriceutical food product of claim 11, wherein the immune modulator comprises a nanofraction immune modulator.

19. A nutriceutical food product, comprising:a solid gel matrix; anda liquid component dispersed throughout the solid gel matrix, the liquid component comprising an immune modulator;at least one of the solid gel matrix and the liquid component comprising acai or an extract of acai.

20. The nutriceutical food product of claim 19, wherein the immune modulator includes at least one of transfer factor and a nanofraction immune modulator. Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is a continuation-in-part of U.S. patent application Ser. No. 11/415,837, filed May 2, 2006, pending, which claims the benefit of priority under 35 U.S.C. .sctn. 119(e) to the filing date of U.S. Provisional Patent Application Ser. No. 60/677,226, filed May 2, 2005, for "Transfer Factor Preparations and Associated Methods." This application is also a continuation-in-part of U.S. patent application Ser. No. 11/855,944, filed Sep. 14, 2007, pending, which claims the benefit of priority under 35 U.S.C. .sctn. 119(e) to the filing date of U.S. Provisional Patent Application Ser. No. 60/848,348, filed Sep. 29, 2006, for "Immune Modulators, Preparations and Compositions Including Immune Modulators, Tests for Evaluating the Activity of Immune Modulators and Preparations and Compositions Including the Same, and Methods."

FIELD OF THE INVENTION

[0002]The present invention relates generally to natural supplements, or nutriceutical food products and, more specifically, to gel-based nutriceutical food products. The nutriceutical food products to which the present invention relates may include an immune modulator, such as transfer factor or a nanofraction immune modulator, which may be distributed throughout a gel-based matrix. The present invention also relates to acai-based nutriceutical products.

SUMMARY OF THE INVENTION

[0003]The present invention includes various embodiments of a nutriceutical food product in the form of a gel, or a semirigid colloidal dispersion of a solid matrix with a liquid. Some embodiments of a nutriceutical food product of the present invention include an immune modulator, which may be distributed throughout the solid matrix. The immune modulator may comprise transfer factor and/or a nanofraction immune modulator of the type described in U.S. patent application Ser. No. 11/855,944, filed Sep. 14, 2007, the entire disclosure of which is, by this reference, incorporated herein. When distributed throughout the gel, the immune modulator may retain substantially all of one or more of its activities (e.g., components of the liquid component may not interfere with one or more activities of the immune modulator), or one or more of the activities of the immune modulator may actually be enhanced by one or more components of the gel component of the nutriceutical food product.

[0004]Various embodiments of nutriceutical food products of the present invention include a fruit component. The fruit component may include at least one oligoproanthocyanidin ("OPC")-containing fruit or an extract thereof The term "extract" is broadly defined herein, including any OPC-including part of a fruit. Examples of extracts include, without limitation, juices (dilute, normal concentration, or concentrate), dehydrated fruit, and powders including one or more components of the fruit. In some embodiments of such a nutriceutical food product, at least some of the fruit component is present in liquid form. In some embodiments, at least some of the fruit component may be included in the nutriceutical food product in solid form, where it may he incorporated into the gel matrix or merely reside within voids of the gel matrix, where it may be distributed throughout the gel matrix (e.g., as a fruit pectin).

[0005]Another aspect of the present invention includes a process for making an edible preparation that includes an immune modulator, such as transfer factor and/or a nanofraction immune modulator. The process includes mixing a fruit component with the immune modulator. Preservatives may also be included in the mixture. The mixture may be chilled to prevent microbial growth. To further prevent microbial growth, the mixture may be pasteurized before chilling. Alternatively, the mixture may be sterilized.

[0006]Other features and advantages of the present invention will become apparent to those of ordinary skill in the art through consideration of the ensuing description and the appended claims.

DETAILED DESCRIPTION

[0007]In an exemplary embodiment of a gel-based nutriceutical food product, the gel comprises a solid polymeric matrix throughout which a liquid is dispersed.

[0008]The solid polymeric matrix may comprise a galactomannan, a polysaccharide (e.g., xanthan, a fruit pectin, etc.), gelatin, or any other suitable gelling agent. In some embodiments, a plurality of gelling agents are used to provide desired properties, such as viscosity, consistency, and edibility.

[0009]Various embodiments of nutriceutical food products that incorporate teachings of the present invention include a fruit component. The fruit component includes at least one fruit that naturally includes OPC or a juice or other extract of such a fruit. By way of nonlimiting example, the fruit component may include one or more of acai, elderberry, grape, and pomegranate or an extract thereof. OPC is a known antioxidant and may, therefore, be useful in neutralizing or otherwise acting against free radicals and other oxidants, which may adversely affect cell membranes, cause accelerated cellular aging, and are known or believed to be at least indirectly responsible for a wide variety of disease states, as well as compromised immunity, in living beings.

[0010]In embodiments of nutriceutical food products that include fruit components, the fruit component may be in solid form, in liquid form, or in some combination of solid and liquid forms. When the fruit component is included in solid form, it may be incorporated into the matrix (e.g., as pectin, such as pectin of an OPC-containing fruit, etc.) or it may be dispersed throughout the matrix (e.g., as chunks, bits, etc.).

[0011]An immune modulator may be present within (e.g., dispersed throughout, dissolved in, etc.) the liquid component of various embodiments of nutriceutical food products of the present invention. When mixed with the liquid component, the immune modulator may retain substantially all of one or more of its activities (e.g., components of the liquid component may not interfere with one or more activities of the immune modulator), or one or more of the activities of the immune modulator may actually be enhanced by one or more components of the liquid component of the nutriceutical food product.

[0012]The immune modulator may include transfer factor. The transfer factor may include any type of transfer factor, as well as a combination of two or more types of transfer factor. For example, avian transfer factor, bovine transfer factor, or any other type of transfer factor may be included in the transfer factor component. The transfer factor of the transfer component factor may be derived from any suitable, acceptable source. For example, avian transfer factor may be obtained from eggs, such as by a process disclosed in U.S. Pat. No. 6,468,534 to Hennen et al. (hereinafter "Hennen"), the disclosure of which is hereby incorporated herein, in its entirety, by this reference. An example of the manner in which bovine transfer factor may be obtained is disclosed in U.S. Pat. No. 4,816,563 to Wilson et al. (hereinafter "Wilson"), the disclosure of which is hereby incorporated herein, in its entirety, by this reference. Compositions that include two or more types of transfer factor, as well as processes for combining and processing two or more types of transfer factor, are disclosed in U.S. Pat. No. 6,866,868 to Lisonbee et al. (hereinafter "Lisonbee"), the disclosure of which is hereby incorporated herein, it its entirety, by this reference.

[0013]Transfer factor is known or believed to improve the oxidative balance of a living being, as well as to enhance the effectiveness of antioxidants, as demonstrated by the disclosure of the international patent application filed pursuant to the Patent Cooperation Treaty and having International Publication Number WO 2004/041071 A2 (hereinafter "Dadali"), the disclosure of which is hereby incorporated herein, in its entirety, by this reference.

[0014]As an alternative to transfer factor, the immune modulator of various embodiments of a nutriceutical food product of the present invention may comprise a nanofraction immune modulator of the type disclosed by U.S. patent application Ser. No. 11/855,944.

[0015]Other embodiments of nutriceutical food products of the present invention may include a plurality of different immune modulators, such as combinations of transfer factor and nanofraction immune modulators. Various examples of compositions that include transfer factor and nanofraction immune modulators are also disclosed by U.S. patent application Ser. No. 11/855,944.

[0016]A nutriceutical food product of the present invention may also include one or more preservatives. Suitable preservatives, such as those accepted for use in foods and beverages, may be used. Any preservatives that are included in a nutriceutical food product of the present invention may be able to withstand pasteurization processes. Examples of preservatives that may be included in an edible preparation of the present invention include, but are not limited to, sodium benzoate and preservatives from the paraben family of chemicals.

[0017]A particular embodiment of gel-based nutriceutical food product that incorporates teachings of the present invention is described in the following example:

EXAMPLE 1

TABLE-US-00001 [0018]TABLE 1 % of % of Total Total Density Juices Ingredient (w/w) (g/ml) (v/v) Water 75.490 1.000 RioVida Juice Blend 15.965 Apple Juice 1.346 19 Purple Grape Juice 1.330 19 Blueberry Juice 1.315 18 Pomegranate Juice Elderberry Juice 1.315 15 Acai Powder 0.325 14 Transfer Factor Tri-Factor Blend 1.880 Glycerine (Vegetable) 4.210 1.249 Grape color concentrate (e.g., 0.510 1.306 MEGANATURAL ™ purple from Canandaigua Concentrates & Colors, a Division of Canandaigua Wine Company of Madera, California) Vitamin C 0.190 Flavorings 0.428 Berry flavor (BE-01407) 1.000 Berry flavor (BE-01271) 1.000 Natural Vanilla (VA-01239) 1.000 Monolaurin (glycerol monolaurate) 0.002 Gums (Xanthan and Guar Gum, at a 1.000 ratio of 1:1 w/w)

[0019]Transfer Factor Tri-Factor Blend includes transfer factor (including bovine transfer factor from cow colostrum and avian transfer factor from the yolk of a chicken's egg) and nanofraction immune modulators.

[0020]The flavorings listed in TABLE 1 are available from Flavors Inc.

[0021]Glycerol monolaurate is a surfactant. A surfactant may be useful for maintaining homogeneity (i.e., for keeping the components of the nutriceutical food product, including, but not limited to, any immune support component present in the nutriceutical food product, dispersed substantially homogeneously throughout the nutriceutical food product).

[0022]A daily dosage of about one fluid ounce (about 30 ml) or more (e.g., about two fluid ounces, or 60 ml, etc.) of a composition with ingredients in the proportions listed in TABLE 1 may be administered to or consumed by a subject. In addition to the numerous known and believed benefits of antioxidants, including the benefits of OPC and OPC-containing fruits such as acai, administration or consumption of a nutriceutical food product that incorporates teachings of the present invention provides the subject with the additional and sometimes synergistic beneficial effects of transfer factor, which are known in the art, as evidenced by the disclosures of Dadali, Hennen, Lisonbee, and Wilson, and of nanofraction immune modulators.

[0023]An edible preparation may be made by mixing components of a food base with transfer factor by processes that are known in the art. Suitable processes that may be used to manufacture edible preparations of a variety of different forms are well known and within the skill of those in the relevant art. Known techniques, such as those disclosed in "Principles and Practices of Small- and Medium-Scale Fruit Juice Processing," Food and Agricultural Organization of the United Nations (FAO) Services Bulletin 146 (Rome, 2001), the entire disclosure of which is hereby incorporated herein by this reference, may be used in one or more parts of a process for manufacturing various embodiments of nutriceutical food products of the present invention.

[0024]Processes that are used to manufacture nutriceutical food products according to the present invention may be effected at a low temperature (e.g., between about 0° C. and about 10° C., at about 4° C., etc.), such as in a refrigerated environment, then transported and stored at such temperatures to reduce the likelihood of microbial growth or proliferation therein.

[0025]Alternatively, a nutriceutical food product that is in a form that is not completely dry may be pasteurized or sterilized. Pasteurization processes, which decrease the number of microorganisms present, but do not entirely eliminate the microorganisms, improve the stability of products that are to be stored at reduced temperatures (e.g., frozen or refrigerated, or "chilled"). When a nutriceutical food product is sterilized, all or substantially all microorganisms therein are killed or inactivated, facilitating prolonged storage of the nutriceutical food product at room temperature or even higher temperatures.

[0026]As an example, a nutriceutical food product that includes transfer factor and/or nanofraction immune modulators may be sterilized by known superheated steam injection processes. The temperatures and durations of such processes depend, of course, upon the form and ingredients of the composition to be sterilized. When making a liquid preparation, the resulting nutriceutical food product may be "flash" heated to a particular temperature (e.g., 250° F.) for a corresponding duration (e.g., two seconds). Alternatively, a sterilization or pasteurization process of different duration and temperature may be used, so long as the duration and temperature of the process are in substantial accord with a practice that has been accepted in the art, such as use of the following equation:

tp=51014e.sup.-0.4353Tmo,

where tp is the minimum duration of the process, and Tmo is the temperature at which the process is effected.

[0027]Of course, processes that reduce microbial load on a nutriceutical food product of the present invention need not comprise heat-treatment techniques. Sterilization or other microbial load-reducing techniques that employ other means (e.g., filtration, antimicrobial ingredients, etc.) may also be used in manufacturing a nutriceutical food product. Examples of suitable processes are disclosed in Hughes, D. E., and Nyborg, W., "Minimally Processed Fruits and Vegetables: Reducing Microbial Load by Nonthermal Physical Treatments," Food Technology 52(6): 66-71 (1997), the disclosure of which is hereby incorporated herein, in its entirety, by this reference.

[0028]It is desirable that, following pasteurization or sterilization, the transfer factor and/or nanofraction immune modulators retain some if not substantially all or all of their activity. A variety of pasteurization or sterilization processes may be employed, including pasteurization or sterilization processes that may be used to reduce microbial counts or completely eliminate microorganisms from foods. As many sterilization processes are known to significantly reduce the activity of certain proteins, including antibodies, a study was performed to determine whether transfer factor retains at least some of its activity following pasteurization.

[0029]In the study, mouse footpad assay techniques, similar to those disclosed in J. Natl. Cancer Inst. 55(5):1089-95 (Nov. 1975), were used to determine the affects of heat pasteurization or sterilization processes (specifically, superheated steam injection processes) on liquid nutriceutical food products including transfer factor. Two sterilized samples were compared with an unsterilized sample, as well as with a negative control and a positive control.

[0030]Separate populations of six mice were tested for each of the five samples and controls. The tests were conducted in two phases, a first that immediately followed heat sterilization of the samples, and a second that was conducted after storing the two heat sterilized samples at a temperature of about 40° C. for about three months, which is well-accepted in the art to be the equivalent of about one year of storage at room temperature. Thirty different mice were used in each phase of the study. The following procedures were followed in each phase of the study.

[0031]In the positive control (i.e., the "fifth group"), fourteen days prior to testing, the footpads of the right rear feet of six BALB/c mice having ages of about nine weeks to about ten weeks were anesthetized with isoflurane. Then 0.02 ml of an about 50/50 (wt/wt) mixture of Freund's adjuvant and bovine rhinotracheitis virus diarrhea vaccine was administered intramuscularly to each mouse by way of two injections at the base of each side of the mouse's tail. This early injection of antigen allows the mice of the positive control group to elicit their own primary immune response and secondary, or delayed-type hypersensitivity response to the antigen. The mice of the other five groups were not preexposed to the antigen in this manner.

[0032]About twenty-four hours before evaluating the hind footpads of the mice, the six BALB/c mice of each group, which were of similar age to the mice of the positive control group, were anesthetized with isoflurane. About 0.5 ml of a sample solution or control solution was then administered by subcutaneous injection at the back of the neck of each mouse.

[0033]In the first group (see EXAMPLE 2 below), which was the negative control group, the back of the neck of each mouse was injected with about 0.5 ml of sterile saline solution.

[0034]In the second group (see EXAMPLE 3 below), the sample solution included 16% solids (w/v) of a reconstituted (in distilled, deionized water) lyophilized colostrum fraction that included transfer factor. The solution was set at a pH of 4.0, which was intended to estimate the pH of a fruit juice preparation (the actual pH of which is about 3.6 or about 3.7). Following reconstitution and pH adjustment, the solution was sterilized by heating the same to a temperature of 250° F. for about two seconds.

[0035]In the third group (see EXAMPLE 4 below), the sample solution included 16% solids (w/v) of a reconstituted (in distilled, deionized water) lyophilized colostrum fraction that included transfer factor. The pH of the resulting solution was not adjusted and, thus, was neutral (i.e., 7.0) or slightly basic (i.e., greater than 7.0)). Following reconstitution, the solution was sterilized by heating the same to a temperature of 250° F. for about two seconds.

[0036]In the fourth group (see EXAMPLE 5 below), the sample solution was a concentrate of a colostrum fraction that included transfer factor, which had been diluted to about 16% solids (w/v) in distilled, deionized water. This solution was not heat sterilized or pH adjusted.

[0037]The mice of the fifth group (see EXAMPLE 6 below), which was the positive control groups, respectively, received sterile saline solution.

[0038]At the start of the mouse footpad assay, the right hind footpad and the left hind footpad of each mouse were measured, such as with a Starrett gauge. The right hind footpad of each of the thirty mice during each phase of the study was then subcutaneously injected with an antigen-containing solution. The footpad on the left hind foot of each of the thirty mice in each phase, which was used as a control, was injected with about the same volume of a control solution, such as a sterile saline diluent, as the volume of antigen-containing solution that was injected into right hind footpad.

[0039]After a sufficient amount of time (e.g., about twenty-four hours) for the secondary immune response components of the immune system of each mouse to respond, each mouse was again anesthetized and the distances across right and left hind footpads were again measured. A significant amount of swelling, determined by an increase in the distance across a right hind footpad of a mouse from the initial measurement to the second measurement, is indicative of the occurrence of a delayed-type hypersensitivity reaction in that footpad.

[0040]The results of the mouse foot pad assays, and some accompanying analysis, are set forth in EXAMPLES 2 through 5 and 7:

EXAMPLE 2

[0041]In the first phase of the study, the footpads on the right hind feet of the six mice of the negative control, or first group, exhibited, on average, about 6.35 micrometers more swelling about twenty-four hours after they were injected with the antigen solution than the swelling measured in the footpads of the left hind feet of these mice, which were merely inoculated with sterile saline.

[0042]The results for the negative control group during the second phase of the study are set forth in the following table:

TABLE-US-00002 TABLE 2 Foot Pad Foot Pad Foot Pad (difference) Foot (untreated) (final) (micro- Mouse (left/right) (micrometers) (micrometers) meters) 1 Left (control) 1930.40 1955.80 25.40 Right (test) 1905.00 1930.40 25.40 2 Left (control) 1981.20 2006.60 25.40 Right (test) 2006.60 2057.40 50.80 3 Left (control) 2057.40 2057.40 0.00 Right (test) 2032.00 2057.40 25.40 4 Left (control) 2006.60 2032.00 25.40 Right (test) 2032.00 2057.40 25.40 5 Left (control) 1955.80 2006.60 50.80 Right (test) 1930.40 1955.80 25.40 6 Left (control) 1905.00 1930.40 25.40 Right (test) 1876.60 1955.80 76.20

[0043]Similar to the results from the first phase, the footpads of the right hind feet of the mice of the negative control group exhibited, on average, only 12.70 micrometers more swelling about twenty-four hours after antigen injection than the footpads of the left hind feet of the same mice exhibited twenty-four hours after sterile saline injection. As twenty-four hours is not a sufficient period of time for a mouse to mount a primary (i.e. , antibody-mediated) immune response to the antigen, these insignificant differences in swelling show that the mice did not exhibit a significant secondary immune response to the antigen.

EXAMPLE 3

[0044]In the first phase of the study, about twenty-four hours after they were injected with the antigen solution, the footpads on the right hind feet of the six mice of the second group of mice (which mice had previously been inoculated with a solution including 16% solids (w/v) colostrum at pH=4.0) swelled, on average, by 50.80 micrometers more than the swelling that was measured in the footpads of the left hind feet of these mice. These results indicate that there was a greater secondary, or delayed-type hypersensitivity, immune response in the footpads into which antigen was injected than in the footpads into which no antigen was injected, which were likely swollen merely because they were pierced by a needle.

[0045]In the second phase of the study, similar results were obtained, as set forth in the following table:

TABLE-US-00003 TABLE 3 Foot Pad Foot Pad Foot Pad (difference) Foot (untreated) (final) (micro- Mouse (left/right) (micrometers) (micrometers) meters) 1 Left (control) 1955.80 2006.60 50.80 Right (test) 1981.20 2057.40 76.20 2 Left (control) 1930.40 2006.60 76.20 Right (test) 1955.80 2108.20 152.40 3 Left (control) 1955.80 2006.60 50.80 Right (test) 1981.20 2082.80 101.60 4 Left (control) 2032.00 2057.40 25.40 Right (test) 2057.40 2108.20 50.80 5 Left (control) 1930.40 2006.60 76.20 Right (test) 1955.80 2032.00 76.20 6 Left (control) 2057.40 2108.20 50.80 Right (test) 2032.00 2159.00 127.00

[0046]More specifically, the footpads of the right hind feet of the six mice of the second group swelled so that they measured, on average, 42.33 micrometers more than the swelling that was measured in the footpads of the left hind feet of these mice before and after inoculation of their foot pads with the antigen solution. The similar results between the first and second phases of the study indicate that, once a liquid solution that includes transfer factor has been heat sterilized, there is little or no change in the activity of the transfer factor after prolonged storage of the solution.

EXAMPLE 4

[0047]The results for the third group of mice (which mice had previously been inoculated with a solution including 16% solids (w/v) colostrum at normal pH) were similar to the results for the second group in the first and second phases of the study.

[0048]In the first phase of the study, about twenty-four hours after the footpad injections, the antigen solution-inoculated footpads on the right hind feet of the six mice of the third group of mice swelled, on average, by 35.98 micrometers more than the swelling that was measured in the sterile saline-inoculated footpads of the left hind feet of these mice. These results indicate that there was a greater secondary, or delayed-type hypersensitivity, immune response in the footpads into which antigen was injected than in the footpads into which no antigen was injected, which were likely swollen merely because they were pierced by a needle.

[0049]In the second phase of the study, similar results were obtained, as set forth in the following table:

TABLE-US-00004 TABLE 4 Foot Pad Foot Pad Foot Pad (difference) Foot (untreated) (final) (micro- Mouse (left/right) (micrometers) (micrometers) meters) 1 Left (control) 2006.60 2032.00 25.40 Right (test) 2032.00 2082.80 50.80 2 Left (control) 2057.40 2057.40 0.00 Right (test) 2006.60 2108.20 101.60 3 Left (control) 1981.20 2006.60 25.40 Right (test) 2057.40 2082.80 25.40 4 Left (control) 2006.60 2057.40 50.80 Right (test) 2032.00 2082.80 50.80 5 Left (control) 2057.40 2082.80 25.40 Right (test) 2082.80 2159.00 76.20 6 Left (control) 2082.80 2108.20 25.40 Right (test) 2108.20 2159.00 50.80

[0050]These results show that the footpads of the right hind feet of the six mice of the third group swelled so that they measured, on average, 33.87 micrometers more than the swelling that was measured in the footpads of the left hind feet of these mice before and after inoculation of the foot pads with the antigen solution. The similar results between the first and second phases of the study indicate that, following prolonged storage, there was little or no change in the activity of the transfer factor in a heat-sterilized solution.

EXAMPLE 5

[0051]These results were confirmed by the results that were obtained from the fourth group of mice. In particular, during the first phase of the study, the footpads of the right hind feet of mice in the fourth group (which included mice that had been inoculated with a diluted liquid colostrum fraction that was not heat sterilized) exhibited, on average, about 35.98 micrometers more swelling than the foot pads of left hind feet of these mice about twenty-four hours after these footpads had been inoculated with antigen solution and sterile saline, respectively.

[0052]Similar results were obtained during the second phase of the study, in which the average difference was 42.33 micrometers, as evidenced by the following data:

TABLE-US-00005 TABLE 5 Foot Pad Foot Pad Foot Pad (difference) Foot (untreated) (final) (micro- Mouse (left/right) (micrometers) (micrometers) meters) 1 Left (control) 1955.80 2032.00 76.20 Right (test) 1981.20 2082.80 101.60 2 Left (control) 2006.60 2057.40 50.80 Right (test) 2032.00 2108.20 76.20 3 Left (control) 1955.80 2006.60 50.80 Right (test) 1930.40 2057.40 127.00 4 Left (control) 1955.80 2082.80 127.00 Right (test) 1905.00 2032.00 127.00 5 Left (control) 2032.00 2082.80 50.80 Right (test) 2057.40 2184.40 127.00 6 Left (control) 1955.80 1955.80 0.00 Right (test) 2006.60 2057.40 50.80

[0053]As these results are comparable to (i. e., not significantly greater than) those obtained with heat-sterilized solutions (see the results from EXAMPLES 3 and 4), it is apparent that heat sterilization of a solution that includes transfer factor does not significantly diminish or reduce the activity of the transfer factor.

EXAMPLE 6

[0054]This conclusion was verified by data from another mouse footpad assay, in which six BALB/c mice were inoculated, behind the neck, with 0.5 ml of a solution including 16% solids (w/v) of a spray-dried colostrum fraction that had been reconstituted in distilled, deionized water. About twenty-four hours later, the mice were anesthetized with isoflurane, then footpads on their hind feet measured and inoculated in the manner described above (i.e., left footpad with sterile saline, right footpad with the antigen solution). After about another twenty-four hours, the footpads were again measured. The right footpads of these mice swelled, on average, about 42.33 micrometers more than the footpads on the left hind feet of these mice. This value is comparable to (i.e., not significantly different from) the differences noted above with respect to the second, third, and fourth groups of mice in both the first and second phases of the study detailed in EXAMPLES 2 through 5 and 7, further supporting the conclusion that heat sterilization of a solution that includes transfer factor, such as the solutions that were tested on the second and third groups of mice (EXAMPLES 3 and 4) does not have a significant adverse affect on the activity of the transfer factor.

EXAMPLE 7

[0055]The fact that the transfer factor with which the mice were inoculated was responsible for the increased secondary immune response is supported by the results from the fifth group, or positive control group, of mice during the second phase of the study, as set forth in the following table:

TABLE-US-00006 TABLE 6 Foot Pad Foot Pad Foot Pad (difference) Foot (untreated) (final) (micro- Mouse (left/right) (micrometers) (micrometers) meters) 1 Left (control) 1981.20 2006.60 25.40 Right (test) 2006.60 2082.80 76.20 2 Left (control) 1828.80 1854.20 25.40 Right (test) 1879.60 2082.80 203.20 3 Left (control) 1905.00 1930.40 25.40 Right (test) 1981.20 2082.80 101.60 4 Left (control) 2006.60 2057.40 50.80 Right (test) 2032.00 2184.40 152.40 5 Left (control) 2032.00 2057.40 25.40 Right (test) 2057.40 2184.40 127.00 6 Left (control) 2108.20 2108.20 0.00 Right (test) 2082.80 2184.40 101.60

[0056]These results, which show on average, 101.60 micrometers more swelling in the footpads that were inoculated with antigen solution over those that were inoculated with sterile saline, are similar to the 124.88 micrometer difference seen in the mice of the positive control group during the first phase of the mouse footpad study. The greater swelling in the antigen solution-inoculated footpads of the mice of the positive control group is indicative of a greater secondary immune response than that induced artificially by administration of transfer factor, as the mice of the positive control group had a sufficient period of time (i.e., two weeks) to generate their own transfer factor and, thus, to mount their own secondary immune response to the antigen.

[0057]Once a nutriceutical food product of the present invention has been manufactured, it may be introduced into a clean or sterile container for subsequent transport and storage.

EXAMPLE 8

[0058]In another study, mouse footpad assays were conducted to determine the effectiveness of transfer factor in heat-treated samples of a liquid solution that included transfer factor that had been stored for one year. In total, four samples were prepared, two each having a pH of about 4 and two each having a pH of about 7. All of the samples had been flash sterilized at a temperature of about 250° F. for about two seconds to about four seconds. The samples were subsequently stored for one year, with one each of the pH=4 and pH=7 samples having been stored at room temperature (which varied from about 65° F. to about 74° F.) and one each of the pH=4 and pH=7 samples having been refrigerated (at temperatures of about 40° F.). After one year, the samples were lyophilized. Prior to testing, the lyophilized samples were reconstituted to desired concentrations, then administered in the manner described above.

[0059]In a first sample, which included liquid having a pH of about 4 that was stored at room temperature, footpad swelling was, on average, 50.80 micrometers greater in footpads that had been injected with antigen versus footpads that had merely been injected with saline.

[0060]These results were repeated in second (liquid of a pH of about 7 that was stored at room temperature), third (liquid of a pH of about 4 that was refrigerated), and fourth (liquid of a pH of about 7 that was refrigerated) samples, in which hind footpads that had been injected with antigen were, on average, respectively swollen 59.27, 67.73, and 63.50 micrometers more than hind footpads that were merely injected with saline.

[0061]Additionally, positive and negative controls were prepared as discussed above. In the positive control, the average difference in swelling between antigen-injected footpads and saline-injected footpads was 114.30 micrometers. In the negative control, the average difference in swelling between antigen-injected footpads and saline-injected footpads was only 38.10 micrometers.

[0062]Taken together, these data indicate that the increased swelling was due to the presence of transfer factor in the mice in the areas (hind footpads) into which antigen was introduced. Additionally, these data indicate that the transfer factor lost little or none of its effectiveness after heat-treatment and prolonged storage. The activity of transfer factor in refrigerated samples appears to have been slightly higher than the activity of transfer factor in the room temperature samples.

[0063]Further, it appears from the foregoing that the pH at which the transfer factor is maintained (about 4 or about 7) has little or no effect on its long term viability.

[0064]Although the foregoing description contains many specifics, these should not be construed as limiting the scope of the present invention, but merely as providing illustrations of some of the presently preferred embodiments. Similarly, other embodiments of the invention may be devised which do not depart from the spirit or scope of the present invention. Features from different embodiments may be employed in combination. The scope of the invention is, therefore, indicated and limited only by the appended claims and their legal equivalents, rather than by the foregoing description. All additions, deletions and modifications to the invention as disclosed herein which fall within the meaning and scope of the claims are to be embraced thereby.



Inventors

* Vaughan, Brent M. * McCausland, Calvin W. * Lisonbee, David A.

Assignee

* 4LIFE PATENTS, LLC

US Class 424/184.1ANTIGEN, EPITOPE, OR OTHER IMMUNOSPECIFIC IMMUNOEFFECTOR (E.G., IMMUNOSPECIFIC VACCINE, IMMUNOSPECIFIC STIMULATOR OF CELL-MEDIATED IMMUNITY, IMMUNOSPECIFIC TOLEROGEN, IMMUNOSPECIFIC IMMUNOSUPPRESSOR, ETC.)

Attorney, Agent or Firm

* TraskBritt, P.C.

International Classes A61K 39/00 A61P 37/04



Abstract text

Compositions, systems, and methods for enhancing the ability of a subject to heal itself following an infection include administering a composition that includes transfer factor to a subject. Administration of such a composition or combination of compositions to a subject may result in improving the subject's overall antioxidant profile, increasing the concentration of chemical antioxidants present in the subject, increasing the efficiency with which the treated subject's enzymatic antioxidants work, increasing the efficiency and/or activity of the treated subject's detoxification enzymes, and improving cellular and molecular health of the subject. Claims

What is claimed is:

1. A system for restoring an oxidative balance of a body of a subject, comprising at least one biologically active agent and a composition including transfer factor, at least the transfer factor included in an amount tailored to restore the oxidative balance of the body of the subject.

2. The system of claim 1, wherein the transfer factor is specific for a pathogen with which the subject has been infected.

3. The system of claim 1, wherein the composition is substantially free of transfer factor specific for a pathogen with which the subject has been infected.

4. The system of claim 1, wherein the at least one biologically active agent includes at least one of an antibiotic agent, an antiparasitic agent, an antiviral agent, and a cytokine.

5. The system of claim 1, wherein the transfer factor comprises at least one of mammalian transfer factor and avian transfer factor.

6. A system for enhancing an efficiency of at least one enzymatic oxidant of a subject, comprising at least one biologically active agent and a composition including transfer factor, at least the transfer factor included in an amount tailored to enhance the efficiency of the at least one enzymatic oxidant.

7. The system of claim 6, wherein the transfer factor is specific for a pathogen with which the subject has been infected.

8. The system of claim 6, wherein the composition is substantially free of transfer factor specific for a pathogen with which the subject has been infected.

9. The system of claim 6, wherein the at least one biologically active agent includes at least one of an antibiotic agent, an antiparasitic agent, an antiviral agent, and a cytokine.

10. The system of claim 6, wherein the transfer factor comprises at least one of mammalian transfer factor and avian transfer factor.

11. A system for increasing an efficiency with which detoxification proteins of a subject remove toxins from the subject, comprising at least one biologically active agent and a composition including transfer factor, at least the transfer factor included in an amount tailored to increase the efficiency with which detoxification proteins remove toxins.

12. The system of claim 11, wherein the transfer factor is specific for a pathogen with which the subject has been infected.

13. The system of claim 11, wherein the composition is substantially free of transfer factor specific for a pathogen with which the subject has been infected.

14. The system of claim 11, wherein the at least one biologically active agent includes at least one of an antibiotic agent, an antiparasitic agent, an antiviral agent, and a cytokine.

15. The system of claim 11, wherein the transfer factor comprises at least one of mammalian transfer factor and avian transfer factor.

16. A system for improving cellular stability in the body of a subject, comprising at least one biologically active agent and a composition including transfer factor, at least the transfer factor included in an amount tailored to improve the stability of cells in the body of the subject.

17. The system of claim 16, wherein the transfer factor is specific for a pathogen with which the subject has been infected.

18. The system of claim 16, wherein the composition is substantially free of transfer factor specific for a pathogen with which the subject has been infected.

19. The system of claim 16, wherein the at least one biologically active agent includes at least one of an antibiotic agent, an antiparasitic agent, an antiviral agent, and a cytokine.

20. The system of claim 16, wherein the transfer factor comprises at least one of mammalian transfer factor and avian transfer factor.

21. A system for improving molecular health of a subject, comprising at least one biologically active agent and a composition including transfer factor, at least the transfer factor included in an amount tailored improve the molecular health of the subject.

22. The system of claim 21, wherein the transfer factor is specific for a pathogen with which the subject has been infected.

23. The system of claim 21, wherein the composition is substantially free of transfer factor specific for a pathogen with which the subject has been infected.

24. The system of claim 21, wherein the at least one biologically active agent includes at least one of an antibiotic agent, an antiparasitic agent, an antiviral agent, and a cytokine.

25. The system of claim 21, wherein the transfer factor comprises at least one of mammalian transfer factor and avian transfer factor.

26. A method increasing a number of chemical oxidants in a body of a subject, comprising administering to the subject a composition comprising a quantity of transfer factor tailored to increase the number of chemical oxidants in the body of the subject.

27. The method of claim 26, wherein said increasing includes increasing a concentration of a reduced form of ascorbate in the subject.

28. The method of claim 26, wherein said increasing includes increasing a concentration of reduced thiols in the subject.

29. The method of claim 26, wherein said administering comprises administering to the subject a composition comprising transfer factor which is specific for a pathogen with which the subject has been infected.

30. The method of claim 26, wherein said administering comprises administering to the subject a composition which is substantially free of transfer factor specific for a pathogen with which the subject has been infected.

31. A method for enhancing an efficiency of at least one enzymatic antioxidant in a body of a subject, comprising administering to the subject a composition comprising a quantity of transfer factor tailored to enhance the efficiency of the at least one enzymatic antioxidant in the body of the subject.

32. The method of claim 31, wherein said enhancing comprises reducing a concentration of said at least one said enzymatic antioxidant in the subject.

33. The method of claim 31, wherein said enhancing comprises enhancing activity of at least one of a superoxide dismutase and a glutathione peroxidase.

34. The method of claim 31, wherein said administering comprises administering to the subject a composition comprising transfer factor which is specific for a pathogen with which the subject has been infected.

35. The method of claim 31, wherein said administering comprises administering to the subject a composition which is substantially free of transfer factor specific for a pathogen with which the subject has been infected.

36. A method for increasing an efficiency with which detoxification proteins of a body of a subject remove toxins from the body of the subject, comprising administering to the subject a composition comprising a quantity of transfer factor tailored to increase the efficiency with which detoxification proteins remove toxins.

37. The method of claim 36, wherein said administering comprises increasing a concentration of said at least one detoxification protein in the subject.

38. The method of claim 36, wherein said administering comprises increasing a concentration of at least one of a catalase and a glutathione S-transferase in the subject.

39. The method of claim 36, wherein said administering comprises administering to the subject a composition comprising transfer factor which is specific for a pathogen with which the subject has been infected.

40. The method of claim 36, wherein said administering comprises administering to the subject a composition which is substantially free of transfer factor specific for a pathogen with which the subject has been infected.

41. A method for improving cellular stability of a body of a subject, comprising administering to the subject a composition comprising a quantity of transfer factor tailored to improve the stability of cells of the body of the subject.

42. The method of claim 41, wherein said administering comprises decreasing a number of red blood cells that are lysed when exposed to a substantially fixed concentration of at least one antioxidant.

43. The method of claim 41, wherein said administering comprises decreasing a concentration the subject of at least one protein indicator of cell lysis.

44. The method of claim 41, wherein said decreasing comprises decreasing a concentration of at least one of alanine amino transferase and aspartate amino transferase in the subject.

45. The method of claim 41, wherein said administering comprises administering to the subject a composition comprising transfer factor which is specific for a pathogen with which the subject has been infected.

46. The method of claim 41, wherein said administering comprises administering to the subject a composition which is substantially free of transfer factor specific for a pathogen with which the subject has been infected.

47. A method for improving molecular health in a body of a subject, comprising administering to the subject a composition comprising a quantity of transfer factor tailored to improve the molecular health.

48. The method of claim 47, wherein said administering comprises increasing a ratio of a reduced form of protein thiol groups to an oxidized form of protein thiol groups.

49. The method of claim 47, wherein said administering comprise decreasing a measure of oxidized lipids in blood of the subject.

50. The method of claim 47, wherein said administering comprises administering to the subject a composition comprising transfer factor which is specific for a pathogen with which the subject has been infected.

51. The method of claim 47, wherein said administering comprises administering to the subject a composition which is substantially free of transfer factor specific for a pathogen with which the subject has been infected. Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of International Patent Application No. PCT/US03/35161, filed Nov. 4, 2003, designating the United States of America and published, in English, as PCT International Patent Application No. WO 2004/141071 A2 on May 21, 2004, which claims priority to U.S. Provisional Application Ser. No. 60/423,965, filed Nov. 4, 2002, the disclosures of both of which are hereby incorporated herein, in their entireties, by this reference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates generally to methods for enhancing or sustaining the ability of a subject to heal itself following an infection and, more specifically, to the use of transfer factor to enhance the ability of a subject to heal itself. More specifically, the present invention relates to systems that include at least one biologically active agent and a composition that includes transfer factor.

Conventional Techniques for Treating Infection

[0003] Conventionally, infections have been treated by use of antibiotics, which affect cells that are exposed thereto in such a way as to kill the exposed cells. In addition to adversely affecting bacterial cells, antibiotics may also induce toxicity and kill beneficial bacteria, as well as damage or kill the cells of a treated subject.

[0004] Antibiotics have been used to treat a wide array of infections. There is a movement, however, to curb or limit their use. This is because, as medical professionals have long been aware, many bacteria evolve in such a way as to develop strains which are resistant to antibiotics. As evidence of the severity of the problem of antibiotic-resistant bacterial strains, great efforts have recently been taken to make the general public aware that antibiotics should be used judiciously.

[0005] The usefulness of antibiotics is also largely limited to bacteria, fungi, and some parasites. Very few substances are considered effective antiviral compounds. Nonetheless, many undesirable pathogenic infections and the diseases that result therefrom are caused by viruses.

[0006] For serious bacterial infections, high doses of antibiotics may be administered to an infected subject. Sometimes, bacterial infections become so severe or unresponsive or inaccessible that surgery is needed to excise the infected areas of a subject's body and, thus, to physically remove the infecting pathogen.

[0007] The use of surgery is somewhat undesirable because of the trauma and increase in oxidative stress caused thereby. As such, surgery is often used as a last resort for eliminating infections.

[0008] Although surgery, the administration of antibiotics, or both of these techniques are useful for removing infections and, thus, for permitting the body of a treated individual to heal itself, neither of these techniques is useful for enhancing the ability of a subject to heal itself.

The Immune System and Transfer Factor

[0009] The immune systems of vertebrates are equipped to recognize and defend the body from invading pathogenic organisms, such as parasites, bacteria, fungi, and viruses. Vertebrate immune systems typically include a cellular component and a noncellular component.

[0010] The cellular component of an immune system includes the so-called "lymphocytes," or white blood cells, of which there are several types. It is the cellular component of a mature immune system that typically mounts a primary, nonspecific response to invading pathogens, as well as being involved in a secondary, specific response to pathogens.

[0011] In the primary, or initial, response to an infection by a pathogen, white blood cells that are known as phagocytes locate and attack the invading pathogens. Typically, a phagocyte will internalize, or "eat" a pathogen, then digest the pathogen. In addition, white blood cells produce and excrete chemicals in response to pathogenic infections that are intended to attack the pathogens or assist in directing the attack on pathogens.

[0012] Only if an infection by invading pathogens continues to elude the primary immune response is a specific, secondary immune response to the pathogen needed. As this secondary immune response is typically delayed, it is also known as "delayed-type hypersensitivity." A mammal, on its own, will typically not elicit a secondary immune response to a pathogen until about seven (7) to about fourteen (14) days after becoming infected with the pathogen. The secondary immune response is also referred to as an acquired immunity to specific pathogens. Pathogens have one or more characteristic proteins, which are referred to as "antigens." In a secondary immune response, white blood cells known as B lymphocytes, or "B-cells," and T lymphocytes, or "T-cells," "learn" to recognize one or more of the antigens of a pathogen. The B-cells and T-cells work together to generate proteins called "antibodies," which are specific for one or more certain antigens on a pathogen.

[0013] The T-cells are primarily responsible for the secondary, or delayed-type hypersensitivity, immune response to a pathogen or antigenic agent. There are three types of T-cells: T-helper cells, T-suppressor cells, and antigen-specific T-cells, which are also referred to as cytotoxic (meaning "cell-killing") T-lymphocytes ("CTLs"), or T-killer cells. The T-helper and T-suppressor cells, while not specific for certain antigens, perform conditioning functions (e.g., the inflammation that typically accompanies an infection) that assist in the removal of pathogens or antigenic agents from an infected host.

[0014] Antibodies, which make up only a part of the noncellular component of an immune system, recognize specific antigens and, thus, are said to be "antigen-specific." The generated antibodies then basically assist the white blood cells in locating and eliminating the pathogen from the body. Typically, once a white blood cell has generated an antibody against a pathogen, the white blood cell and all of its progenitors continue to produce the antibody. After an infection is eliminated, a small number of T-cells and B-cells that correspond to the recognized antigens are retained in a "resting" state. When the corresponding pathogenic or antigenic agents again infect the host, the "resting" T-cells and B-cells activate and, within about forty-eight (48) hours, induce a rapid immune response. By responding in this manner, the immune system mounts a secondary immune response to a pathogen; the immune system is said to have a "memory" for that pathogen.

[0015] Mammalian immune systems are also known to produce smaller proteins, known as "transfer factors," as part of a secondary immune response to infecting pathogens. Transfer factors are another noncellular part of a mammalian immune system. Antigen-specific transfer factors are believed to be structurally analogous to antibodies, but on a much smaller molecular scale. Both antigen-specific transfer factors and antibodies include antigen-specific sites. In addition, both transfer factors and antibodies include highly conserved regions that interact with receptor sites on their respective effector cells. In transfer factor and antibody molecules, a third, "linker," region connects the antigen-specific sites and the highly conserved regions.

The Role of Transfer Factor in the Immune System

[0016] Transfer factor is a low molecular weight isolate of lymphocytes. Narrowly, transfer factors may have specificity for single antigens. U.S. Pat. Nos. 5,840,700 and 5,470,835, both of which issued to Kirkpatrick et al. (hereinafter collectively referred to as "the Kirkpatrick Patents"), disclose the isolation of transfer factors that are specific for certain antigens. More broadly, "specific" transfer factors have been generated from cell cultures of monoclonal lymphocytes. Even if these transfer factors are generated against a single pathogen, they have specificity for a variety of antigenic sites of that pathogen. Thus, these transfer factors are said to be "pathogen-specific" rather than antigen-specific. Similarly, transfer factors that are obtained from a host that has been infected with a certain pathogen are pathogen-specific. Although such preparations are often referred to in the art as being "antigen-specific" due to their ability to elicit a secondary immune response when a particular antigen is present, transfer factors having different specificities may also be present. Thus, even the so-called "antigen-specific," pathogen-specific transfer factor preparations may be specific for a variety of antigens.

[0017] Additionally, it is believed that antigen-specific and pathogen-specific transfer factors may cause a host to elicit a delayed-type hypersensitivity immune response to pathogens or antigens for which such transfer factor molecules are not specific. Transfer factor "draws" at least the non-specific T-cells, the T-inducer and T-suppressor cells, to an infecting pathogen or antigenic agent to facilitate a secondary, or delayed-type hypersensitivity, immune response to the infecting pathogen or antigenic agent.

[0018] Typically, transfer factor includes an isolate of proteins having molecular weights of less than about 10,000 daltons (D) that have been obtained from immunologically active mammalian sources. It is known that transfer factor, when added either in vitro or in vivo to mammalian immune cell systems, improves or normalizes the response of the recipient mammalian immune system.

[0019] The immune systems of newborns have typically not developed, or "matured," enough to effectively defend the newborn from invading pathogens. Moreover, prior to birth, many mammals are protected from a wide range of pathogens by their mothers. Thus, many newborn mammals cannot immediately elicit a secondary response to a variety of pathogens. Rather, newborn mammals are typically given secondary immunity to pathogens by their mothers. One way in which mothers are known to boost the immune systems of newborns is by providing the newborn with a set of transfer factors. In mammals, transfer factor is provided by a mother to a newborn in colostrum, which is typically replaced by the mother's milk after a day or two. Transfer factor basically transfers the mother's acquired, specific (i.e., delayed-type hypersensitive) immunity to the newborn. This transferred immunity typically conditions the cells of the newborn's immune system to react against pathogens in an antigen-specific manner, as well as in an antigen- or pathogen-nonspecific fashion, until the newborn's immune system is able on its own to defend the newborn from pathogens. Thus, when transfer factor is present, the immune system of the newborn is conditioned to react to pathogens with a hypersensitive response, such as that which occurs with a typical delayed-type hypersensitivity response. Accordingly, transfer factor is said to "jump start" the responsiveness of immune systems to pathogens.

[0020] Much of the research involving transfer factor has been conducted in recent years. Currently, it is believed that transfer factor is a protein with a length of about forty-four (44) amino acids. Transfer factor typically has a molecular weight in the range of about 3,000 to about 6,000 Daltons (Da), or about 3 kDa to about 6 kDa, but it may be possible for transfer factor molecules to have molecular weights outside of this range. Transfer factor is also believed to include three functional fractions: an inducer fraction; an immune suppressor fraction; and an antigen-specific fraction. Many in the art believe that transfer factor also includes a nucleoside portion, which could be connected to the protein molecule or separate therefrom, that may enhance the ability of transfer factor to cause a mammalian immune system to elicit a secondary immune response. The nucleoside portion may be part of the inducer or suppressor fractions of transfer factor.

[0021] The antigen-specific region of the antigen-specific transfer factors is believed to comprise about eight (8) to about twelve (12) amino acids. A second highly-conserved region of about ten (10) amino acids is thought to be a very high-affinity T-cell receptor binding region. The remaining amino acids may serve to link the two active regions or may have additional, as yet undiscovered properties. The antigen-specific region of a transfer factor molecule, which is analogous to the known antigen-specific structure of antibodies, but on a much smaller molecular weight scale, appears to be hyper-variable and is adapted to recognize a characteristic protein on one or more pathogens. The inducer and immune suppressor fractions are believed to impart transfer factor with its ability to condition the various cells of the immune system so that the cells are more fully responsive to the pathogenic stimuli in their environment.

Sources of Noncellular Immune System Components

[0022] Conventionally, transfer factor has been obtained from the colostrum of milk cows. While milk cows typically produce large amounts of colostrum and, thus, large amounts of transfer factor over a relatively short period of time, milk cows only produce colostrum for about a day or a day-and-a-half every year. Thus, milk cows are neither a constant source of transfer factor nor an efficient source of transfer factor.

[0023] Transfer factor has also been obtained from a wide variety of other mammalian sources. For example, in researching transfer factor, mice have been used as a source for transfer factor. Antigens are typically introduced subcutaneously into mice, which are then sacrificed following a delayed-type hypersensitivity reaction to the antigens. Transfer factor is then obtained from spleen cells of the mice.

[0024] While different mechanisms are typically used to generate the production of antibodies, the original source for antibodies may also be mammalian. For example, monoclonal antibodies may be obtained by injecting a mouse, a rabbit, or another mammal with an antigen, obtaining antibody-producing cells from the mammal, then fusing the antibody-producing cells with immortalized cells to produce a hybridoma cell line, which will continue to produce the monoclonal antibodies throughout several generations of cells and, thus, for long periods of time.

[0025] Antibodies against mammalian pathogens have been obtained from a wide variety of sources, including mice, rabbits, pigs, cows, and other mammals. In addition, the pathogens that cause some human diseases, such as the common cold, are known to originate in birds. As it has become recognized that avian (i.e., bird) immune systems and mammalian immune systems are very similar, some researchers have turned to birds as a source for generating antibodies.

[0026] U.S. Pat. No. 6,468,534, issued to Hennen et al. on Oct. 22, 2002 (hereinafter "the '534 Patent"), discloses methods for obtaining transfer factor from the eggs of nonmammalian source animals, including chickens. The method that is described in the '534 Patent includes exposing the nonmammalian source animal to one or more antigenic agents. These antigenic agents have been found to elicit a cell-mediated immune response which includes the production of transfer factor. The transfer factor is present in and may be obtained from the eggs of the source animal. Accordingly, the method of the '534 Patent includes collecting eggs from the nonmammalian source animal.

Administration of Transfer Factor

[0027] While transfer factor from such sources is known to facilitate and enhance a subject's cell-mediated immune response to invasion by pathogens, it has been believed that transfer factor enhances the activity of the so-called "T-natural killer" cells, which produce oxidants. It is well known that, by producing oxidants, T-natural killer cells produce conditions which are not favorable to infecting pathogens and, thereby, "kill" the invading pathogens. Additionally, the high oxidant concentration conditions that are created by T-natural killer cells are also damaging to the cells of the infected subject. Thus, in addition to ridding the subject of pathogen, the cell-mediated immune response of a subject increases oxidative stress in the body of the subject (e.g., by increasing the number of oxidants in the body and, thus, production of antioxidants by the body) and has a somewhat adverse affect on the subject's own cells and tissues. By administering transfer factor to a subject, it has been thought that the cell-mediated immune response would be increased, along with a consequent increase in damage to the treated subject's body.

[0028] There are needs for methods and compositions that facilitate the ability of a subject to rid itself of unwanted infections, as well as enhance or sustain, rather than exacerbate, the oxidative balance (i.e., the balance between oxidants and antioxidants) of the subject's body and the ability of the subject's body to heal itself.

SUMMARY OF THE INVENTION

[0029] The present invention includes methods and compositions for focusing the cell-mediated immune response of a subject, such as a mammal (e.g., a livestock, a human, etc.), a bird (e.g., a chicken), or another animal, to an infecting pathogen. The present invention also includes methods and compositions for enhancing or sustaining one or more of a subject's antioxidant profile, detoxification abilities, and general cell and molecular health.

[0030] In particular, a method according to the present invention includes administering transfer factor to an infected individual. The transfer factor, which may be derived from a mammalian or nonmammalian (e.g., avian, amphibian, reptilian, etc.) source, may be administered alone or with other suitable therapies, which are effected with known biologically active agents (e.g., antibiotics, antiparasitics, antiviral agents, cytokines, etc.). It has been unexpectedly discovered that by administering transfer factor to an infected subject the subject's oxidant levels do not increase. Instead, even though transfer factor improves the subject's cell-mediated immune response, the oxidant levels are decreased. Thus, transfer factor is believed to focus the cell-mediated immune response of a subject rather than to generally increase the cell-mediated immune response, while maintaining a healthy oxidative balance.

[0031] In addition, improvements in the antioxidant profiles of various subjects have been accelerated following administration of transfer factor, relative to the rates of improvement in the antioxidant profiles of subjects who were not treated with transfer factor. It has also been discovered that the abilities of the bodies of subjects that have been treated with transfer factor to self-detoxify is enhanced relative to the abilities of the bodies of untreated subjects to detoxify themselves. As such, the present invention includes a method for improving the antioxidant and detoxification profile of a subject by treating the subject with transfer factor.

[0032] The infection-affected cells and tissues of subjects who have been treated with transfer factor also appear to repair themselves more effectively than do the cells and tissues at or near the infection sites of subjects that have not been treated with transfer factor. Accordingly, the present invention includes methods for enhancing the ability of a subject's body to repair its cells by administering transfer factor to the subject.

[0033] Likewise, subjects that have been treated with transfer factor and that are recovering from infections evidence greater molecular health than do untreated subjects who are recovering from similar infections. In particular, the overall "health," as measured by the ratio of reduced forms to oxidized forms, of both proteins and lipids in subjects that are recovering from infections and who have been treated with transfer factor is better than the health of proteins and lipids in subjects who are recovering from similar infections without having been treated with transfer factor. Accordingly, the present invention includes a method for improving the molecular health of a treated subject which includes administering transfer factor to the treated subject. By way of example only and not to limit the scope of the present invention, the invention includes methods for improving the health of a subject's proteins and lipids by administering transfer factor to the subject.

[0034] The present invention also includes compositions that are useful for effecting the method of the present invention. In particular, transfer factor and compositions which include transfer factor are within the scope of the present invention. The transfer factor may be derived from any suitable source, such as from the cells of an animal, the colostrum or milk of a mammal, or from eggs.

[0035] Other features and advantages of the present invention will become apparent to those of skill in the art through consideration of the ensuing description and the accompanying claims.

DETAILED DESCRIPTION

[0036] Those who understand the role of transfer factor in facilitating cell-mediated immune responses know that transfer factor typically increases the activity of T-cells. It has also been recently shown that transfer factor increases the effectiveness of natural killer cells. Additionally, it is believed that transfer factor enhances the response of cytotoxic T-lymphocytes (CTLs) to infections. It is also well known to those in the art that immune cells, such as neutrophils, produce peroxide and other oxidants in infected regions of the body to "kill" invading pathogens. Thus, it would be expected that by administering transfer factor to a subject, the resulting affect on the subject's cell-mediated immune response would increase the levels of oxidants at or near the site of infection and, thus, result in an increase in the levels of antioxidants produced by the subject's body.

[0037] Research has demonstrated otherwise. In particular, it appears that transfer factor may be used to focus the cell-mediated immune response of subjects to invading pathogens. It also appears that administering transfer factor to an subject may enhance and/or increase the efficiency of an subject's various antioxidant systems, permitting the antioxidant systems of the subject to recover more quickly than if transfer factor were not administered. Also, the ability of the subject's body to eliminate toxins appears to be improved by administering transfer factor to the subject. Additionally, it has been discovered that administration of transfer factor to subjects has beneficial affects on the general health of the biomolecules (e.g., proteins, lipids, etc.), cells, and tissues in the treated subject's body.

[0038] The following EXAMPLES summarize studies which have been conducted to show these novel and inventive uses for transfer factor.

EXAMPLE 1

[0039] In a first example, the affects of transfer factor on patients with osteomyelitis were evaluated. Osteomyelitis is caused as pyrogenic (i.e., fever-causing) bacteria infect bones. The presence of such an infection typically causes a significant increase in the cell-mediated (i.e., T-cell or leukocyte) immune response at or near the site of infection, which results in an increase in the number of oxidants (e.g., free radicals, peroxides, etc.) at and near the site of the infection. Moreover, when it becomes necessary to remove osteomyelitis by surgery, the trauma that surgery causes results in a heightened cell-mediated immune response which, in turn, leads to even higher levels of oxidants at and near the site of infection. As a consequence of increased levels of oxidants, cellular and bone tissue damage occurs In addition, the concentration of toxins at the location of infected and decaying cells and bone tissue is usually relatively high.

[0040] Various characteristics of two groups of infected individuals were evaluated and compared with the characteristics of a sampling of "normal" individuals from the same geographic region. The thirteen (13) individuals in the first group were less sick (i.e., had less extensive infections) than the twenty (20) individuals of the second group. Thus, the individuals of the first group were at a different "healthiness baseline" than the individuals of the second group as the study was initiated.

[0041] Administration of transfer factor to each of the individuals of the second group was initiated one week prior to surgery. The treated individuals were each provided with two capsules of TRANSFER FACTOR from 4Life Research, LC, of Sandy, Utah, three times daily, throughout the course of the evaluation.

[0042] The individuals of the second group received no such pre-surgery transfer factor treatment.

[0043] All of the individuals of both the first group and the second group underwent conventional antibiotic treatment and surgery to remove their infections. Following surgery, the osteomyelitis patients of both the first and second groups received four to six weeks of conventional antibiotic treatment (e.g., gentamycin, ampiox, etc.).

[0044] Each of the individuals were evaluated one week before surgery (i.e., at a "baseline" before the individuals in the second group had received transfer factor), one week after surgery, and four weeks following surgery. The ascorbic acid level, thiosulfide antioxidant system (AOS), superoxide dismutase (SOD), glutathioneperoxidase (GPO), catalase, glutathione-s-transferase (G-S-T), malondialdehyde (MDA) level, and protein sulfhydryl (SH) and protein disulfide (SS) groups of each individual were evaluated, as was the cellular membrane integrity as indicated by the erythrocyte stability profiles.

[0045] As shown in TABLE 1, the antioxidant abilities of the individuals in the first and second groups were evaluated. In particular, the ascorbate and thiol antioxidant systems of the individuals, respectively referred to in TABLE 1 as "Ascorbate AOS" and "Thiol AOS," were evaluated. In addition, the levels of various antioxidant enzymes, including SOD, GPO, and catalase, were checked. Levels of G-S-T, an enzyme responsible for removing toxins from the body, were also measured. Protein peroxidation levels were also evaluated.

[0046] The data in TABLE 1 represents average levels of each of the characteristics that were measured in both groups of individuals.

1TABLE 1 Indices of the body non-specific resistance in osteomyelitis patients taking TF Control group Test group 1 Week 4 Weeks 1 Week 4 Weeks Groups Before after after Before after after Indices treatment surgery surgery treatment surgery surgery Low molecular weight antioxidants Ascorbate Tf 26.0 . -. 4.1 13.5* . -. 3.5 17.0* . -. 5.1 14.0● . -. 4.3 18.0● . -. 5.2 16 . -. 4.3 AOS (Mg/l) Of 18.5 . -. 5.0 10.5* . -. 3.1 15 . -. 3.1 12.0 . -. 3.0 11.2 . -. 2.2 15 . -. 3.3 (Mg/l) Rf 5.1 . -. 0.7 4.5 . -. 0.6 2.8* . -. 0.9 2.0● . -. 0.5 4.0 . -. 0.7 3.6 . -. 0.8 (Mg/l) Rf/Of 0.24 0.50 0.21 0.17 0.28● 0.34* Thiol SH 1.36 . -. 0.41 1.28 . -. 0.35 1.24 . -. 0.28 1.20 . -. 0.25 1.12 . -. 0.18 1.38 . -. 0.19* AOS (MM/l) SS 0.50 . -. 0.06 0.52 . -. 0.07 0.44 . -. 0.06 0.44 . -. 0.05 0.44 . -. 0.05 0.38 . -. 0.06 (MM/l) SH/SS 2.5 2.4 2.8 2.7 2.4 3.6 Enzymatic AOS link SOD 63.0 . -. 15.1 32.6* . -. 9.0 59.0 . -. 14.3 53.0 . -. 16.0 47.0 . -. 19.0 34.0* . -. 13.1 (activity/g. sec.) Catalase 1020 . -. 220 757* . -. 186 1200 . -. 235 784 . -. 130 790 . -. 142 1022 . -. 141 (MM/g. sec.) GPO 570 . -. 90 579 . -. 95 535 . -. 105 709● . -. 120 511 . -. 111 542* . -. 123 (MM/g. sec.) G-S-T 53 . -. 9.8 45 . -. 10.6 67 . -. 12.5 21● . -. 11.3 47 . -. 10.1 57.0* . -. 10.7 (MM/g. sec.) General protein 86.0 . -. 12.1 94.0 . -. 14.3 82.5 . -. 13.8 88.0 . -. 12.9 97 . -. 13.0 97 . -. 14.0 hemolysate (×10-4 g/ml) Protein peroxidation SH (MM/l) 7.3 . -. 2.1 7.1 . -. 2.0 7.0 . -. 1.9 6.72 . -. 1.5 6.84 . -. 1.6 7.8● . -. 1.5 SS (MM/l) 3.0 . -. 0.5 2.9 . -. 0.4 2.4 . -. 0.4 2.56 . -. 0.45 2.7 . -. 0.38 2.1 . -. 0.4 SH/SS 2.4 2.4 3.2 2.5 2.35 3.7●* ●statistically significant differences (p ≤ 0.05) as compared with the control group indices *statistically significant differences (p ≤ 0.05) as compared with the indices in the group before the treatment

[0047] From the data in TABLE 1, several of the affects of transfer factor on an infected individual can be seen.

[0048] As one example, the oxidized (Of), reduced (Rf), and total (Tf) ascorbate (i.e., vitamin C) fractions were evaluated. The ratio of the reduced ascorbate fraction to the oxidized ascorbate fraction, or ratio, (Rf/Of) was then determined. The Rf/Of fraction is particularly significant since it provides information about the ability of a subject's body to reduce oxidant levels. More specifically, the reduced form of ascorbate, especially when present in high concentrations, acts as a chemical antioxidant by inactively reacting with oxidants, such as peroxides and free radicals. When oxidants are more likely to react with a chemical antioxidant, such as the reduced form of ascorbate, than proteins, lipids, and other biomolecules, particularly those which are present on or in cell membranes, the incidence of damage to cells and tissues in a subject's body are less likely to be damaged.

[0049] In the geographical region in which these tests were conducted, the Rf/Of ratio of a healthy individual will normally be in the range of about 0.6 to about 0.8. Notably, the Rf/Of ratios in the individuals of the second group (0.17) were initially much lower than the initial Rf/Of ratios of the individuals in the first group (0.24), indicating that, prior to transfer factor treatment, surgery, and antibiotic treatment, the individuals in the second group were initially sicker than the individuals in the first group.

[0050] Moreover, while the Rf/Of ratio does not appear to have increased for the individuals of the first group, who were not treated with transfer factor (the final average was 0.21), which was not unexpected following surgery, a significant, two-fold, increase in the Rf/Of ratio (to 0.34) was seen in individuals who were treated with transfer factor (i.e., those in the second group). This increase in the Rf/Of ratio of the treated individuals was completely unexpected since transfer factor is known to boost the cell-mediated immune response and, thereby, would have been expected to cause an increased oxidant level and, thus, a decrease in the Rf/Of ratio. These results suggest that transfer factor actually enhances the ascorbate AOS of treated individuals.

[0051] When taken in connection with information that suggests that the overall health of the bodies of individuals who have been treated with transfer factor has improved over the same period of time, which is discussed below in reference to TABLE 2, it can be seen that this apparent decrease in oxidant levels is due to a decreased need for a cell-mediated immune response.

[0052] Data that was obtained with respect to the thiol AOSs of the individuals who participated in the study likewise shows that individuals who were treated with transfer factor (i.e., individuals in the second group) exhibited an increase the ratio of reduced thiols (SH), such as glutathione and cysteine, to oxidized thiols (SS), whereas no significant change in this ratio was seen in the individuals of the first group. Again, the increase in the reduced forms (SH) of the molecules that participate in the thiol AOS was unexpected, as transfer factor is known to improve an individual's cell-mediated immune response and, thus, would be expected to result in significantly increased oxidant levels.

[0053] Like the reduced form of ascorbate, reduced thiols (SH) act as chemical "sponges" that react with oxidants in the body to prevent oxidation of proteins and other biomolecules, including those which are present on and in cell membranes. Accordingly, relatively high SH/SS ratios indicate that the general cellular health of an individual is good.

[0054] When taken along with information that indicates that the overall cellular and molecular health of the individual has improved, as discussed in reference to TABLE 2, the increase in the ratio of reduced to oxidized sulfides indicates a decreased need for a cell-mediated immune response.

[0055] Additionally, the information that was obtained about the ascorbate and thiol AOSs of the evaluated individuals indicates that the AOSs of those in the second group, who had been treated with transfer factor, more quickly approach "normal" activity than the antioxidant systems of individuals in the first, untreated group.

[0056] In addition, TABLE 1 shows SOD and GPO levels that were measured in both the first, untreated, and second, transfer factor-treated groups of individuals at one week prior to surgery, one week following surgery, and four weeks following surgery. SOD and GPO levels appear to have decreased slightly in the first group, while levels of these antioxidant enzymes decreased more significantly in the individuals of the second group, who were treated with transfer factor. As known in the art, the production of antioxidant enzymes by a subject is typically increased as the levels of oxidants in the body of the subject increase. Conversely, as oxidant levels in the body of a subject decrease, high levels of antioxidant enzymes are no longer needed and antioxidant enzyme production decreases. Accordingly, the significant decreases in the SOD and GPO levels of the individuals who were treated with transfer factor (i.e., the second group) indicates that transfer factor improved or enhanced (e.g., toward "normal" levels or better) the efficiency with which the antioxidant systems of these individuals worked to remove oxidants from their bodies.

[0057] It is believed that transfer factor may increase the efficiency of a subject's antioxidant systems by one or more of three mechanisms. For example, transfer factor may "lead" natural killer cells to focus more directly on the invading pathogen. As another example, transfer factor may protect the membranes of the cells of an infected subject. Another exemplary mechanism by which transfer factor may increase the efficiency of a subject's antioxidant systems is by actively assisting antioxidants.

[0058] At low levels, catalase works as an antioxidant. At higher levels, however, such as those seen in TABLE 1 with respect to individuals who had been treated with transfer factor, catalase is known to detoxify the body.

[0059] TABLE 1 also shows that the activity of G-S-T, a detoxification enzyme, increased in both the first and second groups of individuals. The mechanism by which G-S-T detoxifies is well known: it binds toxins to glutathione, a solubilizing agent which carries otherwise insoluble toxins out of the body. While the measured increases in G-S-T activity were significant in both the first group and the second group, G-S-T activity increased to a much greater extent in the individuals of the second group than in the individuals of the first group. As GPO and G-S-T share the same intermediate, glutathione, G-S-T levels typically do not increase until there is a corresponding decrease in the amount of GPO present. Accordingly, the increase in G-S-T levels of an individual who has been treated with transfer factor indicates that GPO production is no longer needed to reduce oxidant levels and, thus, that the focus of the body's repair efforts has shifted from reducing oxidant levels to detoxification, or removal of toxins, xenobiotics, "dead" cells, pathogens, and damaged biomolecules. The significantly larger G-S-T levels in the individuals of the second group, to whom transfer factor was administered, indicates that the bodies of these individuals were more efficiently detoxifying themselves. Also, based on the G-S-T measurements that are provided in TABLE 1, it appears that transfer factor decreases the amount of time it takes the body of a treated subject to switch over to the detoxification process.

[0060] In view of these results, the present invention also includes administering transfer factor to a subject to increase the efficiency (e.g., to "normal" levels or better) with which the subject's body detoxifies itself as well as to decrease detoxification time.

[0061] Finally, TABLE 1 includes information about the affect of transfer factor on the "health" (i.e., oxidation) of proteins. In particular, TABLE 1 illustrates that the ratio of reduced sulfhydryl groups on proteins to oxidized sulfhydryl groups on proteins increased in both the first, untreated group and in the second, transfer factor-treated group. The increase in this ratio was more significant, however, in the individuals of the second group, to whom transfer factor was administered, than in the individuals of the first group. As such, it appears that transfer factor is at least partially responsible for preventing protein oxidation and, thus, for improving the overall "health" of the proteins of a subject that has been treated therewith.

[0062] TABLE 2 shows the stability of the membranes of and, thus, the cellular health of erythrocytes (i.e., red blood cells, or rbc's) of the individuals in both the first group and the second group. Erythrocyte stability is an indicator of cellular stability throughout the body of a tested individual. The stability of erythrocytes was measured by exposing them to free radicals, or oxidants. The erythrocyte resistance test is performed to provide an indication of the overall cellular health of an individual who is suffering from a severe infection, such as osteomyelitis. In the erythrocyte resistance test that TABLE 2 illustrates, five categories of erythrocytes are set forth, including prehemolysis, which includes the percentage of erythrocytes that were lysed, or broken, prior to being exposed to free radicals, or oxidants. The remaining four categories of erythrocyte health are based on their relative stabilities when exposed to free radicals, or oxidants over time.

2TABLE 2 Blood erythrocytes resistance (B %) of osteomyelitis patients Control group Test group 4 Weeks 4 Weeks Groups Before 1 Week after after Before 1 Week after after Indices treatment surgery surgery treatment surgery surgery Prehemolysis 1.9 2.5 3.3 2.6 4.5 6.2 Low stable 21 48 68* 63● 58 51● Moderately 58.7 44 25* 31● 23● 38● stable Higher stable 5.2 4.0 3.9● 5.2 4.9 7.9● Highly stable 0.02 0 0 0.02 0.02 0.07● ●statistically significant differences (p ≤ 0.05) as compared with the control group indices *statistically significant differences (p ≤ 0.05) as compared with the indices in the group before the treatment

[0063] The information which is provided in TABLE 2 indicates that, as of one week before surgery, the cellular health of the individuals in the first group, who were not to be treated with transfer factor, was better than the cellular health of the individuals in the second group, who were to be treated with transfer factor. In particular, TABLE 2 indicates that about 66% of the erythrocytes of the individuals in the first group were at least moderately stable, while the about 66% of the erythrocytes of the individuals in the second group were of low stability or worse at the same relative point in time. The overall stability of erythrocytes in the individuals of the first group appears to have decreased four weeks following surgery, as would be expected following a traumatic event such as surgery. In contrast, the overall stability of erythrocytes of the individuals in the second group, who had been treated with transfer factor, appears to have increased by four weeks after surgery. Thus, based on the data which is provided in TABLE 2, treatment with transfer factor appears to improve cellular stability and, thus, cellular health.

[0064] TABLE 3 provides data on the MDA levels of the individuals of the first and second groups, which provides an indication of the blood plasma lipid peroxidation (LPO), or the rate at which fats in the blood are oxidized.

3TABLE 3 Blood Plasma Lipid Peroxidation (LPO) in Osteomyelitis Patients. LPO (by MDA (nmoles/mole)) Before 1 Week after 4 Weeks after treatment surgery surgery Control 2.90 . -. 1.17 3.56 . -. 0.81 3.78 . -. 1.21 Test 3.93 . -. 1.93 3.31 . -. 1.32 3.38 . -. 1.48

[0065] The "Before treatment" levels of MDA shown in TABLE 3 indicate that MDA levels were higher in the patients of the second group prior to being treated with transfer factor and, thus, that the fats in the blood of the individuals of the second group were oxidized to a greater extent than were the fats in the blood of the individuals of the first group. Based on this information, it can be seen that, prior to transfer factor administration and surgery, individuals of the second group were sicker than individuals of the first group. Looking at the data that was obtained one week and four weeks after surgery, opposite trends are seen: oxidation of blood fats in the individuals of the first group increased, while oxidation in the blood fats of the individuals of the second group decreased. From these results, it is evident that the fats of the individuals of the first group became more sickly, while the lipid "health" of the individuals of the second group improved.

[0066] Transfer factor is believed to be responsible for improving (e.g., to "normal" levels or better) the lipid oxidation levels of a subject and, thus, in improving the overall lipid health of a subject. As such, the present invention includes methods for improving the lipid profiles, or health, of a subject by administering transfer factor to the subject.

EXAMPLE 2

[0067] In a second example, the affects of transfer factor on hepatitis patients, including individuals who had been infected with the hepatitis-B virus (HBV) and individuals who had been infected with the hepatitis-C virus (HCV) were studied. The form of viral hepatitis which is caused by HBV causes about two million deaths every year. About two-hundred million people, or about three percent (3%) of the population of the world, are infected with HCV.

[0068] In viral infections, such as viral hepatitis, viruses invade one or more specific types of target cells. In the cases of HBV and HCV, the targeted cells are liver cells, or "hepatocytes." Upon invading a target cell, viruses typically "take over" at least some of the functionality of the cell, often causing the cell to produce more virus particles, then eventually killing the cell as the virus particles are released therefrom.

[0069] In addition, nearby uninfected cells may be indirectly affected by viral infections. This is particularly true in the case of HBV infections, in which most of the damage to the liver is caused by the infected host's own immune system. When cells are damaged by a viral infection or by the host's immune system, the cells release many of their contents, including enzymes, other proteins, nucleic acids, and some of their organelles. As some of the enzymes that are released from a dying or dead cell are typically present only when cell death has occurred, these enzymes may be relied upon a indicators of cell death. Alanine amino transferase (AlAT) and aspartate aminotransferase (AsAT) are two examples of such indicator enzymes. A measure of the amounts of these enzymes in the blood serum of a subject is typically indicative of the level of cell death occurring in that subject.

[0070] Indicator enzyme levels were evaluated in three groups of patients who were suffering from acute HBV infections. The first group included fifteen patients under conventional care (aimed at improving bile secretion and liver metabolism) and to whom one capsule of TRANSFER FACTOR had been administered three times daily for fourteen days. One capsule of TRANSFER FACTOR PLUS, also available from 4Life Research, was administered to the fourteen patients of the second group three times daily for fourteen days. None of the patients of the first or second groups received interferon (a cytokine) treatment. The third group included fifteen patients who received conventional acute HBV infection care, along with interferon treatment. Each group included a similar "cross-section" (i.e., gender, age, etc.) of patients.

[0071] Levels of AlAT and AsAT in the serum of each of these patients were measured during the course of their treatment with TRANSFER FACTOR, interferon, and TRANSFER FACTOR PLUS. On average, the patients of the first group exhibited elevated levels of one or both of AlAT and AsAT for 9.2. -.0.05 days and the levels of AlAT and/or AsAT were above normal in patients of the second group for 10.1. -.0.91 days, while AlAT and/or AsAT levels in the serum of the patients of the third group, who had been treated with interferon, remained elevated for an average of 12.2. -.0.80 days. These results indicate that the transfer factor in both TRANSFER FACTOR and TRANSFER FACTOR PLUS resulted in remission of the symptoms of acute HBV patients in a significantly shorter period of time than interferon treatment caused remission in similar patients.

[0072] These results further indicate that transfer factor improves cell stability, as well as the general cellular health of a treated subject.

[0073] Moreover, treatment regimen that includes transfer factor appears to have been better tolerated by patients than interferon therapy. In particular, all of the patients who had been treated with transfer factor reported a significant improvement of their general state, including lack of excessive fatigue and the absence of discomfort at the locations of their livers.

EXAMPLE 3

[0074] The affects of transfer factor on patients suffering from opisthorchiasis were evaluated in a third example. Opisthorchiasis, which occurs in Eastern and Central Europe, Siberia, and parts of Asia, is caused in mammals, including humans, dogs, and cats, by one of two types of flukes in the infectious metacercaria stage. Mammals typically contract opisthorchiasis by eating raw or undercooked fish.

[0075] An immune imbalance is known to be typical in subjects that are chronically ill with opisthorchiasis.

[0076] Forty-five (45) individuals with chronic opisthorchiasis were split into two groups: a first group including twenty-five (25) individuals and a second group including twenty (20) individuals. The individuals of both groups received conventional praziquantel treatment, an anti-parasitic, or antihelminthic, drug which is used in the treatment of opisthorchiasis and is available under the trade name BILTHRICIDE™ from Bayer AG of Leverkusen, Germany. In addition to praziquantel, two capsules of TRANSFER FACTOR PLUS were administered to the individuals of the first group following praziquantel treatment, three times daily for seven days. The individuals of the second group were only treated with praziquantel.

[0077] Levels of various cytokines, including γ-interferon (IFN-γ), antibodies, and immune complexes were determined, by known processes, for each the individuals prior to therapy and two weeks following TRANSFER FACTOR PLUS therapy in the individuals of the second group was discontinued. The following TABLE 4 lists the collective measures of IFN-γ in both groups, as determined by use of the ProCon IFN-γ assay available from Protein Contour of St. Petersburg, Russia, and photometrically measured at a wavelength of 492 nm. TABLE 4 also includes a collective measure of the IFN-γ levels of fifteen (15) "normal" blood donors.

4TABLE 4 IFN-γ Levels in Chronic Opisthorchiasis Patients First Group Second Group Before Two weeks Before Two weeks Donors treatment after treatment treatment after treatment 46.2 . -. 6.2 43.4 . -. 3.1 96.4 . -. 6.1 42.9 . -. 6.6 51.4 . -. 6.3 p > 0.05 .sup. p > 0.05 p < 0.05 .sup. p > 0.05 p1 > 0.05 p1< 0.05 p2< 0.05 p - statistically significant differences versus blood donors p1 - statistically significant differences prior to and following treatment p2 - statistically significant differences between groups

[0078] These data indicate that, when combined with praziquantel therapy, treatment with TRANSFER FACTOR PLUS resulted in a significant increase in levels of IFN-γ in the individuals of the first group. As is well-known in the art, IFN-γ attracts macrophages, activating them to become more efficient at phagocytosing and destroying invading microorganisms. Stated another way, IFN-γ helps focus the immune system of a treated subject, reducing collateral damage (e.g., in the form increased levels of oxidation or otherwise) that might otherwise be caused by the subject's nonspecific immune response.

EXAMPLE 4

[0079] Similar results were seen in a fourth study, in which the affects of transfer factor on urogenital chlamydiosis patients were determined.

[0080] Among other cytokine levels, levels of IFN-γ were determined for three groups, each including fifteen (15) individuals, and compared with IFN-γ levels of the aforementioned group of fifteen (15) "normal" blood donors. The individuals of a first group were treated with 500 mg of claritomycin twice daily for ten (10) to fourteen (14) days, 100 mg of doxycyclin once daily for ten (10) days, and 200 mg of ofloxacin twice daily for ten (10) days, with the drugs having been administered in succession. The individuals of the second group received 500 mg of claritomycin twice daily for ten (10) to fourteen (14) days and one capsule of TRANSFER FACTOR PLUS three times each day for ten (10) days, with treatment with the claritomycin and TRANSFER FACTOR PLUS beginning on the same day. In the third group, each individual was treated with 500 mg of claritomycin twice daily for ten (10) to fourteen (14) days and one capsule of TRANSFER FACTOR thrice daily for ten (10) days, with administration of the claritomycin and TRANSFER FACTOR PLUS having begun on the same day.

[0081] Known processes were used to determine IFN-γ levels in the individuals of each of the three groups before the treatment regimen started and following completion of the treatment regimen. The following TABLE 5 lists the collective measures of IFN-γ in all three groups, as determined by use of the ProCon IFN-γ assay available from Protein Contour and photometrically measured at a wavelength of 492 nm. TABLE 5 also includes a collective measure of the IFN-γ levels of fifteen (15) "normal" blood donors.

5TABLE 5 IFN-γ Levels in Chlamydia Patients Patients All three Groups First Group Second Group Third Group Before After After After Donors treatment treatment treatment treatment 46.2 . -. 6.2 29.4 . -. 3.1 31.4 . -. 6.1 102.9 . -. 6.6 98.4 . -. 6.3 p < 0.05 .sup. p < 0.05 p < 0.05 .sup. p < 0.05 p1< 0.05 p1< 0.05 p2< 0.05 p - statistically significant differences versus blood donors p1 - statistically significant differences prior to and following treatment p2 - statistically significant differences between groups following treatment

[0082] Similar to the data in EXAMPLE 3, the data of TABLE 5 indicate that, when combined with claritromycin therapy, treatment with transfer factor (in the form of both TRANSFER FACTOR PLUS and TRANSFER FACTOR) resulted in a significant increase in levels of IFN-γ in the transfer factor-treated individuals. Again, it is well-known in the art that IFN-γ is at least partially responsible for focusing the immune system of a treated subject and reducing collateral damage (e.g., in the form increased levels of oxidation or otherwise) that might otherwise be caused by the subject's nonspecific immune response.

[0083] Although the foregoing description contains many specifics, these should not be construed as limiting the scope of the present invention, but merely as providing illustrations of some of the presently preferred embodiments. Similarly, other embodiments of the invention may be devised which do not depart from the spirit or scope of the present invention. Features from different embodiments may be employed in combination. The scope of the invention is, therefore, indicated and limited only by the appended claims and their legal equivalents, rather than by the foregoing description. All additions, deletions and modifications to the invention as disclosed herein which fall within the meaning and scope of the claims are to be embraced thereby.



Inventors

* Dadali, Vladamir A. * Karbisheva, Nina V. * Oganova, Emma A. * McCausland, Calvin W. * Hennen, William J.

US Class 514/1225 or more peptide repeating units in known peptide chain structure

Attorney, Agent or Firm

* TRASK BRITT

International Class 07 A61K038/18



Abstract text

Methods of promoting reproductive health in an animal by administering an effective amount of a composition containing at least one transfer factor are provided. Claims

1. A method of improving reproductive health, comprising the step of administering to a subject in need thereof an effective amount of a composition comprising at least one transfer factor.

2. The method of claim 1 wherein the subject is avian or a mammal.

3. The method of claim 2 wherein the mammal is selected from the group consisting of bovines, porcines, ovines, equines, caprines, felines, canines, and primates.

4. The method of claim 1 wherein the subject is female.

5. The method of claim 1 wherein the subject is male.

6. The method of claim 1 wherein fertility of the subject is improved.

7. The method of claim 1 wherein fecundity of the subject is improved.

8. The method of claim 1 wherein the rate of conception in the subject is increased.

9. The method of claim 1 wherein the number of offspring born alive to the subject is increased.

10. The method of claim 1 wherein the production of ova in the subject is increased.

11. The method of claim 1 wherein the number and/or quality of zygotes recovered from the subject is improved.

12. The method of claim 1 wherein sperm production in the subject is increased.

13. The method of claim 1 wherein the viability of sperm produced by the subject is improved.

14. The method of claim 1 wherein the endocrine function in the subject is improved. Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application claims benefit under 35 U.S.C. .sctn.119(e) of U.S. Provisional Application No. 60/991,681, filed Nov. 30, 2007, the disclosure of which is hereby incorporated by reference herein, in its entirety.

FIELD OF THE INVENTION

[0002]This invention relates generally to the use of compositions and formulations comprising transfer factor in providing health benefits to animals. Such benefits include promoting reproductive health, including aspects of fertility and fecundity.

BACKGROUND OF THE INVENTION

[0003]The present invention relates to the use of compositions and formulations comprising transfer factor, particularly for promoting reproductive health in animals. Other U.S. patents and U.S. patent applications relate to the present invention, including without limitation, U.S. Pat. Nos. 6,506,413 and 6,962,718, U.S. Patent Provisional Application Nos. 60/573,113, 60/649,363, 60/701,860, and 60/814,777, U.S. patent application Ser. No. 11/762,727, U.S. Patent Application Publication Nos. 2006/0029585 A1, 2006/0073197 A1, and 2007/0128253 A1, all of which are incorporated herein by reference. Also related are PCT publications WO/2002/087599 and WO/2005/112891, incorporated herein by reference.

[0004]Transfer factors, which are produced by leucocytes and lymphocytes, are small water soluble polypeptides of about 44 amino acids that stimulate or transfer cell mediated immunity from one individual to another and across species but do not create an allergic response. Since transfer factors are smaller than antibodies, they do not transfer antibody mediated responses nor do they induce antibody production. The properties, characteristics and processes for obtaining transfer factor or transfer factors are discussed in U.S. Pat. Nos. 4,816,563; 5,080,895; 5,840,700, 5,883,224 and 6,468,534, the contents of which are hereby incorporated by reference into the present application.

[0005]Transfer factor has been described as an effective therapeutic for Herpes simplex virus (Viza, et al.), a treatment for acne blemishes, U.S. Pat. No. 4,435,384 and as a treatment against C. albicans (Khan et al.). Transfer factor has also been used to treat intestinal cryptosporidiosis in recipients treated with specific transfer factor (McMeeking, et al.). Still, et al. also showed that chicken pox infections were prevented by pretreatment of children treated with transfer factor from individuals that had chicken pox or who in other words had been sensitized to the varicella antigen. The antigen specific transfer factors are the most well studied and have been demonstrated to be able to convey the antigen recognition ability of the experienced donor to the naive recipient. It may be assumed that the individual or animal that is the source of the transfer factor has been sensitized to the antigen of interest. However, transfer factor as found in commercial bovine colostrum extract coming from a pool of animals (e.g., cows) contains the acquired immunity from all of the pool and therefore provides a type of generalized adoptive transfer of immunity. Transfer factors or transfer factor can be obtained from a dialyzable extract of the lyzed cells or from an extract of extracellular fluid containing transfer factor. Common sources of transfer factors are colostrums and ova. It is common practice to refer to preparations that contain transfer factor by the name of the active component (i.e., transfer factor or TF). Transfer factor extract containing transfer factors is also herein referred to as transfer factor. Transfer factor from bovine colostrum extract is defined as defatted water soluble material from colostrum that will pass through a nominal 10,000 molecular weight filter. The colostral derived transfer factor has been prepared with activity against various organisms including infectious bovine rhinotracheitis virus. One of the specific effects of transfer factor is a significantly increased natural killer (NK) cell activity. Natural killer cells provide protection against viruses as part of the innate immune defense system.

[0006]Although transfer factor is a polypeptide, it has been reported that it is surprisingly stable in the gastrointestinal tract. For example, Kirkpatrick compared oral versus parenteral administration of transfer factor in clinical studies. Kirkpatrick, Biotherapy, 9:13-16, 1996. He concluded that the results refute any arguments that the acidic or enzymatic environment of the gastrointestinal tract would prevent oral therapy using transfer factors.

[0007]When attempts were made to sequence TF, it was reported that an N-terminal end of the transfer factor peptide is resistant to sequential Edman degradation. Kirkpatrick, Molecular Medicine, 6(4):332-341 (2000).

[0008]Accordingly, transfer factor was believed to be stable in the gastrointestinal tract and rumen. However, it has since been shown that transfer factor is not as stable as once believed. It appears to be particularly unstable in the digestive tract of ruminants.

[0009]Transfer factors have been used successfully in compositions for treating animal diseases and syndromes including those in ruminants, as well as in other animals. See, for example, U.S. Pat. No. 6,962,718.

[0010]The present inventor has recognized an unmet need for effective compositions and methods for enhancing reproductive function in an animal.

BRIEF SUMMARY OF THE INVENTION

[0011]In certain aspects, the present invention is directed to methods of promoting reproductive health in an animal comprising the administration to a subject of an effective amount of a composition and/or formulation comprising at least one transfer factor.

[0012]Both the foregoing general description and the following detailed description are exemplary and explanatory, and are intended to provide further explanation of the invention as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0013]It has been found by the present inventor that the administration of compositions comprising transfer factor provides surprising and unexpected improvements in reproductive health and function. These results include, but are not limited to, improvements in fertility and fecundity in both males and females in a variety of animal species.

[0014]In certain embodiments, the present invention is directed to methods for enhancing the reproductive capability of an animal. In preferred embodiments, these methods comprise the administration to an animal of an effective amount of a composition and/or formulation comprising at least one transfer factor.

[0015]As used herein, the term "effective amount" refers to an amount of a compound, material, composition, formulation and/or dosage form as described herein that may be effective to achieve a particular biological result. Such results may include, but are not limited to, enhancement in the reproductive capability of an animal. An effective amount of a composition to enhance reproductive capability refers to an amount that causes an animal to demonstrate greater reproductive capability, including, but not limited to, fertility and/or fecundity, than an animal would otherwise demonstrate in the absence of the composition under otherwise prevailing conditions.

Transfer Factor

[0016]According to certain aspects of the invention, compositions and formulations are provided comprising transfer factor. According to certain embodiments of the invention, various forms of transfer factor may be used. They include, without limitation, excreted transfer factor released from transfer factor containing cells such as lymphocytes, leukocytes, and ova, and collected from extracellular fluids such as colostrums and blood. Another form includes preexcreted transfer factor found within the cell or on the cell surface. In certain embodiments of the invention, substantially purified transfer factor originating from leukocytes, colostrum, or ova and having a molecular weight of less than 10,000 daltons and a specific activity of at least 5000 units per absorbance unit at 214 nanometers, may also be used. The transfer factor used in the Examples of this invention and referred to in the following Tables and further referred to in the rest of the detailed description is generally extracted from colostrum collected from a general pool of lactating cows; although, in some cases, it is derived from eggs. Though bovine colostral derived transfer factor was generally used to develop the formulations of this invention, it is well known to anyone skilled in the art that other kinds and sources of transfer factor could be used.

[0017]Alternative sources of transfer factor include, but are not limited to, avian transfer factor, ova transfer factor, and transfer factor isolated from colostrum collected from non-bovine animals such as goats, pigs, horses and humans. In addition, combinations of transfer factors from any number of sources may be used in the formulations of the instant invention. Transfer factor may also be derived from recombinant cells that are genetically engineered to express one or more transfer factors or by clonal expansion of leukocytes.

Isolation of Transfer Factor

[0018]In certain embodiments of the invention, transfer factor may be obtained from colostrum. In a preferred embodiment, transfer factor is obtained from bovine colostrum. The fraction of colostrum comprising material having a molecular weight of approximately 10,000 daltons (Da) and below is designated as transfer factor. A fraction obtained that is approximately 10,000 to approximately 150,000 Da is designated an antibody fraction, also known as an antibody-colostrum fraction. In certain embodiments, a colostral fraction having a molecular weight of about 10,000 to about 400,000 Da may be used as an antibody fraction. The fraction comprising material having a molecular weight of approximately 10,000 Da and above is designated as the growth factor fraction. The growth factor fraction may include high molecular weight proteins.

[0019]In certain embodiments, an antibody fraction comprises antibodies from about 1% to about 99%, about 5% to about 95%, about 10% to about 90%, about 15% to about 85%, about 20% to about 80%, about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, about 45% to about 55%, or about 50% by weight, the remainder comprising other colostrum components.

[0020]According to certain embodiments of the invention, transfer factor, as used in the formulations described in the Tables, particularly when not defined as obtained from an avian source, may be further defined as defatted water soluble material from bovine colostrum that will pass through a nominal 10,000 molecular weight filter.

[0021]In other embodiments, the transfer factor may be obtained from an avian source. In one embodiment, chickens are given a feed mixture containing excrement from an animal, including without limitation, at least one selected from the group consisting of a human, a fish, a goat, a llama, an alpaca, a pig, a sheep, a cow, and a horse. The excrement will contain a large variety of pathogens and upon administration in a feed to an animal, the animal will develop transfer factor and/or antibodies to such pathogens. Avian transfer factor can then be obtained from the eggs produced by the above-treated chickens. In certain embodiments of the invention, transfer factor may be found in whole egg yolks. As a non-limiting example, the transfer factor of avian source (which is believed to also contain antibodies) listed in the formulation of Table 3 is supplied as powdered whole egg yolks.

[0022]Alternative kinds of transfer factor include, but are not limited to, targeted transfer factors. Target transfer factors include transfer factor collected from sources which have been exposed to (1) one or more viral or otherwise infectious organisms; (2) one or more antigens that produce an immune response; or (3) a combination of organisms and antigens. The term antigen is defined herein is anything that will initiate the cell mediated immune response. Examples of such viral or other infectious organisms include Herpes Simplex Virus 1, Herpes Simplex Virus 2, H. Pylori, Camphobacter and Chlamydia, Bovine Rhinotracheitis Virus, Parainfluenza, Respiratory Syncytial Virus Vaccine, modified live virus, Campylobacter Fetus, Leptospira Canicola, Grippotyphosa, Hardjo, Leterohaemorrhagiae, Pomona Bacterin, Bovine Rota-Coronavirus, Escherichia Coli Bacterin, Clostridium Chauvoei, Septicum, Haemolyticum, Novy, Sordellii, Perfringens Types C & D, Bacterin, Toxoid, Haemophilus Somnus, Pasteurella Haemolytica, Multocida Bacterin. However, one of skill in the art would readily recognize that a wide variety of other viral and otherwise infectious organisms can find use in the instant invention.

[0023]Additionally, transfer factor and antibodies may be derived from any suitable source, as described, for example, in U.S. Pat. Nos. 4,816,563; 5,080,895; 5,840,700; 5,883,224; and 6,468,534; and U.S. patent application Ser. No. 11/762,727, the contents of which are hereby incorporated by reference herein.

[0024]In certain embodiments, the component of a given formulation that is referred to as the "transfer factor" may optionally include a colostral component of higher molecular weight; for example, a portion of the fraction referred to above as an antibody or antibody-colostrum fraction. In certain embodiments, the mammalian "transfer factor" component of a formulation comprises both transfer factor fraction and antibody fraction. In certain preferred embodiments, "mammalian transfer factor" comprises about 70% transfer factor fraction from colostrum (i.e., 10,000 Da or below colostrum fraction) and about 30% antibody colostrum fraction. In other preferred embodiments, "mammalian transfer factor" comprises about 80% transfer factor fraction from colostrum and about 20% antibody colostrum fraction. (The foregoing are in weight percents of the composition). In certain embodiments, the "transfer factor" component of the composition or formulation may include one or both of mammalian and avian transfer factor.

Lyophilization

[0025]The present invention also provides compositions and formulations that have one or more lyophilized component(s). Lyophilization or "freeze-drying" is a process well known to those of ordinary skill in the art. For example, some techniques of lyophilization are described in Akers, Michael J., Chapter 41 in Remington The Science and Practice of Pharmacy, 828 (David B. Troy ed., Lippincott Williams & Wilkins 2006), which is incorporated herein by reference. In certain embodiments, formulations and/or compositions of the present invention may include lyophilized transfer factor. In certain embodiments, transfer factor, which may be lyophilized, may be combined with one or more of: an antibody or an antibody fraction, a growth factor fraction, or some other colostral fraction; one or more of which may, in certain embodiments, be lyophilized. In other embodiments, additional components of formulations and compositions of the invention may be lyophilized, including, without limitation, other peptides and proteins.

Formulations

[0026]In certain embodiments of the invention, transfer factor is provided in a formulation that further comprises one or more additional ingredients. In preferred embodiments, the formulation comprises transfer factor and at least one glucan. In some embodiments, transfer factor fraction and at least one glucan is provided in a formulation that further comprises one or more additional ingredients. In certain embodiments, the transfer factor fraction may be lyophilized. In certain embodiments, the optional antibody or antibody fraction may be lyophilized.

[0027]In a preferred embodiment, transfer factor is present in the formulation in the amount of about 10 mg to about 12 gm/oz, more preferably about 100 mg to about 6 gm/oz and most preferably about 10 mg to about 3 gm/oz. In certain preferred embodiments, such a formulation comprising transfer factor is provided to an animal in an amount of about 1 oz per 1000 lb of animal.

[0028]In certain embodiments of the invention, formulations are provided which comprise glucans. Glucans may be derived from any suitable source, including, but not limited to, fungi, oats, and yeast. Preferably, glucans are present in or derived from fungi. In certain embodiments, the glucans which may be included in the formulations are present in whole fungi. In certain preferred embodiments, glucans are present in or derived from Cordyceps, more preferably, Cordyceps sinensis.

[0029]In certain embodiments, glucans are derived from hybrid strains of fungi. In a preferred embodiment the hybrid glucans used in the invention are present in, or derived from, hybrid strains of Cordyceps and in particular Cordyceps sinensis. One technique to induce the hybridization of Cordyceps involves plating two different strains or species on a single agar plate which has been inoculated with rattlesnake venom as described in, for example, U.S. Patent Application Publication No. 2006/0073197, published Apr. 6, 2006, and U.S. Patent Application Publication No. 2007/0128253, published Jun. 7, 2007, each of which is incorporated herein by reference. In a preferred embodiment, the hybrid strain producing the hybrid glucans that may be used in compositions and formulations of the invention is Cordyceps sinensis Alohaensis, which is available from Pacific Myco Products, Santa Cruz, Calif.

[0030]In addition to Cordycep sinensis hybrids, suitable sources of glucans may include, but are not limited to, Agaricus blazeii, Coriolus, Poira Cocos, Inonotus obliquus, Maitake Mushroom, Shiitake Mushroom, and combinations thereof.

[0031]When glucans are used, the formulation preferably contains about 10 mg to about 18 gm of whole organism/oz, more preferably about 100 mg to about 10 gm of whole organism/oz and most preferably about 100 mg to about 5 gm of whole organism/oz.

[0032]Equivalent amounts of purified or partially purified glucan as well as the nucleosides associated therewith (e.g., Cordycepin (3'deoxyadenosine), adenosine and N6-(2 hydroxyethyl)-adenosine) may also be used.

[0033]In certain preferred embodiments of the invention, formulations that may be administered to a subject, may comprise, in addition to transfer factor, which, in preferred form, may be derived from mammalian and/or avian source, one or more of the following: at least one glucan, mammalian antibody-colostrum fraction, mammalian growth factor fraction, other mammalian colostral fraction, and avian antibodies or antibody fraction (which may, in certain embodiments, be obtained from whole egg yolk).

[0034]In certain preferred embodiments, compositions may further comprise one or more of inositol hexaphosphate (Ip6), mannans, olive leaf extract, and phytosterols. In certain preferred embodiments, mannans are derived from Aloe vera. In certain preferred embodiments, phytosterols may be derived from soya bean.

[0035]In certain embodiments, compositions may further comprise one or more of lactic acid producing bacteria, ascorbic acid, Vitamin A, Vitamin D3, Vitamin E, Vitamin B1, Vitamin B2, Vitamin B12, dipotassium phosphate, potassium chloride, magnesium sulfate, and calcium pantothenate.

[0036]In certain embodiments, compositions and formulations comprising transfer factor may be combined with minerals, antioxidants, amino acids, and other nutraceuticals.

[0037]In certain embodiments, the invention provides compositions and formulations in which one or more components are encapsulated. Encapsulation may be achieved by mixing the component to be encapsulated with a hydrophobic substance or a lipid to form a coating around the component. Encapsulation may protect labile components from inactivation in the gastrointestinal tract. Such encapsulation may be important especially in the case of ruminants where digestion within the rumen has been found to interfere with the administration of certain labile factors. Enhanced bioavailability has been demonstrated, for example, when a transfer factor is encapsulated and administered to ruminants.

[0038]Previous use of non-encapsulated transfer factor in ruminants, e.g., cows, produced significant beneficial results. See, e.g. U.S. Patent Publication 2003/0077254, published Apr. 24, 2003 incorporated herein by reference in its entirety. Subsequently, it was discovered that transfer factor was not stable by oral administration in a stressed population of cattle. After discovering that transfer factor is inactivated in vitro in the presence of rumen fluid and flora, it was determined that prior success with transfer factor in ruminants was due to the presence of the esophageal groove. When not stressed, the esophageal groove provides partial bypass of the rumen. However, in a stressed population the esophageal groove closes and shunts the transfer factor formulation into the rumen. It was discovered that encapsulating transfer factor and/or glucans with a hydrophobic substance or a lipid to form an encapsulated formulation is sufficient to provide substantial by-pass of (e.g., about 85%) of the rumen even in a stressed population.

[0039]While not intending to be bound by any theory or theories of operation, it is believed that encapsulation of transfer factor fraction may increase its bioavailability upon administration to fermenting animals, such as adult ruminants.

[0040]In preferred embodiments, the transfer factor is encapsulated by mixing with a hydrophobic substance or a lipid to form a coating around the growth factor(s). In additional embodiments, one or more additional components such as antibody, antibody fraction, and/or glucans may be encapsulated. Other optional components of compositions and formulations of the invention may be encapsulated, such as, without limitation, inositol hexaphosphate, olive leaf extract, mannans, phytosterol, vitamin C and mixtures thereof. The transfer factor, antibody or antibody fraction and/or additional optional components may each be individually encapsulated or encapsulated as a mixture. Alternatively, the entire formulation can be encapsulated. The encapsulated component(s) and/or formulation can be produced in a variety of ways. In a preferred embodiment, each of the transfer factor, glucans, antibody or antibody fraction and/or additional labile component(s) in the formulation may be encapsulated as described in U.S. Pat. Nos. 5,190,775, 6,013,286 and U.S. Application 2003/0129295, each of which is incorporated herein by reference in its entirety.

[0041]The transfer factor may be encapsulated with a hydrophobic or lipid coating that is preferably between about 25% and about 150 wt % of the transfer factor, about 50-150 wt % and about 75-125 wt %, with an equal weight being most preferred.

[0042]In additional embodiments of the invention, additional components may be used in the formulation administered. Particular components may be encapsulated. For example, IP6, β-sitosterol, olive leaf extract, aloe extract matter and/or vitamin C may be used; in certain embodiments, one or more of these components may be encapsulated. In preferred embodiments, IP6 is present at between 10 mg and 3 gm/oz, or one preferably between 100 mg and 2 gm/oz, and most preferably between 100 mg and 1 gm/oz. The P-sitosterol is preferable in the amount of between 10 mg and 3 gm/oz, or preferably between 100 mg and 2 gm/oz, and most preferably between 100 mg and 1 gm/oz. Olive leaf extract is preferably present in the amount of 2 mg to 2 gm/oz, more preferably between 5 mg and 1 gm/oz, and most preferably between 5 mg and 500 gm/oz. Aloe extract is preferably present at between 2 mg and 1000 mg, more preferably between 5 and 500 mg/oz, and most preferably between 5 and 250 mg/oz. Vitamin C may be present at between 10 mg/oz and 10 gm/oz, or preferably between 100 mg and 8 gm/oz, and most preferably between 100 mg and 5 gm/oz.

[0043]In certain embodiments, glucans of the formulation may be encapsulated, preferably with a hydrophobic or lipid coating. It is preferred that the amount of hydrophobic or lipid coating be between about 25% and 150 wt % of the glucan, about 50-150 wt %, or about 75-125 wt %, with an equal weight being most preferred.

[0044]Columns 2, 3 and 4 of Tables 1-3 show the approximate high, low and preferred amounts, respectively, of the formulation components, in amounts per body weight, to be given to an animal in a single dosage. The formulation represented in Table 2 is designed preferably for livestock. The 5 ounces of the formula listed in column 5 is designed to be given to a 1000 pound animal but that will vary and could be given to a 500 pound animal in some cases. The average horse is around 1000 pounds. However, since these formulas are comprised of nutraceuticals and transfer factor, one skilled in the art will recognize that the ranges are not certain and as critical as the ranges for allopathic drugs.

[0045]Table 3 provides an exemplary encapsulated transfer factor formulation for administration to subjects. In certain embodiments, a transfer factor formulation includes at least encapsulated transfer factor derived from bovine and/or avian sources, and/or one or more of hybrid glucans. It is preferred that the glucan portion of this formulation also be encapsulated. Other components include zinc proteinate, targeted avian transfer factors, β-sitosterol, inositol hexaphosphate (IP6), olive leaf extract, aloe extract powder, probiotics, B. subtlis, B. longum, B. thermophilium, L. acidophilus, E. faecium, and S. cerevisiae. In a preferred embodiment, all of the foregoing are included in this transfer factor formulation.

[0046]In another preferred embodiment, a formulation is provided according to Table 3, but with the following modifications. The component listed as "Transfer factor (mammal source)" is substituted with a composition containing 80% bovine colostrum transfer factor as described herein, combined with 20% bovine colostrum antibody fraction as described herein (both weight percents of the composition). In certain embodiments, the mammalian transfer factor and the colostrum antibody fraction are both lyophilized. In a preferred embodiment, the component listed as "Transfer factor (avian source)" is present in the formulation in an amount of 3000.0 mg/oz. This component may be supplied as powdered whole egg yolk obtained from hyperimmunized chickens, i.e., chickens that had been exposed to pathogens prior to laying the eggs which serve as a source of transfer factor. In various embodiments, the avian transfer factor may be obtained from commercial sources such as, for example, 4LIFE.RTM. Research; Labelle, Inc., Bellingham, Wash.; Troue; and Ghen Corporation, Japan.

[0047]The amount of transfer factor and/or antibody or antibody fraction used in the formulation or the amount of formulation administered will vary depending upon the severity of the clinical manifestations presented. In addition, the amount of transfer factor administered to a recipient will vary depending upon the species from the transfer factor is derived as compared to the species of the recipient. It has been observed that transfer factor derived from bovine species administered to cattle is more efficacious than transfer factor from another species such as avian species. Accordingly, when the source of the transfer factor and recipient are different species, it is preferred that the amount of transfer factor be increased.

[0048]It is preferred in formulations used in the methods described herein that the metal nutraceuticals are proteinated because these forms are easier for the animal to digest and also because the proteinate forms are more stable to pH. The nutraceutical components in the formulations in Tables 1-3 are active components for treating the various described conditions. The fillers and carriers are included to make the formulations more palatable to the animal and also to help preserve the mixture. These include silicon dioxide, maltodextrin, soy and peanut flour, peanut oil, dextrose, whey, spices and flavorings. Mixed tocopherols and choline chloride are nutraceuticals but the effective results described herein can still be achieved by deleting these two components from the formulations.

Methods of Administration

[0049]As discussed herein, it was discovered that encapsulating transfer factor and/or glucans with a hydrophobic substance or a lipid to form an encapsulated formulation is sufficient to provide substantial by-pass of (e.g., about 85%) of the rumen even in a stressed population of ruminants.

[0050]A variety of other methods for rumen by-pass are known. In one embodiment, the encapsulated or non-encapsulated formulation is directly injected (subcutaneously, intramuscularly, or intravenously) to by-pass not only the rumen but also the entire digestive system. Similarly, intravaginal, intrarectal or other direct administration to mucus membranes, such as the eye subconjunctival, by-pass the digestive system and the rumen in particular. Alternatively, the formulation can be mixed with various solvents which allow for direct skin absorption. Furthermore, methods are known in the art to stimulate opening of the esophageal groove in various ruminants and such opening allows for immediate passage of an orally administered formulation to the gastrointestinal tract, by-passing the rumen.

[0051]In certain embodiments, transfer factor compositions and/or formulations may be included in food. Preferred embodiments for human consumption include, but are not limited to incorporation of transfer factor formulations in processed foods such as cereals, snacks, chips, or bars. Preferred embodiments for animal consumption include, but are not limited to, transfer factor formulations admixed in feed pellets, salt licks, molasses licks or other processed feed products.

[0052]Other methods of administration to animals, including, but not limited to, drenching, may also be employed.

[0053]The transfer factor formulations used in the present invention include pharmaceutical compositions suitable for administration. In a preferred embodiment, the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. "Pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.

[0054]The pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers such as sodium acetate; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol. Additives are well known in the art, and are used in a variety of formulations.

[0055]In a further embodiment, the pharmaceutical compositions may be added in a micellular formulation; see U.S. Pat. No. 5,833,948, hereby expressly incorporated by reference in its entirety.

[0056]In certain aspects, the components of the compositions and pharmaceutical formulations of the present invention may have an effect upon administration individually, such as, for example, the enhancement of aspects of reproductive function, but upon administration in one or more combinations, have an effect that is synergistic. By "synergistic" is meant an enhancement of the effect of one or more combined components in a more than additive fashion relative to the effect of each component when used alone.

[0057]In certain embodiments, the present invention provides methods involving administration of compositions and/or formulations comprising transfer factor to a subject. As used herein, the term "subject" is used to mean an animal, including, without limitation, an avian or a mammal. Mammalian subjects include, without limitation, primates, bovines, porcines, ovines, equines, caprines, and carnivores, including, but not limited to, felines and canines. The mammal may be a human.

[0058]Combinations of pharmaceutical compositions may be administered. Moreover, the compositions may be administered in combination with other therapeutics.

[0059]In certain embodiments, the present invention provides methods of treating a subject by administering a composition or formulation comprising transfer factor, as described herein. In certain embodiments, such methods are provided for improving the reproductive health of the subject by administration of transfer factor. In particular embodiments, methods are provided of increasing fertility and/or fecundity in a subject by the administration of transfer factor. As used herein, "fertility" refers to the ability to produce offspring. As used herein, "fecundity" refers to the efficiency of an individual in production of young. Animals that bring forth young frequently, regularly, and, in case of those that bear more than one offspring at a birth, in large numbers, are said to be fecund.

[0060]In certain embodiments of the invention, the subject is female. In other embodiments, the subject is male.

[0061]In certain aspects, methods according to the invention may be used to improve the rate of conception in female subjects. Example 6 demonstrates the positive effects of the administration of a transfer factor formulation on the rate of conception in sheep. Example 8 demonstrates the positive effects of the administration of a transfer factor formulation on the rate of conception in pigs.

[0062]In certain aspects, administration of transfer factor to subject may be used to increase the number of offspring born alive to the subject.

[0063]In certain aspects, methods according to the invention may be used to improve the quality and/or quantity of ova and/or zygotes that may be obtained from female donor animals. Embryos from donor animals are subsequently transferred into recipient females, which give birth to the offspring.

[0064]In a particular embodiment, transfer factor is administered to a donor animal that is a cow. Examples 1 and 3 demonstrate the positive effects of administration of transfer factor formulation on the quantity and quality of eggs produced by donor cows.

[0065]In other embodiments, male reproductive health and function may be improved by the administration of compositions and/or formulations comprising transfer factor. This function may include improvements in the quantity and/or quality of sperm produced by the male. Example 5 demonstrates the positive effect of administration of transfer factor on the viability of sperm produced by the animal. Such effects may increase the commercial value of animals whose sperm is a commodity.

[0066]Other examples demonstrate efficacy of transfer factor formulations in improving reproductive health and function. Example 10 describes the return to reproductive health of a mare diagnosed with an ovarian tumor after a course of treatment involving administration of a transfer factor formulation. In certain embodiments, administration of transfer factor according to the methods described herein may cause an improvement in the endocrine function of the subject.

[0067]Without intending to be bound by any theory or theories of operation, it is postulated that at least some of the beneficial effects of transfer factor on reproductive health may be due to the reduction of (possibly undiagnosed) tumors or cysts in the reproductive organs that may compromise reproductive function in the animal.

[0068]Additional benefits of transfer factor on reproductive health and function may be obtained. Without intending to be bound by any theory or theories of operation, the following benefits may be obtained by the administration of transfer factor to a subject. Administration of transfer factor may improve the balance of cortisol in the subject, thereby reducing the effects of stress, which, in turn, may lead to good endocrine function prior to breeding and/or conception. Administration of transfer factor is thought to lead to improved immune health, thus providing a female subject with a "clean" oviduct and/or endometrium (lining of the uterus), thus providing a fertile bed for implantation of fertilized eggs. Regarding the improvement in egg quality that has been described herein, it is possible that the health benefits of transfer factor may include the induction of a cascade of good endocrine function, leading to luteal activity that produces high-quality ova (eggs).

EXAMPLES

[0069]The following examples serve to more fully describe the manner of using the above described invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes.

TABLE-US-00001 TABLE 1 Transfer Factor Formula (No Encapsulation) (Amounts in mg/lb of body weight unless otherwise stated) Dosage: mg/oz. (unless otherwise noted) of Component formula Transfer factor (mammal 1750.0 source) Transfer factor (avian 750.0 source) β-sitosterol (90% 150.0 phytosterols) Inositol hexaphosphate 175.0 Olive leaf extracts 17.5 Aloe extract powder 8.5 (200:1) Hybridized and non- 2000.0 hybridized Glucans (from Hybridized Cordyceps sinensis, Agaricus blazeii, Miatake, Shitake, Coriolis, Inonotus obliquus, and Poris cocos mushrooms) Vitamin C 1000.0 Vitamin A 4434 IU/oz Vitamin D3 1140 IU/oz Vitamin E 500 IU/oz Vitamin B1 12.77 Vitamin B2 12.77 Vitamin B12 1.5 Di-potassium phosphate 1.5 g/oz Potassium chloride 207 Magnesium sulfate 83 Calcium pantothenate 23 Ascorbic acid 23 Lactic acid bacteria 2.5 × 106 CFU/oz Yeast (S. cerevisiae) 15.0 × 106 CFU/oz Zinc proteinate 10 *These amounts are calculated for livestock animals weighing about 450 to 1,000 pounds, goats weighing about 150 pounds, and dogs and cats weighing from about 8 to about 15 pounds.

TABLE-US-00002 TABLE 2 (Amounts in mg/lb of body weight unless otherwise stated) Dosage: mg/5 oz. Component High Low Preferred of formula 1-Arginine 0.5 0.005 0.05 50.00 *Lacto yeast (4.9% of 69.51 0.6951 6.91 6951.88 blend) Montmorillinite 1 gm/lb 0.24118 2.4118 2411.88 Soybean oil (14.5% mix) 1.5 gm/lb 2.05 20.571 20571.88 Safflower oil (14.5% mix) 1.5 gm/lb 2.05 20.57 20571.88 Flax seed oil (55% Alpha 1.5 gm/lb 2.05 20.571 1418.75 Linolenic Acid) (1.0% mix) Phosphorous (Monosodium 15.750 gm 0.0525 5.08 5080.00 phosphate) 12% Calcium carbonate 8.5% 13.68 gm 0.0485 4.88 4880.00 (38% calcium) Methyl sulfonyl methane 20 0.02 2 2000.00 Vitamin C (ascorbic acid) 21.62 0.2162 2.162 2162.50 d-Biotin (Vitamin H 2%) 9.73 0.000973 0.00973 10.00 Vitamin D3 29.16 IU 0.7298 IU 7.298 IU 7298.38 IU Vitamin B12 0.092 0.000092 0.00092 0.92 Folic Acid 1 0.001006 0.01006 10.06 Niacinimide 12 0.012157 0.12157 121.57 Pantothenic acid (d- 0.324 0.0108 0.108 108.00 Calcium Pantothenate) 91.6% Vitamin B6 (Pyridine Hcl) 1.158 0.001158 0.01158 11.58 82.3%) Vitamin A (Retinol 600 IU 4.02 IU 40.212 IU 40232.50 IU Palmitate) 650M IU/g feed grade Vitamin B2 0.0554 0.002776 0.02776 27.76 Thiamine (Mononitrate) 3.09 0.00308 0.0308 30.80 83% Vitamin E 72.9 IU 0.0729 IU 0.729 IU 729.42 IU Vitamin K 1 0.0007 0.007 7.00 Cobalt (Proteinate) 5% 0.00043 0.000043 0.00043 0.43 Copper (Proteinate) 10% 0.56 0.0112 0.112 112.00 Iodine (Potassiumiodide) 0.005 0.000053 0.00053 0.53 98% Iron (Proteinate) 15% 3.31 0.0331 0.331 331.16 Magnesium (Oxide) 58% 10 0.04 0.4 400.00 Manganese (Proeinate) 1.65 0.04 0.4 332.10 15% Molybdenum (Sodium 0.05 0.001 0.01 10.00 Molybdate Dihydrate) 39% Selenium (Sodium 0.00162 0.000081 0.00081 1.00 Selenite) 44.8% Zinc (Proteinate) 15% 50 0.04987 0.4987 498.72 1-Lysine (Mono HCl) 8.41 0.0841 0.841 841.57 d,1-Methionine 11.03 0.1103 1.103 1103.86 Mixed Tocopherols 300.00 Choline Chloride 2434.00 Sipernat 50 (Silicon 12768.75 dioxide) Lodex-5 (maltodextrin) 7519.38 Soy flour (17.5% mix) 24828.13 Sweet whey 996.00 BF70 spice 146.00 Dextrose powder 750.00 Transfer factor - blend of 750.00 mg/ 70% mammalian and 30% avian 141 gram Olive leaf extract, Ip6, 750.00 mg/ mannins and phytosterol 141 gram blend Poly R blend of Glucans 1000 mg/141 (from Hybridized grams Cordyceps sinensis, Agaricus blazeii, Miatake, Shitake, Coriolis, Inonotus obliquus, and Poira cocos mushrooms) *Lactic acid generating bacteria is two-thirds of component and yeast is one-third; lactic acid generating bacteria is 500,000,000 CFU/gm, yeast (e.g., "Saccharamyces") 250,000,000 CFU/gm

TABLE-US-00003 TABLE 3 Livestock Stress Rumen By-Pass (Amounts in mg/lb of body weight unless otherwise stated) Dosage: mg/oz. (unless otherwise noted) of Component formula Stabilized1 Transfer factor (mammal 3500.0 source) Transfer factor (avian 1500.0 source) β-sitosterol (90% 300.0 phytosterols) Inositol hexaphosphate 350.0 Olive leaf extracts 35.0 Aloe extract powder 17.0 (200:1) Hybridized and non- 4000.0 hybridized Glucans (from Hybridized Cordyceps sinensis, Agaricus blazeii, Miatake, Shitake, Coriolis, Inonotus, Obliquus, and Poris cocos mushrooms) Vitamin C 2000.0 Non-Stabilized Vitamin A 4434 IU/oz Vitamin D3 1140 IU/oz Vitamin E 500 IU/oz Vitamin B1 12.77 Vitamin B2 12.77 Vitamin B12 1.5 Di-potassium phosphate 1.5 g/oz Potassium chloride 207 Magnesium sulfate 83 Calcium pantothenate 23 Ascorbic acid 23 Lactic acid bacteria 2.5 × 106 CFU/oz Yeast (S. cerevisiae) 15.0 × 106 CFU/oz Zinc proteinate 10 *These amounts are calculated for livestock animals weighing about 450 to 1,000 pounds, goats weighing about 150 pounds, and dogs and cats weighing from about 8 to about 15 pounds. 1Stabilized active ingredients are included in a formulation of 50% soybean oil and 50% active ingredient.

Example 1

Effects of Transfer Factor on Yields of Bovine Embryos

[0070]The effects of formulations containing transfer factor was investigated in cows intended to be used as donors for embryo transfer. In this process, fertilized ova are flushed from the uterus of a donor cow and subsequently implanted in a recipient cow. Individual fertilized eggs (referred to herein as "embryos"; also termed "zygotes") are examined via microscope and evaluated for quality; each embryo being classified numerically as to the potential likelihood of success if transferred to a recipient cow. The major criteria for evaluation include: regularity of the shape of the embryo; compactness of the blastomeres (the dividing cells within the boundaries of the embryo); variation in cell size; color and texture of the cytoplasm; overall diameter of the embryo; presence of extruded cells; regularity of the zona pellucida; and the presence of vesicles. Using these subjective criteria, embryos are classified as: Grade 1: Excellent or Good; Grade 2: Fair; Grade 3: Poor; Grade 4: Dead or degenerating. (See, e.g., Embryo Transfer in Cattle, Glenn Selk, Oklahoma State University, No. 3158). Higher grade embryos are also more likely to survive storage by freezing. Overall, a Grade 1 egg has about a 98% probability of leading to conception in recipient cows, while lower grades have about a 60% or lower chance of implanting in the uterus and thus are much less likely to produce a pregnancy in a recipient.

[0071]Nine cows were given 1 oz of the formulation described above in Table 3 on Day 7 and Day 6 prior to the removal of embryos from the donor via flushing. The following table shows the total number of eggs recovered from a given donor animal and the number of eggs graded as number 1 (highest quality embryos), as documented before transfer factor (TF) formulation was supplied, and the increases in both quantity and quality of embryos recovered after administration of formulation comprising transfer factor.

TABLE-US-00004 TABLE 4 Flush Results - No TF Flush Results Post-TF Total Number of Total Number of Number of Grade 1 Number of Grade 1 Animal Embryos Embryos Embryos Embryos 1 9-16 6 27 27 2 6-12 4 27 (1 #2 embryo) 26 3 2-6 4 12 12 4 3-9 6 16 16 5 4-7 4 16 16 6 2-6 3 10 10 7 2-4 2 9 9 8 3-7 2 10 10 9 1-4 2 8 8

Example 2

[0072]A show and professional breeder of cattle has used transfer factor formulation with a cow herd of purebred Angus and Charolais, with unexpected and surprising results, using the following protocol.

[0073]Cows were given two ounces of the formulation of Table 3 at approximately Day 10 prior to breeding. At this time, a "seeder" (CIDR.RTM., progesterone implant, available from Pfizer Animal Health) is placed intravaginally to synchronize estrus. At 72 hours prior to breeding, the CIDR was removed and follicular development was stimulated with an effective dose of LUTALYSE.RTM. (dinoprost tromethamine, available from Pfizer Animal Health). At this time, two ounces of the formulation of Table 3 was again given to the animal.

[0074]Use of the above protocol without administration of transfer factor formulation yielded about 75% conception on first breeding. Addition of the transfer factor formulation resulted in a rate of conception of about 98%.

Example 3

[0075]The breeder of Example 2 also tested transfer factor on donor cows. Without the use of transfer factor formulation, the best flush (donor) cow had averaged a yield of about 6 to 8 eggs; with usually only 1 or 2 eggs attaining Grade 1, the rest being Grade 2 and 3.

[0076]After administration to the cow of one ounce of the formulation of Table 3 at Days 5 and 6 prior to flushing, the number of fertilized eggs recovered was 12 eggs; 10 of these were Grade 1.

[0077]These surprising results are economically significant, as the most valuable cows may yield embryos that are valued at approximately $1000 each.

Example 4

[0078]Transfer factor formulation was administered to dairy cows post calving. One ounce of the formulation of Table 3 was administered on Monday and Friday, starting at 10 days after calving, until the cows came into heat for breeding. After implementation of this protocol, conception increased from about 30% to about 50%.

Example 5

Effect of Transfer Factor on Semen Production in Bull

[0079]A young bull nine months old was evaluated to have no live semen. The animal was then administered one ounce of the formulation of Table 3 daily for one month. After one month of the above protocol, 75 ampules of viable semen were collected from the animal, which is an above average yield.

[0080]Prior to transfer factor administration, the veterinarian who had examined the bull had advised waiting for a year to re-check the animal's semen quality. Without intending to be bound by any theory or theories of operation, it is believed that the unexpected results obtained suggest that administration of transfer factor formulation may stimulate spermatogenesis.

Example 6

Effects of Transfer Factor on Fertility in Sheep

[0081]The following demonstrates the effects of transfer factor on the fertility of sheep in a 35 to 40 ewe herd maintained for show stock.

No TF Protocols

[0082]In Year 1, an about 40% conception rate was achieved on first breeding, with a lamb crop of about 125% on 32 ewes. No TF product was used.

[0083]In Year 2, 32 ewes were bred, resulting in about 40% conception, with a lamb crop of about 140%.

[0084]In Year 3, 36 ewes were bred. These animals were treated with Aureomycin crumbles at 112.50 mg daily in their feed to combat the effect of stress. The rate of conception was about 40% over a 90 day period; the lambing crop was about 160%. No TF product was used.

TF Protocols

[0085]In Year 4, 43 ewes were bred. These animals were administered one ounce of the formulation of Table 3 in individual feed on each of Days 6 and 7 prior to breeding. LA 200 was administered to the ewes at a dosage of 9 mg per pound of body weight as an equivalent to aureomycin crumbles in the feed.

[0086]The bucks used to breed the ewes were administered one ounce of the formulation of Table 3 via drench on Days 29, 30, 13 and 14 prior to breeding.

[0087]Chelated sheep minerals were provided in feed to ewes and bucks.

[0088]Teaser bucks (who were either sterile or they had a breeding ring inserted in the back of the sphigmoid processes so they were unable extend their penises) were placed with ewes 15 days prior to breeding with "good" bucks.

[0089]41 of the 43 ewes conceived on the first breeding for a rate of about 95% conception. Lambing percentage was 85 lambs from 43 ewes, which is about a 198% lamb crop.

[0090]In Year 5, 43 ewes, treated as above, were bred with bucks also treated as above. Conception was completed in 20 days for 41 of the ewes. It is believed that the two ewes that did not conceive were too young and were thus pre-puberty.

Example 7

[0091]Thirty-five (35) mature Hampshire ewes in Santa Rosa with fertility difficulty demonstrated conception at about 40% for several years.

[0092]Administration of one ounce of the formulation of Table 3 on Days 6 and 7 prior to breeding increased conception to about 95%.

Example 8

Effects of Transfer Factor on Breeding of Swine

[0093]A study was conducted on a swine operation in Fordham County, South Dakota to test the efficacy of transfer factor. Prior to the study, conception normally averaged about 83 to about 88%, using sows bred after farrowing and newly bred gilts.

[0094]64 sows were administered 0.5 ounce/head/day of the formulation of Table 1 on Days 6 and 7 prior to breeding and again on two consecutive days 3 weeks prior to farrowing. Gilts were also administered 0.5 ounce/head/day of the formulation of Table 1 on Days 6 and 7 prior to breeding.

[0095]All sows, including controls (no TF formulation administered) and treated were challenged with a serious flu virus during pregnancy.

[0096]61 out of 64 treated sows conceived, for a conception rate of approximately 95.3%.

[0097]The data collected for the sows described above and their litters are summarized below in Table 5. DOA=Dead on Arrival

TABLE-US-00005 TABLE 5 Avg. Average Number wean Days Daily Born Number Average Weight until Gain Alive DOA % DOA Weaned litter/sow (lbs.) weaning (lbs.) Control Sows 518 28 5.12 479 9.979 15.2 20.35 0.746 (48) Treated 697 19 2.65 657 10.770 14.3 18.97 0.753 Sows (64)

Example 9

[0098]This study included 80 sows with second and third litters, 40 control and 40 treated.

[0099]Forty head received treatment consisting of 0.6 ounce of the formulation of Table 1 on Days 5 and 6 before breeding, and 0.6 ounce at Days 21 and 7 prior to farrowing (birthing).

[0100]The results were as follows:

[0101]A) Effect on fertility as measured by approximate rates of conception:

[0102]Controls--90%

[0103]Treated--95%

[0104]B) Death loss from birth through weaning at 17 days:

[0105]Controls--12.7%

[0106]Treated--2.7%

[0107]C) Weight gain thru weaning at 17 days:

[0108]Approximately one half pound per head advantage for the treatment group.

[0109]D) Total weaned pigs:

[0110]Controls--10.7 per litter

[0111]Treated--11.2 per litter

[0112]There were no inputs in this natural study, meaning no vaccines or antibiotics or hormones were administered to the animals.

Example 10

[0113]A 25-year old mare was diagnosed with granulosa cell tumor of the left ovary. The mare suffered from chronic abdominal pain; in addition, her blood tests for progesterone, testerone and inhibin indicated a high suspicion of a granulosa cell tumor. When initially diagnosed, the mare demonstrated a level of progesterone (0.1 ng/ml) consistent with an absence of active luteal tissue; a level of testosterone (64.8 pg/ml) that was marginally elevated for a non-pregnant mare; and an inhibin level (0.70 ng/ml) at the upper limit of normal for a non-pregnant mare.

[0114]After approximately nine (9) months of administration of the formulation of Table 2 at 141 grams daily, the ovary had shrunk about 80% and endocrine indices had returned to within normal limits. At this time, blood test results for progesterone (13.5 ng/ml) were consistent with active luteal tissue. Testosterone levels (27.7 pg/ml) were within normal limits for a non-pregnant mare. Inhibin levels (0.25 ng/ml) were likewise within normal limits for a non-pregnant mare.

Example 11

Effects of Transfer Factor on Yields of Fertilized Eggs from Blackbird Cows (Ovum Flush Study)

[0115]1) The first flush was performed with no TF product given. The animals were treated with effective dosages of Follicle Stimulating Hormone (FSH) (lcc) and LUTALYSE.RTM. (1 cc) on days 7-6-5-4 to stimulate follicular development. The results were 18 non-fertile dead embryos.

[0116]2) The second flush using the same protocol of FSH lcc and lcc LUTALYSE.RTM. on days 7-6-5-4 produced one (1) fertile egg.

[0117]3) Treatment with one ounce of the formula of Table 3 on days 7 and 6 with the same protocol as above (lcc FSH and lcc LUTALYSE.RTM. day 7-6-5-4) resulted in eighteen (18) number one grade eggs and three (3) number 2 grade eggs.

[0118]In both treated animals and controls, LUTALYSE.RTM. was given on day 2 before flush am and pm (l cc) with the flush done on day 1 in the morning.

Example 12

Effects of Transfer Factor on Bucking Bull Stock

[0119]This study contained two groups for recipient of fertilized eggs.

[0120]40 control cows received no TF product. 40 treated animals included mixed heifers and cows.

Serving Protocol:

[0121]40 cows and heifers were treated according to the following:

[0122]Day 1--CIDR.RTM. (synchronizing hormone placed in the vagina) in, administered two 0.5 ounce boluses of the formula of Table 3.

[0123]Day 2--administered two more boluses of the formula of Table 3.

[0124]Day 8--pulled the CIDR.RTM. and administered LUTALYSE.RTM. injection two cc and administered two boluses of the formula of Table 3.

[0125]Day of implantation of egg--two boluses of the formula of Table 3 were given at time of egg implanting.

Flush Cows

[0126]Using the same protocol as above, two boluses were given with CIDR.RTM., and when FSH was administered at lcc, two boluses were given day 7-6, but no administration on day 5-4, only FSH at lcc.

Results:

[0127]1) Flush cows were 15 in number. The numbers of eggs were increased from about 18 to 22%.

[0128]2) Recipient cows (cows the eggs are placed in for gestation) usually average about 50% conception, as measured by proof of pregnancy at 42 days.

[0129]In treated animals, conception increased to about 63% of the total of 40 treated cows and heifers; this represents an increase of about 13% in those who checked in calf at 42 days.

[0130]These data demonstrate effects of the administration of transfer factor formulations on tumors and on reproductive health.

[0131]It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present inventions without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modification and variations of the inventions provided they come within the scope of the appended claims and their equivalents.

[0132]The terms and expressions which have been employed are used as terms of descriptions and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope on this invention.

[0133]In addition, where features or aspects of the invention are described in terms of Markush group or other grouping of alternatives, those skilled in the art will recognized that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

[0134]Unless indicated to the contrary, all numerical ranges described herein include all combinations and subcombinations of ranges and specific integers encompassed therein. Such ranges are also within the scope of the described invention.

[0135]The disclosures of each patent, patent application and publication cited or described in this document are hereby incorporated herein by reference, in their entirety.



Inventor

* Ramaekers, Joseph C.

Assignee

* THE RAMAEKERS FAMILY TRUST

US Class 514/1225 or more peptide repeating units in known peptide chain structure

Attorney, Agent or Firm

* FOX ROTHSCHILD LLP;PRINCETON PIKE CORPORATE CENTER

International Classes A61K 38/17 A61P 15/00

* http://www.patentstorm.us Copyright 2011