CATIONIC WHEY PROTEIN COMPOSITION

[01] This application claims the benefit of U.S. provisional application serial no. 60/780,370 filed March 09, 2006, the disclosure of which is expressly incorporated herein.

TECHNICAL FIELD OF THE INVENTION

[02] This invention is related to the area of dietary supplements generally and anti-cancer dietary supplements. In particular, it relates to a fraction of whey proteins that have beneficial properties in treating and preventing inflammation-related diseases.

BACKGROUND OF THE INVENTION

[03] The proteins appearing in the supernatant of milk after precipitation at pH 4.6 are collectively called whey proteins. These globular proteins are more water soluble than caseins and are subject to heat denaturation. The principle fractions are β-lactoglobulin, α-lactalbumin, bovine serum albumin (BSA), and immunoglobulins (Ig).

[04] Whey contains a variety of proteins and peptides and several studies have shown that collectively, whey proteins are prophylactic in attenuating chemically induced intestinal cancers and inhibit growth and metastasis of transplanted tumors [1-6]. The exact physiological mechanism is not completely understood and studies have focused on elucidating changes in cellular metabolism in the presence of whey proteins and products that decrease cancer cell growth and development. With the many peptides and proteins that are found in whey [7] the natural question has been which protein or peptide is most important or do all of the whey proteins act in concert through a multi-mechanistic approach? What is the cellular mechanism(s) that induces apoptosis or arrests cell growth?

[05] Most of the research regarding whey protein supplementation and cancer cells has been done with whey protein concentrate or whey protein isolate. Both of the products contain

many different proteins and peptides that originate from whey. Fractionating and purifying various peptides and proteins has increased understanding about specific whey proteins and peptides. For example α-lactalbumin has been shown to regulate human colon adenocarcinoma cells [17] and forms a molten globule structure that induces apoptosis in tumor cells [18, 19]. The other protein that has received considerable attention is lactoferrin. Lactoferrin has shown both in vitro and in vivo (rat studies) to inhibit colon cancer and tumor development [20-25].

[06] There is a continuing need in the art to identify nutritional agents which increase general healthfulness, including decreasing tumor incidence and reducing tumor growth. There is a continuing need in the art for treating diseases which are associated with inflammation reactions in the body.

SUMMARY OF THE INVENTION

[07] According to one embodiment of the invention a whey protein composition is provided in which at least 15 % of the composition consists of cationic proteins which bind to a cation exchange resin at pH 6.5. The composition is further characterized by a content of 5-70 % lactoferrin and 5-70 % lactoperoxidase.

[08] Another embodiment of the invention provides a method of treating a cancer patient. A whey protein composition is administered per os to the patient. At least 15 % of the composition consists of cationic proteins which bind to a cation exchange resin at pH 6.5. Moreover, 5-70 % of the composition is lactoferrin and 5-70 % of the composition is lactoperoxidase. The composition is administered in an amount effective to inhibit growth of cancer cells in the patient.

[09] Another embodiment of the invention provides a method of treating a cancer patient. A whey protein composition is administered per os to the patient. At least 15 % of the composition consists of cationic proteins which bind to a cation exchange resin at pH 6.5.

Moreover, 5-70 % of the composition is lactoferrin and 5-70 % of the composition is lactoperoxidase. The composition is administered in an amount effective to reduce tumor volume in the patient.

[10] One aspect of the invention is a food composition which comprises as an ingredient a whey protein composition in which at least 15 % of the composition consists of cationic proteins which bind to a cation exchange resin at pH 6.5. In addition, the composition is and further characterized by a content of 5-70 % lactoferrin and 5-70 % lactoperoxidase.

[11] An additional aspect of the invention is a nutritional supplement comprising a whey protein composition in which at least 15 % of the composition consists of cationic proteins which bind to a cation exchange resin at pH 6.5. Moreover, the composition is further characterized by a content of 5-70 % lactoferrin and 5-70 % lactoperoxidase. The nutritional supplement is in the form of a capsule.

[12] A nutritional supplement in the form of a tablet is also provided as an embodiment of the present invention. The nutritional supplement comprises a whey protein composition in which at least 15 % of the composition consists of cationic proteins which bind to a cation exchange resin at pH 6.5. The composition is further characterized by a content of 5-70 % lactoferrin and 5-70 % lactoperoxidase.

[13] Yet another embodiment of the invention is a prophylactic method for preventing cancer. A human whey protein composition is administered per os. At least 15 % of the composition consists of cationic proteins which bind to a cation exchange resin at pH 6.5, Five to seventy percent of the composition is lactoferrin and 5-70 % is lactoperoxidase.

[14] Still another embodiment of the invention is a method of increasing protein in a diet. A whey protein composition in which at least 15 % of the composition consists of cationic proteins which bind to a cation exchange resin at pH 6.5 is administered per os to a human. The composition has a content of 5-70 % lactoferrin and 5-70 % lactoperoxidase.

[15] Still another embodiment of the invention is a method of preventing inflammation or decreasing chronic inflammation in a human. A whey protein composition in which at least 15 % of the composition consists of cationic proteins which bind to a cation exchange resin at pH 6.5 is administered per os to a human. The composition has a content of 5-70 % lactoferrin and 5-70 % lactoperoxidase. The human may have a family history of inflammatory diseases or may himself have chronic inflammation.

[16] Yet another embodiment of the invention is a method of treating a rheumatoid arthritis patient. A whey protein composition in which at least 15 % of the composition consists of cationic proteins which bind to a cation exchange resin at pH 6.5 is administered per os to the patient. The composition has a content of 5-70 % lactoferrin and 5-70 % lactoperoxidase. The composition is administered in an amount effective to reduce symptoms of rheumatoid arthritis in the patient.

[17] Still another aspect of the invention is a method of treating an Inflammatory Bowel Syndrome patient. A whey protein composition in which at least 15 % of the composition consists of cationic proteins which bind to a cation exchange resin at pH 6.5 is administered per os to the patient. The composition has a content of 5-70 % lactoferrin and 5-70 % lactoperoxidase. The composition is administered in an amount effective to reduce symptoms of Inflammatory Bowel Syndrome in the patient.

[18] Another aspect of the invention is a method of treating a Crohn's Disease or Ulcerative Colitis patient. A whey protein composition in which at least 15 % of the composition consists of cationic proteins which bind to a cation exchange resin at pH 6.5 is administered per os to the patient. The composition has a content of 5-70 % lactoferrin and 5-70 % lactoperoxidase. The composition is administered in an amount effective to reduce symptoms of Crohn's Disease or Ulcerative Colitis in the patient.

[19] Still another aspect of the invention is a method of treating an HIV-infected patient. A whey protein composition in which at least 15 % of the composition consists of cationic proteins which bind to a cation exchange resin at pH 6.5 is administered per os to the

patient. The composition has a content of 5-70 % lactoferrin and 5-70 % lactoperoxidase. The composition is administered in an amount effective to reduce HFV viral load in the patient.

[20] Yet another embodiment of the invention is a method of treating an organ transplant patient. A whey protein composition in which at least 15 % of the composition consists of cationic proteins which bind to a cation exchange resin at pH 6.5 is administered per os to the patient. The composition has a content of 5-70 % lactoferrin and 5-70 % lactoperoxidase. The composition is administered in an amount effective to reduce rejection of the organ in the patient.

[21] According to another aspect of the invention a method is provided for treating a Multiple Sclerosis patient. A whey protein composition in which at least 15 % of the composition consists of cationic proteins which bind to a cation exchange resin at pH 6.5 is administered per os to the patient. The composition has a content of 5-70 % lactoferrin and 5-70 % lactoperoxidase. The composition is administered in an amount effective to reduce symptoms of Multiple Sclerosis in the patient.

[22] These and other embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with compositions and methods for nutritionally affecting the health of humans.

BRIEF DESCRIPTION OF THE DRAWINGS

[23] Fig. 1. HPLC chromatogram of cationic proteins isolated from whey. The peak at approximately 11 minutes corresponds to lactoperoxidase and the peak at 16 minutes corresponds to lactoferrin.

[24] Fig. 2. Inhibition ofCACO-2 colon cancer cells by cationic proteins from whey.

[25] Fig. 3. Inhibition of LnCap prostate cancer cells by cationic proteins in whey.

[26] Fig. 4. Inhibition of Bl 6-F10 skin cancer cells by cationic whey peptides.

[27] Fig. 5. Inhibition of A549 lung cancer cells by cationic proteins.

[28] Fig. 6. Inhibition of MDA-MB 435 mammary cancer cells by cationic proteins.

[29] Fig. 7. Inhibition ofU-87 brain cancer cells by cationic proteins from whey.

[30] Fig. 8. Comparative inhibition of various types of cancer cells' growth in vitro by purified lactoferrin (LF), whey protein co-isolate, and the cationic whey protein composition of the present invention (sometimes referred to as IN or NXP-75C). Cells used were: intestinal (CACO-2), prostate (LnCap), skin (B16-F10), lung (A549), breast (MDA-MB 435), and brain (U87) cancer cells.

[31] Fig. 9. Tumor growth curves (tumor volume) relative to dose of cationic whey protein composition. HT-29 cancer cells were injected into nude mice.

[32] Fig. 10. IL-10 serum levels of mice with colon cancer treated with whey product for 28 days. Control mice were not treated with whey product.

[33] Fig. 11. Effect of Whey Product on Colon Myeloperoxidase Levels in DSS Induced Colitis.

[34] Fig. 12. Effect of Whey Product on Serum IL-lβ Levels in DSS Induced Colitis.

[35] Fig. 13. Effect of Whey Product on Serum IL-6 Levels in DSS Induced Colitis.

[36] Fig. 14. Effect of Whey Product on Serum TNF-α Levels in DSS-Induced Colitis.

[37] Fig. 15. Effect of Whey Product on Serum CRP Levels in DSS-Induced Colitis.

[38] Fig. 16. Effect of Whey Product on Body Weight of mice in DSS-Induced Colitis.

[39] Fig. 17. Effect of Whey Product on Food Consumption by mice in DSS-Induced Colitis.

[40] Fig. 18. Histology of Caecum and Colon of Mice After Treatment with Whey Product.

DETAILED DESCRIPTION OF THE INVENTION

[41] The inventors have developed a whey protein composition which is enriched for cationic proteins which bind to a cationic exchange resin at pH 6.5. The composition shows greater activity as an anti-cancer agent than either purified lactoferrin or whey protein isolate. Two proteins which have been identified in the composition are lactoferrin and lactoperoxidase. However, the beneficial activity which has been observed may be related to other proteins which are present in the cationic whey protein composition.

[42] There are no known toxicity levels for whey proteins. Whey protein isolates have been very popular in sports nutrition as a result of high branched chain amino acid levels. Typically body builders take 30-40 g of whey protein per serving with no known side effects. Even with the minor whey proteins such as lactoferrin there has been no observed toxicity effects in rat studies [28]. Amounts of the present compositions which may be ingested and/or administered range from about 1 mg/kg/day to about 1 g/kg/day. Amounts within the ranges of 50-100 mg/kg/day, within 100-500 mg/kg/day, and within 500-1000 mg/kg/day may be used. As mentioned above, even higher amounts of whey proteins are typically ingested by body builders without adverse side effects.

[43] Purified lactoferrin which is commercially available [Glanbia, Kilkenny, Ireland] comprises greater than 92 % lactoferrin. Whey protein co-isolate which is commercially available [Glanbia] comprises between about 75 and 85 % lactoferrin. The cationic whey protein compositions of the present invention, in contrast, comprise between 5 % and 70 % lactoferrin. Thus, the compositions of the present invention are actually depleted in lactoferrin relative to some other preparations. Therefore lactoferrin cannot be solely

responsible for the improved anti-tumor effects of the cationic whey protein compositions of the present invention.

[44] The compositions of the present invention comprise at least 10, at least 15, at least 25, at least 35, at least 45, at least 55, at least 65, at least 75, at least 85, at least 90, at least 95, at least 97, at least 98, or at least 99 percent cationic proteins. Cationic proteins are those which bind to a cation exchange resin at pH 6.5. Cationic exchange resins are well known in the art and include weak cation exchange resins, such as 115E (Purolite, Philadelphia, PA) and strong cation exchange resins, such as Macro Prep S (BioRad, Hercules, CA). Other suitable cation exchange resins include DOWEX* MAC-3 cation exchange resin and DOWEX G-26 (H) (Dow Chemical Company). Any such cation exchange resin known in the art can be used. Suitable cationic proteins typically have isoelectric points between pH 7.8 and 10.1.

[45] The compositions of the present invention are enriched in cationic proteins and depleted in other whey proteins. For example, the compositions may have less than 20 % β- lactoglobulin, less than 10 % α-lactoglobulin, less than 5 % immunoglobulins, and/or less than 2 % bovine serum albumin. The compositions may have at least 10 %, at least 20 %, at least 30 %, at least 40 %, or at least 50 % of lactoferrin. The compositions may also have at least 10%, at least 15%, at least 20%, or at least 30 % lactoperoxidase. One composition according to the present invention comprises 20-30 % lactoperoxidase and 50-60 % lactoferrin. Transforming Growth Factor-beta is also present in the compositions of the present invention.

[46] The compositions of the present invention can be in any physical form. They can be dried, freeze-dried, in solution, in emulsion, in suspension, etc. They can be mixed in foods, such as baked goods (e.g., wafers, cookies, crackers, bars, breads, and cakes), beverages (e.g., shakes, fruit juices, punches, soft drinks, soups, teas) and frozen desserts (e.g., ice cream, ice milk, sherbet, sorbet, cream pie). They can be used in breakfast cereals and enriched grain flours. The compositions may also be formulated in tablets, capsules or powders and the like which can be taken as supplements to one's diet.

[47] The compositions of the present invention can be used to promote general healthfulness, e.g., by adding protein to the diet, or preventing or maintaining a low level of inflammation. In addition, the biological properties of the proteins which are present in the composition of the present invention, either individually or in combination, are beneficial with respect to treating and/or preventing cancer. The compositions may delay or attenuate cancer initiation and or progression. These effects may occur before cancer cells are detected or diagnosed. The compositions may also act after cancer is detected and diagnosed. Thus volume and/or weight of tumors may be diminished or the growth rate of the cancer cells may be diminished. These effects make the ingestion of the compositions of the present invention beneficial for those who are known to be at risk of cancer and those who are not known to be at risk, as well as those who have already been diagnosed. Those at risk include without limitation those with genetic predisposition to or family history of cancer, those who have previously had cancer who are now in remission, those who have previously had cancer but who have had surgical removal of tumors, those with known exposure to carcinogens, such as asbestos and industrial chemicals, those with exposure to radiation. Particular cancers which are amenable to treatment and/or prophylaxis with the compositions of the present invention include without limitation prostate, skin, lung, breast, brain, and colon. Hematological malignancies, such as lymphomas and leukemia, may also be treated and/or prevented in this manner.

[48] Interleukin-10 (IL-IO) is one of the mediators and activators of natural immunity. It is produced by activated macrophages and Th2 cells. It inhibits the production of IFN-γ by ThI cells which prevents the activation of macrophages due to the elimination of one of the two signals needed for activation. IL-10 is a pleiotropic cytokine that aids in the control of inflammatory responses. It has potent biological effects on the functions of various cells such as lymphocytes, monocytes, natural killer cells, and dendrite cells. It inhibits its own production (IL-10) by monocytes, thus demonstrating the workings of a self-regulatory negative feedback loop (Delves et al., 1998). It inhibits T cell mediated immune inflammation by limiting the cytokine production by macrophages. It inhibits

accessory functions in macrophages in T cell function. IL-IO accomplishes this through the reduction in the expression of MHC (major histocompatibility complex) class II molecules and certain co-stimulators, like B7 for example. The cumulative effect of these functions acts to inhibit T cell-mediated immune inflammation. Numerous studies support the evidence of its strong suppressive effect on ThI lymphocytes, antigen presenting cells (APC) and the production of inflammatory mediators (Zhou X, et al., 2005 Dec 5. Boosting interleukin-10 production: therapeutic effects and mechanisms. Current Drug Targets Immune Endocrine Metabolic Disorders. (4):465-75). Because of these strong anti-inflammatory abilities and its ability to regulate the immune system response, it may be used as a treatment for Rheumatoid arthritis, Crohn's disease, Ulcerative Colitis, and IBD. Increasing IL-10 according to the present invention can also be used to treat these diseases. Other types of inflammatory diseases for which the present invention can be used include: Shoulder tendinitis or bursitis; Gouty arthritis; Polymyalgia rheumatica; Inflammation of the heart (myocarditis); Inflammation of the small tubes that transport air to the lungs causing an asthma attack; Inflammation of the kidneys (nephritis); Inflammation of the large intestine (colitis).

[49] Rheumatoid arthritis is an autoimmune disorder associated with inflammation of the joints. Over time, the inflammation may destroy the joint tissues, leading to disability. Intranasal IL-10 gene delivery significantly reduces bone destruction, shows evidence of reducing joint inflammation, and may be mediated by high levels of IL-10 produced by transfected monocytes trafficking to inflamed joints and draining lymph nodes (Woods AM, et al., 2005 December. Immune modulation of collagen-induced arthritis by intranasal cytokine gene delivery: a model for the therapy of rheumatoid arthritis. Arthritis Rheumatology. 52(12):3761-71). Symptoms of rheumatoid arthritis that are positively affected by the whey composition of the present invention include bone loss and joint inflammation. Other typical symptoms that are ameliorated include stiffness, swelling, and pain in and around joints, including hands, fingers, wrists, ankles, feet, elbows, and knees.

[50] Crohn's disease and Irritable Bowel Syndrome (IBD) are chronic conditions involving inflammation of the intestines. Ulcerative colitis (UC) is a chronic recurring inflammatory condition of the colonic mucosa of unknown aetiology and pathogenesis.Recent studies have suggested that a drug that regulates the responses of the immune system may help quiet the inflammation of IBD and Crohn's disease (McKeown LA. 2000 August 24. New Irritable Bowel Disease Treatments Aim for the Gut. WebMD Medical News). The suppressive abilities of IL-10 in small doses, on the immune system can be utilized. Researchers, led by Lothar Steidler of Gent University in Belgium, administered a genetically engineered bacterium that could secrete the drug (IL-10) when it reached the intestines of one group of animals with colitis and another group of animals that had been engineered to develop colitis. The animals with colitis had a 50% decrease in symptoms and the animals expected to develop symptoms did not develop symptoms (McKeown LA. 2000 August 24. New Irritable Bowel Disease Treatments Aim for the Gut. WebMD Medical News). Symptoms of Crohn's disease include abdominal pain, diarrhea, and weight loss, poor appetite, fever, night sweats, rectal pain, and rectal bleeding. Any of these may be ameliorated by the composition of the present invention Symptoms of Inflammatory Bowel Syndrome include bloating and gas, mucus in the stool, constipation, diarrhea, especially after eating or first thing in the morning, abdominal pain and cramping. Any of these may be ameliorated by the composition of the present invention.

[51] IL-10 has also shown promise in small doses to reduce the viral load in HIV (AIDS) patients. Thus the composition of the present invention also can be used to reduce viral load in HIV-infected persons or persons suspected of being infected due to exposure. Reducing the viral load levels as low as possible for as long as possible decreases the complications of HIV disease and prolongs life.

[52] Recent studies have proven IL-10 gene therapy to significantly reduce organ rejection in heterotopic pancreas transplantation (Ozden H. et al, 2005. Interleukin-10 gene

. transfection of donor pancreas grafts protects against rejection after heterotopic pancreas transplantation in a rat model. European Surgical Research 2005 Jul-Aug; 37(4):220-7).

The composition of the present invention reduces organ rejection, given before and/or after the transplantation. Symptoms of organ rejection which can be ameliorated include organ failure, pain, or swelling in the location of the transplanted organ.

[53] MS is a chronic inflammatory disease of the CNS postulated to be a ThI type cell- mediated autoimmune disease (Balashov, KE, 2000 July 25. Defective regulation of IFN gamma and IL-12 by endogenous IL-10 in progressive MS. Neurology 55(2): 192-8). IL- 10 can reduce the immune responses associated with Multiple Sclerosis. The endogenous effect of IL-10 on the regulation of IL-12 and IFN-γ in progressive MS could be an important factor involved in the transition of MS from the relapsing to the progressive stage and has implications for treating MS patients with exogenous IL-10 (Balashov, KE, 2000 July 25. Defective regulation of IFN gamma and IL-12 by endogenous IL-10 in progressive MS. Neurology 55(2): 192-8). The immune response in MS is thought to involve a delayed-type hypersensitivity (DTH) response mediated by ThI type T cells. IL-10 plays an important role in setting the cellular milieu to produce antibodies in an in vitro immunization (IVI) protocol (Xu, Q, et al., 2004 November. IL-10 augments antibody production in in vitro immunized lymphocytes by inducing a Th2-type response and B cell maturation. Biosci. Biotechnol. Biochem. 68(l l):2279-84). Symptoms of MS include dysesthesia, or altered sensations, such as itching, burning, or pins and needles, electrical shock sensations in the neck and spine, paresthesia or pain, numbness or tingling, weakness in an arm or leg, feeling heavy, loss of strength anywhere in the body, dizziness or vertigo, tightness around the chest, poor balance or staggering, development of a limp or dragging foot, paralysis, seizures, tremors, spasticity, babinski reflex, headaches, cognitive impairments, depression, changes in handwriting, fatigue, slurred speech, bladder or bowel problems, and sexual difficulties. Any of these may be ameliorated by the composition of the present invention.

[54] IL-10 also has stimulatory actions on B cells and may function as a switching factor for the production of IgG4 in humans (homologous to IgGl in mice) (Delves et al., P, Roitt I (eds). 1998. Encyclopedia of Immunology. 2 ^nd Ed. San Diego: Academic Press). IL-10 in combination with IL-12 has shown benefits in cancer immunotherapy. IL-10 has a

wide variety of applications that can be very beneficial in the treatments of many serious autoimmune and/or inflammation-related disorders/diseases. Because the composition of the present invention increases serum IL-IO levels, it can be used to treat and/or prevent such autoimmune and/or inflammation-related disorders/diseases. See Example 4 below.

[55] The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

EXAMPLE 1 — Materials and Methods Materials

[56] Dulbecco's minimum essential medium with 4.5 g glucose was purchased from Sigma, Chemical Co. (St. Louis, MO). A 200 mM L-glutamine solution (catalog # G7513), a 7.5% sodium bicarbonate solution (catalog # S8761), penicillin streptomycin (catalog # P0781), a 100 mM solution of sodium pyruvate (catalog # S8636), non-essential amino acids (cat # M7145) and L- 15 Liebovitz Media (catalog # L-1518) also were purchased from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum and sterile PBS (cat # 14190-250), trypsin-EDTA (Cat # 25200-056) was from Invitrogen Corporation (Carlsbad, CA). RPMI 1640 media (cat # 21870-076) and HEPES (cat # 15630-080) were purchased from Gibco, BRL (Burlington, ON). [ ³ H] thymidine was from ICN Pharmaceuticals (Montreal, PQ). A sample of 1 15E weak cation exchange resin was obtained from Purolite (Philadelphia, PA). The Macro Prep S strong cation exchange resin was purchased from BioRad (Hercules, CA). A Mini S column was purchased from Pharmacia Biotech (Uppsala, Sweden). All other products were of analytical grade and quality.

Cell cultures

[57] Six immortalized cell lines were obtained from American Type Culture Collection (Rockville, MD). Cultures included human colorectal adenocarcinoma (CACO-2), human prostate left supraclavicular lymph node carcinoma (LNCaP), human mammary gland ductal carcinoma (MDA-MB435S), human lung malignant melanoma (A549), human brain glioblastoma (U-87 MG) and mouse skin melanoma (B16-F10).

Media for cell growth

[58] The media used for B16-F10 cell growth was Dulbecco's minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), L-glutamine solution, sodium bicarbonate solution and penicillin streptomycin.

[59] The media for CACO-2 and A549 cell growth consisted of DMEM supplemented with 10% FBS, L-glutamine, sodium bicarbonate solution, sodium pyruvate, non essential amino acids and penicillin streptomycin.

[60] The media for LNCaP cell growth consisted of RPMI 1640 media with 10% FBS, L- glutamine, sodium bicarbonate solution, 100 mM sodium pyruvate, HEPES, and penicillin and/or streptomycin.

[61] The media for MDA-MB-435 cell growth was L-15 Liebovitz media with L-glutamine, 10% FBS and penicillin-streptomycin.

[62] The media for U-87 MG cell growth consisted of DMEM with 10% FBS, L-glutamine, sodium bicarbonate solution, non-essential amino acids and penicillin streptomycin.

[63] Cultures were maintained as per manufacturer protocol.

General instructions for media preparation

[64] Each specific media was made according to cell culture that was used it was sterile filtered with a 0.22 μm filter and then it was tested for sterility. To test for sterility, 2 ml of media was put into a 6-well plate and placed in an incubator for 5 days. After 5 days plates were examined under the microscope for growth and if there was none present the media batch was confirmed as sterile and used.

Passage of adherent cell lines

[65] All media and media components were heated to 37°C in a water bath. Media was removed from the culture flask and 10 ml PBS was added to gently wash the cells. A trypsin-EDTA solution (1-5 ml) was added and the flask was incubated for 5 minutes to detach cells from the surface of the flask. After cells were detached 5 ml of complete media were put into the flask and cells were mixed by pipetting up and down and centrifuged at 1500 RPM for 5 minutes. The supernatant was removed and the cells were resuspended and transferred into a new flask at an appropriate dilution. A hemacytometer was used to determine initial cell counts and determine appropriate seeding concentrations.

Proliferation of cells

[66] Various cancer cells (10xl0 ³ /well) were cultured in the presence or absence of different concentrations of CP in 96 well flat bottom plates (Falcon) in a final volume of 200μl and incubated at 37°C for 48h. Cultures were pulsed with [ ³ H] thymidine (1 μCi/well) after 3 Ih of culture and further incubated for 17h. Cell proliferation was assayed by measuring [ ³ H] thymidine uptake using a Microbeta-Scintiallation counter (Perkin-Elmer, St- Laurent, Quebec, Canada). All samples were plated in triplicate and experiment was repeated to get an average and standard deviation for each sample. The percent inhibition was obtained by calculating the thymidine uptake difference between the control sample and the treatment sample and dividing by the total thymidine uptake of the control sample multiplied by 100.

Preparation ofcationic proteins

[67] Cationic proteins were isolated using weak and strong cation exchange chromatography. Sweet whey was passed over a weak cation exchange resin at pH 6.5 and 15 ⁰ C. Bound peptides were released with 1 N NaCl. The sample was then ultrafiltered to remove the NaCl. A strong cation exchange resin was used to concentrate cationic proteins and bound peptides were released with a 1.5 N NaCl solution. The sample was ultrafiltered to remove salt and concentrated. The purity and peptide profile of the samples were analyzed using a Mini S column on a Waters 2695 HPLC (Milford, MA). The column was equilibrated with a pH 6.8, 50 mM phosphate buffer. The sample was loaded and the elution was achieved by using a 1.5 M NaCl solution in a 50 mM phosphate buffer at pH 6.8. A linear salt gradient from 0 to 1.5 M NaCl over a 30 minute time period was done. Sample elution was monitored at 280 ran. Samples were filtered with a 0.22 μm filter prior to use for both chromatography and cell culture assays.

EXAMPLE 2

[68] The chromatographic profile of the cationic proteins is shown in Fig. 1. The peak at 16 minutes was identified as lactoferrin and the peak at 11 minutes was identified as lactoperoxidase. Total peak integration showed lactoferrin was 56%, lactoperoxidase 23% and the rest were other unidentified cationic proteins or peptides.

[69] A typical microfiltered whey protein isolate contains <1% cationic peptide material at pH 6.5. Thus, a very good enrichment of cationic whey proteins was obtained using cation exchange chromatography.

EXAMPLE 3

[70] Figs. 2 through 7 show the inhibition of CaCo-2 human colon cancer cells, LnCap prostate cancer cells, B16-F10 skin cancer cells, A549 lung cancer cells, MDA-MB 435

mammary cancer cells and U-87 brain cancer cells respectively. The protein concentration that was used (0.215 to 21.5 mg/ml) showed inhibition with the exception of the U-87 brain cancer cells that showed inhibition at higher levels (21.5 and 2.15 mg/ml) but stimulated cell growth at lower levels. CaCo-2 colon cancer cells showed complete inhibition at higher levels. Based upon these results it appears that the cationic protein fraction from whey displays a wide inhibition to various cancer cells lines.

EXAMPLE 4

[71] Figure 10 shows the effect of supplementation with whey product on IL-10 levels in serum. 11-10 levels in serum show a significant increase (p=0.0267) in mice treated with whey product (50 mg/day/mouse) compared to vehicle. Similar measurements of IFN-γ, IL- lβ, TNF-α, and TGF-β demonstrated no significant effect by the whey product. The significant increase in IL-10 in serum of mice with colon cancer treated with whey product at 50 mg/day/mouse suggests an upregulation of anti-inflammatory Th2 response. These results suggest that there was a trend towards decrease in inflammation.

EXAMPLE 5

[72] Exposure to dextran sulphate sodium (DSS) induces acute colitis, which is normally resolved after DSS removal. The following study was undertaken to determine the effect of a whey product on DSS induced colitis and inflammatory markers. It was observed that treatment of female C57/BL6J mice with the whey product at 50mg/kg dose significantly inhibited myeloperoxidase levels in colonic tissue. The whey product also inhibited serum levels of IL-6 and TNF-α suggesting a systemic inhibition of inflammatory response at 50mg/kg dose. However, treatment with the whey product increased the secretion of IL- lβ at both of the doses tested. The whey product did not have any effect on the serum CRP levels at any dose tested. No significant differences were observed in the histology between the different groups. These results suggest that treatment with the whey product reduces inflammation at the local and systemic levels in a DSS induced colitis model.

[73J Ulcerative colitis (UC) is a chronic recurring inflammatory condition of the colonic mucosa of unknown aetiology and pathogenesis. Dextran sulfate sodium (DSS)-induced murine colitis represents an experimental model for human inflammatory bowel disease. Oral administration of DSS induces colitis resembling UC in hamsters and mice (1, 2).

[74] Myeloperoxidase (MPO) is one of a number of enzymes located in the azurophil granules of polymorphonuclear leukocytes (PMNs, also called neutrophils) and, to a lesser extent, in monocytes. MPO catalyzes the formation of hypochlorous acid (HOCl), a powerful oxidant formed from chloride ions and hydrogen peroxide. MPO generates reactive oxygen species as part of its function in innate host defense mechanisms; however, in an unregulated state, it is capable of causing tissue destruction or inflammation, and has been implicated in numerous diseases, including atherosclerosis, cancer, and Alzheimer's disease.

[75] In response to infection or tissue damage, the host initiates a series of events referred to as the acute phase response. The acute phase response is characterized by both local and systemic events. Both local and systemic events are mediated in part by cytokines. Interleukin 6 (IL-6) is a 21-kDa cytokine that is produced by a variety of cells including fibroblasts, endothelial cells, mononuclear phagocytes, neutrophils, hepatocytes, and T and B lymphocytes and is involved in the acute phase response and the immune response. IL-6 has been shown to contribute to polymorphonuclear neutrophilic granulocyte (PMN) production in response to infection (3, 4) and to contribute to the activation of PMN effector functions (5, 6). IL-6 has been reported to have both proinflammatory and antiinflammatory effects (7, 8). Increased interleukin- 1 beta (IL- lβ), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) concentrations have been reported in subjects with chronic inflammatory bowel diseases (9-13).

[76] The present study was undertaken to determine the effect of a whey product on DSS- induced colitis and various markers of inflammation.

[77] Experimental Protocol

[78] C57BL/6J mice (10 weeks old, females, Charles River) were acclimatized for a week and randomly assigned to 4 groups with 10 mice in each group:

[79] 1.) Control, (with DSS vehicle)

[80] 2.) 4% DSS

[81 ] 3.) 4% DSS + Test Agent ( 1 mg/kg body weight)

[82] 4.) 4% DSS + Test Agent (50mg/kg body weight)

[83] Mice in the appropriate groups were given the 4% Dextran Sulfate Sodium (DSS) in drinking water for 7 days to induce colitis. One group of mice received the test product by oral gavage at 1 mg/kg body weight and one group at 50mg/kg body weight for the duration of the study in addition to the DSS. During this period the general appearance of the animals (e.g., activity level, body weight, presence of occult blood in feces) and animal mortality were monitored on a daily basis.

[84] At the end of the study, blood was collected by heart puncture and serum was separated for cytokine analysis. The remaining sera was stored at -8O ⁰ C for future analysis. The following cytokines and CRP were tested:

[85] 1. Cytokines:

[86] a) IL-lβ

[87] b) IL-6

[88] c) TNF-α

[89] 2. C-Reactive Protein (CRP)

[90] Following euthanasia using CO2, the colon was cleaned of its lumenal content with cold saline and weighed using a semi-analytical balance. Five-cm colonic segments from the mice were collected and tested for myeloperoxidase activity. Cecum, proximal and distal colon segments were fixed in formaldehyde for histological evaluation.

[91] Test Article and Vehicle Control Material

[92] Test Material: One preparation of whey product.

[93] Vehicle: Water and 75% Glycerol

[94] Receipt, Identification and Storage: The whey product was received by KGK Synergize Inc. on June 26, 2006 from Glanbia Nutritionals.

[95] Preparation and Analysis: The whey product was dissolved in water and diluted with 75% glycerol to obtain the desired concentrations.

[96] Statistical Analysis: Student's t-test was used to compare differences between groups.

Effect of the Whey Product on Myeloperoxidase Levels:

[97] The mice were treated with two concentrations of the whey product together with DSS. Parts of their colon were collected at the end of the treatment period and myeloperoxidase levels were measured. The data presented in Figure 11 show that the treatment of the mice with DSS significantly up regulated the MPO levels compared to the control mice (p=0.0006). Treatment with the whey product at 50mg/kg dose together with DSS significantly decreased the MPO levels compared to DSS-treated mice (p=0.0001). These results suggest that the whey product at high dose can reduce the colitis associated inflammation.

Effect of Whey Product on Serum IL- lβ Levels:

(98] The treatment of the mice with DSS did not induce any significant up regulation of IL-I β levels in serum. On the other hand, treatment with the whey product together with DSS significantly (p<0.02) up regulated IL- lβ levels compared to the DSS-treated mice at both the doses tested (Figure 12).

Effect of Whey Product on Serum IL-6 Levels:

[99] A slight increase in IL-6 levels was observed in the mice treated with DSS alone and with DSS and the lower dose (lmg/kg) of the whey product but the increase was not significant p>0.07). However, the treatment of the mice with the high dose (50mg/kg) of the whey product significantly (p=0.02) down regulated the IL-6 levels compared to the DSS-treated group again suggesting a reduction in inflammatory response (Figure 13).

Effect of Whev Product on Serum TNF-ct Levels:

[100] The data presented in Figure 14 show that the treatment of the mice with DSS significantly up regulated the secretion of TNF-α compared to the control mice (p=0.05). The treatment with the lower dose (lmg/kg) down regulated the TNF-α levels slightly but the decrease was not significant (p=0.28). On the other hand, the treatment with the high dose (50mg/kg) significantly (p=0.039) down regulated the TNF-α secretion compared to the DSS control (Figure 14).

Effect of Whev Product on Serum CRP Levels:

[101] Further, the effect of the treatment with DSS and the whey product on serum CRP levels was determined. It was observed that there was no significant (p>0.48) effect on the serum CRP levels after treatment with DSS or the whey product (Figure 15).

Effect of Whev Product on Body Weight of Mice:

[102] The data presented in Figure 16 show that the treatment with DSS and the whey product did not have any significant effect on the body weight of the mice. This suggests that the normal growth of mice was not affected.

Effect of Whey Product on Food Consumption by Mice:

[103] It was observed that the treatment with DSS and the whey product did not alter food consumption by the mice significantly (Figure 17).

Effect of Whey Product on Histology of Caecum and Colon of Mice:

[104] Treatment with DSS did not induce histological damage in the caecum and colon of the mice compared to the control mice. No significant differences were observed between any of the groups (Figure 18).

[105] The incidence of inflammatory bowel diseases (IBD) such as Crohn's disease (CD) and ulcerative colitis (UC) is increasing. During IBD, a marked invasion of inflammatory cells is observed, resulting from the production of inflammatory cytokines and chemokines in the inflamed area.

[106] The present study was undertaken to determine the effect of a whey product on DSS induced colitis and inflammatory markers. It was observed that the treatment with the whey product at 50mg/kg dose significantly inhibited myeloperoxidase levels in colonic tissue suggesting that the local inflammatory response induced by DSS was inhibited by the whey product. The whey product also inhibited serum levels of IL-6 and TNF-α suggesting a systemic inhibition of inflammatory response at 50mg/kg dose. The lower dose (lmg/kg) was not found to be effective. It has been shown that serum IL-6 levels are substantially elevated in patients with active Crohn's disease (14—17). Moreover, serum IL-6 is a clinically relevant parameter for Crohn's disease that correlates with inflammatory activity (18). In patients suffering from Crohn's disease with colonic or ileocolonic involvement, serum IL-6 concentrations were higher than in patients with

disease restricted to small bowel (18). Therefore, inhibiting IL-6 production by whey product may prove beneficial in the treatment of the disease.

[107] The whey product did not have any effect on the serum CRP levels at any dose tested. Several sub-clinical markers are used to monitor disease activity in humans. One of these markers is serum C reactive protein (CRP), which is generally elevated in 95% of patients with active CD. However, a weak correlation has been found between serum CRP levels and symptom values (19). Interestingly, the whey product increased the secretion of IL- lβ at both the doses. It has been shown that α-lactalbumin, a fraction of whey can enhance IL- lβ secretion (20). It is possible that this whey preparation contains higher concentrations of α-lacalbumin resulting in an increase in IL- lβ secretion. No significant differences were observed in the histological sections of the caecum and colon between groups. This may be due to the fact that treatment with DSS for 7 days initiates the colitis represented by the up regulation of inflammatory markers but the duration may not be long enough to see histological changes. These results suggest that the whey product reduces inflammation at the local and systemic levels in a DSS-induced colitis model.

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The disclosure of each reference cited is expressly incorporated herein.

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