NZ567126A - A dairy derived composition comprising beta-lactoglobulin, IGF and TGF-beta for use in treating chronic intestinal inflammatory disease - Google Patents

Abstract

Disclosed is a composition comprising TGF-beta1 at a concentration between about 0.1 and 5ìg/g of composition, TGF-beta2 at a concentration between about 5 and 50ìg/g of composition, the composition further comprises IGF-1 and is least 15% (w/w) of whey proteins from which at least 60% is beta-lactoglobulin. Further disclosed is the use of the composition in the manufacture of a medicament for the treatment of chronic intestinal inflammatory diseases such as Crohn's disease.

Description

P567126 A DAIRY DERIVED COMPOSITION ENRICHED IN TGF- (3 FOR TREATING INFLAMMATION TECHNICAL FIELD The present application is related to the treatment of inflammation, and especially chronic intestinal inflammation. The present application more particularly relates to a 5 non-toxic oral and topical formulation that includes a pharmaceutically effective amount of dairy proteins and its use in the treatment of inflammation.

BACKGROUND OF THE INVENTION Sumerian and antic Egyptian cultures described inflammatory processes as abscesses and ulcers. Greek culture introduced the term edema and the vocabulary of 10 inflammation began its development. The first comprehensive description of inflammatory symptoms can be found in the writing of Aulus Celsus (died AD 38). He introduced the cardinal symptoms of inflammation: redness, swelling, heat and pain.

Inflammation is usually a local protective reaction that serves to protect the organism against infections or trauma. Excessive inflammation or unusual inactivity of an 15 otherwise normal inflammatory process leads to inflammatory diseases.

Inflammatory processes are a combination of very complex mechanisms in which many factors are implicated. These factors are cellular (endothelial cells, macrophages, leucocytes, etc) and/or humoral (coagulation factors, cytokines, eicosanoid acids, adhesion molecules, free radicals, etc). In general, inflammatory processes are 20 associated with immune cell proliferation, infiltration of cells and accumulation of exudates at the inflammation site, and degradation of the injured tissue. Liberation of mediators like cytokines and eicosanoid acids lead to a more global reaction like fever. At the end of the inflammation process, the injured tissue is destroyed by the organism.

Exposure of mammalian tissue or cells to harmful stimuli such as lipopolysaccharides 2 5 (LPS, a compound found in bacterial structure), toxic chemicals, radiations or inflammatory mediators, are known to activate immune cells to produce inflammatory mediators such as tumor necrosis factor-a (TNF-a), interleukin-1 (IL-1), IL-6, prostaglandins (PG), leukotrienes and nitric oxide (NO). Activated immune cells can eventually lead to inflammatory diseases (e.g., arthritis and septic shock), diseases P567126 linked to immunity (e.g., graft rejection, autoimmune diseases, and diabetes mellitus), and the death of some cell types (neurons for example).

Prostaglandins are molecules originating from arachidonic acid and involved in various physiological activities and pathologies. Their production is regulated by the enzyme 5 cyclooxygenase (COX). Non-steroidal anti-inflammatory drugs (NSAIDs) and analgesic agents such as aspirin and indomethacin, which inhibit prostaglandins synthesis by inhibiting COX activity, are known to provide an excellent therapeutic effect for inflammation-related diseases.

The activation of nuclear factor kB (NF-kB) is required for the production of NO, 10 prostaglandins, and TNF-a. NF-kB is also known to regulate the expression of a variety of proteins involved in the various cellular responses such as apoptosis, immune responses, and inflammatory reactions.

In abnormal inflammation processes, the inflammatory process remains out of control and induces persistent diseases. Many diseases are described with an important 15 inflammatory component, but chronic inflammatory diseases are recognized to be polysystemic diseases with an unknown origin. They are as diversified as psoriasis, Crohn's disease, rheumatoid arthritis. In general, an immune component in all of these diseases can be seen.

The description of Crohn's disease made hereafter should be viewed only as an 2 o illustration of the topic and will not represent a limit to the possible applications of this application.

Crohn's disease is an inflammatory bowel disease that causes persistent or recurring inflammation of one or more parts of the intestine. Crohn's disease can affect any part of the gastrointestinal system, from the mouth to the anus. There is no known cure for 2 5 Crohn's disease. The causes are unknown and are not only due to stress as was once thought. Inflammatory bowel diseases (IBD) can occur at any age, but usually begins in the late teens or early adulthood. It is sometimes associated with medical problems outside of the intestine, including arthritis, cancer, gallstones, kidney stones, and skin conditions.

P567126 Crohn's disease symptoms include abdominal pains, diarrhea, and weight loss. Pain is most often felt around the navel or the lower right part of the abdomen. The pains may occur during eating or soon after a meal. Steady dull aches of the abdomen may also be felt and may be worsen with activities like jogging and sports. Other Crohn's disease 5 symptoms that patients have noted are fatigue and loss of appetite.

Drugs having mesalamine as a main ingredient are often first prescribed to a patient with Crohn's disease. Mesalamine helps in controlling the inflammation. However, possible side effects of mesalamine preparations include nausea, vomiting, heartburn, diarrhea, and headache. Corticosteroids may also be administered to those patients. 10 Patients taking corticosteroids to control inflammation face a difficult trade-off between effective treatment and serious side effects. For example, corticosteroids increase the likelihood of getting infections. Crohn's disease can also be controlled with drugs suppressing the immune system such as 6-mercaptopurine and azathioprine. Immunosuppressive agents block the immune reaction related to inflammation. These 15 drugs may have side effects like nausea, vomiting, and diarrhea. In addition, the drugs may lower patient's resistance to infection. The combination of immunosuppressive drugs and corticosteroids allows the dosage of corticosteroids to be reduced over time. There is evidence that immunosuppressive drugs can increase the effectiveness of corticosteroids. Infliximad is a recently approved drug for treating Crohn's disease 2 0 patients. The purpose of infliximad is to prevent inflammation by inhibiting the activity of TNF.

One important medical need is the effective treatment of inflammation and autoimmune diseases. These diseases are treated with drugs that have high side effects profiles and limited efficacy. Currently, the commonly used therapeutic alternatives available to 2 5 treat chronic inflammations consist in the use of corticosteroids or NSAIDs.

Unfortunately, all of these anti-inflammatory agents are associated with highly significant side effects, including gastrointestinal irritations and bleeding, bone loss, and fluid retention, some of which can reduce significantly the well being of the patients. 3 0 Several drugs have been designed to treat inflammatory diseases by targeting specific cells, specific cytokines, or specific interactions between ligands and receptors. The main advantage of these biological agents over cyclosporin and methotrexate is the P567126 absence of nephrotoxicity and hepatotoxicity, but toxic effects can take years to develop.

Current treatments are directed towards either cell proliferation or the immune component of inflammation, for example in approaches to psoriasis treatment. 5 Japanese patent application JP 6145069 describes angiogenesis inhibitors comprising ganglioside GM3 or a GM3 analog as an active agent. At 100 MQ/mL, GM3 inhibited the growth of normal human endothelial cells (4.5x104 on day 5, compared with 76x104 in controls. US Pat. No. 5,330,977 describes n-deacetyl-lysoganglioside derivatives for use as phospholipase A2 inhibitors for the treatment of proliferative and autoimmune 10 diseases, including various forms of cancer, psoriasis, and rheumatoid arthritis.

Anti-inflammatory agents such as glucocorticoids or aspirin inhibit the activation of NF-kB and/or repress the production of NO or prostaglandin. Some herbal drugs widely used as anti-inflammatory agents in oriental natural medicine are known to exert their effects by the inhibition of NF-kB activity (Kopp and Ghosh, Science, 265: 956-959 15 (1994); Ray and Prefontaine, Proc. Natl. Acad. Sci. U.S.A., 91, 752-756 (1994)).

There are several drugs which show inhibitory activity for TNF-a production, including IL-4, IL-10 and transforming growth factor-p (TGF-P), and clinical trials using antibody against TNF-a and soluble TNF-a receptor are underway. As synthetic compounds, for example, new derivatives of 1-thiadibenzoazulene (US patent 6,897,211) and 2 0 dibenzoazulene compounds (US patent 6,897,211) have been developed for their TNF- a inhibitory activity. Because these agents are TNF-a inhibitors, concerns have been raised about the risks of infection resulting from such an immunosuppression.

Inhibitors of NO production have focused on the development of agents that specifically inhibit the enzymatic activities of inducible NO synthase (iNOS), the enzyme 25 responsible for NO synthesis. For example, analogues of NO precursors such as L-arginine and L-citrulline, and derivatives of aminoguanidine and isothiourea are under investigation (Babu and Griffith, Current Opinion in Chemical Biology, 2, 491-500 (1998)).

Curcumin, capsaicin, caffeic acid derivatives, and some other natural compounds have 3 0 been reported to inhibit the activation of NF-kB, while dexamethasone, a representative of the anti-inflammatory agent of the glucocorticoid family, inhibits NO production by P567126 inhibiting the expression of the iNOS gene. The molecular mechanism of dexamethasone in the NO synthesis has been known to be the suppression of iNOS gene transcription by the way of the inhibition of NF-kB activation (De Vera et al., Am. J. Physiol., 273, 1290-1296 (1997)).

About 200 diterpene compounds have been isolated from Isodon japonicus or close relatives. However, there has been no report indicating that these compounds not only inhibit the production of NO, PGE2, or TNF-a in cell lines stimulated by LPS, but also that these compounds can inhibit the activation of NF-kB. US Patent 6,894,073 described kamebanin, kamebacetal A, kamebakaurin, and exisanin A, which are 1 o members of the kaurane diterpene compounds family isolated from Isodon japonicus, in inhibiting not only the activation of NF-kB, but also the production of NO, prostaglandin, and TNF-a in the LPS-stimulated macrophage cells. These four compounds have been proposed as therapeutic agents for the treatment of inflammatory diseases, immunological diseases, and cancers.

Human and bovine milks contain many biologically active polypeptides including growth factors (West, Exp. Clin. Endocrinol. 8: 145-146, (1989)). One of these factors, milk growth factor (MGF), was recently identified as being identical to, or having close homology to, members of the TGF-3 family, notably TGF-32 (Cox et al. Eur. J. Biochem. 197: 353-358, (1991)). TGF-Ps are a group of polypeptides consisting of at least 5 distinct but closely related members having considerable structural and biological homologies (Roberts, A. B., et al. In: Peptide Growth Factors and their Receptors Vol. 1, pp. 419-472, Eds. Sporn M. B. et al., Springer, (1990)). TGF-Ps are homodimeric proteins of about 25 kDa comprising identical 12.5 kDa polypeptide chains linked through disulphide bridges. They may form latent complexes with other 2 5 proteins, and these complexes may be activated by acid treatment or mild proteolysis (Roberts, A. B. et al., Supra). They are multipotent, having a number of biological activities dependant upon the type of target cell, the state of differentiation of the cell and the presence of other factors. Their effects include stimulation or inhibition of cell proliferation and differentiation, regulation of extracellular matrix deposition, 3 0 immunomodulation, steroidogenesis and angiogenesis (Roberts, A. B. et al., Supra).

Suitable methods are known for purifying TGF-ps from various species such as human, mouse, green monkey, pig, bovine, and chick, and from various cellular sources such as bone cells, platelets, or placental cells, as well as for producing it in recombinant cell P567126 cultures and for determining their activities (European Patents 200,341; 169,016; 268,561 and 267,463; G.B. Patent. Application 2,146,335; U.S. Patent 4,774,322).

TGF-p is multifunctional, as it can either stimulate or inhibit cell proliferation, differentiation, and other critical processes in cell functions (M. Sporn, Science, 5 233:532-534 (1986)). TGF-P has been shown to have numerous regulatory actions on a wide variety of cells. Recent studies indicate an important role for TGF-P in immune cells (J. Kehrl et al., J. Exp. Med., 163: 1037-1050 (1986); H-J. Ristow, Proc. Natl. Acad. Sci. U.S.A., 83: 5531-5533 (1986); A. Rook et al., J. Immunol., 136: 3916-3920 (1986)) and in proliferation of connective and soft tissues for wound healing 10 applications (U.S. Pat. No. 4,774,228). Moreover, TGF-p has been described as a suppressor of cytokines production (interferon-y (IFN-y), TGF-P), indicating a possible use as an immunosuppressant for treating inflammatory disorders (European Patent 269,408; U.S. Patent 4,806,523). Further, TGF-p induces collagen secretion in human fibroblast cultures (Roberts et al., Proc. Nat. Acad. Sci. USA 83: 4167-4171 (1986)), 15 stimulates the release of PGs and mobilization of calcium (A. Tashjian et al., Proc. Natl. Acad. Sci. U.S.A., 82: 4535-4538 (1985)) and inhibits endothelial regeneration (R. Heimarketal., Science, 233:1078-1080 (1986)).

TGF-p is produced by both immune and non-immune cells, and it exhibits a broad range of functions, including the modulation of immune responses. Oral administration 2 0 of TGF-P2 is well known to decrease the inflammatory process. For example, Fell et al. (Aliment Pharmacol. Ther. 14 (3): 281-289, (2000)) have demonstrated the antiinflammatory effect of a rich polymeric TGF-P2 diet (CT3211: Nestl6 Vevey, Switzerland) in children with IBD. In this study, 29 children with IBD were treated with CT3211 during 8 weeks. Following the treatment, the results shown that 79% of the 2 5 children were in complete remission from their disease. The macroscopic and histological healing of the ileum and colon was associated with a decrease of proinflammatory cytokines mRNA (IL-1 p, IFN-y, IL-8) in ileum and colonic mucosa as well as an increased TGF-P2 mRNA in ileum tissue. This study confirms the interest in using TGF-P2 solution as a therapeutic agent. 3 0 TGF-Ps, including TGF-p2, have a number of immunoregulatory properties and act at several stages of the inflammatory and immune reactions. For example they inhibit the proliferation of T and B lymphocytes (Kerhl, J. H., et al. J. Immunol. 137: 3855-3860, P567126 (1986); Kerhl, J. H., et al. J. Exp. Med. 163: 1037-1050, (1986)). They also antagonize the effects of interleukins including IL-1, IL-2 and IL-3 and other immunoregulatory agents such as TNF-a and interferons (Roberts, A. B. et al. , Supra). TGF-3s interfere with certain accessory cell functions important in antigen presentation and they were 5 specifically shown to suppress major histocompatibility complex (MHC)-Class II expression by melanomas, glial cells and astrocytes (Czarniecki, et al J. Immunol. 140: 4217-4223, (1988); Schlusener, J. Neuroimmunol. 24: 41-47, (1990); Zuber, et al. Eur. J. Immunol. 18: 1623-1626, (1988)). The regulation of MHC-Class II expression on epithelial cells in the intestine by TGF-3s or MGF has been reported in European 10 patent EP0527283.

US patent 6,649,168 describes a pharmaceutical dry powder composition comprising a TGF-3 in a water soluble salt (calcium chloride, calcium phosphate, potassium acetate, lithium acetate, ammonium acetate or ammonium bicarbonate) for therapeutic applications such as promotion and acceleration of wound healing, bone repair, soft 15 tissue repair, nerve repair, stroke therapy, cancer treatment, bone marrow protection, cardioprotection, angiogenesis induction, mammalian cell growth regulation and induction of anti-inflammatory and immunosuppressive activity.

In US patent 6,403,562, methods for treating autoimmune-related diseases, such as multiple sclerosis (MS), are presented. Those methods comprise administering IL-10 2 0 and TGF-3 in combination to a person afflicted with or predisposed to an autoimmune disease. When administered in combination, these cytokines act in a synergistic manner as suppressor factors in order to inhibit the activation of self-reactive T cells that are involved in autoimmune diseases.

U.S. Pat. No. 5,461,033 describes the use of an acid casein fraction isolated from 2 5 bovine milk and containing TGF-32 in the treatment of Crohn's disease. The data reported in the patent is based upon in vitro experimentation and the patent gives no specific description of nutritional formulas that can be used in the treatment of Crohn's disease. U.S. Pat. No. 5,952,295 gives a precise formulation of enteral preparation for the treatment of inflammatory conditions in the gastro-intestinal tract using a casein 3 0 fraction with TGF-32 and reports improvement of such inflammatory conditions in an open-label study with 11 young patients.

P567126 European Patent 0313515 describes a milk growth factor fraction obtained from milk, and the use of this fraction for treating trauma, suppressing the immune response or treating cancer in mammals. Patent application WO 02/05828 proposes the treatment of skin disorders by neutral or basic growth factors from milk or colostrum. Japanese 5 Patent 14308090 describes a fraction from colostrum with TGF-p activity.

A dairy growth factors fraction obtained from chromatographic methods, especially with an ionic exchange step, is described in US patent 6,447,808. This fraction can be used for the treatment of gastrointestinal injuries, ulcers, or surface wounds. Patent application WO 01/25276 also describes isolation of growth factors from dairy products 10 by the use of chromatography. Different fractions can be produced by this process, especially fractions enriched in TGF-3 and IGF-1.

Patent application WO 03/006500 describes a process to obtain a dairy fraction enriched in TGF-3- This process is based on a precipitation step induced by a thermal treatment in acidic conditions followed by the recovery of the precipitate by filtration. 15 The fraction contains 45 to 80% of alpha-lactalbumin protein and more than 5 pg of TGF-3 per gram of proteins. This fraction is proposed for the treatment of auto-immune diseases, arthritis, osteoporosis, wound healing and graft rejection.

Considering the state of the art described above, all of the methods of the prior art are complex, expensive and time consuming. Thus, there is still a significant need for a 2 0 new and simple method for obtaining a dairy fraction enriched in TGF-3 that can be used effectively in the treatment of chronic inflammatory disorders (such as chronic intestinal disorders) without the disadvantages associated with the currently available treatments. Products containing growth factors, especially TGF-32, present a very interesting approach to the treatment of chronic inflammatory disorder, and especially 2 5 of Crohn's disease.

SUMMARY OF THE INVENTION One aim of the present application is to provide a new and simple method for obtaining a dairy fraction enriched in TGF-3 that can be used as an effective chronic intestinal P567126 inflammatory disorders treatment, a composition containing such a fraction and uses thereof.

In accordance with the present application, there is provided a dairy-derived composition for prophylaxis or treatment of an inflammatory disease or a related 5 disorder in a subject, wherein said composition comprises TGF-p1, TGF-p2, IGF-1, beta-lactoglobulin, and at least 15% (w/w) of whey derived proteins.

Preferably the concentration of TGF-pi is between about 0.1 and 5 pg per gram of composition, and more preferably between about 0.2 and 1.2 pg per gram of composition.

In one embodiment of the application, the concentration of TGF-02 is preferably between about 5 and 50 pg per gram of composition, and more preferably between about 10 and 18 pg per gram of composition.

In one embodiment, the concentration of whey derived proteins is at least 50% (w/w), and more preferably at least 80% (w/w).

The composition may further comprise between 0.1 and 15% of minerals.

In one embodiment, the composition comprises at least 35%, and preferably at least 70% of water-soluble proteins.

The inflammatory disease may be for example chronic inflammatory disease, or chronic intestinal inflammatory disease, such as Crohn's disease or ulcerative colitis. 2 o The subject can be an animal or a human.

In accordance with the present application, there is provided the use of a dairy-derived composition comprising TGF-pi, TGF-P2, IGF-1, beta-lactoglobulin and at least 15% (w/w) of whey derived proteins for prophylaxis or treatment of inflammatory diseases or related disorders. 2 5 Still in accordance with the present application, there is provided a method for treating or preventing an inflammatory disease or a related disorder, comprising the step of administering to a subject a therapeutically effective amount of a dairy-derived P567126 composition comprising TGF-p1, TGF-(32, IGF-1, beta-lactoglobulin, and at least 15% (w/w) of whey derived proteins.

Still in accordance with the present application, there is provided a method for treating or preventing an inflammatory disease or a related disorder, comprising the step of • combining TGF-(31, TGF-02, IGF-1, beta-lactoglobulin and at least 15% (w/w) of whey derived proteins to obtain a dairy-derived composition; and • administering a therapeutically effective amount of said dairy-derived composition to a subject in need thereof.

Someone skilled in the art will recognize that various types of chronic inflammatory 10 diseases can be prevented or treated with the method and composition of the present application. For example, without limitation, the term chronic inflammatory disease includes rheumatoid arthritis, psoriasis, cutaneous inflammation, atopic dermatitis, encephalitis, hypersensitivity pneumonitis, chronic lung inflammation, ischemia-reperfusion injury, systemic lupus erythematosus, myositis, ankylosing spondylitis, 15 scleroderma, acute inflammatory demyelinating polyradiculoneuropathy, vasculitis, appendicitis, arachnoiditis, myocarditis, acute cholecystitis, chronic airflow obstruction, chronic hepatitis, chronic obstructive pulmonary disease, conjunctivitis, dermatitis, enteritis, gingivitis, hepatitis, ileitis, asthma, and/or intestinal chronic inflammation diseases such as Crohn's disease, inflammatory bowel disease or ulcerative colitis . 2 0 Accordingly, one aspect of the present application contemplates a method for reducing the effects of a proliferative and/or inflammatory disorder in a subject, the method comprising treating a chronic inflammatory disease with an effective amount of a composition capable of preventing or treating chronic inflammatory diseases and related disorder, as described herein. 2 5 For the purpose of the present application the following terms are defined below.

The term "inflammation" as used herein is intended to represent the normal response of the immune system to infection or irritation. The present application only addresses the cellular compartment of the immune response, also referred to as the Th1 component of the immune system, and is not addressing the humoral, or exudative P567126 portion of the immune response, also referred to as the Th2 component of the immune system.

The terms "inflammatory disease" and "inflammatory disorders" as used herein are intended to mean any disease or disorder affecting the normal response of the immune 5 system. The disease or disorder can involve either an excessive or an insufficient response of the immune system to infection or irritation, a lack of response following infection or irritation, and a response in the absence of infection or irritation. Such disorders can be for example, without limitation, ulcerative proctitis, proctosigmoiditis, left-sided colitis, pan-ulcerative (total) colitis, psoriatic arthritis, ankylosing spondylitis, 10 juvenile ankylosing spondylitis, seronegative enthesopathy, arthropathy syndrome, Reiter's syndrome, spondyloarthropathies, and/or endogenous uveitis The term "inflamed tissue" as used herein refers to a tissue affected by inflammation and affected cells contained within the tissue. Cells that have migrated to the inflammation site, such as leukocytes, are excluded from the definition of this term. 15 Preferably, the inflamed tissue is from a mammalian species, preferably a human.

The expressions "effective amount" and "therapeutically effective amount" as used herein are intended to mean an amount sufficient to initiate a beneficial or desired clinical result, such as an improvement of the condition of the patient. An effective amount can be administered in one or more doses. For the purposes of this 2 0 application, an effective amount of the present composition is an amount that induces a therapeutic or prophylactic response against at least one factor responsible for inflammation. Such amount will vary according to the nature of the inflamed tissue, the severity of the inflammation, the mode of administration, etc. One skilled in the art can easily and without difficulty monitor the inflamed tissue so as to determine what will be 2 5 such effective amount.

The terms "individual", "subject" or "patient" according to this application is intended to mean a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, rodents, primates, and pets.

The term "minerals" as used herein is intended to mean salt constituents and minerals 3 0 normally found in milk. For example, calcium, phosphorus, magnesium, potassium, sodium, chloride, sulfur, and citric acid can be used in various concentrations. This also P567126 means that trace elements known in the art as being present in milk can be assimilated or inferred therein.

The term "treatment" as used herein is intended to mean a medical remedy to improve or alleviate in part or totally the condition of a subject suffering from inflammation or 5 inflammatory disorders.

The term "prophylaxis" as used herein is intended to mean any procedure directed to the prevention of inflammation or inflammatory disorders. It will be understood that this term represents both primary prophylaxis, i.e. prevention of the apparition of the disease or disorder, and secondary prophylaxis, i.e. prevention of the worsening of an 10 already developed disease or disorder.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows, a dose-dependent inhibition of ConA-induced murine splenocytes proliferation by the composition of the present application; Fig. 2 shows a dose-dependent inhibition of ConA-induced murine splenocytes IL-2 production by the composition produced in example 1 and described in example 2; Fig. 3 shows a dose-dependent inhibition of ConA-induced murine splenocytes IFN^y production by the composition produced in example 1 and described in example 2; Fig. 4 illustrates the effect of colorectal administrations of the composition produced in 2 o example 1 and described in example 2 on daily body weight variations of Sprague-Dawley rats treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS), a chemical inflammatory inducer, as described in detail in example 4; Fig. 5 illustrates the effects of colorectal administrations of the composition produced in example 1 and described in example 2 on weight-to-length ratios of the colons of 25 Sprague-Dawley rats treated with TNBS, as described in detail in example 4; Fig. 6 illustrates the effects of colorectal administrations of the composition produced in example 1 and described in example 2 on macroscopic damages of rat colon tissues previously irritated with TNBS, as described in detail in example 4; P567126 Fig. 7 illustrates the effects of the composition produced in example 1 and described in example 2 on the macroscopic appearance of lesions in TNBS colitis, as described in detail in example 4; Fig. 8 shows hematocrit levels before and during oral treatment with different doses of 5 the composition produced in example 1 and described in example 2, where A is a placebo, B is the composition (7.82 mg/kg), C is the composition (78.2 mg/kg), and D is the composition (782 mg/kg), as described in detail in example 5; and Fig. 9 shows hemoglobin levels before and during oral treatment with different doses of the composition produced in example 1 and described in example 2, where A is a 10 placebo, B is the composition (7.82 mg/kg), C is the composition (78.2 mg/kg), and D is the composition (782 mg/kg).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present application now will be described in more details hereinafter with reference 15 to the accompanying drawings, in which preferred embodiments of the application are shown. This application, may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the application to those skilled in the art. 2 o All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby incorporated by reference.

In accordance with the present application, there is provided a composition and a method for treating chronic inflammatory diseases in an individual, wherein the composition effective in stimulating a specific immunological response against a 25 physiological imbalance aberrantly expressed in inflamed tissues. This composition comprises a cocktail of products that shares growth and regeneration characteristics of a growth factor that is aberrantly expressed in inflamed tissues (such as human inflamed tissues). Particular growth factors included in the composition of the present application are, but not limited to, TGFs, such as TGF-pi and TGF-P2, beta- 3 0 lactoglobulin and at least 15% of dairy derived proteins.

P567126 The growth factors present in the composition are distinguished by having a strong anti-inflammatory action. The concentration of the compounds in the composition of the present application may be adjusted depending of the needs. TGF-(31 can be present at concentration of between 0.1 to 5 pg/g of composition and TGF-(32 can be present at 5 concentration of between 5 and 50 pg/g of composition. Preferably, TGF-{31 is present at concentration of 0.2 to 1.2 pg/g, and TGF-(32 at between 10 to 18 pg/g of composition. The preparation can be used daily in a customary manner.

In another embodiment of the present application, the composition comprises at least 70% (w/w) of whey derived proteins. The total concentration of proteins in the 10 composition would normally be of at least 80%. Fat concentration can be generally found at concentration between about 0.5 to 10 % (w/w) in the composition.

In one particular embodiment of the present application, at least 35%, preferably 70%, of the proteins are water-soluble. The proteins may be comprised of at least 60% (w/w) of p-lactoglobulin, and/or between 0.1 to 30% (w/w) of lactoferrin.

The active compounds according to the application, such as the growth factors mentioned earlier, can be mixed with customary pharmaceutically tolerable diluents or vehicles and, if appropriate, with other auxiliaries molecules, and administered, for example, topically, orally, parenterally or colorectally. This formulation can preferably be administered orally in the form of granules, capsules, pills, tablets, film-coated 2 0 tablets, sugar-coated tablets, syrups, emulsions, suspensions, dispersions, aerosols, solutions, and/or liquids. It can also be administered as suppositories, vaginal suppositories, and/or parenterally, e.g. in the form of solutions, emulsions, creams or suspensions. It can be administered in preparation for time delayed release, or protected from gastric acid by coating to be released in the intestinal part of the gut. 25 Preparations to be administered orally can contain one or more additives such as sweeteners, aromatizing agents, colorants and preservatives. Tablets can contain the composition mixed with customary pharmaceutically tolerable auxiliaries, as for example inert diluents such as calcium carbonate, sodium carbonate, lactose and talc; granulating agents and agents which promote the disintegration of the tablets on oral 3 0 administration, such as starch or alginic acid; binding agents such as starch or gelatin; and lubricants such as magnesium stearate, stearic acid and talc.

P567126 The formulations are prepared, for example, by extending the composition with solvents and/or excipients if appropriate, using emulsifiers and/or dispersants, it being possible, for example, in the case of the use of water as a diluent optionally to use organic solvents as auxiliary solvents.

Administration is carried out in a customary manner, preferably orally or parenterally, or prelingually, sublingually, colorectally, topically or intravenously. In the case of oral administration, apart from the excipients mentioned, tablets can also contain additives, such as sodium citrate, calcium carbonate and dicalcium phosphate together with other various additives, such as starch, preferably potato starch, gelatin and the like. 10 Furthermore, lubricants such as magnesium stearate, sodium lauryl sulphate and talc can additionally be used for tableting. In the case of aqueous suspensions and/or elixirs, which are intended for oral administration, the active compounds can be mixed, apart from with the above-mentioned auxiliaries, with various flavors enhancers or colorants. In the case of parenteral administration, solutions of the composition using 15 suitable liquid excipients can be employed.

Capsules can contain the composition as a single constituent or mixed with a solid diluent such as calcium carbonate, calcium phosphate or kaolin. The injectable formulations are also formulated in a manner known perse.

Furthermore, antioxidants can be added to the composition and to the pharmaceutical 2 o and topical formulations. The use of natural or naturally identical compounds such as, for example, tocopherols is particularly preferred here. The antioxidants mentioned are contained in the compositions according to the application, for example, in amounts from 0.01-5% by weight, in particular from 0.5-2% by weight, based on the total composition.

The preferred formulation according to the application can be formulated as liquid, pasty or solid preparations, for example as aqueous or alcoholic solutions, aqueous suspensions or emulsions.

Estimates of the prevalence of chronic inflammatory diseases vary according to each specific disease. For example, intestinal inflammatory diseases are considered to affect 3 0 0.3% of American population.

P567126 In all types of chronic inflammatory diseases, the composition can be used prophylatically and for the treatment of the disorders.

While a detectable immune or tissue response is likely to be beneficial, efficacy can also be deduced by an improvement in symptoms, or control of the chronic 5 inflammatory condition beyond what would be expected without treatment.

For prophylaxis, the composition is administered in order to decrease manifestations of the disease in frequency and strength. Treatment in the manifest stage leads to its curtailment and to the alleviation of the symptoms.

The composition can also be administered colorectally or protected with enteric coating 10 for delivery in the intestinal part of digestive tractus. The composition can also be incorporated into customary oral or pharmaceutical bases for preferred oral administrations.

The application also relates to the use of the composition according to the application for the production of a pharmaceutical composition, in particular oral and 15 pharmaceutical compositions for the prophylaxis and treatment of chronic inflammatory diseases.

Without wishing to be bound to theory, the composition also addresses the underlying T-cell disorder that results in an inflammatory condition. The Applicants have recognized that most, if not all, of the current therapies in the prior art for T-cell 2 0 mediated inflammatory conditions are designed to eliminate T-cells and to thereby improve the inflammation condition. A major problem with those treatments is that there is a possibility that the therapy itself is so toxic that it may promote recurrence during healing. The toxicity of these treatments can unleash some or all of the cytokines that are associated with the promulgation of these chronic and often rebounding diseases.

The synergy of the two TGF-P factors of the composition results in a non-toxic, highly effective treatment for chronic inflammatory diseases that is without the side effects observed with virtually all other therapies for those diseases.

The present application will be more readily understood by referring to the following examples which are given to illustrate the application rather than to limit its scope.

P567126 EXAMPLE 1 : Preparation of composition 1000 kg of a whey protein isolate was rehydrated up to 5% in deionized water and stabilized overnight at 4°C under agitation. The pH of the solution was adjusted to between 4.8 and 5.2 by addition of concentrated HCI. This adjustment induced protein 5 precipitation. The precipitate was then recovered by centrifugation on a Westfalia separator at 25 m3 per hour and washed with acidified water. The precipitate was rehydrated to obtain 800 liters, which were thermally treated at 75°C during 15 seconds, before concentration and spray drying. Approximately 60 kg of composition was obtained.

EXAMPLE 2 : Analysis of the composition Composition of the ingredient is presented in table 1. Proteins were analyzed by the Dumas method, while fat was analyzed by the Mojonnier method, humidity by drying in a dry oven, beta-lactoglobuline by RP-HPLC and growth factors by ELISA methods.

Table 1: Typical analysis of the composition Compound Quantity Proteins 86% water-soluble proteins Fat 1.9% Beta-lactoglobulin 66% Humidity 4.2 % IGF-1 54 pg/mg TGF-(32 11.700 pg/mg TGF-pi 300 pg/mg P567126 EXAMPLE 3 : Inhibition of proliferation of mice splenocytes TGF-p has multiple immunosuppressive effects at the cellular level and has been described as inhibiting type 1 and type 2 cells, B cells, CD8+ T cells, macrophages and natural killer cells. TGF-p blocks cell-cycle progression and may have a direct effect on 5 expression of the gene encoding IL-2 (Brabletz et al., Mol. Cel. Biol., 13(2): 1155-1162, 1993). Importantly, it can suppress the expression of IL-12 and IL-2 receptors, as well as downregulate MHC class II expression on macrophages (specifically by antagonizing TNF-a and IFN-y) (Harber et al., Exp. Rev. Mol. Med., 27 Nov., http://www-ermm.cbcu.cam.ac.uk/00002143h.htm. 2000). The importance of TGF-P in 10 the induction and maintenance of normal tolerance has been illustrated by TGF-p-knockout mice, who incur severe, spontaneous, multi-organ, autoimmune disease associated with autoantibody production, which proves fatal in early life (Harber et al., Exp. Rev. Mol. Med., 27 Nov., http://www-ermm.cbcu.cam.ac.uk/00002143h.htm. 2000). T lymphocytes isolated from murine spleen (splenocytes) are immune cells that 15 respond to ConA (mitogenic stress) by proliferation and cytokine production (Mercier, et al. Internal Dairy J., 14: 175-183, (2004); Wong, et al. J. Dairy Sci., 81: 1825-1832, (1998)). The purpose of this example was to evaluate the inhibition effect of the composition produced in example 1 and described in example 2 on splenocytes proliferation and production of proinflammatory cytokines. 2 0 Female BALB/c mice, 6-8 weeks old, were obtained from Charles River (Montreal, Canada). Mice were sacrificed by C02 inhalation and single-cell suspensions were prepared individually from murine spleen under aseptic conditions. Murine lymphocytes (1.25 x 106 cells/ml) were treated with ConA (1.25 pg/ml) for 72 hours. Alamar blue® was added 24 hours before measuring ConA-induced lymphocytes 25 proliferation. Cytokines (IL-2 & IFN^y) levels were also determined in the supernatant following the incubation period. BSA was used as a protein control (1-1000 pg/ml).

The composition produced in example 1 and described in example 2 (1, 10, 50, 100, 500, and 1000 pg of the total composition per ml of culture medium), was added to the cells for the whole incubation period and dose-dependently decreased 1) the 3 0 lymphocyte proliferation (Fig. 1), 2) the production of IL-2 (Fig. 2), and 3) the production of IFN-y (Fig. 3). As shown in Fig. 1, there was a significant inhibition (SI) of induced proliferation by the composition. Without wishing to be bound to theory, these results P567126 suggest that the composition presents immunomodulatory functions and that it can be use to address immune-mediated chronic inflammatory diseases such as psoriasis, inflammatory bowel diseases and related arthropathies.

EXAMPLE 4 : Test on TNBS-induced inflammation in healthy rats Inflammatory bowel diseases (IBD) is a term used to describe a collection of diseases that involve the intestine and are characterized by chronic inflammation, sometimes accompanied by ulceration in the small or large intestine. Cytokines play an important role in modulating the immune system since they are rapidly synthesized and secreted from activated inflammatory cells and induce the production of adhesion molecules and 10 other inflammatory mediators, such as reactive oxygen metabolites, nitric oxide, and lipid mediators (De Winter et al., Am J Physiol 276: G1317-1321, 1999). Cytokines induce, amplify, prolong, and inhibit inflammation. Recently, several murine models of colitis have been developed which have highlighted the important role that abnormalities of the immune system, particularly those affecting T cells, may play in 15 disease pathogenesis. The purpose of this example was to characterize the effect of local application of the composition produced in example 1 and described in example 2 on TNBS-induced colitis in rats.

Male Sprague Dawley (Charles River Laboratories, Montreal), were kept in individual cages, at 20°C and 55% relative humidity with 12h light/12-h dark cycles, in a facility 2 o meeting the Canadian Council on Animal Care guidelines. The rats were subjected to a 24-hour starving period prior to inflammatory stress induction. Colonic inflammation was induced by using a technique from Togawa et al. (Am. J. Physiol., 283:187-195, 2002). Briefly, the rats were lightly anesthetised with isoflurane after overnight food deprivation, and a polyethylene catheter was inserted 8 cm into the colon via the anus. 2 5 TNBS (Sigma-Aldrich, Canada) dissolved in 50% (vol/vol) aqueous ethanol (25mg/ml) was injected into the colon (total volume of 1 ml/rat). The control rats received 1 ml of PBS intracolonically in place of TNBS and ethanol.

Four groups of rats were treated in the following manner: Group 1: Non-stressed control receiving 2 ml of PBS (n=2) once a day throughout the 3 o protocol; P567126 Group 2: Stressed control: TNBS (stress agent) in the intestinal lumen receiving 2 ml of PBS once a day, 5 days prior to administration of the stress agent in the lumen and 24 hours following the administration of the stress agent in the lumen (n=8); Group 3: Colorectal administration of the composition produced in example 1 and 5 described in example 2 (78,2 mg/kg in 2 ml of PBS, n=7) once a day, 5 days prior to administration of the stress agent in the lumen and 24 hours following the administration of the stress agent in the lumen.

Animals were weighed every day for the entire time of the protocol. Forty-eight hours following the TNBS administration, the rats were sacrificed and a 1 cm excision of the 10 intestine was sampled. Samples were weighed and macroscopic colonic damage was scored by the following scale: 0. No damage 1. Localized hyperemia, no ulcer 2. Ulcerations without hyperemia of bowel wall thickening 15 3. Ulceration with inflammation at one site 4. Two or more sites of ulceration/inflammation . Major sites of damage extending more than 1 cm along the length of colon 6-10. If damage extends more than 2 cm along length of colon, score is 2 0 increased by one for each additional 1 cm Body weight. As shown in Fig. 4, body weights of the animal increased constantly prior to fasting and TNBS injection in all groups. Following PBS injection (for control animals only), animals regained the weight lost during the fasting period. In TNBS-treated animals, however, the animals did not regain the body weight lost during the fasting 2 5 period. As for the animals treated with the composition produced in example 1 and described in example 2, body weight was increased following the TNBS injection.

P567126 Weight-to-length ratio. TNBS injection increased weight-to-length ratio in colon for both TNBS and the composition produced in example 1 and described in example 2 groups, indicating the presence of inflammation and edema. However, the increase was significantly (P< 0.05) inferior in animals treated with the composition produced in 5 example 1 and described in example 2 than in TNBS-treated animals (Fig. 5).

Macroscopic scores. TNBS injection increased macroscopic scores of inflammation in both groups treated with either TNBS and the composition produced in example 1 and described in example 2, indicating the presence of inflammation and edema. However, the increase was significantly inferior (P< 0.05) in animals treated with the composition 10 produced in example 1 and described in example 2 than in TNBS-treated animals (Fig. 6 and Fig. 7).

EXAMPLE 5 : HLA-B27 rats HLA-B27 transgenic rat, expressing HLA-B27 and human H2m, exhibits a phenotype similar to humans suffering B27 related rheumatic disorders such as reactive arthritis, 15 ankylosing spondylitis, psoriasis and inflammatory bowel disease. This rat model is useful for research and pharmacological studies of spontaneous systemic inflammation, arthritis, inflammatory bowel disease and skin diseases including psoriasis and alopecia.

The primary goal of this example was to evaluate the in vivo efficacy of the composition 2 0 produced in example 1 and described in example 2 in IBD present in HLA-B27 rat model. Basically, four group of ten (10) male HLA-B27 transgenic rats (7 to 20 weeks old) were formed following randomization which was be based on the week of first symptoms of IBD (i.e. positive Hemoccult™). After one week of adaptation, the composition produced in example 1 and described in example 2 (7.82, 78.2 and 782 2 5 mg/kg) was orally administered daily via a gastric needle (total volume of 1,5 ml).

Hematocrits. As shown is Fig. 8, hematocrit levels were higher in rats treated with 7,82 and 78,2 mg/kg of the composition produced in example 1 and described in example 2 compared to the value observed before the beginning of the treatment. Also, animals treated with 7,82 and 78,2 mg/kg of the composition produced in example 1 and 3 0 described in example 2 had a higher level of hematocrit compared to placebo (PBS).

P567126 Hemoglobin. During the course of the disease, hemoglobin levels decreased significantly in all groups. However, the decrease was significantly lower in rats treated with 7,82 and 78,2 mg/kg of the composition produced in example 1 and described in example 2 compared to placebo or even to 782 mg/kg of the composition produced in 5 example 1 and described in example 2 (Fig. 9).

While the application has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures 10 from the present disclosure as come within known or customary practice within the art to which the application pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

P567126 Recieved at IPONZ 3 August 2010

Claims (14)

WHAT IS CLAIMED IS:

1. A composition for prophylaxis or treatment of chronic intestinal inflammatory disease in a subject, wherein said composition comprises TGF-(31 at a concentration between about 0.1 and 5 pg per gram of composition, TGF-|32 at a concentration between about 5 and 50 |ag per gram of composition, IGF-1 and at least 15% (w/w) of whey proteins from which at least 60% is beta-lactoglobulin.

2. The composition of claim 1, wherein the concentration of said TGF-(31 is between about 0.2 and 1.2 |_ig per gram of composition.

3. The composition of claim 2, wherein the concentration of said TGF-(32 is between about 10 and 18 pg per gram of composition.

4. The composition of claim 1, wherein the concentration of said whey proteins is at least 50% (w/w).

5. The composition of claim 4, wherein the concentration of said whey proteins is at least 80% (w/w).

6. The composition of claim 1, wherein the chronic inflammatory disease is Crohn's disease or ulcerative colitis.

7. The composition of claim 1, wherein said subject is human.

8. Use of a composition for the manufacture of a composition for the prophylaxis or treatment of chronic intestinal inflammatory disease in a subject, wherein said composition comprises TGF-(31 at a concentration between about 0.1 and 5 ng per gram of composition, TGF-|32 at a concentration between about 5 and 50 |_ig per gram of composition, IGF-1 and at least 15% (w/w) of whey proteins from which at least 60% is beta-lactoglobulin.

9. The use of claim 8, wherein the concentration of said TGF-|31 is between about 0.2 and 1.2 |jg per gram of composition. P567126 Recieved at IPONZ 3 August 2010 - 24-

10. The use of claim 8, wherein the concentration of said TGF-|32 is between about 10 and 18 |ag per gram of composition.

11. The use of claim 8, wherein the concentration of said whey proteins is at least 50% (w/w).

12. The use of claim 11, wherein the concentration of said whey proteins is at least 80% (w/w).

13. The use of claim 8, wherein the chronic inflammatory disease is Crohn's disease or ulcerative colitis.

14. The use of claim 8, wherein said subject is human.