WO2009135306A1 - Colostrum-derived fraction for wound healing and skin care - Google Patents

Abstract

The present invention relates to a colostrum-derived mixture enriched in growth factors, particularly, a bovine colostrum fraction in topical or oral formulations for the treatment of skin injury, diseases, wounds or ulcers. The invention also provide a process for the preparation or the production of a composition enriched in growth factors from colostrum, and its uses thereof for the treatment of surface wounds and ulcers.

Description

COLOSTRUM-DERIVED FRACTION FOR WOUND

HEALING AND SKIN CARE

FIELD OF INVENTION

[0001] The present invention relates to a colostrum-derived mixture enriched in growth factors. Particularly, the invention provides a bovine colostrum fraction for the treatment of skin injury, diseases, wounds or ulcers. More particularly the invention provides a topical or oral composition that includes a pharmaceutically effective amount of bioactive growth factors and antimicrobial peptides originating from bovine colostrum, for the treatment of skin injury, diseases, wounds or ulcers.

BACKGROUND Skin function

[0002] Skin, as the outermost organ in the human body, continuously confronts the external environment and serves as a primary defense system or protective barrier against the environment (Lee et al. 2006). In addition to keeping the harsh external environment away from our own critically sensitive internal biological environments, skin acts as a regulator of body temperature and a sealant against fluid loss. The protective functions of skin can be classified as physical, thermal, immune, ultraviolet, oxidant radical, antimicrobial and a permeability barrier. [0003] Following an injury (ulcer, cut, laceration, gash, tear, scrape, abrasion, scratch, floor burn, bruise, bite, UV radiation or excessive pressure), loss of the integrity of large portions of the skin may lead to major disability or even death due to an increased susceptibility to fungus, bacteria, and viral infections.

Skin wound healing [0004] A scar is the end result or end point of the body to close a wound, and it is a normal process that occurs whenever an injury involves the dermis. The ideal end point is total regeneration, with new tissue having the same structural, aesthetical, and functional attributes as the original uninjured skin. However, in man and domestic animals, unstructured scarring in the skin after trauma, surgery, burns or

DOCSQUE: 756309\l sports injury is a major medical problem, often resulting in adverse aesthetics, loss of function, restriction of tissue movement and/or growth and adverse physiological effects. Current treatments are empirical, unreliable and unpredictable; there are no prescription drugs for the prevention or treatment of scarring (Bayat et al. 2003). [0005] Skin wounds normally heal in a very orderly and efficient manner characterized by four distinct, but overlapping phases; 1) hemostasis, 2) inflammation, 3) proliferation and 4) remodeling (Singer & Clark 1999).

1) Hemostasis

[0006] Tissue repair begins with clot formation and platelet degranulation, which release the growth factors necessary for wound repair. Platelet-derived growth factors are biologically active substances that enhance tissue repair mechanisms such as chemotaxis, cell proliferation, angiogenesis, extracellular matrix deposition, and remodeling (Werner & Grose 2003).

2) Inflammation [0007] Infiltrating neutrophils cleanse the wounded area of foreign particles and bacteria and are extruded with eschar or phagocytosed by macrophages. In response to specific chemoattractants, such as fragments of extracellular-matrix proteins, growth factors, and monocytes chemoattractants protein 1 , monocytes also infiltrate the wound site and become activated macrophages that release various growth factors (Werner & Grose 2003).

3) Proliferation

[0008] Within hours after injury, the proliferation phase begins. The proliferative phase is characterized by the increased formation of granulation tissue where fibroblasts lay bed of collagen and produces new capillaries, the contraction of the wound (wound edges pull together to reduce defect) and finally epithelialization of the affected area. Growth factors in concert with extracellular-matrix molecules, presumably stimulate fibroblasts of the tissue around the wound to proliferate (Werner & Grose 2003). The formation of new blood vessels is necessary to sustain the newly formed granulation tissue. Wound contraction involves a complex and

DOCSQUE: 756309U superbly orchestrated interaction of cells, extracellular matrix, and cytokines. The contraction probably requires stimulation by growth factors (Werner & Grose 2003). 4) Remodeling

[0009] Collagen remodeling during the transition from granulation tissue to scar is dependent on continued synthesis and catabolism of collagen at a low rate. The degradation of collagen in the wound is controlled by several proteolytic enzymes termed matrix metalloproteinases, which are secreted by macrophages, epidermal cells, and endothelial cells, as well as fibroblasts.

[0010] Chronic wounds may be stopped in any of the four phases, commonly; however, disruption occurs in the inflammatory or proliferative phases. Many mediators including inflammatory cells, growth factors, proteases and cellular and extracellular elements play an important role in different stages of the healing process. Alterations in one or more of these components may account for the impaired healing observed in chronic wounds. Growth factors and wound healing

[0011] As suggested above, and as reviewed by Werner & Grose (2003), growth factors are involved in all steps of wound healing. Furthermore, multiple studies have demonstrated a beneficial effect of exogenous growth factors like PDGF, FGF, EGF, VEGF, TGF and IGF for wound healing (Werner & Grose 2003). In fact, PDGF was the first growth factor to be approved for the treatment of human ulcers (Regranex gel^®).

Insulin-like growth factor

[0012] IGF-I and -Il are small peptides, approximately 7 kDa in size, that are structurally similar to insulin. IGF-I and -Il are growth factors that have both mitogenic and metabolic actions that participate in the growth, survival and differentiation of a number of cell types and tissues (Cohick & Clemmos 1993, Stewart & Rotwein 1996). IGFs are unique among growth factors in that they can act both synergistically, as a hormone, and locally, as autocrine/paracrine factors (Cohick & Clemmos 1993, Stewart & Rotwein 1996, Butler & LeRoith 2001 ).

DOCSQUIi: 756309M - A - Although liver is the principal source of circulating IGFs, it is now believed that brain, muscle and bone also produce IGFs (Cohick & Clemmos 1993, Stewart & Rotwein 1996, Butler & LeRoith 2001 ).

[0013] In vivo IGFs are found complexed with specific IGF-binding proteins (IGFBPs) (Shimasaki & Ling 1991 ). Most IGF-I present in the circulation as part of a 150-kDa complex, comprising IGFBP-3 and an acid-labile subunit, which is thought to regulate the bioavailability of IGF to the tissues and to prolong the half-life of IGFs (Jones & Clemmons 1995).

[0014] IGFs are specifically expressed in granulation tissue and several studies have suggested a role of the IGF system in the wound healing abnormalities associated with diabetes and glucocorticoids treatment (Bitar &Labbad 1996, Bitar 2000, Blakytny et al. 2000). More specifically, these studies suggested that a reduced expression of IGFs and/or their receptors leads to impaired wound healing, although this hypothesis has yet to be confirmed by functional studies. Additionally, IGFs act in synergy with other growth factors to promote healing (Werner & Grose 2003).

[0015] The role of IGFBPs is less clear. It has been shown that the co administration of IGFBP-I with IGF-I can enhance the stimulatory wound-healing properties of IGF-I (Tsuboi et al. 1995) and IGF complexes with IGFBP-3 has a greater effect on wound tissue formation than IGF alone (Campbell et al. 1999), suggesting a vital role of IGFBPs in the regulation of IGF-I action at the cellular level.

Antimicrobial peptides and wound healing

[0016] Antimicrobial peptides (AMPs) are predominantly small cationic polypeptides that are classified together due to their capacity to inhibit the growth of microbes. As effectors of innate immunity, AMPs directly kill a broad spectrum of bacteria, fungi, and viruses. In addition, these peptides modify the local inflammation response and activate mechanisms of cellular and adaptive immunity (Reddy et al. 2004).

[0017] Human skin produces antimicrobial agents that form an innate epithelial chemical shield. These endogenous AMPs are molecules produced by the epithelial

DOCSQUL 756309M surface of the host. In addition skin peptides play an integral role in stimulating and regulating wound healing and inflammation. Two major classes of peptides have been identified in mammalian skin; (1) defensins and (2) cathelicidins.

[0018] The defensins are cationic peptides (MW 3-5 kDa) divided in three subfamilies, α-, β-, and circular θ-defensins based on the alignment of the disulphide bonds. AMPs of the defensins family exhibit broad activity against gram- negative bacteria, fungi, mycobacteria and enveloped viruses and have been isolated form neutrophils granules, macrophages and some specialized epithelial cells of the small intestine (Yang et al. 2007). [0019] More specifically, α- and β-defensins show a broad antibacterial activity against gram positive and negative bacteria (Ehcksen et al. 2005, Ganz et al. 1985), and have antifungal activity (Hoover et al. 2003). Defensins also have antiviral properties against adenovirus (Bastian & Schafer 2001), papilloma virus (Buck et al. 2006), human immunodeficiency virus (HIV) (Chang et al. 2005), and herpes simplex virus (Hazrati et al. 2006). Although directly antimicrobial in vitro, the effect of defensins on mammalian cells is an important component of how these peptides affect immunity, α-, β-defensins modify cell migration and maturation, β-defensin are chemoattractive for immature-dentritic cells and memory T-cells (Yang et al. 1999), and also induce cytokines and other molecules secreted from host cells, α-defensin up-regulate the expression of TNF-α and IL-β in monocytes (Chaly et al. 2000).

[0020] The amphipathic structure and cationic charge of cathelicidin enable the latter to interact in the aqueous environment, the lipid-rich membrane, and bind negatively charged bacterial membranes. Cathelicidin peptides have broad antimicrobial activity against gram-positive and negative bacteria (Nizet et al. 2001 ), vaccinia virus (Howell et al. 2004), and fungi (Lopez-Garcia 2005). Cathelicidin peptides are cationic and, like defensins, thought to directly bind to anionic cell wall and membrane of the microbe, increasing the permeability of the microbe cell wall. Cathelicidin also induces cellular signaling and activates keratinocytes and leucocytes. Cathelicidin are chemoattractive to neutrophils, monocytes, and T cells and also promote angiogenesis (De Y et al. 2000). Transactivation of epidermal

DOCSQULi: 756309M growth factor receptors by cathelicidin was observed in human epidermal keratinocytes, and this induces keratinocytes migration (Tokumaru et al. 2005).

[0021] All Proline rich peptides (PRPs) isolated from mammals derive from the cathelicidin. PRPs are very small peptide chains (3.5-6 kDa) of less then a dozen amino acids, with proline predominating (up to 50%). PRPs isolated from early sheep colostrum have been shown to have regulatory effects on the immune response (Wieczorek et al. 1979). PRP acts both in vitro and in vivo, and is not species specific. PRP increases permeability of skin vessels (Wieczorek et al. 1979), and causes differentiation of murine thymocytes into functionally active T cells (Januz & Lisowski 1993). It can simultaneously change surface markers and functions of the cell (Wieczorek 1989).

[0022] Evidence is accumulating that AMPs enter cells without membrane lysis and, once in the cytoplasm, bind to, and inhibit the activity of specific molecular targets essential to bacterial growth, thereby causing cell death. AMPs from mammals, exerts other potentially exploitable biological activities, such as induction of syndecan expression in mesenchymal cells and inhibition of the NADPH oxidase activity of neutrophils, suggesting a role of this peptide in wound healing and inflammation (Gennaro et al. 2002).

Colostrum [0023] Colostrum is the early milk produced by mammals during the first several days post-parturition. Colostrum provides passive immunity to protect the newborn from opportunistic infections while the immune system is developing, as well as to facilitate the growth and immune maturation of the digestive tract and most certainly other tissues (Gopal & Gill 2000). [0024] Colostrum has a nutrient profile and immunological composition that differs substantially from mature milk. In addition to macronutrients found in milk such as protein, carbohydrates, fat and micronutrients including vitamins and minerals, colostrum is rich in immunoglobulins, growth factors cytokines and nucleosides but also in oligosaccharides, antimicrobials, and immune-regulating factors (Thapa 2005). The typical composition of bovine colostrum is enumerated in Table 1.

DOCSQUE: 7₅6309\l Colostrum as a source of growth factors

[0025] The presence of growth factors was first demonstrated in human colostrum and milk during the 1980's and then in bovine colostrum, milk and whey (Gauthier et al. 2006). Of significant interest is the fact that bovine growth factors found in colostrum are almost identical to that of human in both structures and functions. Specifically, colostrum represents an important source of EGF, IGF-I and II, FGF, PDGF, TGF, but also of interleukins and interferons (Table 1).

Colostrum as a source of antimicrobial peptides

[0026] Colostrum provides numerous antimicrobial factors that exhibit both specific and nonspecific bacteriostatic/bactericidal properties. Immunoglobulins are among the first line of protection that are delivered to the neonate through suckling and provide passively acquired immunity. Other proteins, such as the iron-binding protein, lactoferrin (Clare et al. 2003), and enzymes, including lysozyme, and lactoperoxidase play more of a direct role in inhibiting bacterial invasion (Clare & Swaisgood 2000). Recently, soluble defensins, cathelicidins, and toll-like receptors (TLRs) have been identified in human milk (Armogida et al. 2004).

[0027] Immunocompetent cell types, including macrophages, granulocytes, T and B-lymphocytes are also present in colostrum. One thing of interest is the fact that the total antibacterial effect of colostrum is greater than the sum of the individual contributions of immunoglobulin and nonimmunoglobulin defense proteins. This is most likely due, at least in part, to their synergy.

Clinical uses of colostrum

[0028] The use of colostrum for the treatment of illness and for the maintenance of well-being dates back thousands of years. The Ayurvedic physicians and the Rishis of India have been using colostrum for medicinal purposes since cows have become domesticated. The remarkable thing about colostrum is that all these substances act synergistically in such a way as to enhance the overall effect of each individual component.

[0029] Available evidence suggests a beneficial effect of oral supplementation of colostrum in improving body composition, aspects of athletic performance, diarrhea

DOCSQUC 756309U in person with immune-deficiency syndromes, NSAID-induced gastrointestinal disturbances and aspects of the acute phase response that occurs secondary to surgery (Kelly 2003). Furthermore, some authors have claimed that topical application of colostrum has benefic effects on surface wound and other skin related diseases. Other milk-derived products when taken orally or when applied topically have demonstrated therapeutic benefits (Kelly 2003).

Table 1. Composition of human and bovine colostrum. ^afrom Solomon 2002, ^bfrom Gauthier et al. 2006. Components

Water 78 %^a

Carbohydrates 3.1 % ^a

Fat 3.6 % ^a

Protein 14.3 % ^a

Immunoglobulins

IgG 77 mg/ml ^a

IgM 4.9 mg/ml ^a slgA 4.4 mg/ml ^a

Lactoferrin + ^a

Lysozyme + ^a

Lactoperoxidase + ^a

Growth factors

SUMMARY OF THE INVENTION

[0030] One aspect of the present invention is to provide a process for the preparation or the production of a composition enriched in growth factors from colostrum, and its uses thereof for the treatment of surface wounds and ulcers.

[0031] More particularly, the process of the invention comprises the following steps of: a') optionally thawing frozen whole colostrum to a temperature of at least about 4⁰C;

DOCSQUC. 756309U a) precipitating casein from said colostrum and separating curds from colostrum whey; b) heating said colostrum whey to a temperature of greater than 4O⁰C; c) submitting said heated whey to ultra-filtration with a molecular weight cut- off of about 14kDa or less and collecting permeate; d) acidifying said permeate obtained in step c) to a pH of about 3 or above.

[0032] Optionally, the acidified permeate obtained in step d) is further concentrated with nanofiltration to obtain a liquid composition. Particularly, this liquid composition comprises at least 20 ng/ml of IGF-1 ; less than 5% (w/w) of protein and is substantially free of lysozyme and TGF-β.

[0033] Further optionally, this liquid composition is freeze-dried to obtain a solid composition. Particularly, this solid composition comprises at least 1 ng of IGF-1 per mg of solid; less than 5% (w/w) of protein and is substantially free of lysozyme and TGF-β. [0034] A particularly aspect of the invention provide a composition enriched in antimicrobial peptides of molecular weight of less than about 15kDa, preferably less than 12 kDa, more preferably less than 1OkDa. Particularly, the composition of the invention comprises physiological amount of these low molecular weight peptides.

[0035] More particularly, the composition of the invention is substantially free of lysozyme and/or TGF-β.

[0036] Particularly, the composition of the invention comprises a total protein concentration of less than 5% (w/w), preferably less than 2% (w/w).

[0037] More particularly, the liquid composition of the invention comprises at least 20 ng/ml of IGF-1 , particularly, at least about 40 ng/ml. [0038] More particularly, the solid composition of the invention comprises at least 1 ng of IGF-1 per mg of solid, particularly, at least about 1.8 ng/mg.

[0039] A further aspect of the invention provides the use of the composition as defined herein for the manufacture of a formulation for the treatment of a wound

DOCSQUb 756309M and/or facilitating and/or accelerating wound healing while inducing minimal collagen contraction. Particularly, the composition is a topical or an oral formulation.

[0040] A further aspect of the invention provides a method for healing a wound while inducing minimal collagen contraction, said method comprising treating said wound with the formulation as defined herein.

[0041] Another aspect of the present invention is to provide a method for treating skin injury and ulcers comprising application to the wound with the liquid composition "as is" or with the topical or oral formulation as defined herein.

[0042] Accordingly, one aspect of the present invention contemplates a method for accelerating wound healing and improving aesthetical presentation of the scars formed in the wound healing process, the method comprising application on the wounded skin an effective amount of a composition or formulation capable of improving wound healing properties.

[0043] Someone skilled in the art will recognize the type of skin injury or ulcers that can be treated with the method and/or composition and/or formulation of the present invention and may include, amongst others, skin surface wounds, surgery wounds, burns, diabetic ulcers and pressure sores.

DETAILED DESCRIPTION OF THE INVENTION [0044] The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention, 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 invention to those skilled in the art.

Brief description of the drawings

[0045] Figure 1. Effect of colostrum-derived fraction on fibroblasts metabolic activity

(XTT assay).

DOCSQUE. 756309M [0046] Figure 2. Effect of colostrum-derived fraction on collagen contraction.

[0047] Figure 3. Effect of colostrum-derived fraction on collagen synthesis/deposition and soluble collagen release.

[0048] Figure 4. Effect of colostrums-derived fraction on wound contraction. [0049] Figure 5. Effect of colostrums-derived fraction on wound tissue areas. [0050] Figure 6. Effect of colostrums-derived fraction on wound thickness. [0051] Figure 7. Effect of colostrums-derived fraction on collagen deposition [0052] Figure 8. Effect of colostrums-derived fraction on collagen density.

[0053] Figure 9. Effect of colostrums-derived fraction on tissue ingrowth in PVA sponge.

[0054] Figure 10. Effect of colostrums-derived fraction on scar formation.

[0055] Figure 11. Effect of colostrums-derived fraction on the prevention of hypertrophic scar.

Definitions

[0056] For the purpose of the present invention the following terms are defined below. The expression "effective amount" as used herein is intended to mean an amount sufficient to induce a beneficial or desired clinical result. An effective amount can be administered in one or more doses. For purposes of this invention, an effective amount of growth factors and/or dairy derived proteins, or other composition is an amount that induces a treatment or prophylactic response against at least one wound healing and/or collagen "anti-contraction" responsible factor, in vitro and/or in vivo.

[0057] The terms "individual" or "subject" treated according to this invention is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, rodents, primates, and pets.

DOCSQUE 756309\l [0058] The term "substantially free" as used herein is intended to mean an amount that is substantially reduced from the original amount in which this constituent is found in colostrum. Particularly, when referring to lysozyme of TGF-β, the term "substantially free" means less than 10% of the original amount, preferably, less than 5% of the original amount, more preferably, less than 2% of the original amount, most preferably in trace amount.

[0059] The terms "polypeptide" and "peptide" are used interchangeably to refer to polymers of amino acids of molecular weight of about 15kDa or less, and may be interrupted by non-amino acids. [0060] The term "protein" refers to polymers of amino acids of molecular weight of more than 15kDa, and may be interrupted by non-amino acids.

Description of particular embodiments

[0061] Particularly, the present invention provides the process as defined herein, wherein the colostrum in step a) is obtained from thawing frozen whole colostrum to a temperature of at least about 4⁰C; preferably about 1O⁰C.

[0062] Particularly, the present invention provides the process as defined herein, wherein, in step a), the casein and curds are separated by treatment with microbial rennet; or by acid treatment, preferably at pH 4.6. [0063] Particularly, the present invention provides the process as defined herein wherein the acidified permeate obtained in step d) is concentrated to obtain a concentrated liquid fraction.

[0064] Particularly, the present invention provides the process as defined herein, wherein the concentrated liquid fraction is freeze-dried to obtain a solid fraction (powder).

[0065] Particularly, the present invention provides the process as defined herein, wherein in step b), said colostrum whey is heated to a temperature of at least 45⁰C, preferably at a temperature of at least about 5O⁰C.

DOCSQUE: 756309U [0066] Particularly, the present invention provides the process as defined herein, wherein in step c), said heated whey is submitted to ultra-filtration with a molecular weight cut-off of about 12kDa or less; preferably about 1OkDa.

[0067] Particularly, the present invention provides the process as defined herein, wherein in step d) the permeate is acidified at a pH of about 3.5; preferably at a pH of 3.2.

[0068] Particularly, the present invention provides the process as defined herein, wherein the acidified permeate is concentrated with nanofiltration or any other concentration method. [0069] Particularly, the present invention provides the process as defined herein, wherein said colostrum is obtained from: cow, ewe, jenny (donkey's female); goat or buffalo.

[0070] In a particular aspect of the invention, there is provided a composition derived from colostrum comprising at least 60% of antimicrobial peptides of MW less than 500Da; IGF-1 and a total protein concentration of less than 5% (w/w).

[0071] Particularly, the present invention provides the composition as defined herein, comprising at least 70% of antimicrobial peptides of MW less than 500Da and further comprising at least 20% (w/w) of antimicrobial peptides of molecular weight between 500Da and 1OkDa of which between 2% and 10% (w/w) is constituted of IGF-1.

[0072] Particularly, the present invention provides the composition as defined herein, wherein the composition is liquid and comprises at least 20 ng/ml of IGF-1 ; a total protein concentration of less than 2% (w/w) and is substantially free of lysozyme and TGF-β. Particularly, the present invention provides the composition as defined herein, wherein IGF-1 is present at at least about 40ng/ml.

[0073] Particularly, the present invention provides the composition as defined herein, wherein the composition is solid (powder) and comprises at least 1 ng of IGF-1 per mg of solid; a total protein concentration of less than 2% (w/w) of protein and is substantially free of lysozyme and TGF-β. Particularly, the present invention

DOCSQUL 756309M provides the composition as defined herein, wherein IGF-1 is present at at least about 1.6ng/mg total solid.

[0074] Particularly, the present invention provides the composition as defined herein, wherein said peptides below 500Da are present in physiologically effective amounts.

[0075] Particularly, the present invention provides the composition as defined herein, wherein said peptide of MW less than 500Da is selected from the group consisting of: PDGF; FGF; EGF; VEGF; defensins, cathelicidins, anti-microbial peptides (AMP) and proline- rich peptides (PRP). [0076] Particularly, the present invention provides a formulation comprising a composition as defined herein, further comprising a physiologically acceptable excipient; particularly, the formulation is topical or an oral formulation.

EXAMPLES Example 1 -Preparation of composition

[0077] Frozen whole bovine colostrum (less than 48 hrs post-parturition) is slowly thawed to 1O⁰C until complete melting. The fat fraction is then separated from this raw colostrum using a milk separator. The skimmed colostrum is then heated to 35°C and caseins are precipitated with the addition of double strength microbial rennet (Danisco, Madison, Wl, USA) for 20 minutes. The curds are separated by centrifugation from colostrum whey at 65°C. After the casein fines are removed at 37°C from the colostrum whey with a milk separator, the colostrum whey is heated to 50°C and submitted to an ultrafiltration step using a 10,000 molecular weight cut off (MWCO) spinal wound membrane (Parker Process Advanced Filtration, Oxnard, CA, USA). The ultrafiltration permeate is collected and acidified to pH 3.2. The acidified permeate is then concentrated with nanofiltration. Finally, the concentrated acidified permeate is freeze-dried.

DOCSQUE: 756309M Example 2-Analysis and composition

Liquid Solid

Proteins <2% <2%

IGF-I 43 ng/mL 1.95 ng/mg total solids

Antimicrobial Presence Presence peptides (<1 O kDa) (<1 O kDa)

Molecular weight distribution (HPSEC)

[0078] Samples were solubilized in HPLC grade water and then filtrated on 0.2μm pore size filters. Samples were then passed trough a TSK-GEL, Guard SWXL (6.0 i.d. x 40 mm) pre-column (Tosoh Biosep LLC, Montgomeryville, PA, USA) and a TSK-GEL, G2000 SWXL (7.8 i.d. x 300 mm) column (Tosoh Biosep LLC, Montgomeryville, PA, USA) at a flow rate of O.δmL/minute using a Waters HPLC (Mississauga, Ont., Can) containing two pumps (model 600), a controller (model 600E) and a UV detector (model 486). The results were analyzed with Millennium 32 software.

DOCSQUE: 756309M Example 3-Fibroblast functions

[0079] Human fibroblasts were derived from foreskins and used at passages between 10 and 25. They were maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma Chemical Co.) supplemented with 5% FBS and antibiotics/antifungic. For the cultures in the presence of the compositions to be tested the concentration of FBS was decreased to 0.5%. In monolayer and 3-D culture experiments, fibroblasts were seeded for a few hours in the presence of

5%FBS and then rinsed and the medium was changed to 0.5%FBS in the presence of the different concentrations of the compositions to be tested. Cultures were performed in 5% CO₂ and humid atmosphere.

[0080] The XTT Cell Proliferation Assay is a colorimetric assay system which measures the reduction of a tetrazolium component (XTT) into soluble formazan product by the mitochondria of viable cells. The samples are read using an ELISA plate reader at a wavelength of 450nm. The amount of color produced is directly proportional to the number of viable cells.

Metabolic activity measurements (XTT assay) in monolayer cell cultures

[0081] XTT (2,3-bis{2methoxy-4-nitro-5-sulfophenyl}-2H-tetrazolium-5- carboxyanilide inner salt) assay was performed according to the manufacturer's procedure (TOX2, Sigma-Aldrich Canada Ltd., Oakville, Ontario). For the XTT assay, cells were seeded at 2.5x10⁴ cells/cm² for fibroblasts and endothelial cells both in 24 multi-well plates. After 2-4 hrs, cell cultures were rinsed once and media supplemented with only 0.5% FBS containing the different dilutions (1 :1000, 1 :100, 1 :10 and 1 :1 ) of each composition to be tested were introduced. Cells were grown for 4 days with one medium change at day 2. However, each composition was added every day in culture medium. Afterwards, cell cultures were rinsed in PBS. A PBS solution of XTT (1 mg/ml) was mixed with a PBS solution of phenazinemethosulfate and incubated at 37⁰C for 1 hr. Optical densities were then

DOCSQUL 756309M measured with a fluorometer (Spectra Max 340Pe molecular Devices, Sunnyvale,

CA) set at 450 nm absorbance, and values were subtracted from the blank values. For each condition, cultures were performed in triplicate. In addition, a standard curve of increasing cell densities was used to correlate the optical density value with the cell numbers.

[0082] As demonstrated in Figure 1 , the 1 :1 dilution of the fraction enhanced significantly the cell growth compared to control value (P<0.05). The lowest dilution tested (1 :0.5) did also enhance cell growth significantly in comparison to the control (P<0.05). The highest dilutions tested (1 :10 and 1 :5) were not different from the control value. These data suggest that the present colostrum-derived fraction increased fibroblast metabolic activity in a bell-shape manner with a peak activity at a dilution of 1 :1.

Example 4-Collagen contraction [0083] Fibroblasts were mixed in collagen solution prior to gel formation at a density of 5 x 10⁴ cells per 500μl gel in wells of 24 multiwell plates. Collagen gels were made by mixing a rat tail tendon collagen solution (3.0 mg/ml) with a solution containing culture medium (DMEM 5x sans NaHCO₃), FBS, 0.26M NaHCO₃, 1 N NaOH and the suspension of cells as previously described by Elsdale & Bard (1972). Briefly the formula was the following:

1ml DMEM 5x without NaHCO₃ 500μl FBS

500μl NaHCO₃ at 0.26M 20μl NaOH at 1 N 100μl H₂O

Add 0.88ml cell dispersion (5.7 x 10⁵ cells/ml) and then 2ml rat tail collagen. The mixed solution was transferred to wells. Gels were formed after 15mn incubation. After 2-4hrs of incubation in medium with serum (5%)-supplemented medium, the

DOCSQUE 756309M medium was changed, cell cultures rinsed and the different compositions were diluted in culture medium supplemented with 0.5% FBS. Composition was introduced daily for 4 consecutive days. Triplicate samples were analyzed. By 24 hrs the gels were released from wells and left floating in order to trigger the collagen contraction by fibroblasts. Medium was changed at day 2. After 4 days, images of the gels were taken and quantified using an image analysis system (Image J). The values of the areas occupied by gels were expressed in mm² according to scale setting.

[0084] As shown in Figure 2, all dilutions prevent collagen contraction, compared to control value (P<0.05), which suggest that in presence of the colostrum-derived fraction collagen will not contract to form an hypertrophic scar.

Example 5-Collagen synthesis/deposition

[0085] Fibroblasts were seeded at 1x10⁵ cells per well of 6 multi-well plates in serum-supplemented medium. As described earlier, medium was changed after 2- 4hrs of incubation with a low-serum-supplemented (0.5% FBS) medium containing the composition to be tested. Fibroblast cell cultures were labeled by introducing 1.5μCi/ml [³H]-proline (Perkin Elmer) in medium after 4 hrs post-cell seeding and then every other day at medium changes for 7 days in monolayer cell cultures. The labeling medium contained 10μg/ml of ascorbate. At each medium change, media were pooled (medium pool) corresponding to most soluble collagen that has been released form the cells. At day 5, media were pooled with the previous respective pools, and the cell-matrix layers were scraped from wells with a rubber policeman and collected to be pooled (cell-matrix pool). The latter consisted mainly of insoluble collagen that has been deposited by the cells forming the extracellular matrix around the fibroblasts. To avoid any biodegradation during these maneuvers, both pools were treated with a protease inhibitor cocktail (EDTA, NEM, PAB, and PMSF in 1 M TrisHCI at pH 7.5). Pools were then promptly frozen until further use. The two frozen pools were thawed, vortexed, and then dialyzed against water for 3-4 days until the free radioactivity was released as determined by periodic counts. Dialyzed pools

DOCSQUL 756309M were frozen to be freeze-dried in a freeze-drying apparatus (Labconco). Dried samples were rehydrated in a 4%SDS solution in 5mM TRIS (pH 8.0) while keeping an equal final volume to all samples. For each condition, duplicate samples from the cell-matrix pool and those from the medium pool were counted separately in a liquid scintillation counter (Ultima Gold, Perkin-Elmer). Values were counted in dpm. The compositions were changed on a daily basis for 6 days.

[0086] As shown in Figure 3, the two highest dilutions tests (1 :10 and 1 :5) of the colostrum-derived fraction increased amount of collagen and deposition in the cell- matrix pool rather than releasing it in soluble form. [0087] The effect of the colostrum-derived fraction seems to have two distinct activities depending on its dilutions. The proliferative activity is prone at lower dilutions, whereas collagen metabolic activity is expressed at higher dilutions.

[0088] The colostrum-derived fraction appears to have interesting properties not only on fibroblast growth and synthetic activities, but also on the remodeling of pre- existing collagen. Collagen gel contraction is a phenomenon that has been reported as a physical arrangement of pre-existing collagen fibrils {Guidry and Grinnell, 1985). This is due to direct contact between fibroblasts that adhere strongly to the extracellular matrix and remodel the fibrils. In addition, the collagen synthesis in collagen gel has been found slightly diminished, as well as the DNA replication, compared to that observed in monolayer cell culture (Mauch et ai, 1988; Nusgens et al., 1984). This phenomenon may be largely due to the presence of collagen fibrils around the cells that down-regulate cell activity as observed in normal connective tissue.

[0089] Inhibition or limitation of collagen gel contraction has been reported previously with specific molecules such as prostaglandins E1 and E2, and the incorporation of proteoglycans, heparin, or plasma fibronectin in the collagen gel (Anderson et al, 1990; Guidry and Grinnell, 1987). Serum deprivation is also an important factor as demonstrated also in our assessments. Moreover, it has been reported that high doses of bFGF inhibited collagen gel contraction (Finesmith et al. 1990) whereas TGF-β facilitated it. Withouit wishing to be bound by theory, it is

DOCSQUE: 7S6309M thought that the present colostrum-derived fraction contains some of these molecules that correspond to antagonist on the matrix contraction and remodelling.

[0090] Since the collagen gel contraction assay is a model relative to the physiopathology of wound healing, scar formation and tissue remodeling, the research and development of agonists and antagonists is incomplete. The present fraction is a potential antagonist product, especially as it facilitates also cell growth and collagen synthesis as shown in specific in vitro experimental conditions. This composition has potential applications in plastic and reconstructive surgery to treat scar tissues. Indeed, recurrence of excessive remodeled scar tissues (e.g., keloids, hypertrophic scar tissues) after surgery represents an important issue. Furthermore, such composition may find application not only in wound healing but also in fibrotic reaction. The present fraction may also find an application in skin ulcers since rebuilding collagen matrix in the wound is also important to consider in such cases.

Example 6- Large open wounds on guinea pigs

[0091] The healing effect of compounds such as the colostrum-derived fraction was studied using a model of large full-thickness open wounds in guinea pigs. This model allows investigating the long term wound healing effect of the colostrum- derived fraction. Since the colostrum-derived fraction is in solution, different dilutions were applied daily for a period of 3 weeks.

[0092] Female Hartley guinea pigs weighing between 250-35Og (3-4 weeks old) were purchased at Charles River. Animals were kept in separate cages, with water and food ad libitum. All animal experiments were performed according to the guidelines of the Canadian Council for Animal Care and approved by our institutional Animal Care Committee.

[0093] The wound model consisted in the creation by scalpel incision of one large open full-thickness wound on each flank of each animal, close to the spinal cord. Each wound area had a 2.5 cm length along the cranio-caudal axis and 2cm large. The whole connective tissue was removed to insure no residue left at the bottom. The animals received, 1hr before surgery, a subcutaneous injection of an analgesic

DOCSQUB: 756309M (Buprenorphine®, 0.05mg/kg) and a Ringer's lactate solution at a volume corresponding to 1/10^th of their weights. The animals were anesthetised by isofluorane under oxygen. Surgery was performed in sterile conditions. The skin was aseptized with hibitane, 70% alcohol, and proviodine. Animals received for 2 days after surgery an analgesic at the same dose as described earlier at 12hrs interval. Animals were weighed periodically at dressing change without the dressing to compare with the initial weight performed one day before surgery. All animals grew in an exponential manner as expected in a normal guinea pig development.

[0094] Thereafter, the respective wound was filled with the solutions to be tested. Five compositionPBS dilutions were tested: 1 ) 1 :0.5, 2) 1 :1 , 3) 1 :10, 4) 1 :100 et 5) 1 :1000. The control animals received PBS. The wounds were then covered with a transparent self-adhesive polyurethane film (Tegaderm®, 3M), followed by a sterile gaze. The trunk of each animal was wrapped with a self-adhesive elastic bandage (Elastoplast®) to maintain the bandage in place and to avoid any trauma. Each animal was identified by a label on its cage. The bandage was left in place and periodically changed at days 5, 9, 14, and 19. A photograph of each wound was taken at days 5, 9, 14 and 23 (last sacrifice). A cm scale ruler and an identified label were inserted in each photograph. Quantification of macroscopic view of the wound areas was performed directly on photographs. Wound contraction

[0095] Wound contraction as measured represents the size of the wound as viewed from above the back of the animals as determined by tracing over the photographs. The trend curves of contraction were generally similar for all conditions, with an important drop by day 9 (Figure 4). At days 5 and 9, there were no significant difference statistically (equal variance test failed). However, statistic analyses (ANOVA and Fisher LSD method) show a significant difference by day 14 in the wounds treated with the 1 :1 and 1 :0.5 dilutions compared to control wounds and those treated with 1 :10 and 1 :1000 dilutions (p<0.05) By day 23, there was also a significant difference between the highest doses (1 :0.5) and control wounds. (Figure 4).

DOCSQUL 7₅6309\l Wound tissue areas

[0096] As shown in Figure 5 the treatment of large open wound with the composition of the invention increases formation of wound tissue. Quantification of the surface areas occupied by the wound tissue by day 23 showed significant increase with 1 :1 dilution (Fisher LSD method of statistic analysis was used with a failed equal variance test).

Wound thickness

[0097] Quantification of the thickness of the wound tissue also showed a significant increase by day 16 with the wounds treated with 1 :1 dilution compared to control and the lowest dose (1 :1000). By day 23, the thickness of wounds treated with a 1 :100 dilution was significantly higher than the other conditions using the Student- Newman-Keuls method as statistical comparisons methods (normality and equal variance test passed). (Figure 6)

Collagen deposition and density [0098] The composition increased the areas occupied by collagen with a tendency to increase its density. By day 23, there was an increase in surface area occupied by collagen where the composition was applied at a dilution of 1 :100 (Figure 7). However, this increase in surface area was not significantly different compared to control (PBS). Treatment of large open wound with the composition also induced an increased of collagen density compared to control (Figure 8).

Example 7- Subcutaneous implantation of PVA sponges

[0099] The PVA (Polyvinyl alcohol) sponge model is used for studying granulation and reparative tissue ingrowth. Tissue grows within its interstices shortly after implantation. This model is highly reproducible and is a biologically valid model for studying healing responses.

[00100] Spraque Dawley rats were used for this implantation model. A dorsal median incision (rostral) was performed in the skin down to the aponevrosis, and

DOCSQUE: 756309M then two subcutaneous pockets (one on each side) were made. Each wound area was 1x1 cm. A PVA sponge was introduced in each pocket and the skin was sutured (4/0 monofilaments). Sponges were far from the skin incision to avoid any interference. Specimens were retrieved at days 8 and 15 to see the speed of tissue ingrowth. After 8 and 15 days, implants were retrieved, fixed in formaldehyde and processed for histological sections which was stained with hematoxylin, phloxin, saffron and Hoechst 33342 to mark the nuclei and the sections of day 15 were also stained with picro-sirius to visualize the collagen deposition. Quantification was performed by measuring the length of tissue invasion from the host-implant interface to the invasive front, in multiple areas in a random manner. Moreover, using Hoechst-stained tissue sections, the number of nuclei present in the sponge was counted using the image analysis system and reported to the total surface of the void spaces of PVA sponge present on each histological section. This ratio was used for comparison between the different conditions. [00101] As shown in Figure 9, the composition significantly (p=0.001 ) increases the tissue ingrowth in PVA sponges. An increase of 42% of the tissue growth in PVA sponges was observed in animal treated with the composition. This result demonstrates that the composition of the invention induces tissue formation in wounds.

Example 8- Hypertrophic scar

[00102] Wound healing is a fundamental complex-tissue reaction leading to skin reconstitution. Alterations in the orchestrated wound healing process result in hypertrophic or keloid scarring. Although hypertrophic scarring commonly occurs following burns, many aspects such as incidence of an optimal treatment for scar hypertrophy remain unclear. Hypertrophic scar is associated with polarized Th2 systemic response to injury that leads to increased T cells and their Th2 fibrogenic cytokines in tissues and the development of fibrosis and hypertrophic scars.

Concentration of the composition

DOCSQUE: 756309M [00103] Bioactive compounds of the composition were concentrated by dehydration with speed-vacuum. The samples were frozen and applied in a speed vacuum with a pressure of 100 milliTorr. A concentration of almost 30 times was achieved. This concentrated fraction was named speed-vaccum IM. [00104] The wound model consisted in the creation by scalpel incision of two open full-thickness wounds on each flank of each animal, close to the spinal cord. Each wound area had a 1x1 cm. If necessary, haemostasis was insured by compression with a gaze and thermocoagulation. The whole connective tissue was removed to ensure no residue left at the bottom. The day of surgery, the animals received, 1 hr before surgery, a subcutaneous injection of an analgesic (Buprenorphine®, 0.05mg/kg) and a Ringer's lactate solution at a volume corresponding to 1/10^th of their weights. For surgery, the animals were anesthetised by isofluorane under oxygen. Surgery was performed in sterile conditions. The skin was aseptized with hibitane, 70% alcohol, and proviodine. The respective wound was covered with a polyurethane sheet (Tegaderm) and the animals wrapped in gauzes and elastoplast to protect the wounds for 5 days. After 5 days the dressing was renewed in the same manner. After 15 days, the wounds were left at air-free environment until epithelialisation was almost completed (>90%). This was reached at 19 days and iconography was performed as image prior to treatment. Wounds on each animal were then treated (day 19 corresponds to day 0 of treatment) by speed- vacuum IM and speed-vacuum BSA on one side and on the other side, respectively. Wounds were treated for 14 days by daily application (except Sundays) and photographs were taken at day 7 (Day 26) and at sacrifice at day 14 (day 33). Macroscopic evaluation of the wound area included the scar tissue, thickening of the scar, etc. and microscopic observation with quantification (see Figure 10 for an example).

[00105] Scar formation. As shown in Figure 10, treatment of a wound by the composition reduces scar formation. An untreated wound may lead to the formation of a scar. Indeed, a scar was formed in 7 out of 8 wounds treated with control vehicle (BSA) (Figure 10). However, following 14 days of treatment with the composition, the majority of wounds (5 of 8) have no scar.

DOCSQUE 756309M [00106] Hypertrophic wound Treatment with the composition prevented the formation of hypertrophic wound (Figure 11) No hypertrophic scar was observed in wounds treated with the composition However, in wounds treated with the BSA (control), hypertrophic wounds were formed (3 out of 8)

DOCSQUL 7₅6309\1 [00107] REFERENCES

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DOCSQUE 756309M

Claims

CLAlMS

1. A process for the preparation of a colostrum-derived fraction enriched in growth factors, said process comprising the steps of: a) precipitating casein from colostrum and separating curds from colostrum whey; b) heating said colostrum whey to a temperature of greater than 4O⁰C; c) submitting said heated whey to ultra-filtration with a molecular weight cutoff of about 14kDa or less and collecting permeate; d) acidifying said permeate obtained in step c) to a pH of between 3 and 4.

2. The process according to claim 1 , wherein said colostrum in step a) is obtained from thawing frozen whole colostrum to a temperature of at least about 4⁰C.

3. The process according to claim 2, wherein said colostrum in step a) is obtained from thawing frozen whole colostrum to a temperature of about 1O⁰C.

4. The process according to claim 1 , wherein, in step a), said casein and curds are separated by treatment with microbial rennet or by acid treatment.

5. The process according to claim 4, wherein, in step a), said casein and curds are separated by treatment at pH 4.6.

6. The process according to claim 1 , wherein the acidified permeate obtained in step d) is concentrated to obtain a concentrated liquid fraction.

DOCSQUL 756309M

7. The process according to claim 6, wherein the concentrated liquid fraction is freeze-dried to obtain a solid fraction.

8. The process according to claim 1 , wherein in step b), said colostrum whey is heated to a temperature of at least 45⁰C.

9. The process according to claim 8, wherein in step b), said colostrum whey is heated to a temperature of about 5O⁰C.

10. The process according to claim 1 , wherein in step c), said heated whey is submitted to ultra-filtration with a molecular weight cut-off of about 12kDa or less.

11. The process according to claim 10, wherein in step c), said heated whey is submitted to ultra-filtration with a molecular weight cut-off of about 1OkDa.

12. The process according to claim 1 , wherein in step d) the permeate is acidified at a pH of about 3.5.

13. The process according to claim 12, wherein in step d) the permeate is acidified at a pH of 3.2.

14. The process according to claim 6, wherein the acidified permeate is concentrated with nanofiltration or any other concentrating method.

DOCSQUE 756309M

15. The process according to claim 1 , wherein said colostrum is obtained from: cow, ewe, jenny (donkey's female); goat or buffalo.

16. A composition derived from colostrum comprising at least 60% of antimicrobial peptides of MW less than 500Da; IGF-1 and a total protein concentration of less than 5% (w/w).

17. The composition according to claim 16, comprising at least 70% of antimicrobial peptides of MW less than 500Da and further comprising at least 20% (w/w) of antimicrobial peptides of molecular weight between 500Da and 1OkDa of which between 2% and 10% (w/w) is constituted of IGF-1.

18. The composition according to claim 16, wherein the composition is liquid and comprises at least 20 ng/ml of IGF-1 ; a total protein concentration of less than 2% (w/w) and is substantially free of lysozyme and TGF-β.

19. The composition according to claim 18, wherein IGF-1 is present at at least about 40ng/ml.

20. The composition according to claim 16, wherein the composition is solid and comprises at least 1 ng of IGF-1 per mg of solid; a total protein concentration of less than 2% (w/w) of protein and is substantially free of lysozyme and TGF-β.

21. The composition according to claim 20, wherein IGF-1 is present at at least about 1.6ng/mg total solid.

DOCSQUE 756309U

22. The composition according to claim 16 or 17, wherein said peptides below

500Da are present in physiologically effective amounts.

23. The composition according to claim 16 or 17, wherein said peptide of MW less than 500Da is selected from the group consisting of: PDGF; FGF; EGF; VEGF; defensins, cathelicidins, anti-microbial peptides (AMP) and proline- rich peptides (PRP).

24. A formulation comprising a composition according to claim 16 or 17, further comprising a physiologically acceptable excipient.

25. The formulation according to claim 24, wherein said formulation is topical.

26. The formulation according to claim 24, wherein said formulation is an oral formulation.

27. Use of the composition according to claim 16 or 17, for the manufacture of a formulation for the treatment of a wound and/or facilitating and/or accelerating wound healing while inducing minimal collagen contraction.

28. A method for healing a wound while inducing minimal collagen contraction, said method comprising treating said wound with the formulation according to claim 24.

29. A method for treating skin injury and ulcers comprising application to the wound with the liquid composition "as is" or with the formulation as defined in claim 24.

DOCSQUE: 756309M

30. A method for accelerating wound healing and improving aesthetical presentation of the scars formed in the wound healing process, the method comprising application on the wounded skin an effective amount of a formulation as defined in claim 24.

DOCSQUE 756309M