(12) United States Patent (10) Patent No.: US 6,541,447 B1 Dawson (45) Date of Patent: Apr

USOO6541447B1 (12) United States Patent (10) Patent No.: US 6,541,447 B1 Dawson (45) Date of Patent: Apr. 1, 2003

(54) WOUND HEALING COMPOSITION AND OTHER PUBLICATIONS METHOD FOR USE THEREOF Gasior-Chrzan, B., Przeglad Dermatologiczny 75(6) (75) Inventor: Monica E. Dawson, Lenexa, KS (US) 431–434 (1988) “Wplyw lizozymu bialkajaja kurzego na gojenis StandaryZowanych ran Skory Swnek morskich (73) Assignee: B & M Healthcare Technologies, Inc., (Effect of ovalbumin lyZozyme on healing of Standard skin Lenexa, KS (US) wounds in guinea pigs)'. Katz, M. H., et al., J. Am. Acad. Dermatol., “Human Wound (*) Notice: Subject to any disclaimer, the term of this Fluid From Acute Wounds Stimulates Fibroblast and Endot patent is extended or adjusted under 35 helia Cell Growth", 25: 1054–1058 (Dec. 1988). U.S.C. 154(b) by 0 days. Stanley, A. C. et al., J. Vasc. Surg., “Reduced Growth of Dermal Fibroblasts From Chronic Venous Ulcers Can Be Stimulated With Growth Factors”, 26(6) 994–999 (Dec. (22) Filed: Sep. 1, 1999 Lynch, S. E., et al., Proc. Natl Acad. Sci. USA, “Role of Platelet-derived Growth Factor in Wound Healing: Syner (51) Int. Cl." ...... A61K 38/00; A61K 38/24; gistic Effects With Other Growth Factors”, 84(21): A61K 38/27; CO7K 14/00 7696–7700 (Nov. 1987). (52) U.S. Cl...... 514/2; 514/12; 530/350; Steed, “The Role of Growth Factors in Wound Healing”, 530/399; 530/388.24; 435/69.4; 424/400; Surgical Clinics of North America, vol. 77, No. 3, pp. 424,581 575.586 (Jun. 1997). (58) Field of Search ...... 530/350, 399, Martin, et al., “Growth Factors and Cutaneous Wound 530/388.24; 514/2, 12; 435/69.4; 424/400, Repair”, Progress in Growth Factor Research, vol. 4, No. 1, 581 pp. 25. 44 (1992). Hom, et al., “Angiogenic Growth Factors: Their Effects and (56) References Cited Potential in Soft Tissue Wound Healing”, Ann. Otol. Rhinal. Laryngol, vol. 101, No. 4, pp. 349-354 (Apr. 1992). U.S. PATENT DOCUMENTS Katz, "Human Wound Fluid From Acute Wounds Stimulates 115,032 A 5/1871 Crossman Fibroblast and Endothelial Cell Growth”, Journal of the 122,621 A 1/1872 Masters American Academy of Dermatology, vol. 25, No. 6 (Part 1), 240,150 A 4/1881 Lawrence et al. pp. 1054–1058 (Dec. 1991). 247,818. A 10/1881 Gilman Stanley, et al., “Reduced Growth of Dermal Fibroblasts from 451,307 A 4f1891 Miles Chronic Venous Ulcers Can Be Stimulated with Growth 3,194,7322,555,731 A 6/19517/1965 CooperNeuhauser Factors.actors”, JJournal | ofOf V.Vascular lar Surgery,S vol.1. 26,26. No. 6,6 pp. 3,558,771. A 1/1971 Balassa 994–1001 (Dec. 1997). 3,624.201. A 11/1971 Balassa Lynch, et al., “Role of Platelet-Derived Growth Factor in 4,219,544 A 8/1980 Burg Wound Healing: Synergistic Effects With Other Growth 3. E. A F. test al. Factors”, Proc. National Academy of Sciences USA, Vol. 84, 4670.257 A 6/1987 GEast al. No. 21, pp. 7696–7700 (Nov. 1987). 4,719,111 A 1/1988 Wilson Schultz, et al., “EGF and TGF-Alpha in Wound Healing and 4,808.402 A 2/1989 Leibovich et al. Repair”, Journal of Cellular Biochemistry, vol. 45, No. 4, 4,832,946 A 5/1989 Green pp. 346-352 (Apr. 1991). 4,839,164 A 6/1989 Smith 4,843,063 A 6/1989 Seyedin et al. (List continued on next page.) 4929,442 A 5/1990 Powell 4.959,353 A 9/1990 Brown et al. 5,023,090 A 6/1991 Levin Primary Examiner Karen Cochrane Carlson 5,064,655 A 11/1991. Uster et al. ASSistant Examiner-Chih-Min Kam 5,104,977 A 4/1992 Sporn et al. (74) Attorney, Agent, or Firm-Mayer, Brown, Rowe & 5,130,298 A 7/1992 Cini et al. Maw; Joseph A. Mahoney; Thomas R. Stiebel 5,219,998 A 6/1993 Levin et al. 5,460.832 A 10/1995 Yamaguchi et al. (57) ABSTRACT 5,618,544 A 4/1997 Brown 5,637,318 A 6/1997 Gross et al. A composition for wound healing and hair growth is dis 5,676.975 A 10/1997 Dezes et al. closed. The composition comprises at least 5.0% by weight 5,849,273 A 12/1998 Bonda et al...... 424/59 of ovalbumin, about 1.0% phenoxyethanol, about 0.5% carbomer, and about 0.3% triethanolamine. The composition FOREIGN PATENT DOCUMENTS may be in the form of a cream, powder, lotion, gel, emulsion, EP O338459 A2 * 10/1989 ...... A61 K/7/06 or ointment, Its method for use in wound healing and for hair EP O444.638 9/1991 growth is also disclosed. FR 2654342 5/1991 WO 91.18999 12/1991 20 Claims, No Drawings US 6,541,447 B1 Page 2

OTHER PUBLICATIONS Knighton, et al., “Amputation Prevention in an Indepen dently Reviewed At-Risk Diabetic Population Using a Flamme, et al., “Overexpression of Vascular Endothelial Growth Factor in the Avian Embryo Induces Hypervascu Comprehensive Would Care Protocol”, The American Jour larization and Increased Vascular Permeability Without nal of Surgery, vol. 160, pp. 466-472 (Nov. 1990). Alterations of Embryonic Pattern Information', Develop Van Brunt, et al., “Growth Factors Speed Wound Healing”, mental Biology, vol. 171, No. 2, pp. 399–414 (Oct. 1995). Biotechnology, vol. 6, pp. 25-30 (Jan. 1988). “Pasteurization Keeps Eggs Bacteria-Free”, USA Today, Hosgood, “Wound Healing. The Role of Platelet-Derived vol. 124, No. 2611, p. 11 (Apr. 1996). Growth Factor and Transforming Growth Factor Beta', Fremont, et al., “Crystal Structure of an H-2K (super Veterinary Surgery, vol. 22, No. 6, pp. 490–495 (Nov.-Dec. b)-Ovalbumin Peptide Complex Reveals the Interplay of 1993). Primary and Secondary Anchor Positions in the Major Bennett, et al., “Growth Factors and Wound Healing: Bio Histocompatibility Complex Binding Groove', Proceedings chemical Properties of Growth Factors and Their Recep of the National Academy of Sciences of the United States, tors”, American Journal of Surgery, vol. 165, No. 6, pp. vol. 92, No. 7, pp. 2479-2483, (Mar. 28, 1995). 728-737 (Jun. 1993). Niedermann, et al., “The Proteolytic Fragments Generated Eisinger, et al., “Growth Regulation of Skin Cells by Epi by Vertebrate Proteasomes: Structural Relationships to dermal Cell-Derived Factors: Implications for Wound Heal Major Histocompatibility Complex Class I Binding Pep ing, Proc. National Academy of Science USA, Vol. 85, No. tides”, Proceedings of the National Academy of Sciences of 6, pp. 1937–1941 (Mar. 1988). the United States, vol. 93, No. 16, pp. 8572-8577, (Aug. 6, Falanga, “Growth Factors and Chronic Wounds: The Need 1996). to Understand the Microenvironment”, The Journal of Der Qui, et al., “Spatiotemporal Expression Patterns of Chicken matology, vol. 19, No. 11, pp. 667-672 (Nov. 1992). Ovalbumin Upstream Promoter-Transcription Factors in the Hudson-Goodman, et al., “Wound Repair and the Potential Developing Mouse Central Nervous System: Evidence for a Use of Growth Factors”, Heart& Lung., vol. 19, No. 4, pp. Role In Segmental Patterning of the Diencephalon”, Pro 379–384 (Jul 1990). ceedings of the National Academy of Sciences of the United Greenhalgh, “The Role of Growth Factors in Wound Heal States, vol. 91, No. 10, p. 4451–4455, (May 10, 1994). ing', The Journal of Trauma: Injury, Infection, and Critical Kawamoto, et al., “Transforming Growth Factor Type B Care, vol. 41, No. 1, pp. 159-167 (Jul. 1996). Stimulates Epidermal Growth Factor-Dependent Cell Hom, “Growth Factors in Wound Healing”, Otolaryngologic Growth in vivo and in vitro Senescent Human Skin Fibro Clinics of North America, vol. 28, No. 5, pp. 933–953 (Oct. blasts”, Cell Structure and Function, vol. 13, No. 6 (1524) 1995). (Dec. 1988). Kingsnorth, et al., “Peptide Growth Factors and Wound Satoh, et al., “Charcterization of Endothelial Cell Growth Healing', British Journal of Surgery, vol. 78, No. 11, pp. Factor from Serum-Free Culture Supernatant of Human 1286–1290 (Nov. 1991). Diploid Fibroblast Cells”, Cell Structure and Function, vol. Moulin, “Growth Factors in Skin Wound Healing”, Euro 13, No. 6 (1536) (Dec. 1988). pean Journal of Cell Biology, vol. 68, No. 1, pp. 1-7 (Sep. Brown, et al., “Acceleration of Tensile Strength of Incisions 1995). Treated with EGF and TGF-B”, Ann. Surg., vol. 208, No. 6, Otau, et al., “Cosmetics Containing Urogastone/Epidermal pp. 788–794 (Dec. 1988). Growth Factor', Chemical Abstracts-Essential Oils, CoS Brown, et al., “Stimulation of Healing of Chronic Wounds metics, vol. 104, #192928u, p. 417 (1986). by Epidermal Growth Factor”, Plastic and Reconstructive Ogawa, et al., "Skin Conditioners Containing Epidermal Surgery, vol. 88, No. 2, pp. 189-194 (Aug. 1991). Growth Factor', Chemical Abstracts-Essential Oils, CoS Brown, et al., “Enhancement of Wound Healing by Topical metics, vol. 106, #182467v, p. 395 (1987). Treatment with Epidermal Growth Factor”, The New Nonokawa, et al., “Cosmetics Containing Urogastrone”, England Journal of Medicine, vol. 321, No. 2, pp. 76–79 Chemical Abstracts-Essential Oils, Cosmetics, vol. 104, (Jul 13, 1989). #174376h, p. 369 (1986). Chua, et al., “Receptor for Epidermal Growth Factor Retains Nishiyama, et al., "Skin Cosmetics Containing Saikosaponin Normal Structure and Function in Aging Cells’, Mecha b1 and/or b2 and Cell Growth Factors', Chemical nisms of Ageing and Development, vol. 34, pp. 35-55 Abstracts-Essential Oils, Cosmetics, vol. 116, #241736p, (1986). p. 407 (1992). Hollenberg, et al., “Receptors For Insulin and Epidermal Matauo, et al., “Preparation of Polypeptides for Cosmetics Growth Factor-Urogastrone In Adult Human Fibroblasts Do and Pharmaceuticals”, Chemical Abstracts-Essential Oils, Not Change with Donor Age”, Mechanisms of Ageing and Cosmetics, vol. 117, #219741k, p. 495 (1192). Development, vol. 11, pp. 37-43 (1979). Kadowaki, et al., “Development of New Host for Gene Allen, “Cosmetics-Chemical Technology or Biotechnol Recombination and Possibility of Application to Cosmetic ogy?”, International Journal of Cosmetic Science, vol. 6, Products”, Chemical Abstracts, vol. 115, #176096t, p. 232 No. 2 (Apr. 1984). (1991). Sprugel, et al., “The Effects of Different Growth Factors In “Testimonials From PolygenTM Users”, www.polygencare Subcutaneous Wound Chambers', Growth Factors and Other Aspects of Wound Healing. Biological and Clinical .com/testimonials (Dec. 2001). Implications, pp. 77-91 (1988). * cited by examiner US 6,541,447 B1 1 2 WOUND HEALNG COMPOSITION AND domains: an extracellular domain, which binds to the growth METHOD FOR USE THEREOF factor ligand; a transmembrane domain; and an intracellular domain. When the ligand binds to neighboring receptors, conformational changes occur that are transmitted to the FIELD OF THE INVENTION intracellular domain and elicit a Series of cytoplasmic changes leading to the initiation of the nucleic acid tran This invention pertains to wound healing compositions Scription. In many cases, these intracytoplasmic events are and, particularly, to Such a composition comprising of a enacted through an enzyme, tyrosine kinase, that phospho growth hormone and growth factor in a balanced mixture rylates cytoplasmic proteins, Some of which remain in the that will deliver the maximum therapeutic results with cytoplasm and Some pass to the nucleus with a correspond non-healing wounds, burns, trauma, and certain dermato ing gene activation. logical disorders. Studies have shown that fluid factors isolated from chronic wounds lack the presence of growth factors. BACKGROUND OF THE INVENTION Moreover, in vitro studies have shown that growth factors 1. Overview of the Invention 15 added to wound fluid extracted from postoperative or trau There have been recent dramatic strides with the discov matic wounds have accelerated the wound healing cascade. ery of growth factors as wound healing agents. Indeed, the These facts Strongly Suggest the important role of growth discovery of growth factorS has triggered great optimism factors. into the possibility of mastering the art of wound healing and One in vitro study examined the effect of human wound intense effort has been launched on the part of medical fluid on the growth of human dermal fibroblasts and umbili researchers and pharmaceutical companies to procure, cal vein endothelial cells. Katz M H et al., J. Am. Acad. characterize, and harvest these healing enhancement agents. Dermatol., Human Wound Fluid from Acute Wounds Stimu Because it is believed that administering growth factors to lates Fibroblast and Endothelial Cell Growth, patients with dermal and Subdermal wounds enhances the 25:1054–1058 (December 1988). Katz et al. collected Speed by which wounds heal, as discussed in greater detail 25 wound fluid from Six patients undergoing Split-thickneSS below, it is the object of this invention to provide a topical skin and wound fluid from postoperative patients. After composition containing growth factors to increase the Seeding the wound fluid in optimal growth media (control) body's wound healing properties. on day 0, cultures of human dermal fibroblasts and umbilical vein endothelial cells were supplemented with or without 2. Background and Prior Art acute wound fluid on days 1 and 3. The study found that 2% Since their discovery almost 30 years ago, growth factors acute wound fluid Stimulated the growth of human dermal have been shown to Stimulate neovascularization in Vitro fibroblasts and umbilical vein endothelial cells when these and in animal Studies. In the past 10 years, knowledge of cells were cultured in 2% fetal bovine serum and endothelial growth factors has grown immensely. Broadly defined, growth medium, respectively. Wound fluid from the post growth factors are multifunctional, locally acting, intercel 35 operative patients caused the same level of Stimulation. lular Signaling polypeptides which, among other things, Furthermore, when anti-platelet-derived growth factor anti organize and coordinate cellular proliferation. Most growth body was added to wound fluid, there was a 45% mean factors are large peptides or glycoproteins Secreted by many reduction in the stimulatory effect on fibroblast growth. This cells as a base function, or in response to a challenge, Such result further Suggests that platelet-derived growth factor as a wound or carcinogen. These peptides represent a System 40 contributes to the fibroblast growth effect. of Signals that mediate physiologic and pathologic cellular It has also been Suggested that ulcer healing may be growth and repair, including embryogenesis, wound healing improved by the exogenous provision of Specific growth and carcinogenesis. factors. Research has shown that fibroblasts isolated from Currently, there are two known classes of intercellular wound Sites on patients proliferated at a slower rate and are Signaling proteins: (i) endocrine proteins which are long 45 morphologically distinct (larger and polyglonal in shape) range Signaling proteins released into the circulation or other from normal fibroblasts cells. Stanley A C et al., J. Vasc. body fluids, and (ii) paracrine proteins which are short range Surg., Reduced Growth of Dermal Fibroblasts from Chronic Signaling proteins that act locally within tissues. Growth Venous Ulcers can be Stimulated with Growth Factors, factors are generally considered as paracrine proteins Since 26(6):994–999 (December 1997). It was also found that the they are predominantly short range locally acting, intercel 50 decreased growth of wound fibroblasts were stimulated by lular signaling proteins. growth factors FGF, EGF, IL-1. There exist six known varieties of growth factors: A combination of growth factors produce a magnified platelet-derived growth factor (PDGF), epidermal growth effect in Stimulating protein Synthesis and decreasing protein factor (EGF), fibroblast growth factor (FGF), insulin-like degradation. They have also been shown to have additive growth factor (IGF), transforming growth factor beta 55 effect in improving whole-body and muscle kinetics. For (TGFB), and TGFB Superfamily. In addition to their local example, it has been shown that the addition of pure PDGF mode of action, the different varieties of growth factors to a wound Site involving the epidermis and dermis has little share common biological properties. For example, growth effect on the morphology or biochemistry of wound healing. factor action is mediated by association with Specific, high Lynch S E et al., Proc. Natl. Acad. Sci. USA, Role of affinity receptorS expressed by the target cells, and growth 60 Platelet-Derived Growth Factor in Wound Healing. Syner factors are able to exert their biological effect at low gistic Effects with Other Growth Factors, 84(21):7696–7700 concentrations (10 to 10'M). (November 1987). In contrast, the addition of partially Furthermore, on the cellular level, growth factors bind purified PDGF results in significant dose-dependent with ligands on the cell Surface to generate an intracellular increases in the width of the newly Synthesized connective signal on the inside of the cell to modify cellular behavior. 65 tissue and epidermal layers. Further, the addition of partially Growth factors function by binding to Specific cell-Surface purified PDGF results in significant increases in the rate of receptors that are composed of three distinct regions or protein and DNA synthesis and the total content of these US 6,541,447 B1 3 4 components in biopsies taken from the wound Site. Similar In a preferred embodiment of the invention, the wound effects were obtained when IGF was added in combination healing composition comprises about 1.0% by weight of with pure PDGF. Combining the growth factors caused a 2.4 phenoxyethanol, about 0.5% carbomer, about 20% ovalbu fold increase in the width of newly formed connective tissue min and about 0.3% triethanolamine. These compounds are layer and a 95% increase in epidermal thickness compared blended to produce a eutectic mixture in gel, lotion, cream with controls. Id. On the other hand, IGF applied alone did or ointment form. The inventive composition can also be in not cause Similar morphological changes, thus indicating powder form as well. that the synergistic actions of other factors with PDGF are It is therefore an object of the present invention to treat important in the modulation of the wound healing process. mammalian nonhealing wounds manifested by diabetes, Therefore, a combination of growth factors and growth burns, trauma and Subcutaneous trauma, Osteomyelitis, Vari hormones have a potential amplified effect on accelerated ous Surgical procedures, and various forms of dermatitis. non-healing of ulcers, open wound, osteomyelitis, Skeletal It is another object of the present invention to treat muscle injuries Such as Sports and trauma, and bums and wounds with a topical agent that is easily administered, dermatological disorders. economical and well-tolerated by patients. Animal and clinical trials using growth factor therapy 15 It is another object of this invention to provide a compo have produced outstanding results. Healing of a variety of Sition to Stimulate fibroblast production for tissue repair. wounds in animals and patients was enhanced by treatment with EGF or TGF-alpha. Several different studies of topi It is yet another object of this invention to provide a cally applied growth factors have shown to accelerate heal composition for the Stimulation of hair follicle growth. ing by Stimulating granulation tissue formation and enhanc It is still another object of the present invention to provide ing epithelialization. Epidermal regeneration of partial a composition for treating non healing wounds by the thickneSS bums on pigs or dermatome wounds on patients addition of an individual or combination of growth factors was accelerated with topical application of EGF TGF-alpha, including IGF, PDGF, FGF, EGF, TGFB and TGFB Super and EGF treatment accelerated healing of gastroduodenal family. ulcers. Schultz, G. et al., J. Cell. Biochem., EGF and 25 These and other features and advantages of the present TGF-Alpha in Wound Healing and Repair, 45(4):346-352 invention will be found in the following description of the (April 1991). EGF also increased tensile strength of skin preferred embodiments and in the claims. incisions in rats and corneal incisions in rabbits, cats, and DESCRIPTION OF THE PREFERRED primates. Id. EMBODIMENT Specific use of particular factorS is becoming an accepted mode of therapy for many patients with non-healing Before the present composition for wound healing is wounds. Accordingly, there is ample Scientific and clinical disclosed and described, it is to be understood that this evidence Suggesting that the biological actions of growth invention is not limited to the particular configurations, factorS regulate cell division, differentiation, migrations and process Steps, and materials disclosed herein as Such 35 configurations, proceSS Steps, and materials may vary Some gene expression. what. It is also to be understood that the terminology The present invention is a composition comprising a employed herein is used for the purpose of describing mixture of growth factors and growth hormones in a der particular embodiments only and is not intended to be matologically Safe vehicle, Such as a liquid, gel or cream. It limiting Since the Scope of the present invention will be is therefore an object of the present invention to provide a 40 limited only by the appended claims and equivalents thereof. wound-healing composition comprising a protein growth AS described above, the present invention is directed to a factor to treat healing resistant wounds, dialetic ulcers, composition for Stimulating cell growth and the healing of bedsores, burns, Osteomyelitis, trauma wounds, Subcutane wounds by administering a pharmacologically effective ous trauma and various forms of dermatitis. The composi amount of growth factors directly to the affected tissue. The tion is also useful in reducing the formation of Scar tissue. 45 composition may be used to treat any type of nonhealing wound whereby fibroblast activity is reduced or ineffective. SUMMARY OF THE INVENTION Such wounds include diabetic non-healing wounds, burns, In accordance with the present invention, it has now been Osteomyelitis, trauma wounds, Subcutaneous trauma and discovered that wounds may be effectively treated with the various forms of dermatitis. The composition may also be growth factors that are contained in egg whites of hens. 50 employed to Stimulate hair growth on the Scalp and other Specifically, topically administered growth factors in phar body areas. macologically effective amounts are useful for increasing The growth factors of the present invention have been the local activity of the body's wound healing process. identified in hen's egg whites. Ovalbumin is the major Accordingly, the present invention is a low cost, over-the constituent of egg whites from the hen comprising about counter, composition containing growth factors isolated 55 75% by weight of the egg white. The molecular weight of from a naturally occurring Source-egg whites. The com ovalbumin is approximately 4,500, and Ovalbumin is pro position may be used to treat any disease State where growth duced under hormonal control by the bird Oviduct. It may be factors play a role including diabetic ulcers, nonhealing isolated and crystallized readily from the filtrate of an wounds, burns, osteomyelitis, trauma wounds, Subcutaneous acidified mixture of egg white and an equal Volume of trauma and various forms of dermatitis. 60 Saturated ammonium Sulfate. Sorensen et al., C. R. Trav. In particular, the wound healing composition is comprised Lab. Carlsberg 12, 12 (1917). Alternative methods of iso of phenoxyethanol, carbomer, protein and triethanolamine. lation of ovalbumin are disclosed by Kekwick et al., Bio The protein is ovalbumin from egg whites, which includes Chem Journal 30: 227 (1930). Ovalbumin can also be a combination growth factors including epidermal growth Separated by electrophoresis and chromatography from factor (EGF), transforming growth factor alpha (TGF 65 about 10 other minor components found in egg whites Alpha), fibroblast growth factor (acidic) (FGF-a), fibroblast including avidin (qv), lysozyme (qV), conalbumin (qv), and growth factor (basic) (FGF-b). OVOmucoid. US 6,541,447 B1 S 6 The structure of ovalbumin is that of a complex protein behenic acid, isoStearic acid and oleic acid, the above alkali consisting of a Single polypeptide chain of about 460 resi Salts of ether carboxylic acids, Salts of N-acylamino acids, dues (about half of which are hydrophobic), a maximum of N-acylsalconates, higher alkylsulfonates and other anionic 2 phosphate residues per mole, and a oligosaccharide side Surfactants, alkylamine Salts, polyamine, aminoalcohol fatty chain composed of only mannose and glucosamine residues. acids, organic Silicone resin, alkyl quaternary ammonium See Narita, J. Biochem. 52:367 (1962); Thompson et al., Salts and other cationic Surfactants, and lecithin, betaine Aust. J. Biol. Science 24: 525 (1971). derivatives and other amphoteric Surfactants. Examples of oils and fats include vegetable oils and fats Ovalbumin is soluble in electrolyte free water and com Such as castor-oil, olive oil, cacao oil, camelia oil, coconut bines with Salts, acids and bases. Denaturation can be oil, wood wax, jojoba oil, grape Seed oil and avocado oil; induced by heating to 56° C., by Vigorous Shaking, by animal oils and fats Such as mink oil and egg yolk oil, waxes electric current and by various chemicals. Such as acids, Such as beeswax, whale wax, lanolin, carnauba wax and ammonium Salts, heavy metal Salts and alcohols. Such candelilla wax, hydrocarbons Such as liquid paraffin, methods produce complete and irreversible denaturation. Squalene, microcrystalline wax, ceresine wax, paraffin wax The isoelectric point of ovalbumin is 4.63. See Merck Index and Vaseline; natural or Synthetic fatty acids Such as lauric (12th Ed. 1996). 15 acid, myristic acid, Stearic acid, oleic acid, isoStearic acid In accordance with the compositions and method of the and behenic acid; natural or higher alcohols Such as cetanol, present invention, Ovalbumin may be administered in the Stearyl alcohol, hexyldecanol, octyldecanol and lauryl alco form of a pharmaceutical composition additionally compris hol; and esterS Such as isopropyl myristate, isopropyl ing a pharmaceutically acceptable carrier. One skilled in the palmitate, octyldodecyl myristate, octyldodecyl oleate and art will appreciate that Suitable methods of administering the cholesterol oleate. ovalbumin compositions to an animal, Such as a mammal, Examples of polyhydric alcohols include ethylene glycol, are available and, although more than one method can be polyethylene glycol, propylene glycol, 1,3-butyrene glycol, used to administer a particular composition, a particular 1,4-butyrene glycol, dipropylene glycol, glycerol, method and dosage can provide a more immediate and more diglycerol, triglycerol, tetraglycerol and other polyglycerols, effective reaction than others. Pharmaceutically acceptable 25 glucose, maltose, maltitose, Sucrose, fructose, Xylitose, carriers are also well known to those skilled in the art. The Sorbitol, maltotriose, threitol and erythritol. choice of carrier will be determined, in part, both by the Examples of thickening agents include naturally particular composition and by the particular method used to occurring high molecular Substances Such as Sodium administer the composition. Accordingly, there is a wide alginate, Xanthene gum, aluminum Silicate, quince Seed variety of Suitable formulations of the pharmaceutical com extract, gum tragacanth, Starch, collagen and Sodium hyalu positions of the present invention. ronate, Semi-Synthetic high molecular Substances Such as The present invention is preferable in the form of a topical methyl cellulose, hydroxyethyl cellulose, carboxymethyl dosage form Such as creams, ointments, lotions, gels or cellulose, Soluble Starch and cationized cellulose; and Syn thetic high molecular Substances Such as carboxyvinyl poly powders. 35 The present invention may be formulated as necessary mer and polyvinyl alcohol. with additives used commonly in the pharmaceutical Examples of UV absorbents include p-aminobenzoic acid, 2-ethoxyethyl p-methoxy cinnamate, iSopropyl Sciences, Such as Surfactants, oils and fats, polyhydric p-methoxycinnamate, butylmethoxybenzoylmethane, alcohols, lower alcohols, thickening agents, UV absorbents, glycery 1 - mono-2-ethyl he X a noyl-di-p- light Scattering agents, preservatives, antioxidants, 40 antibiotics, chelating agents, pH regulators, flavoring agents, methoxybenzophenone, digalloyl trioleate, 2,2'-dihydroxy 4-methoxyben Zophe none, ethyl-4- pigments and water. bishydroxypropylaminobenzoate, 2-ethylhexyl-2-cyano-3, Examples of Surfactants include polyoxyethylene 3'-diphenyl acrylate, ethylhexyl p-methoxycinnamate, (hereinafter abbreviated as POE-branched alkyl ethers such 2-ethylhexyl Salicylate, glyceryl p-aminobenzoate, homom as POE-octyldodecyl alcohol and POE-2-decyltetradecyl 45 ethyl Salicylate, methyl O-aminobenzoate, 2-hydroxy-4- alcohol, POE-alkyl ethers such as POE-oleyl alcohol ether methoxybenzophenone, amyl p-dimethylaminobenzoate, and POE-cetyl alcohol ether, Sorbitan esters such as Sorbitan 2-phenylbenzoimidazole-5-Sulfonic acid and 2-hydroxy-4- monooleate, Sorbitan monoisoStearate and Sorbitan methoxybenzophenone-5-Sulfonic acid. monolaurate, POE-Sorbitan esters such as POE-Sorbitan monooleate, POE-Sorbitan monoisoStearate and POE 50 Examples of preservatives include benzoates, Salicylates, Sorbitan monolaurate, fatty acid esters of glycerol Such as Sorbates, dehydroacetates, p-oxybenzoates, 2,4,4'-trichloro glyceryl monooleate, glyceryl monoStearate and glyceryl 2'-hydroxydiphenyl ether,3,4,4'-trichlorocarbanilide, benza monomyristate, POE-fatty acid esters of glycerol Such as Ikonium chloride, hinokitiol, resorcinol and ethanol. POE-glyceryl monooleate, POE-glyceryl monostearate and Examples of antioxidants include tocopherol, ascorbic POE-glyceryl monomyristate, POE-dihydrocholesterol 55 acid, butylhydroxyanisole, dibutylhydroxytoluene, nordihy ester, POE-hardened castor oil, POE-hardened castor oil droguaiaretic acid and propyl gallate. fatty acid esterS Such as POE-hardened castor oil isoStearate, Examples of chelating agents include Sodium edetate and POE-alkylaryl ethers such as POE-octylphenol ether, glyc Sodium citrate. erol esterS Such as glycerol monoisoStearate and glycerol Examples of antibiotics include penicillin, neomycin, monomyristate, POE-glycerol ethers such as POE-glycerol 60 cephalothin, potassium permanganate, Selenium Sulfide, monoisoStearate and POE-glycerol monomyristate, polyg erythromycin, bacitracin, tethacyclin, chloramphenicol, lycerol fatty acid esterS Such as diglyceryl monoStearate, Vancomycin, nitrofurantoin, acrisorcin, chlorodontoin, and decaglyceryl decastearate, decaglyceryl decaisoStearate and flucytosine. diglyceryl diisoStearate and other nonionic Surfactants, Some of these additives function to enhance the efficacy potassium Salts, Sodium Salts, diethanolamine Salts, trietha 65 of the composition by increasing the Stability or percutane nolamine Salts, amino acid Salts and other Salts of higher ous absorbability of the essential components of the present fatty acids Such as myristic acid, Stearic acid, palmitic acid, invention. US 6,541,447 B1 7 8 Also, any dosage form is acceptable, whether in Solution, Note: Percentage wound acceleration in days=(1-TD/ emulsion, powder dispersion, or others. Applicability is CD)x100%, where TD and CD are the days required for wide, including fundamental dosage forms Such as lotions, 80% wound healing in the treatment and control animals, emulsions, creams and gels. respectively. Percent wound acceleration in size=(1-TA/ The composition of the present invention is preferrably CA)x100%, where TA and CA are the areas of the wounds formulated according to Formula 1: in the treatment in control animals, when 80% wound Phenoxyethanol 1.0% (w/w) healing is reached on the former. Carbomer 0.5% (w/w) EXAMPLE 3 Ovalbumin 20.0% (w/w) Triethanolamine 0.3% (w/w) A male patient routinely plagued by muscle and joint Suitable vehicle 79.2% (w/w) SoreneSS and Stiffness related to Sports activities on the The method for processing and pasteurizing hen egg morning following Such activities, applied the inventive whites is well known in the art and generally involves composition to the affected areas and found that he experi reverse osmosis, heating, and drying StepS resulting in Solid enced no joint Soreness the mornings following the Sports egg whites ready for compounding. 15 activities. In addition to those Stated above, Suitable vehicles, car EXAMPLE 4 riers and adjuvants include water, Vaseline, petrolatum, mineral oil, vegetable oil, animal oil, organic and inorganic Patient number 964885 has been applying the inventive waxes, polymerS Such as Xanthanes, gelatin, cellulose, compositions to a diabetic ulcer on the left foot and has collagen, Starch, kaolin, carrageenan, gum arabic, Synthetic experienced a shrinkage of the wound. polymers, alcohols, polyols, and the like. The carrier can also include Sustained release carrier Such as lypizomes, EXAMPLE 5 microSponges, microSpheres, or microcapsules, aqueous A female applied the inventive composition in a cream base ointments, water in oil or oil in water emulsions, gels form to a third degree burn on her left inner forearm. After or the like. 25 daily application of the cream to the affected area, the blister The dose administered to an animal, particularly a human, reabsorbed readily, and the area healed within Seven days in the context of the present invention should be sufficient to with no residual Scar tissue. effect a therapeutic response over a reasonable time frame. The dose will be determined by the strength of the particular EXAMPLE 6 compositions employed and the condition of the perSon. The An 88-year-old patient Sustained a fall resulting in Several Size of the dose and the frequency of application also will be facial abrasions and a 1.5 inch laceration over the right determined by the existence, nature, and extent of any eyebrow. With the use of the inventive cream to the affected adverse side effects that may accompany the administration area, these lacerations healed in about 72 hours leaving no of a particular composition. Scar tissue. The composition of the present invention may be 35 employed to treat diabetic ulcers, healing resistant wounds, EXAMPLE 7 bed Sores, burns, osteomyelitis, trauma wounds, Subcutane A male who has had diabetes for more than 30 years ous trauma and various forms of dermatitis. Sustained various skin tears and Small ulcers on his upper The following examples illustrate the inventive compo 40 and lower extremities. Daily application of the inventive Sitions and methods, but should not be regarded as limiting composition in a cream form has kept the affected areas the invention in any manner. Additional Studies are in clean and painfree and healing took place in a relatively progreSS as of the filing date of this application. Short time frame. EXAMPLE 1. EXAMPLE 8 In March 1998 a female patient who underwent laminec 45 tomy and fusion of T3 and T4 began applying the inventive A wound on a dog's hind leg was So extensive that the pad composition to the Surgical wound. This patient experienced of the paw was almost detached. The inventive composition no Scaring from the Surgical wounds and continued to be cream was applied and the wound bandaged. A few days without Scars 10 months post Surgery. This patient had a later, the veterinarian noticed remarkable healing in that the previous history of radical Surgery in May 1960 for removal 50 wound was clean and dry with a good granulation bed, and of a right breast tumor. The wound from that Surgery healed there was the beginning of granulation acroSS the wound. in approximately one month, but left a large Scar. Eighteen The extent of the healing was So great that the dog was able months after the May 1960 Surgery she had major abdominal to go home with no bandage on its paw. surgery, and in 1985 had reconstructive of the right and left Similarly, a filly who had experienced a cut foreleg to the breast, but with both Surgeries experienced significant Scar 55 cannon bone was administered the inventive cream and the ing. Thus, with the recent Surgery, the inventive composition wound was wrapped. Two days later, upon removal of the prevented Scaring. bandage and checking of the wound, it had healed nicely acroSS with a base of granulation over the periosteum and EXAMPLE 2 showed a good, clean, dry wound bed with moist, Supple The wound healing characteristics of the inventive com 60 edges. position was demonstrated on Sprague-Dawley rats using a cream according to Formula 1 applied to a wound once daily EXAMPLE 9 for five days per week. The percentage of wound accelera Several patients have used the inventive composition for tion in days and in Size was compared to control. The percent arthritis, foot calluses, dry skin, back pain, bruises, allergies of wound acceleration in days was determined to be 5.19, 65 (including latex allergies), and inflammation. All Such and the percent of acceleration in Size was determined to be patients reported Symptomatic relief using the inventive 22.07. composition. US 6,541,447 B1 9 10 Analysis of the Composition plier of the primary antibody. All other Samples and controls were run under reducing conditions. The Samples were The composition of the invention (formula 1) was tested electrophoresed for 45 minutes at 200 V. using antibodies directed against bovine and other mamma The gel was placed on a PVDF membrane and electro lian derived growth factors. In particular, the antibodies used phorectically transferred for one hour at 25-30 amps. The were targeted toward PDGF, FGF a, FGFb, TGFB, TGFC, membrane was blocked for one hour in PBS containing 5% and EGF. The following antibodies were used for detection nonfat dry milk. The primary antibodies were diluted in of the growth factors. Unless noted otherwise the antibodies preparation for incubation with the membrane. Unless oth were purchased from Research Diagnostics Inc., Flanders, erwise noted the antibodies were diluted in PBS. The N.J. concentration of the antibodies used was as follows:

Antibodies Catalog Number Identification Specificity According to Label RDI-PDGFABabg goat anti-PDGF Recognizes PDGF-AA, -AB, -BB chains of human, primate, bovine and porcine. RDI-BFGFAabm mouse anti-FGF Recognizes bovine acidic FGF, human acidic Acidic FGF and human basic FGF. RDI-BRGFBabm2 mouse anti-bovine Recognizes bovine, rat, mouse and human FGF FGF Basic RD-TGFBams mouse anti-human Recognizes human, mouse and bovine TGFB1 TGF-B and B2, also Xenopus TGFB3. RDI-TOFAabmb mouse anti-human Recognizes mouse, rat and human TGF-C. TGF-C. RDI-MSEGFCabg goat anti mouse EGF Recognizes mouse and human EGF at carboxy C-term terminus

Secondary Antibodies From Jackson Laboratories 3O Donkey anti-goat IgG (H & L) linked to biotin SP used for Goat anti-Human PDGF-aB 10 ug/ml EGF and PDGF antibodies. Mouse anti-human TGFB 1 ug/ml in PBS with 1 mg/ml BSA Mouse anti-bovine basic FGF 1 ug/ml From Vectastain Mouse anti-bovine acidic FGF 1 ug/ml in PBS with 5 mg/ml BSA Horse anti-mouse (IgG (H & L) linked to biotin used for Mouse anti-human TGF a 10 ug/ml all other antibodies. 35 Mouse anti-goat EGF 10 ug/ml Other Reagents Lanes were cut from the membrane and incubated over From Zymed night at 4 C. with the primary antibody of interest. The Streptavidin linked to Horse Radish Peroxidase (HRP) membrane strips were washed for one hour in PBS-tween (1:4000) 40 (0.05% Tween 20) and incubated for two hours with the Protein Determination secondary antibody of choice diluted in PBS. For lanes incubated with goat derived primary antibodies, biotin Protein concentration WS determined by the BCA assay linked donkey anti-goat IgG at 1:10,000 dilution was used as (Pierce) according to manufacture's instructions. secondary antibody. For lanes incubated with mouse derived Samples 45 primary antibody, biotin linked horse anti-mouse IgG at 7.5 Samples Provided by B&M Technologies ug/ml diluted in PBS (1:200) with 1% normal horse serum was used as Secondary antibody. The membrane Strips were washed for one hour in PBS and incubated with either streptavidin-HRP for one hour (Zymed 1:4000, for donkey Protein conc. By BCA 50 anti-goat IgG stained Strips) or Vectastain AB reagent ppt MGS4-2 21 mg/ml (Avidin DH linked to Biotinylated peroxidase) for 30 min 1. MGS4-2 7-24-97 115 mg/ml utes (for horse anti-mouse IgG Stained Strips). The Strips 2 MGS4-3 9-8-97 55 mg/ml were washed for 30 minutes in PBS and developed using Sample #2 was selected for use in the Western Blot diaminobenzadine tetrahydrochloride (DAB). The mem p 55 brane strips were washed for 10 minutes in PBS and air Crude Rat Brain Homogenate Ris For control samples crude rat brain homogenates were A preliminary Western run to test the response of the generated and run in adjacent lanes to Samples of the antibodies was run without controls. Antibodies to PDGF inventive composition. 60 and TGF f showed little activity against the inventive Western Blot composition (lane 6 PDGF, 7 TGF B). Antibodies to EGF, Separation of proteins was carried out in modified form TGF C, FGF C, FGF f, (lanes 1-4, respectively) had from the procedures of Laemmli (Nature 227:680-685 identical binding to the invention with bands at 70 kDa, (1970)). Samples and controls were electrophoresed on a 32-34 kDa and 15-17 kDa. 15% SDS-PAGE gel. 40 ug of sample or control was loaded 65 While the specific invention has been described with an onto each lane. Samples to test for PDGF and TGFB were emphasis upon preferred embodiments, it will be obvious to run under non-reducing conditions as Specified by the Sup those of ordinary skill in the art that variations in the US 6,541,447 B1 11 12 preferred methods of the present invention may be used and ing growth factor alpha, fibroblast growth factor alpha, and that it is intended that the invention may be practiced fibroblast growth factor basic; about 1.0% by weight otherwise than as Specifically described herein. Accordingly, phenoxyethanol, about 0.5% by weight carbomer; and about this invention includes all modifications encompassed 0.3% by weight triethanolamine; wherein the composition within the spirit and scope of the invention as defined by the stimulates hair follicle growth on the skin of the subject. following claims. 8. The composition of claim 1, wherein the growth factor I claim: is epidermal growth factor. 1. A composition for accelerating wound healing in a 9. The composition of claim 1, wherein the growth factor subject in need thereof, comprising: about 5.0% to 20% by is transforming growth factor alpha. weight crude Ovalbumin containing a growth factor Selected from the group consisting of epidermal growth factor, trans 10. The composition of claim 1, wherein the growth factor forming growth factor alpha, fibroblast growth factor alpha, is fibroblast growth factor alpha. and fibroblast growth factor basic; about 1.0% by weight 11. The composition of claim 1, wherein the growth factor phenoxyethanol, about 0.5% by weight carbomer; and about is fibroblast growth factor basic. 0.3% by weight triethanolamine. 15 12. The composition of claim 1, wherein the wound is 2. The composition according to claim 1, in the form of Selected from the group consisting of an ulcer, burn, a gel. Osteomyelitits, trauma wound, Subcutaneous trauma wound, 3. The composition according to claim 1, in the form of dermatitis, Sports injury, muscle Soreness, joint Soreness, a CC. muscle Stiffness, joint Stiffness, laceration, Scarring, Surgical 4. The composition according to claim 1, in the form of wound, arthritis, foot calluses, dry skin, back pain, bruise, a lotion. and inflammation. 5. The composition according to claim 1, in the form of 13. The composition of claim 1, wherein the composition an ointment. is in a form of a topical composition. 6. A method for Stimulating wound healing in a Subject in 14. The composition of claim 1, wherein the composition need thereof, comprising: applying a pharmacologically 25 is in a form of a Solution, emulsion, powder, or dispersion. effective amount of a composition to the wound of the 15. The composition of claim 1, wherein the crude oval subject, the composition comprising about 5.0% to 20% by bumin is from hen egg white. weight crude Ovalbumin containing a growth factor Selected 16. The composition of claim 1, wherein the composition from the group consisting of epidermal growth factor, trans further comprises a Surfactant, oil, fat, alcohol, thickening forming growth factor alpha, fibroblast growth factor alpha, agent, UV absorbent, light Scattering agent, preservative, and fibroblast growth factor basic; about 1.0% by weight antioxidant, chelating agent, or antibiotic. phenoxyethanol, about 0.5% by weight carbomer; and about 17. The method of claim 6, wherein the composition is in 0.3% by weight triethanolamine; wherein the amount of the a form of a topical composition. composition applied to the skin increases local wound 18. The method of claim 6, wherein the composition is in healing activity of the Subject's skin. 35 a form of a gel, cream, lotion, ointment, or powder. 7. A method for Stimulating hair growth in a Subject in 19. The method of claim 7, wherein the composition is in need thereof, comprising: applying a pharmacologically a form of a topical composition. effective amount of a composition to the Skin of the Subject, 20. The method of claim 7, wherein the composition is in the composition comprising about 5.0% to 20% by weight a form of a gel, cream, lotion, ointment, or powder. crude Ovalbumin containing a growth factor Selected from 40 the group consisting of epidermal growth factor, transform