WO2010050980A1

WO2010050980A1 - Topical formulations comprising hydroxyapatite particles for stimulation and maintenance of collagen fibers

Info

Publication number: WO2010050980A1
Application number: PCT/US2008/082142
Authority: WO
Inventors: Zahra Mansouri
Original assignee: Laboratory Skin Care, Inc.
Priority date: 2008-10-31
Filing date: 2008-10-31
Publication date: 2010-05-06

Abstract

A composition that includes a calcium phosphate carrier agent and a compound selected from a collagen stimulator, a collagenase inhibitor, an enzyme based component, and a peptide component, present in a vehicle formulated for application to a topical surface of a subject is disclosed. The subject composition can further comprise a compound selected from collagen and procollagen.

Description

TOPICAL FORMULATIONS COMPRISING HYDROXYAPATITE PARTICLES FOR STIMULATION AND MAINTENANCE OF COLLAGEN FIBERS

INTRODUCTION

During aging, the levels of collagen in the skin not only decrease but the collagen fibers begin to cross-link and this is often due to the effect of external factors such as exposure to the sun, especially UVA radiation. The result of this cross-linking is that the skin loses its elasticity. In aging skin a higher proportion of cross-linked insoluble collagen is found, while younger, healthy, taut skin possesses a higher level of fresh-formed, soluble collagen. In the past, topical application of soluble animal collagen was used in an attempt to stimulate the formation of collagen in the skin. However these tests were unsuccessful as collagen cannot penetrate the epidermis. In attempting to maintain a youthful appearance, one option is to stimulate the production of collagen fibers or to maintain collagen fibers to achieve an optimal clinical end result of tissue response that is more consistent with normal healthy tissue.

SUMMARY

A composition that includes a calcium phosphate carrier agent and a compound selected from a collagen stimulator, a collagenase inhibitor, an enzyme-based component, and a peptide component, present in a vehicle formulated for application to a topical surface of a subject is disclosed. The subject composition can further comprise a compound selected from collagen and procollagen.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them unless specified otherwise. The terms "polypeptide" and "protein" and "peptide", interchangeably used herein, refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.

In certain instances, amino acid residues are identified by conventional three- letter amino acid code.

The terms "subject," "individual," "host," and "patient" are used interchangeably herein to refer to a mammal, including, but not limited to, murines (rats, mice), felines, non-human primates (e.g., simians), humans, canines, ungulates, etc.

The terms "treatment," "treating," "treat," and the like are used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.

DETAILED DESCRIPTION

A composition that includes a calcium phosphate carrier agent and a compound selected from a collagen stimulator, a collagenase inhibitor, an enzyme-based component, and a peptide component, present in a vehicle formulated for application to a topical surface of a subject is disclosed. The subject composition can further comprise a compound selected from collagen and procollagen. Further aspects include methods in which the compositions are topically administered to a subject. The subject methods and compositions find use in a variety of different applications, including but not limited to general skin care and the treatment of a subject for wrinkles, discoloration, puffiness, and scarring.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

Methods recited herein may be carried out in any order of the recited events which is logically possible, as well as the recited order of events.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

REPRESENTATIVE EMBODIMENTS

One embodiment provides a composition comprising a compound selected from a collagen stimulator, a collagenase inhibitor, an enzyme based component, and a peptide component; and a calcium phosphate carrier agent, present in a vehicle formulated for application to a topical surface of a subject.

The calcium phosphate carrier agent can comprise a ceramic calcium phosphate. In a related embodiment, the ceramic calcium phosphate is ceramic hydroxyapatite. In certain instance, the ceramic hydroxyapatite is sintered.

The calcium phosphate carrier agent can comprise a particulate composition of sintered ceramic hydroxyapatite.

The particulate composition can comprise particles ranging in size from 1 μm to 10 μm. In certain instances, the particulate composition comprises particles ranging in size from 1 μm to 8 μm. In other instances, the particulate composition comprises particles ranging in size from 1 μm to 6 μm. In other instances, the particulate composition comprises particles ranging in size from 2 μm to 6 μm. The calcium phosphate carrier agent can be present in an amount ranging from 0.001 to 10% by weight of said composition. In certain instances, the calcium phosphate carrier agent is present in an amount of 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10% by weight of said composition. The calcium phosphate carrier agent can be present in an amount ranging from from 0.01 to 5% by weight of said composition. In certain instances, the calcium phosphate carrier agent is present in an amount of 0.1 , 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5% by weight of said composition.

The collagen stimulator can be a small molecule or a polypeptide. In one case, the collagen stimulator is a small molecule. In another case, the collagen stimulator is a polypeptide. The polypeptide can be natural, semi-synthetic, synthetic, or recombinant.

The collagen stimulator can be a growth factor. In certain embodiments, the collagen stimulator is selected from epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF), and insulin-like growth factor (IGF). In certain instances, the collagen stimulator is epidermal growth factor (EGF). In other instances, the collagen stimulator is platelet- derived growth factor (PDGF), including PDGFA, PDGFB, PDGFC, PDGFD, and PDGFAB. In other instances, the collagen stimulator is fibroblast growth factor (FGF), including FGF1 to FGF14 and FGF16 to FGF23. In other instances, the collagen stimulator is transforming growth factor (TGF), including TGF-α and TGF-β. In other instances, the collagen stimulator is insulin-like growth factor (IGF), including IGF-1 and IGF-2.

The collagen stimulator can be a polypeptide from EGF family. Members of the EGF family include epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), epiregulin (EPR), epigen, betacellulin (BTC), neuregulin-1 (NRG1 ), neuregulin-2 (NRG2), neuregulin-3 (NRG3), neureguline-4 (NRG4). In certain embodiments, the collagen stimulator is epidermal growth factor (EGF).

In certain instances, the collagen stimulator can be selected from vitamin C and derivatives, palmitoyl oligopeptide, elastin, hyaluronic acid, hyaluronic acid salts (such as sodium salts), procollagens (such as procollagen type I for example), allantoin, hydroxyproline, proline, lysine, centella asiatica, and mimosa pudica.

The collagenase inhibitor can be a peptide having proline, leucine, and glycine.

The composition can comprise a peptide component, where the peptide component is peptide PLA. In one case, peptide PLA is a dermal stimulating peptide selected from the group consisting of palmitoyl tetrapeptide, derivatives of palmitoyl tetrapeptide, palmitoyl tripeptide, palmitoyl oligopeptide, palmitoyl pentapeptide, and mixtures thereof. In a certain case, peptide PLA is palmitoyl tripeptide. Palmitolyl tripeptide is also known as palmitoyl tripeptide-3 and palmitoyl tripeptide-5. In another case, peptide PLA is cyclic branched peptide.

The composition can comprise an enzyme-based component, where the enzyme-based component is collagenase. In one case, the enzyme-based component is CoIH-FM or CoIH-SR.

Further, the composition can have a compound selected from collagen and procollagen.

The composition can have additives, such as sunscreen. Examples of compounds used as sunscreen include p-aminobenzoic acid, Padimate-O, phenylbenzimidazole sulfonic acid, cinoxate, dioxybenzone, oxybenzone, homosalate, methyl anthranilate, octocrylene, octyl methoxycinnamate, octyl salicylate, sulisobenzone, trolamine salicylate, avobenzone, ecamsule, titanium dioxide, zinc oxide, 4-methylbenzylidene camphor, Tinosorb M, Tinosorb S, Neo Heliopan AP, Mexoryl XL, Uvinul T 150, Uvinol A Plus, Uvasorb HEB, Parsol SLX, and isopentenyl-4- methoxycinnamate.

The vehicle can be a lotion, a cream, or a gel.

COMPONENTS OF SUBJECT COMPOSITION COLLAGEN STIMULATORS

Many studies involving human subjects have shown that collagen type I is decreased with increasing severity of photodamage (See Kligman, A., JAMA, (1969), 210, pp. 2377-2380; Lavker, R., J. Inv. Derm., (1979), 73, 79-66; Smith, J. et al., J. Inv. Derm., (1962), 39, pp. 347-350; and Shuster, S. et al., Br. J. Dermatol., (1975), 93, pp. 639-643); and some correlation in the histology of wrinkles and reduction in collagen levels in the sun-exposed skin has been reported. (See Chen, S.; Kiss, I., J. Inv. Derm., (1992), 98. pp. 248-254.)

It may be possible to stimulate the proliferation of collagen-producing fibroblasts and to induce them over a limited time to produce more collagen. A "collagen stimulator" is a compound that can stimulate collagen production. A collagen stimulator can be a small molecule or a polypeptide.

The subject compositions can comprise growth factors as collagen stimulators. Cellular growth and differentiation appear to be initiated, promoted, maintained and regulated by a multiplicity of stimulatory, inhibitory and synergistic factors and hormones. Growth factors are implicated in a wide variety of pathological and physiological processes including signal transduction, cell communication, growth and development, embryogenesis, immune response, hematopoiesis cell survival and differentiation, inflammation, tissue repair and remodeling, atherosclerosis and cancer. Peptide growth factors in epidermal growth factor (EGF) family influence epithelial and epidermal cells through autocrine and paracrine mechanisms. They also play important roles in normal wound healing in tissues such as skin, cornea and gastrointestinal tract and all share substantial amino acid sequence homology including the conserved placement of three intra-chain disulfide bonds. Growth factors, such as EGF, can stimulate the synthesis of collagen. (Creely et al, American Journal of Pathology, VoI 136, 1247-1257 (1990))

Examples of growth factors in subject compositions include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF), and insulin-like growth factor (IGF). In certain embodiments, the collagen stimulator is a polypeptide from EGF family. In certain embodiments, the collagen stimulator is epidermal growth factor (EGF).

The collagen stimulator can be a natural product or small molecule. Voorhees and colleagues supported findings of utility of stimulation of collagen synthesis by showing the restoration of collagen type I in photo-damaged human skin by a topical treatment with tretinoin. (See Christopher, E., et al., The New Eng. J. of Medicine (1993), 329, pp. 530-535.) These results provide in vivo evidence that retinoic acid restores collagen type I. Many etiology studies showed a parallel effect between collagen synthesis and wrinkle effacement. It is also believed that the strengthening of the dermal matrix by collagen stimulation may have some beneficial effect for treatment of cellulite. (See U.S. Pat. No. 5,051 ,449 (Kligman)). There are natural products which are known to stimulate collagen synthesis.

One of these is ascorbic acid, a cofactor for collagen synthesis, known to activate the enzyme prolyl-hydroxylase (See Peterkofsky, B., Arch. Biochem. Biophys., (1972), 152, pp. 318-328) and to increase procollagen mRNA. (See Cardinale, G. et al. Adv. Enzymol., (1974), 41 , p. 245; Geesin, J. et al., J. Inv. Derm., (1988), 90, p. 420; Tajima, S. et al., Biochem. Biophys. Res. Comim., (1982), 106, pp. 632-637.) Stimulation of collagen synthesis by betel nut alkaloids, arecolin and arecaidine, has also been described. (See Vermillo, A. et al. Arch. Oral Biol., (1986), 31 , pp. 819-823.) Forskolin which is a common component of Coleus forskolli has been shown to increase the production of collagen in cloned osteoblastic cells. (See Hakeda, Y. et al., J. Biochem., (1987), 101 , pp. 1463-1469.)

Some studies indicate that Centella asiatica promotes greater elasticity and suppleness to the skin by its action on the collagen synthesis by the fibroblasts. (See Tenni, R. et al., J. Biochem, (1988), 38. pp. 69-77; Maquart, F. et al., Actualite Therapeutique, (1989), pp. 1571 -1574.) Centella asiatica also acts as a regulating agent in the connective tissue, promoting its regeneration and preventing its excessive proliferation, producing supple, healthy connective tissue. (See Lawrence J. Eur. J. Pharmacol., (1967), pp. 414-425; Rosen, H. et al., J. Proc. Soc. Exp. Biol. Med., (1967), 125, pp. 279-280; Bosse, J. et al.; Ann. Plast. Surg., (1979), 3, pp. 13-21.) Vogel et al. have shown a significant increase of collagen in scar tissue leading to an increased resistance to traction and rupture. (See Vogel, H. et al., Acta Therapeutica, (1990) 16, pp. 285-296.) This increased resistance of the collagen fibers is only noticeable in collagen which has reached a certain maturation stage. The triterpenes of Centella asiatica increase both the collagen synthesis and its maturation level.

The plant, Centella asiatica, grows in Madagascar and around the Indian Ocean. Traditionally, this plant has been used for wound healing. (See Chopra, R. et al.,

"Indigenous Drugs of India", Dhur & Sons Pvt. Ltd. (1985), Calcutta.) In Europe, a drug prepared from this plant is used for the treatment of ulcers and wounds. Centella asiatica is also suitable for cosmetic use, i.e., skin conditioning improvement, anti- cellulite effect, and improvements in skin color. (See Adolphe, M. et al., Int. J. Cosmetic Soc, (1984), 6, pp. 55-58.) Centella asiatica contains asiatic acid, madecassic acid, asiaticoside and madecaside, all of which belong to the class of triterpenoids. Maquart et al., Conn. Tissue Res., (1990), 24 pp. 107-120 showed that the triterpene extract from Centella asiatica stimulated collagen synthesis in fibroblast monolayer cultures, and asiatic acid was found to be the major component responsible for collagen synthesis stimulation. A number of small molecules and natural products can stimulate the synthesis of collagen. In certain embodiments, the collagen stimulator is selected from vitamin C and derivatives, palmitoyl oligopeptide, elastin, hyaluronic acid, hyaluronic acid salts (such as sodium salts), procollagens (such as procollagen type I for example), allantoin, hydroxyproline, proline, lysine, centella asiatica, mimosa pudica, betulinic acid, tretinoin, betel nut alkaloids, and forskolin.

COLLAGENASE INHIBITORS

Collagenases are proteolytic enzymes which initiate the degradation of collagen. Not being bound by theory, the mechanism of action of mammalian collagenases on the molecular level is fairly understood. Tissue collagenases hydrolyze a specific peptide bond at a single cleavage site on each of the three collagen chains of triple helical collagen. This cleavage site is contained within the amino acid sequence Pro- Gln-Gly-Leu-(lle)-Ala-Gly-Gln-Arg, with cleavage occurring between glycine 775 and leucine or isoleucine 776, in Types I, Il and III collagen, the predominant collagen in skin, bone, tendon, dentin, fascia and cartilage. Type IV collagenase (gelatinase) degrades basement membrane (Type IV) collagen. The collagenases are metallopeptidases which contain zinc at the active site. The zinc is assumed to function by interactions with the scissile carbonyl of the substrate, thus facilitating hydrolysis of the peptide bond. Collagen degradation can be inhibited, for example by using TIMP proteins

(tissue inhibitors of metalloproteinases). Certain tripeptides may be employed as collagenase inhibitors (see e.g., U.S. Pat. Nos. 4,687,841 and 4,720,486 to Spilburg, et al.). U.S. Pat. Nos. 4,687,841 and 4,720,486 disclose peptides having proline, leucine, and glycine as having collagenase inhibitory activity. In other instances, peptides having proline, leucine, and alanine can also have collagenase inhibitory activity. Furthermore, the scientific literature contains references to various further collagenase inhibiting compounds. For example, Clark, et al. (Life Sciences 37: 575- 578 (1985)) refer to N[[5-chloro-2-benzothiazo- lyl)thiophenyl]acetyl]-L-cysteine, said to be a powerful mammalian collagenase inhibitor. Deleaisse, et al. (Biochem Biophys. Res. Comm. 133: 483-490, 1985) also refer to an inhibitor N-[3-N-(benzyloxy-carbonyl)- amino-1 -(R)-carboxypropyl]-L-leucy-1 -O-methyl-L-tyrosine-N-methylamide. Gray, et al. (Biochem. Biophys. Res. Comm. 101 : 1251 -1258, 1981 ) disclose a number of thiol- containing analogues of the collagen cleavage site. Additional thiol-containing peptides are disclosed by Gray, et al. in J. Cell Biochem., 32: 71 -77, 1986. Carboxyalkyl peptide analogues are described in Gray, et al. in Federation Proc. 44: 1431 , 1985. Miller, et al. and Gray, et al. also disclose thiol-containing peptides in abstracts. [Fed. Proc. 45: 1859 (1986) and FASEB J. 2: A345 (1988), respectively]. Mookhtiar, et al. also discloses phosphonamidate inhibitors of collagenase. (see Biochemistry, 26, 1962 (1987)).

ENZYME-BASED COMPONENT

Other components include enzyme-based component. One enzyme-based component is CoIH-FM protein. CoIH-FM protein can increase body firmness and reshape contours of a body.

Another enzyme-based component is CoIH-SR. CoIH-SR is a bioengineered enzyme that targets excessive scar collagen. CoIH-SR can smooth, soften, and flatten scars.

Collagen degradation occurs during various physiological and pathological conditions. However, only a limited number of proteases with unique characteristics can trigger collagen degradation. Collagenase is an enzyme that has the specific ability to digest collagen and collagenase injections have been proposed for the treatment of conditions associated with collagen plaques or cords. The collagenase enzyme has been used to treat a variety of collagen-mediated diseases and collagenase for use in therapy has been obtained from a variety of sources including mammalian (e.g. human), crustacean (e.g. crab, shrimp), fungal, and bacterial (e.g. from the fermentation of Clostridium, Streptomyces, Pseudomonas, or Vibrio). One common source of crude collagenase is from a bacterial fermentation process, specifically the fermentation of C. histolyticum (C. his) which must then be purified. Collagenase can be used to prevent scar formation and to treat conditions, such as keloids, hypertrophic scars, Peyronie's Disease and Dupuytren's Disease.

Collagenase gene colH can be expressed in E. coli. The corresponding amino acid sequence of the gene is published in Genbank by Matsushita et al. 1999

(accession number D87215) and Yoshihara et al. 1994 (accession number D29981 ). for Clostridium histolyticum strain JCM 1403 (ATCC 19401 ). The production of recombinant collagenase CoIH in E. coli is described in U.S. Patent Publication No.

2008233614.

PEPTIDE COMPONENT

Other components include a peptide component. One peptide component is protein PLA.

In one case, peptide PLA is a dermal stimulating peptide selected from the group consisting of palmitoyl tetrapeptide, derivatives of palmitoyl tetrapeptide, palmitoyl tripeptide, palmitoyl oligopeptide, palmitoyl pentapeptide, and mixtures thereof. Peptide PLA can be contained within a commercial composition SYN^®-COLL.

(commercially available from Valeant Pharmaceuticals, Costa Mesa, CA). Peptide PLA can be a compound selected from palmitoyl tetrapeptide (N-palmitoyl-Gly-Gln-Pro-Arg) and derivatives thereof and palmitoyl tripeptide (N-palmitoyl-Gly-His-Lys) and derivatives thereof. Of interest is palmitoyl tripeptide, also known as palmitoyl tripeptide-3 and palmitoyl tripeptide-5.

Naturally occurring peptides, such as peptide PLA, can be instrumental in stimulating the healing process following a wound to the skin. Robinson teaches (U.S. Pat. No. 6,492,326) various formulations containing combinations of palmitoyl pentapeptide-3, derivatives of pentapeptides, and mixtures thereof. Lintner (U.S. Pat. No. 6,620,419) discloses peptide formulas of the general sequence palmitoyl-lysyl- threonyl-lysyl-serine that increase the synthesis of collagen and glycosaminoglycans. They act synergistically to heal wrinkles and other forms of skin aging far more effectively than earlier formulations. The key difference in the Lintner teaching to that of Robinson is the addition of a fatty acid chain onto the terminal end of a pentapeptide that makes this lipophilic modified peptide very efficient at penetrating the epidermis and thus more effective reaching the formative layers of the dermis. Other peptide formulations, i.e., oligopeptides, tetrapeptides and tripeptides are described by Lintner in (US Publication No. 20040132667). In another case, peptide PLA is cyclic branched peptide. Cyclic peptides can exhibit an affinity for collagen, and can be used to treat, prevent, ameliorate, or evaluate physiologic functions, manifestations, or disorders where collagens are involved.

COLLAGEN

Collagen is a protein of connective tissue in animals. A distinctive feature of collagen is the regular arrangement of amino acids in each of the three chains of these collagen subunits. The sequence can often follow the pattern Gly-Pro-Y or Gly-X-Hyp, where X and Y can be any of various other amino acid residues, and Hyp is hydroxyproline.

Collagen can refer to any one of the known collagen types, including known collagen types, such as collagen types I through XIX, as well as to any other collagens, whether natural, synthetic, semi-synthetic, or recombinant. The term "collagen" can also encompass procollagens. The term "collagen" specifically encompasses variants and fragments thereof, and functional equivalents and derivatives thereof, which preferably retain at least one structural or functional characteristic of collagen.

Collagens are classified into several types based on sequence identity and function. Types I, II, and III collagen molecules make up the main fibers of most animal extracellular structures. Type I forms a large percentage of the body's collagen and is the primary component of bone, skin and tendons. Type Il makes up the major fibers of cartilage. Collagen fibers are arranged in rigid plates in bones, in parallel bundles in tendons, and in a dense meshwork in cartilage. Type I and lesser amounts of type III make up tendons and skin. Type IV collagen molecules make up very fine, unstriated fibers present in basal laminae. Over a dozen other collagen types are known but are less well characterized. Collagen plays a role in maintaining skin structure and accounts for as much as

70% of the weight of the skin. The formulation of new collagen fibers is helpful for healthy, firm skin. Type I and type III collagen are present in the highest levels in the skin, forming 80% and 15% of the total collagen present respectively. Type IV collagen is the main component of the lamina densa, a 50nm thick layer in the basement membrane zone and type VII collagen is a major constituent of the anchoring fibrils beneath the lamina densa, at the dermal-epidermal interface. Type V collagen is found pericellularly. During aging, fibroblasts in the skin produce less collagen. Not being bound by theory, it has been generally believed that type III collagen is found in much higher levels in young skin but decreases significantly with age. Its role has been correlated with tissue extensibility, being replaced in the first instance by type I collagen which forms a more rigid structure.

PROCOLLAGEN

Procollagen is a biosynthetic precursor to collagen. Biosynthesis of the procollagen molecule involves a large number of post-translational modifications, using at least eight procollagen-specific enzymes and several non-specific enzymes. Over a hundred amino acids in each a chain can be modified post-translationally. After procollagen is assembled, it is secreted from cells. Extracellularly, the N-propeptide is cleaved from the procollagen molecule by an enzyme and the C-propeptide is cleaved from the procollagen molecule by another enzyme, yielding an individual mature collagen subunit. The solubility of the collagen subunit is two thousand times lower than the solubility of the corresponding procollagen subunit. Low collagen solubility drives spontaneous polymerization of collagen subunits into collagen fibrils. In vitro assembly of collagen subunits formed by enzymatic cleavage of procollagen subunits has been demonstrated (Prockop et al., 1989, In: Cytoskeletal and Extracellular Proteins, Aebi et al., eds., Springer Series in Biophysics, Vol. 3, pp. 81 -89; Kadler et al., 1990, Biochem J. 268:339-343).

Procollagen refers to a procollagen corresponding to any one of the collagen types I through XIX, as well as to a procollagen corresponding to any other collagens, whether natural, synthetic, semi-synthetic, or recombinant, that possesses additional C- terminal and/or N-terminal propeptides or telopeptides that assist in trimer assembly, solubility, purification, or any other function, and that then are subsequently cleaved by N-proteinase, C-proteinase, or other enzymes, e.g., proteolytic enzymes associated with collagen production. The term procollagen specifically encompasses variants and fragments thereof, and functional equivalents and derivatives thereof, which preferably retain at least one structural or functional characteristic of collagen.

CALCIUM PHOSPHATE CARRIER AGENT

The macroporous calcium phosphate particles of the subject compositions are macroporous particles made up of calcium and phosphate. As the calcium phosphate particles are macroporous, the include pores that are 50 nm in diameter or larger. In addition, they have a porosity of 30% or more, such as 40% or more, including 50% or more, where the porosity may range from 30% to 75%, such as from 40% to 60%, including from 45% to 55%, as determined using a mercury intrusion porosimeter porosity determination protocol as described in ASTM D 4284-88 "Standard Test Method for Determining Pore Volume Distribution of Catalysts by Mercury Intrusion Porosimetry". In some cases, the hydroxyapatite particles have a porosity such that their internal surface area ranges from 10 m²/g to 150 m²/g, such as from 20 m²/g to 100 m²/g, including 30 m²/g to 80 m²/g, as determined using a BET gas adsorption surface area determination protocol as described in ASTM D3663-03 Standard Test Method for Surface Area of Catalysts and Catalyst Carriers. In certain embodiments, the particles have a tapping density ranging from 0.2 g/cm³ to 0.5 g/cm³, such as from 0.25 g/cm³ to 0.45 g/cm³, including from 0.3 g/cm³ to 0.4 g/cm³. The tap density can be measured by using standard ASTM WK13023 - New Determination of Tap Density of Metallic Powders by a Constant Volume Measuring Method. As the particles are calcium phosphate particles, they are mineral compositions that include calcium and phosphate. In certain embodiments, the calcium phosphate is hydroxyapatite (i.e., Cai₀(PO₄)₆(OH)₂) or substituted versions thereof. In certain embodiments, the particles are chemical pure, by which is meant that they are made up of a single type of calcium phosphate mineral, e.g., hydroxyapatite. Of interest is ceramic hydroxyapatite, where the term ceramic is used to denote inorganic non- metallic materials which are formed by the action of heat.

In certain embodiments, the particles are spheres having a controlled diameter. Of interest are particulate compositions in which the median diameter of the particles in the composition is dimensioned to penetrate human skin, e.g., where it ranges from 0.5 to 20 μm, such as from 1 to 10 μm, and including from 2 to 6 μm.

The macroporous calcium phosphate particles employed in the invention may be prepared using any convenient protocol or obtained from a commercial source. For example, ceramic hydroxyapatite spheres may be manufactured by the agglomeration of small crystals (50-100 nm size range) followed by sintering of the agglomerated crystals at high temperature to provide mechanically and chemically stable spheres. Ceramic hydroxyapatite which is of interest as a penetration agent is exemplified by that manufactured by the Asahi Optical Company, Tokyo. Also of interest is the ceramic hydroxyapatite describe in, for example, R. Kasai et al. J. Chromatography 407, 205 (1987); S. Tsuru et al. J. Immunol. Methods 106, 169 (1988); T. Kadoya et al. J. Liquid Chromatography 9,3543 (1986); T. Kadoya et al. J. Liquid Chromatography 1 1 ,2951 (1986). See also U.S. Patent No. US Pat. No. 5,158,756, the disclosure of which is herein incorporated by reference. Of particular interest in certain embodiments is are Hydroxysomes® calcium phosphate particulate compositions commercialized by Laboratory Skin Care, Tahoe City, CA.

FORMULATIONS

The compositions comprising the calcium phosphate carrier agent and a compound selected from a collagen stimulator, a collagenase inhibitor, an enzyme- based component, and a peptide component are formulated in a manner convenient for topical application. Thus, the subject compositions can be formulated as stable solutions or suspensions of the components. Alternatively, the components can be combined with one or more carrier materials to form a solution, suspension, gel, lotion, cream, ointment, aerosol spray or the like, as in known in the art.

Gel vehicles can be formulated to produce a topical application useful in the subject methods are physiologically acceptable and can comprise a solvent in combination with a thickening agent. The solvent can be an alkanol, such as an alcohol or polyol, including: ethanol, isopropanol, propylene glycol, glycerol, and the like. These alcohols and polyols can be used individually or in combination. In the gel vehicle, the solvent will generally be present in from 1 to 80 weight %, more commonly 10 to 40 weight % of the topical composition. Conventional gelling or thickening agents can be employed to provide for a formulation which can be conveniently applied to the skin. Gelling agents which have been found to be effective and are illustrative of conventionally used gelling agents for skin application include Carbomer 940 (neutralized with diisopropanolamine), neutralized polyacrylic acid, etc. The gelling agent will be used in an amount sufficient to provide the appropriate viscosity, generally being in the range of 0.1 -5 weight percent of the formulation. Non-ionic surfactants can be included in the compositions, where the nonionic surfactants can serve as cosolvents and epidermal penetration enhancers, in addition to the eucalyptol and any optional penetration enhancing agent described above. Conventional surfactants can be employed, which are physiologically acceptable, such as sorbitan esters, etc. When present, the nonionic surfactant will generally be present in an amount of from 2-20 weight percent of the formulation.

The topical composition can also contain other physiologically acceptable excipients or other minor additives, particularly associated with organoleptic properties, such as fragrances, dyes, emulsifiers, buffers, cooling agents (e.g. menthol), antibiotics, stabilizers or the like. The excipients and minor additives will be present in conventional amounts ranging from 0.001 % to 5%, more commonly 0.001 -2%, by weight, usually not exceeding a total of 10% by weight.

A wide variety of pharmaceutically acceptable excipients is known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7^th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3^rd ed. Amer. Pharmaceutical Assoc.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

Where convenient, e.g. with a gel formulation, the topical application can be covered with an occlusive dressing, which can be porous or non-porous, so as to protect the gel from mechanical removal during the period of treatment. Various inert coverings can be employed, which can include various materials which may find use in plasters, described herein. Alternatively, non-woven or woven coverings can be employed, particularly elastomeric coverings, which can allow for heat and vapor transport. These coverings allow for cooling of the pain site, which provides for greater comfort, while protecting the gel from mechanical removal.

Instead of a gel, a plaster can be employed, where the composition comprising the components can be formulated into the adhesive of the plaster. In the case of plasters, the coverings can include polyvinyl chloride, polyvinylidene chloride, polyethylene, synthetic rubber, woven or nonwoven polyester fabric, etc. The components can be combined with the adhesive with the aid of a cosolvent, or a combination of cosolvents, such as propylene glycol, glycerin, methyl salicylate, glycol salicylate, or the like.

Optionally the formulations can further include a preservative. Suitable preservatives include, but are not limited to, a benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. Formulations may also be lyophilized, in which case they generally include cryoprotectants such as sucrose, trehalose, lactose, maltose, mannitol, and the like. Lyophilized formulations can be stored over extended periods of time, even at ambient temperatures. METHODS OF USE

Aspects of the embodiments include methods of delivering a composition comprising a collagen stimulator, a collagenase inhibitor, an enzyme-based component, and a peptide component to a subject. Embodiments of the methods include applying to a topical region of the subject an effective amount of a composition that includes a compound selected from collagen stimulator and collagenase inhibitor.

The topical region is, in certain embodiments, a keratinized skin region. The keratinized skin region may be present at a variety of locations, e.g., limbs, arms, legs; torso, e.g., chest, back, stomach; head, e.g., neck; etc. In certain embodiments, the skin region is a facial region. The topical region to which the composition is applied may vary with respect to area, ranging in certain embodiments from 1 cm² to 20 cm², such as from 2 cm² to 15 cm², and including from 3 cm² to 10 cm².

Following application, the topical composition is maintained at the site of application for a period of time sufficient for an amount of the collagen stimulator to be delivered to the subject. The period of time may vary, and in certain embodiments ranges from 1 minute to 24 hours, such as from 30 minutes to 12 hours and including from 1 hour to 12 hours.

In practicing the subject methods, a subject can be administered a single dose or two or more doses over a given period of time. For example, over a given treatment period of one month, 1 or more doses, such as 2 or more doses, 3 or more doses, 4 or more doses, 5 or more doses, etc., may be administered to the subject, where the doses may be administered weekly or daily or even multiple times per day.

The subject methods and compositions may be used in a variety of different kinds of animals, where the animals are typically "mammals" or "mammalian," where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), lagomorpha (e.g., rabbits) and primates (e.g., humans, chimpanzees, and monkeys). In certain embodiments, the subjects or patients are humans. The subject methods and compositions find use in a variety of different applications, including but not limited to general skin care and the treatment of a subject for wrinkles, discoloration, puffiness, and scarring.

KITS

Also provided are kits that find use in practicing the subject methods, as described herein. For example, kits for practicing the subject methods may a quantity of the composition, present in unit dosages, e.g., ampoules, or a multi-dosage format. As such, in certain embodiments the kits may one or more unit dosages (e.g., ampoules) of the formulation. In yet other embodiments, the kits may include a single multi dosage amount of the formulation.

In addition to the above components, the subject kits may further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.

Provided is a kit that includes at least two of the following components: 1 ) a cream for daytime use; 2) a cream for daytime use for area around the eyes; 3) a cream for nighttime use; and 4) a cream or serum for nighttime use for area around the eyes. The cream for daytime use and the cream for daytime use for area around the eyes can include sunscreen. The cream for nighttime use and the cream or serum for nighttime use for area around the eyes does not include sunscreen. A cream is a preparation comprising the composition in the form of a thick consistency. A serum is a preparation comprising the composition in the form of a cream or liquid. The cream can be applied to facial region. Thus, it is a cream applied to the skin in the facial or neck region. The facial region includes the front part of the head that in humans extends from the forehead to the chin and includes the mouth, nose, cheeks, and eyes. The neck region includes the part of an animal that connects the head with the body.

The cream or serum can be applied to skin area around the eyes. The skin area is on the face around the eyes, such as eyelids, area under the eyes extending to cheeks, area between the eyes and bridge of nose, and area around the eyes on the opposite end of the area between the eyes and bridge of nose (normally where crow's feet appear).

The daytime cream can be used during the day. In one instance daytime is period of time when there is daylight. Thus, the daytime cream can be used for an applied region that comes into contact with light, such as outdoors. The daytime cream can include sunscreen.

The nighttime cream or serum can be used during the night. In one instance nighttime is period of time when there is no daylight. Thus, the nighttime cream or serum can be used during sleep at night. The nighttime cream or serum does not include sunscreen.

In one instance, the cream for daytime use and the cream for daytime use for area around the eyes includes sunscreen with UVA/UVB coverage. For example, the sunscreen protection can be SPF 15, 45 or 60. The components: 1 ) a cream for daytime use; 2) a cream for daytime use for area around the eyes; 3) a cream for nighttime use; and 4) a cream or serum for nighttime use for area around the eyes, can include peptide PLA. In one instance, these components include peptide PLA, which is palmitoyl tripeptide, palmitoyl tripeptide-3, palmitoyl tripeptide-5. In certain instances, the kit comprises all four components.

Provided is a cream for reviving damaged cells and promoting younger skin containing a composition comprising: a compound selected from a collagen stimulator, a collagenase inhibitor, an enzyme-based component, and a peptide component; and a calcium phosphate carrier agent; present in a vehicle formulated for application to a topical surface of a subject, wherein the collagen stimulator is EGF. Provided is a cream for body contouring containing a composition comprising: a compound selected from a collagen stimulator, a collagenase inhibitor, an enzyme- based component, and a peptide component; and a calcium phosphate carrier agent; present in a vehicle formulated for application to a topical surface of a subject, wherein the enzyme-based component is CoIH-FM.

Provided is a cream for scar reduction containing a composition comprising: a compound selected from a collagen stimulator, a collagenase inhibitor, an enzyme- based component, and a peptide component; and a calcium phosphate carrier agent; present in a vehicle formulated for application to a topical surface of a subject, wherein the enzyme-based component is CoIH-SR.

METHODS OF PREPARATION

Methods of producing a calcium phosphate carrier agent of the embodiments are disclosed in U.S. Patent No. 6,096,324, which is incorporated by reference in its entirety.

Ceramic hydroxyapatite spheres are manufactured by the agglomeration of small crystals (50-100 nm size range) followed by sintering at high temperature. As a result of this process, each sphere is porous and can act as a miniature sponge. This characteristic of ceramic hydroxyapatite spheres allows it to absorb, carry, and subsequently release components of the composition to which it has been bound.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising: a compound selected from a collagen stimulator, a collagenase inhibitor, an enzyme-based component, and a peptide component; and a calcium phosphate carrier agent; present in a vehicle formulated for application to a topical surface of a subject.

2. The composition according to Claim 1 , wherein the calcium phosphate carrier agent comprises a ceramic calcium phosphate.

3. The composition according to Claim 2, wherein the ceramic calcium phosphate is ceramic hydroxyapatite.

4. The composition according to Claim 3, wherein the ceramic hydroxyapatite is sintered.

5. The composition according to Claim 4, wherein the calcium phosphate carrier agent comprises a particulate composition of sintered ceramic hydroxyapatite.

6. The composition according to Claim 5, wherein the particulate composition comprises particles ranging in size from 1 μm to 10 μm

7. The composition according to Claim 1 , wherein the calcium phosphate carrier agent is present in an amount ranging from 0.001 to 10% by weight of said composition.

8. The composition according to Claim 7, wherein the calcium phosphate carrier agent is present in an amount ranging from 0.01 to 5% by weight of said composition.

9. The composition according to Claim 1 , wherein the collagen stimulator is a small molecule or a polypeptide.

10. The composition according to Claim 1 , wherein the collagen stimulator is a growth factor.

1 1. The composition according to Claim 10, wherein the collagen stimulator is selected from epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF), and insulin-like growth factor (IGF).

12. The composition according to Claim 1 , wherein the collagen stimulator is a polypeptide from EGF family.

13. The composition according to Claim 12, wherein the polypeptide from EGF family is epidermal growth factor (EGF).

14. The composition according to Claim 1 , wherein the collagenase inhibitor is a peptide derivative comprising proline, leucine, and glycine.

15. The composition according to Claim 1 , wherein peptide component is peptide PLA.

16. The composition according to Claim 15, wherein peptide PLA is a dermal stimulating peptide selected from the group consisting of palmitoyl tetrapeptide, derivatives of palmitoyl tetrapeptide, palmitoyl tripeptide, palmitoyl oligopeptide, palmitoyl pentapeptide, and mixtures thereof.

17. The composition according to Claim 16, wherein peptide PLA is palmitoyl tripeptide, palmitoyl tripeptide-3, or palmitoyl tripeptide-5.

18. The composition according to Claim 15, wherein peptide PLA is cyclic branched peptide.

19. The composition according to Claim 1 , wherein the enzyme-based component is collagenase.

20. The composition according to Claim 1 , wherein the enzyme-based component is selected from CoIH-FM and CoIH-SR.

21. The composition according to Claim 1 , further comprising a compound selected from collagen and procollagen.

22. The composition according to Claim 1 , further comprising sunscreen.

23. The composition according to Claim 1 , wherein the vehicle is a lotion.

24. The composition according to Claim 1 , wherein the vehicle is a cream.

25. The composition according to Claim 1 , wherein the vehicle is a gel.

26. A method of transdermal^ administering a compound to a subject, said method comprising: applying to a topical region of said subject an effective amount of a composition comprising: (i) a compound selected from a collagen stimulator, a collagenase inhibitor, an enzyme based component, and a peptide component; and

(ii) a calcium phosphate carrier agent; present in a vehicle formulated for application to a topical surface of a subject; to treat said subject for said skin condition.

27. The method according to Claim 26, wherein the topical region is a facial region of said subject.

28. The method according to Claim 26, wherein the administration results in treating the subject for a skin condition.

29. The method according to Claim 28, wherein the skin condition is wrinkling.

30. The method according to Claim 28, wherein the skin condition is discoloration.

31. The method according to Claim 28, wherein the skin condition is puffiness.

32. The method according to Claim 28, wherein the skin condition is scarring.

33. The method according to Claim 26, wherein the administration results in caring for the skin of the subject.

34. A kit comprising: a) a composition comprising: i) a compound selected from a collagen stimulator, a collagenase inhibitor, an enzyme based component, and a peptide component; and ii) a calcium phosphate carrier agent; present in a vehicle formulated for application to a topical surface of a subject; and b) instructions.

35. The kit of Claim 34, which is a day-time skin cream.

36. The kit of Claim 34, which is a day-time skin cream for application to area around eyes.

37. The kit of Claim 34, which is a night-time skin cream.

38. The kit of Claim 34, which is a night-time skin cream for application to area around eyes.

39. A kit comprising at least two creams of Claims 35-38.

40. A kit comprising four creams of Claims 35-38.

41. The kit of Claim 34, wherein the collagen stimulator is EGF.

42. The kit of Claim 34, wherein the enzyme-based component is CoIH-FM.

43. The kit of Claim 34, wherein the enzyme-based component is CoIH-SR.