AU2010249270B2 - Methods for treating skin pigmentation - Google Patents

Abstract

METHODS FOR TREATING SKIN PIGMENTATION This invention relates to methods and compositions for bringing about changes in 5 skin pigmentation. More particularly, this invention relates to compounds which affect melanogenesis and can be used as depigmenting agents or as agents for darkening skin utilizing the PAR-2 pathway.

Description

- 1 AUSTRALIA PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT ORIGINAL Name of Applicant: Johnson & Johnson Consumer Companies, Inc. Actual Inventors: Stanley S Shapiro and Susan Niemiec and John Kung and Miri Seiberg Address for Service is: SHELSTON IP 60 Margaret Street Telephone No: (02) 9777 1111 SYDNEY NSW 2000 Facsimile No. (02) 9241 4666 CCN: 3710000352 Attorney Code: SW Invention Title: METHODS FOR TREATING SKIN PIGMENTATION Details of Original Application No. 2006252275 dated 22 Dec 2006 The following statement is a full description of this invention, including the best method of performing it known to melus: File: 37491AUP02 - ]a METHODS FOR TREATING SKIN PIGMENTATION The present application is a divisional application of Australian Application No. 2006252275, which is incorporated in its entirety herein by reference. 5 Field of the Invention This invention is related to methods and compositions for bringing about skin pigmentation and/or for causing skin depigmentation. More particularly, this invention relates to compounds which affect melanogenesis and can be used as depigmenting agents or as agents for darkening skin. 10 Background of the Invention Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. Skin coloring has been of concern to human beings for many years. In particular, 15 the ability to remove hyperpigmentation, such as found in age spots, freckles or aging skin generally, is of interest to individuals desiring a uniform complexion. In certain areas of the world, general body whitening is desirable. There are also hypopigmentation and hyperpigmentation disorders that are desirable to treat. Likewise, the ability to generate a tanned appearance without incurring photodamage due to solar 20 radiation is important to many individuals. There have been many methods proposed to accomplish depigmentation, as well as to accomplish darkening of the skin. For example, kojic acid, hydroquinone, retinoids and other chemical compounds have been used for depigmentation. Dihydroxyacetone and like chemical compounds have been utilized for their ability to "tan" the skin without exposure to the sun. 25 Many of these previous solutions have not been found acceptable. There is often a distinct line of demarcation between the areas of skin to which such previous compositions have been applied. Therefore, precise application of all these compounds is necessary in order to achieve the desired result. Many of these compounds have been found to be quite irritating to the skin and therefore undesirable for use. 5 The understanding of the chemical and enzymatic. basis of melanogenesis is heavily documented. Melanocytes migrate from -the embryonal neural crest into the skin to produce secretory granules, melanosomes, which produce melanin. Melanogenesis occurs within the 10 melanosome, and the melanin is later distributed to keratinocytes via the melanocyte dendrites. The key enzyme in melanogenesis is tyrosinase, which initiates a cascade of reactions which convert tyrosine to the biopolymer melanin. Two tyrosinase-related proteins 15. (TRP's) are known, TRP- and TRP-2. These proteins share, with tyrosinase about 40% homology and have catalytic activities as well as regulatory roles in melanogenesis. TRP-1 is the most abundant glycoprotein in melanocytes. 20 In spite of the fact that the chemical and enzymatic -basis of melanogenesis is well-documented, its regulation -at the cellular level is only partially understood. Tyrosinase and the TRP's share structural and biological properties with the lysosomal-associated 25 membrane protein (LAMP) gene family, therefore their targeting to the melanosomal membrane might induce their activation. A phosphorylation/dephosphorylation reaction at the cytoplasmic tails of these proteins could be involved in the regulation of melanogenesis. 30 - The beta isoform of the Protein Kinase C (PKC) family has been shown to regulate human melonogenesis through tyrosinase activation. Gene expression of tyrosinase, TRP-l and TRP-2 is coordinated- All three enyzmes are -3 expressed in human epidermis. In melanocytes co cultured with keratinocytes, these transcripts are expressed at a ratio of 45:45:10, respectively. In 5 melanocytes cultured alone, only TRP-1 transcripts are present, indicating that a keratinocyte-derived signal is involved in the coordinate expression of these genes. The regulation of keratinocyte-melanocyte interactions and the mechanism of melanosome transfer into 10 keratinocytes are not yet understood. -The Protease-activated receptor-2 (PAR-2) is a seven transmembrane G-protein-coupled receptor, that is related to, but distinct from the thrombin receptors (TR, 'also named PAR-., and PAR-3) in its sequence. Both 15 receptors are activated proteolytically by an arginine serine cleavage at the extracellular domain. The newly created N-termini then activate these receptors as tethered ligands. Both receptors could be activated by trypsin, but only the TRs are activated by thrombin. 20 Only PAR-2 is activated by mast cell . tryptase. Both receptors could also be activated by the peptides that correspond to their new N-teimini, independent of receptor cleavage. SLIGRL, the mouse PAR-2 activating peptide, is equipotent in the activation of the human 25 receptor. While the function of the TR is well documented, the biology of the PAR- 2 has not yet been fully identified. A role fog PAR-2 activation in the inhibition of keratinocyte growth and differentiation has~ been recently described (Derian et al., 30 "Differential Regulation of Human Keratinocyte Growth and Differentiation by a Novel Family of Protease activate Receptors", Cell Growth & Differentiation, Vol. 8, pp. 743-749, July 1997).

-4 Summary of the Invention In accordance with this invention, we have found a method for affecting changes in mammalian skin 5 pigmentation comprising topically applying to the skin of a mammal a compound which affects the PAR-2 pathway. The compositions of this invention may contain one or more compounds that act as trypsin, as tryptase, as serene protease or as PAR-2 agonists, for increase in 10 pigmentation. Alternatively, they may contain one or more compounds that act as serine protease inhibitors, trypsin inhibitors, thrombin inhibitors, tryptase inhibitors,' as PAR-2 pathway inhibitors or as a PAR-2 antagonist for decrease in pigmentation, or 15 "depigmentation". As used herein, "mammal" means any member "of the higher vertebrate animals comprising the class "Mammalia", . as defined in Webster's Medical Desk Dictionary 407 (19e6), and includes but is not limited 20 to humans. As used herein, "receptor" 'shall include both intracellullar and extracellular receptors and shall mean those molecules capable of receiving and transducing a -signal. The term PAR-2 refers to the protease-activared receptor-2 or a related protease 25 activated receptor. The Protease-activated receptor-2 (hereinafter, "PAR-2") is a serine-protease activated receptor that is expressed in numerous tissues, including keratinocytes and fibroblasts. The thrombin receptor (also named PAR-1, hereinafter, "TR") is a 30 serine-protease activated receptor that is expressed in numerous tissues, including keratinocytes. The biological roles of PAR-2 and TR in skin are not entirely known. However, we have found that interactions between keratinocytes and melanocytes, via -5 the PAR-2 pathway, affect melanogenesis. We have found that thrombin inhibitors, and/or tryptase inhibitors, and/or trypsin inhibitors and PAR-2 antagonists can be used as depigmenting agents without irritation of the skin. PAR-2 agonists and serine proteases such as trypsin and tryptase can be used as darkening agents. Furthermore, 5 PAR-2 could be useful as a target for whitening and darkening agents. Summary of Invention According to a first aspect, the present invention provides a method of effecting changes in mammalian skin pigmentation comprising administering to a mammal a pigmentation-changing effective amount of a compound which is: 10 (i) a PAR-2 antagonist which binds to or blocks but does not activate PAR-2; or (ii) a PAR-2 agonist which binds to and activates PAR-2. According to a second aspect, the present invention provides a composition when used to affect changes in mammalian skin pigmentation comprising a pigmentation 15 changing effective amount of a compound according to the first aspect. According to a third aspect, the present invention provides use of a compound which is: (i) a PAR-2 antagonist which binds to or blocks but does not activate PAR-2; or 20 (ii) a PAR-2 agonist which binds to and activates PAR-2 in the manufacture of a medicament for affecting changes in the mammalian skin pigmentation. Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an 25 inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". Brief Description of the Drawings Fig. IA is a graph depicting the increase or decrease in relative pigmentation of epidermal equivalents containing melanocytes treated with known pigmenting and 30 depigmenting agents in accordance with the methods of this invention. Fig. 1 B is a graph depicting the increase or decrease in relative pigmentation in epidermal equivalents containing melanocytes treated in accordance with the methods and compositions of this invention.

- 5a Fig. 2 is a group of images of epidermal equivalents containing melanocytes treated with PAR-2 agonists and Compound I. Fig. 3 is a graph depicting the increase or decrease in relative pigmentation in epidermal equivalents containing melanocytes treated in accordance with the methods 5 and compositions of this invention. Fig. 4A is a graph depicting the dose/response with respect to pigmentation in epidermal equivalents containing melanocytes when treated with compositions of this invention. Fig. 4B is a graph depicting the response of epidermal equivalents containing 10 melanocytes after -6 exposure to ultraviolet light followed by treatment with compositions of this invention. Fig. SA is a photograph depicting gels showing the 5 expression of TR and PAR-2 in skin, melanoma cells and epidermal equivalents containing melanocytes. Fig. 5B is a photograph depicting gels showing the expression of TR and PAR-2 by primary human- melanocytes. Fig. 6A and 6B are photographs depicting gels 10 showing the expression of various genes after treatment with different c-oncentrations of.Compound.I. and SLIGRL. Fig. 7 is a graph showing the effects of different compositions of this invention on the brightness of guinea pig nipple pigmentation. 15 Fig.. 8 is a photograph of Yucatan Swine skin which has been treated with compositions of this invention for depigmentation of skin. Fig. 9 is a graph depicting the brightness of Yucatan Swine skin during the course of treatment in 20 accordance with the methods and compositions of this invention. Fig. 10A, 10B, 10C and 10D are photographs'of F&M stained histological sections of Yucatan Swine skin treated with compositions, containing C;-ipound I in 25 accordance with methods of this invention at concentrations of- 0, 10 gM, 50 pM and 250 pM respectively. Figs. 1lA, 11B and 11C are photographs of electron micrographic views of epidermal equivalents containing 30 melanocytes treated with compositions of this invention. Figs- 11E, 11' and llH are photographs of electron micrographic views of Yucatan Swine skin treated witb compositions of this invention.

-. 7 Figs. llD and 11G are photographs of electron micrographic views of untreated sites of Yucatan Swine skin. Figs. 12A, 12B, 12C, 12D and 12E are photographs 5 of histological F&M stained sections of Yucatan Swine skin, as follows: 12A shows untreated skin; 12B shows skin treated with compositions of this invention after eight weeks of treatment; 12C shows skin one week after stopping treatment; 12D shows skin two weeks after 10. stopping treatment and 12E shows skin four weeks after stopping treatment. Fig. 13 is a photograph of F&M stained histological sections taken from Yucatan Swine skin treated with compositions of this invention. 15 Fig. 14 contains ultraviolet and visible light digital photographs of human skin prior to treatment and subsequent to treatment with compositions of this invention. 20 Detailed Description of the Preferred Embodiments We have discovered that trypsin, tryptase and PAR 2 agonists can be used to increase pigmentation and that trypsin inhibitors, and/or tryptase inhibitors, and/or thrombin inhibitors and PAR-2 antagonists act to 25 decrease pigmentation in mammalian skin. In our opinion, some of the compounds described in U.S. Patent No. 5,523,308, which is hereby incorporated herein by reference, and behave as thrombin and/or trypsin and/or tryptase inhibitors, will be useful in methods of this 30 invention. some of these compounds are also described in Costanzo, et al., "Potent Thrombin Tnhibitors That Probe the S1' Subsite: Tripeptide Transition State Analogues Based on a Heterocycle-Activated Carbonyl -- 8 Group", J. Med. Chem., 1996, Vol. 39, pp. 3039-3043 and have the following structural formula: 5 H 10 wherein: A is selected from the group consisting of Cj ealkyl, carboxycI--4alkyl, 15 CI-4alkoxycarbnylCi-.alkyi, phenylCI- 4 alkyl, substitut-ed phenylCI..

4 alkyl (where the phenyl substituents are independently selected' from one or more of, Ci- 4 alkyl, perfluoroCI.

4 alkyl, C1..aalkoxy, hydroxy, halo, amid, nitro amino, CI...alkylamino, 20 Ci-dialkylamino, carboxy or C1- 4 alkoxycarbonyl), formyl, C1..4alkoxycarbonyl, CI-2alkylcarbonyl, pheny],Ci-alkoxycarbonyl, C3 7cycloakylcarbonyl, phenylcarbonyl, substituted phenylcarbonyl (where the phenyl substituents are 25 independently selected from one or more of, CI- 4 alkyl, perfluoroC..

4 alkyl, CL-4alkoxy, hydroxy, halo,- amido, nitro, amino, Ci- 4 alkylamino, Ci-4dialkylamino, carboxy or CI, 4 alkoxycarbonyl), CI-4alkylsulfonyl,

C

1 . 4alkoxysulfonyl, perfluoroCI- 4 alkyl-sulfonyl, 30 Phenyisulfonyl, substituted phenylsulfonyl (where the phenyl substituents are independently selected from one or more of, Ci-alkl, perfluoroC- 4 alkyl,

CI-

4 alkoxy, hydroxy, halo, amido, nitro, amino, Ci..4alkylamino, Cj 4dialkylamino, carboxy or ,CI-4 alkoxycarbonyl), 10 35 camphrsulfonyl, phenylCI- 4 alkysulfonyl, substituted phenylCi- 4 alkysulfonyl, Ci-4alkylsulfinyl, perfluroCi- 4alkylsulfinyl, phenylsulfinyl, substituted. phenylsulfinyl (where the phenyl substituents are independently selected from one or more of, ci-,alkyl, 5 perfluorocI- 4 alkyl, CI- 4 alkoxy, hydroxy, halo, amido, nitro, amino, C14alkylamino, C..4dialkylamino, carboxy or CI-4alkoxycarbonyl), phenylC.

4 alkylsulfinyl, substituted pehnylCI-4alkylsulfinyl, 1-naphthylsulfonyl, 2-naphthylsulfonyl or substituted naphthylsulfonyl 10 (where the naphthyl substituents are independently selected from one or more of, Ci-4alkyl,perfluoroci. 4alkyl, Ct.

4 alkoxy, hydroxy, halo, amido, nitro, amino, carboxy or Ci-4alkoxyy-carbonyl) , 1-naphthylsulfinyl, 2 naph'thylsulfinyl or substituted naphthylsulfinyl (where 15 the naphthyl substituents are independently selected front one or more of, C 1 4 alkyl, perfluorocI- 4 alkyi, C.. 4 alkoxy, hydroxy, halo, amido, nitro, amino, C1 4alkylamino, Ci...4dialkylamino, carboxy or C.. 4alkoxycarbonyl); 20 a D or L amino acid which is coupled as its carboxy terminus to the nitrogen depicted in formula I and is selected from the group consisting of alanine, asparagine, 2-azetidinecarboxylic acid, glycine, N-Ct.. Ralkyglycine, praline, 1-amino-1-cycloC 3 25 aalkylcarboxylic acid, thiazzolidine-4-carboxylic acid, 5, 5-dimethylthiazolidine-4-carboxylic acid, oxadolidine-4-carboxylic acid, pipecolinic acid, valine, methionine, cysteine, serine, threonine, norleucine, leucine, tert-leucine, isoleucine, 30 phenylalanine, 1-naphthalanine, 2-naphthalamine, 2 thienyl-alanine, 3-thienylalanine, (1,2,3,4] tetrahydroisoquinolin.e- 1-carboxylic acid and 1, 2, 3, 4, 1 -tetrahydroisoquinoline-2-caroboxylic acid -10 where the amino terminus of said amino acid is connected to a member selected from the group consisting of C 4 alkyl, tetrazol-5yl-C 1 2 alkyl, carboxytC.

4 alkyl, CI- 4 alkoxycarbonylCl4alkyl, phenylC.

5 4 alkyl, substituted phenyl Ci.

4 alkyl (where the phenyl substituents are independently selected from one or more of, CI..

4 alkyl, perfluoroC,- 4 alkyl, CI- 4 alkoxy, hydroxy, halo, amido, nitro, amino, CI- 4 alkylamino, C 1 . 4dialkylamino, carboy or Cl-4alkoxycarbonyl), 1,i 10 diphenylCI- 4 alkyi, 3 -phenyl-2-hydroxypropionyl, 2,2 diphenyl- 1-hydroxyethylcarbonyl, [1,2,3,41 tetrahydroisoquinoline- 1-carbonyl, (1,2,3,4] tetrahydroisoquinoline-3,carbonyl, 1-methylamino-1 cyclohexanecarbonyl, 1-hydroxy-1-cyclohexanecarbonyl, 15 i-hydroxy-1-pheny-lacetyl, 1-cyclohexyl-l hydroxyacetyl, 3 -phenyl-2-hydroxypropionyl, 3,3 diphenyl-2-hydroxypropionyl, 3 -cyclohexyl-2 hydroxypropionyl, formyl, CI-alkoxycarbonyl, Ci-ialkylcarbonyl, perflucroC 1 4 alkyl, 20 ci-4alkylcarbonyl, phenylCi 4 alkylcarboni, substituted phenylCi 4 alkylcarbonyl (where the phenyl substituents are independently.selected from one or more of, Ci 4alkyl,. perfluoroct- 4 alkyl, C-- 4 alkoxy, hydroxy, halo amido, nitro amino, CL4alkylamino,

C

1

..

4 dialkylamino, 25 carboxy or CI- 4 alkoxycarbonyi) 1,1-diphenylC 4alkylcarbonyl, substituted 1,1-dihenylCl 4alkylcarbonyl (where the phenyl substituents are independently selected from one or more of, Ci- 4 alkyl, perfluoro

C

1

.

4 alkyl, C- 4 alkoxy, hydroxy, halo, amido, 30 nitro, amino, Ci 4 alkylamino, Ci- 4 dialkylamino, carboxy or CI- 4 alkoxy-carbonyl), perfluoroC, 4 alkysulfonyl, CI-. 4alkysulfonyl,

CI.

4 alkoxysulfonyl, phenysulfonyl, substituted phenylsulfonyl (where the phenyl substituents are independently selected from one or more of, C-lalkyl, perfluoro Ci- 4 alkylamino, Cj. 4dialkylamino, carboxy or CI-4alkoxycarbonyl), 10 cxamphorsulfonyl, phenylCt-<alkylsulfonyl, substituted 5 phenylCi- 4 alkylsufonyl, perfluroCi- 4 alkysulfinyl,

C

1 4 alkysulfinyl, phenylsulfinyl, substituted phenysulfinyl (where the phenyl substituents are independently selected from one or more of, Ci- 4 alkyl, perfluoro Ci..

4 alkyl,

C..

4 alkoxy, hydroxy, halo,- amido, 10 nitro, amino, Ci- 4 alkylamino, Ci- 4 dialkylamino, carboxy or CI-4 alkoxycarbonyl), 1-naphthysulfonl, 2 naphthylsulfonyl, substituted naphthylsulfonyl (where the naphthyl substituents are independently selected froi one or more of, Ct- 4 alkyl, perfluroct- 4 alkyl, CI_ 15 4alkoxy, hydroxy, halo, amido, nitro, amino, C._ 4alkylamino, Ci- 4 dialkylamino, carboy or Ci 4alkoxycarbonyl) , 1-naphthysul finyl, 2-naphthysulfinyt, and substituted naphthylsulfinyl (where the naphthyl substituents are independently selected from one or 20 more of, Cil- 4 alkyl, perfluoroCk- 4 alkyl, Ci- 4 alkoxy, hydroxy, halo amido, nitro, amino, Ci- 4 alkylamino, C10 4 dialkylamono, carboxy or C-14alkoxycarboryl): or a poly peptide- comprised of two amino acids, where-the first amino a-cid is a D or L amino acid, 25 bound via i.ts carboxy terminus tot he nitrogen depicted in Formula I and is selected from the group consisting of glycine, N-CI-ealkylglycine, alanine., 2 azetidinecarboxylic acid, proline,. thiazolidine-4 carboxylic acid, 5.5-dimethylthiazolidine-4-carboxvlic 30 acid, oxazolidine-4-carboxylic acid, 1-amino-1-cycloC 3 - alkylcarboxylic acid, 3 -hydroxypropoline, 4-. hydroxyproline, 3- (C-4alkoxy) proline, -12 4 (Ct..4alkoxy)proline, 3

,

4 -dehydropriine, 2, 2-)iimethyl-4 thiazolidine carboxylic acid, 2.2-dimethyl-4 oxadolidine carboxylic acid, pipecolinic acid, valine, methionine, cysteine, asparagine, serine, threonine, 5. leucine, tert-leucine, isoleucine, phenylalanine, 1 naphthalanine, 2 -naphthalanine, 2 -thienylalanine, 3 thienylalnine, [1, 2

,

3

,

4 1-tetrahydroisoquinoline 2 carboxylic acid,' aspartic acid-4-C- 4 alkyl ester and glutamic acid 5-C1- 4 alkyl ester 10 and the second D or L amino acid, is bound to the amino terminus of said first amino acid, and is selected from the group consisting of phenylalanine, 4 benzolyphenylalanine, 4 -carboxyphenylalanine, 4 (Carboxy Cl- 2 alkyl)phenylalanine, substituted 15 phenylalanine. (where the phenyl substituents are independently selected from one or more of CI- 4 alkyl, perfluoroCt- 4 alkyl, CI- 4 alkoxy, hydroxy, halo, amido, nitro, amino, CI-4alkylamino, C.-4dialkylamino, carboxy or Cl-Aalkoxycarbonyl), 3 -benzothienylalanine, 4 20 biphenylalanine, homophenylalanine, octahydroindole-2 carboxylic acid, 2 -pyridylalanine, 3-pyridylalanine, 4 thiazolyalanine, Z-thienylalanie, 3-(3 benzothienyll alanine, 3 -thienylalanine, tryptophan, tyrosine. asparagine, 3-tri 25 Ct.4alkylsilylalanine, cyclohexyiglycine, diphenylglycine, phenylglycine, methionine sulfoxide, methionine sulfone, 2, 2 -dicyclohexylalanine, 2-(1 naphthylalanine), 2- ( 2 -naphthylalanine), phenyl. substituted phenylalanine (where the subst.ituents are 30 selected from CI.

4 alkyl, perfluoroCi' 4 alkyl, C..

4 alkoxy, hydroxy, halo, amid, nitro, amino, Ci.

4 alkylamino,

C

4dialylamino, carboxy or Ct.. alkoxycarbonyl), aspartic acid, aspartic acid-4C- 4 alkyl, perfluoroci...alkyl, Ct 4alkoxy, hydroxy, halo, amido, nitro, amino,

CI.

-13 4alkylaminfl, C . .4dialkylamino, carboxy or C 1

.

4alkoxycarbony), aspartic acid, aspartic acid-4-C 1 .. 4 alkyl ester glutaxaic acid, gltmcai-- 1 . lyl ester, CYCIOC3-salkylaalanine, substituted cycloC 3 5 ealkylalanine (where the ring substituents *are carboxy,.

CI-..

4 alicyl ester, cycloC3-salkylalanine, substituted cycloC3.ealkylaanine (where the ring substituents are carboxy, Cl-4alky-IcarboXY,- CL-.4a-koxycarbonyl or aminocarbonyl), 2, 2 -dipheriylalanine and all alpha-C 1 .. 10 salkyl of' all amino acid derivatives thereof, where the amino terminus of said second amino acid is UnSUbStitUted or !fofloSubstituted with a member of the group consisting of formyl, Cl-12 alkyl, tetrazol-5 y1CI-2alkyl, carboxyCI-8 alkcyl, carboalkxyC..

4 alkyl, 15 pheny. CI..alkyl, substituted phenylCI- 4 alkyl (where the phenyi- substituents or independently selected fromione or more of;~ CI- 4 alkyl, perfluorOCL- 4 alkyl, CI-4alkoxy, hydroxy, halo, axaido, nitro, amino, CL.4alkylamino,

C

1 .. 4dialkylamina, carboxy or Cj-.aIkoxycarb6ny1I, 1,1 20 dipehnyl.CL.

4 alkyl, C1- 6 alkoxycarbonyl,-phenylol Ealioxvcarbonyl, Ci-.

2 alkylcarbonyl, perfluoroC 1 .alky'LCo-4 alkvlcarbonyI, phenyCj- 4 alkyIcarbonyI, substituted .phenyCI-..akyIcarbonyI (where the phenyl substizuents are independently selected from one or 25 more Of CJ- 4 alky., perfluoro C--akl

C-

4 ' ailkoxy, hydroxy, halo, amnido, nitro, amino, CI-4alkylamino, ,Ci 4dialkylamino, carboxy or ci-4alkoxycarbonyi) 1, 1 diphenvlC.

4 alkyl, perfluor6Ct-alkyi.,

C

1 .. 4aikoxycarbonyl) , 1O-camphorsulfonyl±. phenylCl.. 30 4 a1 kysulfonyl, substit'uted phenylC 1 4alkylsulfornyl,

C

1 .. 4alkysulflinyj, perfluoro C-4alkylsulfinfyl, phenylsulffinyl, substitut-ed phenylsulfinyl (where the phenyl substitijents are independently selected* from one or more of, C-- -4aiky1, perf~uoroC,.;alkvl,

C

1

-

4 alkoxy, -14 . hydroxy, halo, amido, nitro, amino, C.- 4 alkylamono, C1. 4dialkylamono, carboxy or Ci-4alkoxycarbonyl) , phenyC 1 . 4alkylsulfinyl, substituted phenylCi- 4 alkylsulfinyl 1 naphthylsulfonyl, 2-naphthylsulfonyl, substituted 5 naphthylsulfonyl (where the naphthyl substituent is selected from C 1

-

4 alkyl, perfluoroCI- 4 alkyl, C 1

.

4 alkoxy, . hydroxy, halo amido, nitro, amino, C1- 4 alkylamino, C.. 4dialkylamino, carboxy or C..-alkoxycarbonyl), 1 haphthyl-sulfinyl, 2-haphthylsulfinyl and substituted 10 naphthyl-sulfinyl (where the naphthyl substituent is selected from CI- 4 alkyl, perfluoroC

.

.

4 alkyl, CI- 4 alkOxy, hydroxy, halo, amid, nitro, amino, CI-alkylamino,

C

14dialkylamino, carboxy or Ci-4alkoxycarbonyl); Rx is selected from the group consisting of hydrogen and 15 alkyl; R 2 is selected from the group consisting of aminoC2-salkyl, guanidinoC2-salkyl, CI.alkylguanidinoa salkyl, diCt..

4 alkylguanidinoC 2 ..salkyl, amidinoC 2 -salkyl, C.. 4alky-lamidinoCz-salkyl, diCI-4alky-lamidinoC 2 -salkyl, Ci.. 3alkoxyC2..alkyl, phenyl, substituted phenyl (where the 20 substituents are independently selectedc from one or more of amino, amidino, guanidino, CI-alkylamino,

C

1 .dialkylamino, halogen, perfluoro CI- 4 alkyl,- C\.

4 alkyl, C1.. alkoxy or nitro) , benzyl, phenyl substituted benzyl (where the substituents are independently selected from 25 one or more of, amino, amidino, guanidino,

C

1 aalkylamino,

C..

4 dialky-lamino, halogen, perfluoro Ct.. 4alkyl, Cl-04alkyl,

CL-

3 alkoxy or nitro), hydroxyC2 salkl, C'1-5alkylaminoC 2 -salkyl, C1..dialkylaminoC 2 ..salkyl, ' 4 -aminocyclohexyICo..2alkyl and CI-5alkyl; -15 p is 0 or 1; B is 50 10 where n is 0-3, R 3 is H or Cl-Salkyl and the carbonyl moiety of B is bound to E: E is a het.eracycle selected from the group consisting 15 of oxazolin-2-yl, oxazol-2-yl, thiazol-2-yl, thiazol-5 yl, thiazol-4-yl, thiazolin-2-yl, imidazol-2-yl, 4-oxo 2-quinoxalin-2yl, 2-pyridyl, 3-pyridyl, benzo(b thiophen-2-yl, triazol-4-yl triazol-6-yl,. pyrazol-2-yl, 4,5,6, 7 -tetrahydrobenzothiazol-2yl, 20 naphtho[2, 1-di thiazol-2-yl, naphtholi-2-d thiazoI-2-yI qruinoxalin- 2-yl, isoquinolin-1-yl, isboquinolin-3-yl, benzo [b) furan-2-yl, [pyrazin-2-yl, quinazolin-2-yl, isothiazol-5-yl, isothiazol-3-yI, purin-Byul and a substituted heterocycle where the substituents are 25 selected from CI- 4 from C-14alky, perfluoro

CI-

4 alkyl,Cl 4alkoxy, hydroxy, halo, amido, nitro, amino, Ci 4alkylamino, Ci4dialkylamino, carboxy, Ci.. 4alkoxycarbonyl, hydroxy or phenylcI-4 alkylaminocarbonyl; 30 or pharmaceutically acceptable salts thereof. More particularly, -in our opinion, some of the compounds of * the foregoing formula containing a d phenylalanine-proline-argjinine motif should be effective in inhibiting the PAR-2 pathway and causing depigmentation. One Particularly preferred compound which acts as a thrombin and trypsin inhibitor and is active in depigmenting mammalian skin is (S)-N-Methyl-D phenylalanyl-N-.(4- ((aminoiminomethyl) amino ]-1- (2 5 benzothiazolylcarbonyl) butyl] -L-prolinamide (Chemical Abstracts name) (hereinafter referred to as "Compound I"). We suggest that other compounds which are analogs or function similarly to Compound I and are set forth in U.S. Patent No. 5,523,308 may be active in the methods 10 and compositions of this invention. Other compounds that inhibit trypsin, such as serine protease inhibitors, and in particular, soybean trypsin inhibitor (STI) will also be useful in methods of this invention. Soybean, limabean - and blackbean extracts, and other 15 natural products made from these beans, such as, but not limited to, bean milk, bean paste, miso and the like, also serve to reduce pigmentation by this mechanism. Additional sources of serine protease inhibitors may be extracted from the species belonging to the 20 following plant families: Solanaceae (e.g.,* potato, tomato, tomatilla, and the like); Gramineae (e.g., rice, buckwheat, sorahum, wheat, barley, oats and the like); Cucurbitaceae (e.g., cucumbers, squash, gourd, luffa and the like) ; and, preferably, Leguminosae - (e.g., beans, 25 peas, lentils, peanuts, and the like) While not willing to be bound by the following theory, we theorize that the compounds capable of affecting the pigmentation of the skin do so by interacting directly or indirectly with the keratinocyte 30 PAR-2 or with its activating protease, and thereby affect melanogenesis, directly or indirectly. Possibly, the compounds of this invention induce, in the case of increased pigmentation or reduce, in the case of decreased pigmentation, the signal to transport- -17 melanosomes by melanocytes, or to receiire melanosomes by keratinocytes in the skin. The compounds which are active in the compositions and methods of this invention may be delivered topically 5 by any means known to those of ski4l in the art. If the delivery parameters of.the .topically active pharmaceutical or cosmetic agent so require, the topically active composition of this invention may preferably be further composed of a pharmaceutically or 10 cosmetically acceptable vehicle capable of functioning as a delivery system to enable the penetration of the topically active agent into the skin. One acceptable vehicle for topical delivery of some of the compositions of this invention, 15 particularly proteins such as trypsin and STI, may contain liposomes. The liposomes are more preferably non-ionic and contain a) glycerol dilaurate (preferably in an amount of between about 5% and about 70% by weight) ; b) compounds having the steroid 20 backbone found in cholesterol (preferably in an amount of between about 5% and about 45% by weight); and c) one or more fatty acid ethers having from about 12 to about 1.8 carbon atoms (preferably in an amount of between about St and about-70% by weight collectively), 25 wherein the constituent compounds of the liposomes are preferably in a ratio of about 37.5:12.5:33.3:16.7. Liposomes comprised of glycerol dilaurate / cholesterol/ polyoxyethylene -lO-stearyl ether/polyoxyethylene-9-lauryl ether (GDL liposomes) 30 are most preferred. Preferably the liposomes are present in an amount, based upon the total volume of the composition, of from about 10 mq/mL to about 100 mg/mL, and more preferably from about 20 mg/mL to about 50 mg/mL. A ratio of about 37.5:12.5:33.3:16.7 is most -18 preferred. Suitable liposomes may preferably be prepared in accordance with the protocol set forth in Example 1, though other methods commonly used in the art are also acceptable. The above described 5 composition .may be prepared by combining the desired components in a suitable container and mixing them under ambient conditions in any conventional high shear mixing means well known in the art for non-ionic liposomes preparations, such as those disclosed in Niemiec et 10 al., "Influence of Nonionic Liposomal Composition On Topical Delivery o.f.- Peptide. D-rugs Into Pilosebacious Units: An In Vivo Study Using the Hamster Ear Model," 12 Pharm. Res. 1184-88 (1995) ("Niemied"), which is incorporated by reference herein in its entirety. We 15 have found that the presence of .these liposomes in the compositions of this invention may enhance the depigmenting capabilities of some of the compositions of this invention. Other preferable formulations may contain, for 20 example, soybean milk or other liquid formulations derived directly from legumes or other suitable plant. For example, such a formulation may contain a large proportion of soybean milk, an emulsifier that maintains the physical stability of the soybean milk, and, 25 - optionally a chelating agent, preservatives, emollients, humectants and/or thickeners or gelling agents. Oil-in-water emulsions, water-in-oil emulsions, solvent-based formulations and aqueous gels known to those of skill in the art may also be utilized as 30 vehicles for the delivery of the compositions of- this invention. The source of active compound to be formulated will generally depend upon the particular form of the compound. Small organic molecules and peptidyl -19 fragments can be chemically synthesized and provided in a pure form suitable for pharmaceutical/cosmetic usage. Products of natural extracts can be purified according to techniques known in the art. Recombinant sources of 5 compounds are also available to those of ordinary skill in the art. In alternative embodiments, the topically active pharmaceutical or cosmetic composition may be optionally combined with other ingredients such as 10 moisturizers, cosmetic adjuvants, anti-oxidants, bleaching agents, tyrosinase inhibitors and other known depigmentation agents, surfactants' foaming agents, conditioners, humectants, fragrances, viscosifiers, buffering agents, preservatives, sunscreens and the 15 like. The compositions of this invention may also contain active amounts of retinoids (i.e., compounds that bind to any members of the family of retinoid receptors) , including, for example, tretinoin, retinol, esters of tretinoin and/or retinol and the like. 20 The topically active pharmaceutical or cosmetic composition should be. applied in an amount effective to affect changes in the pigmentation of mammalian skin. As used herein 'amount effective" shall mean an amount sufficient *to cover the region of skin surface .where a 25 change in pigmentation is desired. Preferably, the composition is liberally applied to the skin surface such that, based upon a square cm of skin surface, from about 2 pl /cm 2 to about 200 l11 /cm2 of topically active agent is present when a change in pigmentation 30 - is desired. When using a thrombin and trypsin inhibitor such 'as Compound I or its analogs, .whether synthetically- or naturally-derived in a formulation, such an active compound should be 'present in the amount of from about 0.0001% to about 15% by weight/volume of -20 the composition. More preferably, it should be present in an amount from about 0.0005% to about 5% of the composition; most preferably, it should be present in an amount of from about 0.001 to about 1% of the 5 composition. Of course, these ranges are suggested for the foregoing components.. The lower set of ranges is intended to be efficacious for PAR-2- pathway agonists/antagonists and/or inhibitors having high therapeutic indices and which do not require 10 significantly larger concentrations or doses to be effective in the methods of this invention. Such compounds may be synthetically- or naturally-derived. Liquid derivatives and natural extracts made directly from plants or botanical sources may be 15 employed in the compositions of this invention in a concentration (w/v) from about 1 to about 99%. Fractions of natural extracts and naturally-derived protease inhibitors such as STI may have a different preferred range, from about 0.01% to about 20% and, 20 more preferably, from about 1% to about 10% of the composition. Of course, mixtures of the active agents of this invention may be combined and used -together in the same formulation, or in serial applications of different formulations. 25 We have unexpectedly found that when topically active agents, such as PAR-2 agonists and/or inhibitors and trypsin and/or thrombin and/or tryptase and/or their inhibitors, are topically applied to an animal's skin, a significant-change in pigmentation was 30 achieved. Preferably, depigmenting agents (as well as other pigmentation-affecting agents of this invention) are applied to the. skin of a mammal at a relatively high concentration and dose (from about 0.005% to about 1% for compounds having high therapeutic indices such -21.

as Compound I and related compounds; from about 20% to about 99% for liquid derivatives and extracts of botanical materials; and from about 1% to about 20% for fractions of natural extracts and naturally-derived 5 protease inhibitors such as STI or mixtures thereof) between one and two times daily for a period of time until the skin evidences a change in pigmentation. This may be for from about four to about ten weeks or more. Thereafter, once the change in pigmentation has 10 been achieved, a lower concentration and dose (from about 0.00001% to about 0.005% for compounds having high therapeutic indices such as Compound I and related compounds; from about 10% to about 90% for liquid derivatives and extracts of botanical materials; and 15 from about 0.01% to about 5% for fractions of natural extracts and naturally-derived protease inhibitors such as STI or mixtures thereof), of active ingredient may be applied on a less frequent time schedule, e.g., about once per day to about twice per week. The 20 effects of the active agents of this invention are reversible, therefore, in order to maintain these effects, continuous application or administration should be performed. The invention illustratively disclosed herein suitably may be practiced in the 25 absence of any component, ingredient, or step which is not specifically disclosed herein. Several examples are set forth below to further illustrate the nature of the invention and the manner of carrying it out, but do not serve to limit the scope of 30 the methods and compositions of this invention. Example 1: Protease Inhibitors Affect Pigmentation In order to study the possible roles of the PAR-2 pathway in pigmentation, an in vitro epidermal -22 equivalent system was used. The- epidermal equivalent system used contained melanocytes. One epidermal equivalent system which is - useful in performing this study is the MelanoDerm system, available commercially 5 from MatTek Co. This system contains human normal melanocytes, together with normal, human-derived epidermal keratinocytes, which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. In the following examples, equivalents 10 were treated with test compounds for three days and samples were harvested on the fourth day after beginning of treatment. The harvested equivalents were stainedwith DOPA (a substrate for tyrosinase) and H&E (a standard histological stain) or with Fontana-Mason (F&M) 15 staining, another stain known to those of skill 'in the art. F&M staining is a silver staining technique that clearly and cleanly marks melanins which have high silver nitrate reducing activity. Multilayered human epidermal equivalents containing melanocytes were used 20 as an in vitro model system to study the effect of protease inhibitors on melanogenesis. Epidermal equivalents used were commercially available as MelanoDerm from MatTek of Ashland, MA. These equivalents are known to respond to ultraviolet B 25 ("UvB") irradiation and known whitening agents such as benzaldehyde and hydroquinone by increasing and reducing piginentation, respectively. The MelanoDerm epidermal equivalents were exposed to benzaldehyde (available from Sigma of St. Louis, MO) , hydroquinone (available from 30 Sigma) and UVB' irradiation. tV irradiation was performed with -a UVB FS light source in an exposure chamber, with late covers removed and Phosphate buffered saline (PBS, from Gibco-BRL, Gaithersburg, MD) present in the lower chamber. UVB intensity was -23measured with a UVX radiometer (UVP Inc., San Gabriel, CA) . Equivalents were treated with 0.1-0.12 J/cmZ ' No loss of viability was observed in equivalents treated with up to 0.3 Jcmz 5 -On the fourth day of exposure to the test compounds/ultraviolet irradiation, the equivalents were fixed, sectioned and stained, or stained as whole without sectioning. MelanoDerm equivalents were formalin fixed and put in paraffin blocks, and sections 10 from the MelanoDerm equivalents were stained in accordance with the following standard procedures: (1.) H&E, (2) DOPA + H&E and (3) Fontana-Mason ("F&M") using standard techniques known to those of, skill in the art. Alternatively, whole MelanoDerm equivalents were stained 15 and their images were captured for image analysis. At least three sections per equivalent, three equivalents per experiment were processed. Each experiment was repeated three time. DOPA is a . substrate for tyrosinase. F&M identifies silver nitrate reducing 20 molecules, which identifies primarily melanins. F&M stained sections were used for image analysis using Optomax !mace A nalysis systems, from Optomax Inc., Hollis, NH. Alternatively, Empire Images database 1.1 was used on a: Gateway 2000 25-100 computer (Media 25 Cybernetics, Silver Springs, MD) for capturing images. Image Pro Plus version 4.0 was used for image analysis. Parameters measured were as follows: (1) level of pigmentation within individual melanocytes. and (2) number of pigmented melanocytes per field, for the 30 Optomax system, or (1) the surface area of silver deposits within melanocytes and (2) the number of pigmented melanocytes for the Image Pro system. Using the Optomax system, surface area of silver deposits within individual melanocytes was measured in 60 -24 melanocytes, using multiple sections front triplicate equivalents per treatment. The number of melanocytes per field was calculated in these sections. A "pigmentation factor" was defined as the average surface 5 area of silver deposits within an individual melanocyte, multiplied by the number of pigmented melanocytes per field. A value of one was assigned to untreated controls, and values of treatment groups were normalized to their relevant controls. Using the Image Pro system, 10 surface area of silver nitrate deposits and number of melanocytes were measured for whole equivalents. A value of one was assigned to untreated controls and values of treatment' groups were normalized to their relevant controls. 15 Figure 1A is a graph depicting the increase or decrease in relative pigmentation, as measured and calculated by the whole equivalent/Image Pro system, as set forth above, when exposed to benzaldehyde (500), hydroquinone (500M) and UVB irradiation. (0.12 J/cm'). 20 The human epidermal equivalents were also exposed to mixtures of protease inhibitors, said protease inhibitors are set forth in Table A below. The protease inhibitors were available from Boehringer Mannheim of Indianapolis, IN. CompleteD Protease Inhibitor Cocktail 25 tablets available from Boehringer Mannheim were used, containing inhibitors of chymotrypsin, thermolysin, papain, pronase, pancreatic extract and trypsin. Soybean trypsin inhibitor ("STI") was available from Sigma and was dissolved in a 50 mg/ml liposome vehicle or in lx 30 OBS. All other protease inhibitors used in this in vitro example were dissolved in lxPBS. GDL liposomes were prepared as set forth in Niemic, et al., above, with the exception of the following changes: the non ionic liposomal formulation contained glycerol dilaurate -25 (E.mnlsynt GDL, ISP Van Dyk)/cholesterol (Croda) /polyoxyeth'ylene-10-ste aryl ether (Brij 76, ICI) /polyoxyethylene-9-lauryl ether, as at ratio of 32.5:12.5:33.3:16.7. Hepes buffer, 0.05, pH 7.4 5 (Gibco-BRL of Gaithersburg, MD) was used as the aqueous phase in the preparation of the liposomes. These mixtures of protease inhibitors and different combinations of serine protease inhibitors were tested for their ability to affect melanogenesis. As set forth 10 in Figure 1, some of the serine protease inhibitors, particularly STI (soybean trypsin inhibitor), were very effective in inhibiting melanogenesis. TABTE A Test Ingredients Formulation Complete& Total protease inhibitor mixture - x25 Mix-1 Serine Protease inhibitors - 9.0 pg/mL Phenylmethyl-sulfonyl fluoride ("PMSF") and 50 pg/mL L-1-Chloro-3--[4 tosylamidol-4-henyl-2 butanone ("TPCK") Mix-2 Serine protease inhibitors - 0.1 gg/mL aprotinin, 50 pig/mL Soybean trypsin inhibitor ("STI"), 0.5 ig/mL leupeptin and 0.25 4g/mL (L-1-Chloro-3-(4 tosylamido)-7-amino-2 heptanone-HCl) ("TLCK ") STI Soybean trypsin inhibitor - 1 mQ/Ml 15 -26 Example 2: A Protease-activated Receptor Is Involved _In Pigmentation Example 1- demonstrates that STI reduces 5 pigmentation. STI inhibits trypsin. Because trypsin is known to activate TR and .PAR-2, we tested the possible involvement of TR and PAR-2'in pigmentation. MelanoDerm human epidermal. equivalents were treated with the TR and PAR-2 agonists and antagonists set forth in Table B 10 below daily for three days. On the fourth day, the samples were harvested, fixed, and DOPA, H&Z or F&M staining.was performed. Histological and whole equivalent examination revealed ~changes in pigmentation following the treatments- Figure 2 depicts the results 15 of this example.. As shown therein, the PAR-2 peptide agonist SLIGRL induced pigmentation in ---individual melanocytes. Treatment with Compound I, an inhibitor of thrombin and trypsin, resulted in decreased pigmentation. 20 Figure 3 shows the results of the studi-es set forth in this example, representing the level of pigmentation in MelanoDerm equivalents - treated with TR and EAR-2 reagents. SLIGRL, a PAR-2 agonist, dramatically increased pigmentation, indicating that 25 PAR-2 might be involved in pigmentation. Hirudin, a thrombin-specific inhibitor, and TFLLRNPNDK, a TR selective agonist had no effect on pigmentation. However, SFLLRN, a less specific TR agonists, showed a trend of lightehing or reducing pigmentation. This 30 indicates that TR is less likely- to be involved in pigmentation.

-27 TABLE B m an M-2 um Thromoin Actvates TR Trypzin Actvates TR ana PAR-2 TELLRNPNOK TR peptide aqonist - activates TR only SLIGRL PAR-2 peptide agonist - activates eAR-2 only SFLLRN TR peptite ago.st - activates TR, TR, crosa-reacts with PAR-2 F5LLRN Scrantlea peptiae - inactive Hirudin Specif.ic inhibitor of thcomtun compound I Trcostain and t clpsin intubitoar Example 3: A Dose-response Relation Between Protease 5 activated Receptors Signaling and Melanogenesis MelanoDerm equivalents were treated with increasing concentrations of SLIGRL, the PAR-2 peptide agonist, at 0, 10 and 50 pM in the same manner as set forth in Example 2. F&M staining was performed in the fourth 10 day. As shown in Figure 4A, increasing concentrations of SLIGRL, the PAR-2 activator, result i.n increased pigmentation. Trypsin, a PAR-2 activator, has the same effect. Treatment with increasing. concentrations of Compound I, the thrombin and . trypsin inhibitor,. from 15 0.lpM to 1pM resulted in decreasing pigmentation (see Figure 4A). Pretreatment of the equivalents with UVB irradiation increased melanogenesis, relative to untreated controls. Compound I was able to reduce this UVB-induced pigmentation as well (Fig. 4B).. This 20 example demonstrates a dose-response -relation for increasing .and decreasing pigmentation with - the modulation cf ?AR-2 signaling. This example also demonstrates that Compound I can inhibit pigmentation and prevent UV--induced pigmentation.

-28 Example 4: PAR-2 is Expressed in Keratinocytes, But Not In Malanocytes PAR-2 and TR expression bhave been demonstrated 5 previously in keratinocytes and fibroblasts. This example demonstrates that PAR--2 is expressed in keratinocytes, but not in melanocytes. Furthermore, it demonstrates that TR is expressed in both keratinocytes and melanocytes. In order to demonstrate this, 10 MelanoDerm human epidermal equivalents, human primary melanocyte cultures (neonatal and adult, from Clonetics of San Diego, CA) and Cloudman 591 mouse melanoma cells from ATCC of Rockville, MD were grown in culture and total RNAs were extracted using "PNA Stat-60" reagent 15 available from "Tel-Test B", Incorporated as described in Chomczymski, "Single Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-phenol-chloroform extraction," 162 Anal. Biochem. 156--69 (1987). A sufficient amount of RNase-free Dlase available from 20 Promega Corporation under the tradename "RQl RNase-free DNase" was then added to the extracted RNA from each sample such that each respective product will yield 200ng of DNased-RNA'using the procedure set forth in "RNase-free DNase", protocol published by Promega 25 Corporation (May, 1995) . The resulting 200ng of DNased-RNA was reverse transcribed ("RT") via the procedure set forth in "Superscript II Reverse Transcriptase" a protocol published by Gibco-BRL (now Life Technologies, Incorporated) (April 1992), using 30 random hexamers such as the random primers which are commercially available from Life Technologies, Incorporated. The resulting RT products were then amplified via polymerase chain reaction ("PCR") using about a 0.5 -29 unit (per 100 pl reaction) of a thermostable DNA polymerase which is commercially available from Perkin Elmer-Cetus Corporation under the tradename "Taq 5 polymerase" and about 0.1 imol/reaction of TR and PAR 2 specific primers as described in Table C and in Marthinuss et al., 1995 which is hereby .incorporated herein by reference or of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) primers, available from Clontech 10 Laboratories, Inc. of Palo Alto, CA in accordance with the procedures set forth in Marthinuss et al., 1995 or in the protocol accompanying the primers 'from Clontech Laboratories. The PCR products were then analyzed using 2% 15 agarose/ethidium bromide gels according to methods well-known in the art in order to compare the level of expression of certain genes. in keratinocytes and melanocytes. When necessary for better visualization, the resulting PCR products were precipitated with 20 ethanol according to well-known procedures. When primers for G3PDH were used, only 10% of the PCR reaction products we're used. An RNA sample from epidermal equivalents that was not reverse-transcribed was used as a negative control for'each PCR 25. amplification. The lack of genomic DNA contaminants was indicated by the lack of a band on the relevant lanes in the gels. A human skin RNA sample which was reverse-transcribed was used as a positive control when commercial positive controls were not available. The 30 migration of the RT-PCR products on the gels was always identical to that of the positive controls-, and to that of the reported amplimer sizes.

-30 The relative quality of each respective RT-PCR reaction product was then compared by analyzing the mRNA level of G3PDH, a "housekeeping" gene, in each respective product. As illustrated in FIG. 5 and 6, 5 G3PDH gene expression was found to be similar at all the time points examined, which thereby enabled the comparison of the relative levels of gene expression for the desired genes. Figure SA shows that, as expected, TR and PAR-2. 10 are expressed in total skin and in the MelanoDerm equivalents ("MD") . However, 391 melanoma cells ("S91") did not express PAR-2 or TR. To _investigate this further, we tested primary newborn ("mel-NB") and adult ("mel-A") melanocytes for TR and PAR-2 expression. AS 15' shown in Figure 5B, primary human melanocytes express TR but not PAR--2. Therefore, we suggest that PAR-2 agonists and antagonists can interact with keratinocytes, but not with melanocytes, in the MelanoDerm equivalents, and that TR agonists and 20 antagonists could interact with both keratinocytes and melanocytes. A keratinocyte-melanocyte interaction is, therefore, suggested, during which the keratinocyte-PAR 2 signal is converted into a pigmentation end-point. Table C illustrates some of the DNA primers used, 25 the amount of Mgcl 2 required for the PcR reaction, and the lengt-h of the PCR cycle.

Table C: DNA~ Primers Utilized in RT-PCR Assay Primer Aint1. Cycle No. f DNA (See of Imin) Cyc.les Seq. at-tached MCIS a *c ID No. Sequenee tml4 tLIt~nal Tyuosinase 1..25 3 9435 sens 2 e* 55 TCAG=CAGC 3. .9 '72 ATCCIICTT TY.rOasnase 1.25 1 435 2 antisense 2 0 55 CAGCCATGT 3 (1 '12 'rCAAAAATAC TRP-I sense 2.5 1. a 54 35 S'CCA A 2 e 55 TAAr.CCCAAAC 3 8 72 ThP-I 4.b I @ 94 35 4 antisense 2 a 55 51 CTCAGCCAT 3 e 12 TCATCAAMGAC TRP-2 sense Z..5 1 9 94 35 5'AAAAGACAT 2 e S5 ACGACAT-,aCC 3 e 72 TP22-5 1 e 94 356 A~I6n ~2 0 55 5'CACAPAAAG 3 e -7i IACCAACCAAAG Trypsin 2.5 1 Lk 94 3 sense 2 C! 45 5/A7CCITACT 3 e 9 rCCTGATCC-.7A Tryps.n 1.6 1. d 94 35 antLsense 2 645 5, rTrcATTcGT 3 72 TI CCAGAGTCI CTlGCIGC PAR-Z sense .1.5 0 94 30 9 I. e 55 GGGAAAGGG- 3 72 TCGGGTAkGAA CCAGGCtMC C (511 PA1R-2 .b.5 0594 20 1 antisense 1 3. 5 GCC,C~VCGr 3 e6 12 -32 ATGTrTGCCr? CT1C=GsGa Tht-aenSe - 2.5 .5 a 94 30 U ccrTcTGAGTGC 1 855 CAGGGTAG-' 3 1 ~7 2 TerACAIS TR- 2.5 .5 4 94 30 12 antj~ane - . 655 CcAAGrrAC 3 72 AGMAILI"GTA ATG~rrA CAGG (' Throwbxn- 2.5 .5'0 94 35 13 Sense- 55s AACCTGAAGGA 3 6 72 GACGTGGAC Thronuln- 2.5 .5 9 94 35 1 antizenue - 1 8 55 CAGGAGCCCAG 3 a 12 AAT5G*2 T -33 Example 5: Karatinocyte-Melanocyte Contact is Raquirad for Compound I Depigmenting Effect The results of Example 4 suggest that melanocytes 5 alone might not respond to the depigmenting effect of PAR-2 antagonists. Indeed, the level of pigmentation of human primary melanocytes or choleratoxin-induced 591 cells, which is reduced by hydroquinone and benzaldehyde, was not affected by Compound I. 10 Since PAR-2 is not expressed in melanocytes, we tested the possible requirement of keratinocyte-melanocyte interactions for the depigmenting effect of Compound I. Primary melanccyte cultures were compared to identical cultures plated under epidermal equivalents (EpiDerm, 15 lacking melanocytes) to create a co-culture with no contact between keratinocytes and melanocytes. These were also compared to MelanoDerm equivalents, where melanocytes are present in the basal layer of the equivalent. Cultures were treated for three days with 20 Compound I, with the PAR-2 agonist SLIGRL, and with the T, agonist TFLLRNPNDK, as set forth in Table 0, and DOPA stained on the fourth day. In Table D, keratinocytes are indicated by "K",' melanocytes are indicated by "" and lack of keratinocyte-melanocyte contact is indicated 25 as "no K-M contact". As shown in Table D, no effect on pigmentation was observed in primary melanocytes and in co-cultures treated with these agents. In MelanoDerm equivalents, compound I reduced and SLIGRL induced pigmentation, while TFLLRNPNDK had no effect. These -34 results demonstrate that keratinocyte-melanocyte contact is required for the PAR-2 effect on pigmentation. 5 TABLE D Tzeatmnnn Melanoayees Co-culturen. too K- contact) (K-M taact) compound z no no lighening affect effect SLGRL no no dartening 'TiLLRNPNOK no no noC ettect effect effect 10 Exaple 6: Compound I Affects Melanocyte Gene Expression MelanoDerm equivalents were treated with increasing concentrations of the thrombin and trypsin inhibitor, Compound I, or with increasing concentrations of the 15 PAR-2 agonist SLIGRL. RNAs extracted from untreated and Compound I-treated equivalents were analyzed for gene expression by RT-PCR in the manner set forth above in Example 4. Gene-specific primers were designed as set forth in Table C above, and Clontech primers for human 20 G3PDH were used as .in Example 4. Melanogenic genes tested for expression level were tyrosinase, TRP-1, and TRP-2. A dose-dependent decrease in TRP-l and a dose dependent increase in TRP-2 mRNA levels were observed in 25 Compound I-treated samples, as shown in Figure 6A Tyrosinase expression, however, was not affected. These changes correlated with the. dose-dependent whitening effect of this inhibitor. Both patterns of gene expression result in a lightening effect. TRP-2 enzyme 30 processes dopaquinone to 5, 6-dihydroxyindole carboxylic acid (DHICA) , rather, than to 5.6-dihydroxyindole

(DHI).

-35 This process results in brown, finely dispersed eumelanin, as opposed to insoluble black eumelanin, and results in a lighter skin tone. TRP-1 stabilizes the 5 melanogenic complex, enabling pigment production. Reduced levels of TRP-1 result in reduced tyrosinase activity and reduced pigmentation. Lack of this protein results in albinism. Increasing concentrations of SLIGAL, however, did not affect melanogenic gene 10 expression (Fig. 6B). TRP-1 and TRP-2 are melanocyte-specific. Compound I inhibits trypsin and. thrombin. Hirudin, a specific thrombin .inhibitor, had no effect on' pigmentation, as seen above in Example 2. Thus, we decided to test 15 whether trypsin and thrombin are expressed in skin. A probe designed to detect both brain and gastric trypsins, as described in Table C, detected the expression of both mRNAs in a total skin mRNA sample available from Invitrogen of Carlsbad, CA, as well as in 20 Melanonerm equivalents. The same expression pattern was detected for thrombin. Both trypsin and thrombin were not expressed in normal melanocytes (Figs. 5A', B) These data suggest that if trypsin 'activates PAR-2, it could be produced by the keratinocytes only. As shown in 25 Figure 6A, treatment with Compound I resulted in increased expression of trypsin. SLIGRL, which did rot affect melanogenesis gene expression (Fig. 6B) also increased trypsin expression in the equivalents. We conclude that while trypsin is a possible natural 30 activator of PAR-2 in skin and possibly affects pigmentation, its mRNA levels do not correlate. with pigmentation- This suggests that another, yet unidentified serine protease, which is inhibited by compound I, STI and the like, is the natural activator -36 of PAR-2 in the epidermis. Compounds that induce or inhibit this protease would serve as. darkening and lightening agents; respectively. 5 Example 7: Thrombin and Trypsin Inhibitors and PAR-2 Agonists Affect Pigmentation In Vivo Two guinea pigs were treated twice daily, five days/week for seven weeks with Compound I at I and 10 10 pM in 70:30 ethanol:propylene glycol vehicle on one pigmented nipple. - The other nipple of each animal was treated' with vehicle only and served as a control. Chromameter measurement after seven weeks of treatment revealed a dose-dependent lightening effect of +9.6 L+ 15 and nearly 18 L* units respectively. No visible signs of irritation were observed at that time. - Four groups of three guinea pigs each were treated respectively with Compound I, SFLLRN, FSLLRN and SLIGRL at 10 pM, twice daily five days per week for eight 20 weeks. Chromameter measurement. after six weeks demonstrates a lightening effect by Compound I and a darkening effect by SLIGRL, the PAR-2 agonist. The results of this example are set forth in Figure 7. 25 Example 8: Thl-ombin and Trypsin Inhibitors and PAR-2 Agonists Affect Pigmentation In vivo A Yucatan microswine was treated with Compound I, SFLLRN,- FSLLRN and SLIGRL at 10 pM. Each compound was applied to two sites on the pig twice daily, five days 30 per week for eight weeks-. After eight weeks of treatment; chromamerer measurements were taken. The application of Compound I resulted in a visible lightening effect. The PAR-2 agonist SLIGRL resulted in 37.a darkening effect as measured by chromameter. SFLLRN and FSLLRN had no significant effects. Two Yucatan swine were treated for seven and a 5 half weeks, or for ten weeks, twice daily, five days per week, with increasing concentrations of Compound I. Four concentrations of active compound were used, as follows:- 0, .10, 50 and 2 50pM. Two sites per concentration were placed on opposite sides of the swine 10 dorsum. Chromameter measurements were taken before treatment started and every two weeks thereafter. Pictures were taken periodi-cally and at the end of the experiment. A visible lightening effect was- observed during the 4th, 5th and 6th weeks of treatment, for the 15 250, 50 and 10 pm treatments, respectively. By the eighth week, the whitening effect of the two highest doses was similar. 'These results are illustrated in Figure 8. The chromameter readings (L*, measuring brightness) during the treatment course of one swine are 20 shown in Fig. 9. A saturation effect is observed, which is a time and concentration dependent. This example demonstrates a visual depigmenting ef-ect by Compound I, in. the animal model system most resemble pigmented human skin. 25 At the end of these experiments, biopsies were taken for histological and electron microscopy (EM) analyses. Histological samples were stained with R&E and F&M. H&E staining showed that there was no irritation, inflammatory response or changes in skin 30 architecture, demonstrating the safety of using Compound I over long periods of time- F&M staining demonstrated that there was reduced pigmentation in the treated samples, both in the basal layer and throughout the -38 epidermis. These results are illustrated in Figure. 10. Untreated and vehicle-treated samples (Fig. 10A) were identical and darkest. The 10 pM treatment (Fig. lOB) 5 showed reduced pigmentation and the 50 and 250 pM treatments (Fig. 1OC, 10D, respectively) were the lightest. The results of this example suggest that the maximal whitening effect of Compound I could be achieved 10 with higher concentration over a shorter period of time or with lower concentration over a longer period of time. Thus, at least two difference regimens may be used -to achieve the desired skin whitening results. 15 Example 9: Ultrastructural Studies Demonstrate the Effect of Compound I On the Skin In Vitro and In Vivo Ultrastructural analysis was performed on MelanoDerm equivalents and swine skin sites treated with Compound 20 I. MelanoDerm equivalents treated with Compound I were analyzed for melanosome formation and distribution using electron microscopy. Treated samples contained more. melanosomes, but less mature maelanosomes, i.e., melanosomes which evidence reduced melanin production, 25 within the melanocytes, relative to untreated controls (Fig. 11A, 1IB). Dendrites containing melanosomes were easily identified within treated keratinocytes (Fig. 1IC), but were difficult to find within control keratinocytes. This suggests abnormal melanosome 30 formation and slow or impaired melanosome transfer into keratinocytes in the treated samples. Skin samples from Yucatan swine treated with compound I for -eight weeks, as described in example 8, -39 were also analyzed by electron microscopy. Melanosomes within keratinocytes of treated sites were smaller and less pigmented, compared to controls (Fig.llD, 11E and 5 11F). Moreover, the distribution of melanosomes within the treated * skins was abnormal. Melanosomes were detected mainly at the epidetmal-dermal border, compared to a random distribution in untreated controls (Fig. 11G, 11H) . While we cannot rule out other mechanisms, 10 we suggest that Compound I treated keratinocytes were unable to actively take or receive melanosomes from the presenting dendrites. Example 10: The in vivo depigmenting effect of Compound 15 1 is reversible A Yucatan swine was treated with Compound I, 250 GM, for eight weeks, twice daily, five days a week, on eight sites. All sites showed visible depigmentation by the. end of the treatment period, as set forth- in 20 Figure 12B. For the following four weeks (starting at week nine of the experiment), the color of the treated sites was monizored, and two bioDsies were taken each week from two reactedd sites. Untreated sites were biopsies as well. The depigmenting effect could be 25 visualized at one and two weeks post treatment, and a complete reversal was observed by the forth week. Histological examination of F&M stained skin sections confirmed the repigmentation observed visually (as indicated in Fig. 12) . As early as one week post 30 treatment, repigmentation was demonstrated histologically. The visual observations correlate with the histological demonstration of stratum corneum pigmentation. This example demonstrates that Compound I does not induce a permanent damage to the pigmentation -40 machinery, and its effect is reversible in vivo. Figure 12A shows two histological F&M. stained sections of sites which were not treated with Compound I. Figure 12B shows two histological F&M stained sections of sites 5 which were treated with Compound I for eight weeks. Figure 12C shows sections of sites which were treated for eight weeks with Compound I, one week after treatment was stopped. Figure 12D shows sections of sites which were treated for eight weeks with Compound 10 I, two weeks after treatment was stopped. Figure 12E shows sections of sites which were treated for eight weeks with Compound I, four weeks after treatment was stopped. As indicated in Figure 12E, the sections were fully repigmented four weeks after the end of treatment. 15 Example 11: Preparation of naturally-derived products containing STI Example 1 demonstrates that ,the presence of 20 soybean trypsin inhibitor in any lightening formulation is desirable for its depigmenting activity. Based on analytical testing, it has been determined that soybean milk and. soybean paste are rich sources of soybean trypsin inhibitor. 25 To make soybean paste, soybeans were first soaked in deionized cr purified water for several hours. The soybeans were ground after they were fully hydrated, with the addition of small quantities of water, if needed, to smoothen the paste. To make soybean milk, 30 the same procedure was performed with the addition of more water. (The grinding process allows the soybean milk to be extracted) . After collection, .the soybean .milk was filtered to remove any residual parts. of the bean husk.

-41 Soybean milk, soybean paste and miso were prepared to be used as naturally-derived materials that contain STI and are able to lighten skin color. 5 Example 12: Treatnent With Naturally-Derived aterials that affect the PAR-2 Pathway Induces Depigmntation Two Yucatan swine were treated for eight and ten weeks, twice a day, five days a week, with different 10 soybean- and lima-bean-derived products. These natural products include soybean paste, soybean protein acid hydrolysate, miso, native and boiled soybean milk, and a commercially available extract of soybean (Actiphyte t m of Active Organics,. Dallas Texas), as well as purified 15 STI, and different preparations of trypsin inhibitors from soybeans and limabeans. At seven weeks of treatment, all sites were visually lighter than the surrounding skin, except for the boiled soybean milk and the soybean protein acid hydrolysate treated sites. 20 Histological analysis of biopsies from' the treated sites following F&M staining confirmed the depigmenting effect of the 'ovbean and limabean products. An example of such histological data is given in Fig. 13. The lack of depigmenting activity in the boiled soybean milk and in 25 the soy protein acid hydrolysate is explained by the denaturation or the degradation of the soy proteins in these preparations, respectively. We theorize that the active depigmenting agents in the soybean and limabean products are soybean trypsin - inhibitor (STI) and 30 limabean txypsin inhibitor, respectively. (Example i shows the .depigmenting effect of STI in vitro) . This -example demonstrate that natural extracts containing trypsin inhibitory activity could. be used as whitening agents which affect the PAR-2 pathway.

-42 Example 13-: An STI in liposome formulation can lighten human age spots 5 An individual with three age spots on the. dorsum of their hand was treated for eight weeks, twice a day, with the following: The age spot locateck closest to the arm was treated with placebo, containing 20 mg/ml of 10 liposomes. The middle age spot was not treated. The ' third age spot was treated with STI, 1%, in liposomes (20 mg/ml). GDL liposomes were prepared as set forth in Niemiec, et al., above, with the exception of the 15 following changes: the non-ionic liposomal formulation contained glycerol dilaurate (Emulsynt GDL, ISP Van Dyk) /cholesterol (Croda) /polyoxyethylene--10-stearyl ether'(Brij76, ICI)/polyoxyethylene-9-lauryl ether, as at ratio of 37.5:12.5:33.3:16.7. Hepes buffer, 0.05M, 20 pH 2.4 (Gibco-BRL of Gaithersburg, MD) was used as the aqueous phase in the preparation of the liposomes. UV and visible light digital pictures were taken at time 0, 4 and 8 weeks of treatment. L* (brightness) values were -calculated from the images using Adobe Photoshop. 25 - As shown in figure 14, the age spot treated with - STI became lighter following 8 weeks of treatment. Figure 14 is a composite of four pictures. The left panel is the visible light pictures of the hand, before (upper) and after (lower) 8 weeks of treatment. At 30 this orientation the top age spot is the placebo treated, the middle age spot is untreated, and the lower age spot is the STI-treated. The right panel shows the same hand at the same time points, using UV photography. UV light enables the visualization of 35 pigment deeper in the skin, demonstrating that the STl whitening effect was not superficial- Ligure 14 -43 clearly demonstrates that the STI formulation was able to lighten the lower age-spot. An increase of 15 L* units was. calculated for this STI-treated site, further 5 demonstrating the ability of this treatment to lighten age spots. Example 14: Depigmenting formulations with soybean milk 10 In making the soybean milk, it was discovered that the rich emolliency of the milk would be desirable in a skin -care formulation. Because water is used as the predominant ingredient of any oil-in-water emulsion, and in. many other skin-care formulations we 15 hypothesized that the soymilk could be used to substitute for the deionized water in such formulations. However, we expected that this type of formulation would not be physically stable due to the immiscibility of the oil and water components of the 20 soybean milk. Surprisingly, we found that this substitution of soybean milk for water was physically stable. Formulations utilizing soybean milk should contain between about 11 and about 99% of soybean milk, more preferably from about 80%. to about 95% soybean 25 milk. Preferably, this an similar formulations should include a viscosity builder in an amount from about 0% to about 5% (more preferably, from about .0.1 to about 2%), one or more emollients in an amount up to about 20% and/or emulsifiers in an amount from about 0.1% to 30 about 10% (more preferably from about 3 to about 5%), and, optionally, a spreading agent in an amount from about 0 to about 5% (more preferably from about 1 to about 2%), a preservative, a chelating agent or a humectant. The preservative should be present in an -44 effective amount in order to preserve integrity of the milk and maintain the composition's activity. Sufficient thickener should be present to imnpart body to the formulation without causing it to become so 5 viscous that it would hinder spreadabilit'y, e.g., from about 0 to about 10%, more preferably from about 3 to about 5%. Sunscreen, antioxidants, vitamins other depigmenting agents and other skin care topical ingredients may also be incorporated into the 10 compositions of this invention. A particularly preferred example of a depigmenting formulation substituting' s6ymilk for water is shown in table E below. 15 t soybean mi Vehicle, 84.9% depiamentinet alumizluz tarch viscolCY . 0.75% octenyl builder SUCCInate cyloethcone 3preadinc agent 29 P,. 6- emoXlent/ 3 capric/caprylic ifier ~riolycerides fe eethaol I reservative 0.75% 3ucxosac cocoare mlin/-u 1 ifier NaEDTA chelating aaenC 0 lycerin humeczant 1 2.5%1 polyacrylamde:. thickener 5% isoparaffin, 1aureth-I STI, soybean paste and other trypsin inhibitor - containing natural extracts can be incorporated into 20 such formulations to provide increasing concentrations of the .serine protease inhibitor. Use levels of the added active ingredient can range between 0.01% to 15% in a formulation. Other depigmenting agents, including PAR-2- inhibitors, -tyrosinase inhibitors, hydroquinones, 25 soy products, ascorbic acid .and its derivatives, as -45 well as other ingredients with skin care benefits could also be incorporated into this formulation. Example 15: An Oil-in-water Emulsion depiamenting formulation Two examples of a 'depigmenting formulation with oil in-water emulsion are presented in Table F. A formulation with STI, where STI could be replaced with 10 any naturally-derived serine protease inhibitor, or with any naturally-derived extract or fraction thereof containing serine protease inhibitors, is 'described in column 4 of Table F. A similar formulation with Compound I is presented in column 5 of Table F. 15 Compound I in this composition could be replaced with similar compounds, or with serine protease inhibitors or with any 1AR-2 inhibitor. materials having high therapeutic indices, whether derived synthetically or naturally, as the active ingredient. Suggested ranges 20 for the ingredients in such formulations are also listed in Table F. The deionized water content of these formulations could be replaced-with soybean milk.

-46 Table F CEM Hnam Functon W/W W/W ag a Cetearyl Glucoalde Surfactant 1.4 1.4 0.1~ 2.8 C12-15 Alkyl Surfactant 4.0 4.0 1-6 Bentoate octyi . Emollient 1.0 1.0 0-5 Nvroxystearate Dimethicone. Spreading 1.0 1.0 0-5 A t 'II CyClomethicone Spreading 1.0 1. .0 0-5 Cetvl Alcohok EmaLL tent 2.5 2.5 0-4 Butylated Anti-oxidant 0.1 0.1 0-0.5 Hyaroxytoluene OctyL Sunacreen 6.0 6.0 0-10 Methoxycinnamate Pronyiparaoen Preservative 0.5 0.1 0-0.5 VitamLn C acetate Arti-oxxdant O.S 0.5 0-0.5. Tocoonerol Acetate Anti-tex dant. 0..5- 0.5 -. 0-0.5 Giveerine 44umeectant 3.0 3.0 1 0-20 D-Pathenot Pro--vitamin . .0.5. 0..5 0.

- i Dsootum EDTA Chelator. Z,.L 0..1 0.01 whLtaning aoent MetnytV Paraben Preservartve 0.2 0.2 .0-0.3 Car ooer ThickeneC 0.35 0.35 0-3 0eionizea Water or carrier 7o.35 77.5 50-s0 Soybean Milk Whitening STI or natural Whitening . 1.0 0 0-15 extract Auent Compound L Whitening 0 : 0.25 0-1 Agent 5 To prepare this formulation, the ingredients of the lipid phase were combined and mixed at 85 0 C, and then cooled to 60 0 C. In a separate vessel, the carbopol was slowly added to the water or to the soybean milk. 10 After mixing for ten minutes the rest of the aqueous phase ingredients were added and the mix was'heated to 6.0 0 C. The two phases were then combined, mixed for ten minutes, and cooled to room temperature. Of course, one or more depigmentation agents may. be combined 15 within the same formulation, in this Example and in the following examples and other embodiments of the methods and compositions of this invention.

-47 Examnle 16: Deiarmenting Commosition (Oil-in-Water Emulsion) Two additional examples of an oil-in-water emulsion depigmenting formulation are presented in 5 Table G. A formulation with STI, where STI could be replaced with any naturally-derived serine protease inhibitor, or with any naturally-derived extract or fraction thereof containing serine protease inhibitors, is.described in column 3 of Table G. A similar 10 formulation with Compound I is presented in column 4 of Table G. Compound I in this composition could be replaced with similar compounds or with ser-ine protease inhibitor or with any PAR-2 inhibitor materials having high therapeutic indices, whether derived synthetically 15 or naturally, as the active ingredient. Suggested ranges for the ingredients in such formulations are .also listed in Table G. The deionized water content of these formulations could be replaced with soybean milk. Table G CA Ma'antin4/ Pn' Ltnanot Selv5nt Pcopyiene Solvent 3.D 1-10 HyarxyetnyLce ThicKener 1 .2 ' -3 ILulos9 PoLvme r. - .l~riatestThCr~entC f ! .UUu . C10-30 Alkyl Polymer Acrylate E csoovme - P.nthenoi eco-vitamin . 0.1-3 1oqjU I Humecrant F ragrance Fragrance 5 0-0.5 *shexacer-an .Soteaadnflr Arnent j.0 0.-S Vitamin E Antx-ox ant 1.0 0-2 acetato sodium 'NeutralizCr u.35 0.1-0.5 Glucerine wimectant " . 0-2 0 OCtonL7=a Carrier i 1.5 0 Water or whItening Agentt Zovnean MI I k Comoouna : niteni no Aen 25ng :TI or natural Whitening / 0 20tract Aent 20) -48 To prepare this formulation, the hydroxyethylcellulose was slowly added to the water or to the soybean milk and-stir until completely dissolved. In a separate 5 container the Acrylates/ C10-30 Alkyl Acrylate Crosspolymer was added and stir until completely dissolved. The content of the two.containers was combined and mixed for 20 minutes. Vitamin E acetate was then added and mixed, following by' the addition of 10 .. Isohexadecane and Panthenol (98%) . After mixing for five minutes the STI, or the natural extract, or Compound I were added together with Propylene Glycol, and stirred for 5 minutes. Next, glycerine was added and the formulation was stirred for 20 minutes. 15 Finally, the pH was adjusted with sodium hydroxide to 8 for STI (range is 6-8.5) or to 7 for Compound I (range is 5.5-8.5). Exaxmle 17: Depiamenting Comosition (Water-In-Oil 20 FMU.lsion) An -example of a depigmenting formulatiot with water-in-oil emulsion is presented in Table H. A formulation with STI, where S'I could be replaced with 25 any naturally-derived serine protease inhibitor, or with any naturally-derived extract or fraction thereof containing serine protease inhibitors, is described in column 4 of Table H. A similar formulation with Compound I is presented in column 5 of Table H. 30 Compound I in. this composition could be replaced with similar compounds or with serine protease inhibitor or with any PAIR-2 inhibitor materials having high therapeutic indices, whether derived synthetically or naturally, as the active ingredient. Suggested ranges 35 for the ingredients in such formulations are also -49 To prepare this formulation, the hydroxyethylcellulose was slowly added to the water or to the soybean milk and stir until completely dissolved. In a separate 5 container the Acrylates/ ClO-30 Alkyl Acrylate Crosspolymer was added and stir until completely dissolved. The content of the two containers was combined and mixed for 20 minutes. Vitamin E acetate was then added and mixed, following by the addition of 10 Isohexadecane and Panthenol (98%). After mixing.for five minutes the STI,. or the natural extract, or Compound I were added together with Propylene Glycol, and 'stirred for 5 minutes. Next, glycerine was added and the formulation was stirred for 20 minutes. 15 Finally, the pH was adjusted with sodium hydroxide to 8 for STI (range is 6-8.5) or to 7 for Compound.I (range is 5.5-8.5). Exam=le 17: Depiamenting Comoosition (Water-In-Oil 20 Emulsion) An example of a depigmenting formulation with water-in-oil emulsion is presented in Table H. A formulation with STI, where STI could be replaced with 25 any naturally-derived serine prorease inhibitor, or with any naturally-derived extract or fraction thereof containing serine protease inhibitors, is described in column 4 of Table H. A similar formulation with Compound I is presented in column 5 of Table H. 30 Compound I in this composition could be replaced with similar compounds or with serine protease inhibitor or with any PAR-2 inhibitor materials having high therapeutic indices, whether derived synthetically or naturally, as the active ingredient. Suggested ranges 35 for the ingredients in such formulations are also -50 listed in Table H. The deionized water content of these formulations could be replaced with soybean milk. 5 Table H4 Phase CTFA Name Function I %WJW vW4W Pefd Ranqes 10 OIL I Minerai Oil Emotient 25.0 25.0 40-80 Somitan Sustactant 5.0 5.0 1-6 I Monceaeate . I Steann Alconot Emoitten - 25.0 25.0 I 20-60 I Dimeinicane I Soreaona-Aent I 10 A0 15-5 I Cetvi Alcanot ' Erottent 2.0 2.0 1 0.1-10 Hyarogenateci Anti-ox3ant .0 0-10 iLeein I Persal McX Sunscreen ' 3.0 3.0 04 IProavioaraoen Preservauve 0:5 0.5 1 0.01-0.5 Vitamin E Ant-oicmant 0.5 0.5 0.01-0.5 acetate AQUEO Glycerne . umacrant 3.0 3.0 0-20 Metnii Paraoen Preservative 0.2 0.2 1 0.01-0 3 Watr !Or Say Camer i 30.5 31.55 20-45 20 uilk whierna A 10ent 0 ISTI I Whtennq Acent 1 1 I : 0 )-0 i Cod I ' Whitertma Acent 1 0 i 0.25 0-1 To prepare this formulation the stearyl alcohol and mineral oil were melted at 70*C. The other oil phase 25 ingredients were added and the mixture heated to 75*C. The aqueous phase ingredients, which have been previously dissolved in the bulk phase water or Soy Milk and warmed to 70*C, were then added and the mixture was stirred until it congealed. 30 Example 18: Depigmentation Composition (Aqueous Gel) Two examples of a depigmenting formulation with aqueous gel are presented in Table J. A formulation 35 with STI, where STI could-be replaced with any naturally-derived serine protease inhibitor, or with any naturally-derived extract or fraction thereof containing serine protease inhibitors, is described in column 3 of Table J. A similar formulation with -51- Compound I is presented in column 4 of Table J. Compound I in this composition could be replaced with similar compounds or with serine protease inhibitor oi with any PAR-2 inhibitor materials. having high 5 therapeutic indices, whether derived synthetically or naturally, as the active ingredient. Suggested ranges for the ingredients in such formulations are also listed in Table J. The deionized water content of these formulations could be replaced with soybean milk. 10 TABLE J CTFA Name I Function *AWNW i %WIW I Octoxvnol-13 I Surfactant 0.2 1 0-2 1 0.05-0.5 15 2,4Hexadienotc Preservave 0.1 0.1 0-0.3 Acid Benzenemethan Preservatuve 1.0 1.0 - 0-2 Disoaium EDTA Chetator I 0.05 0.05 0.01-0.2 Preservative Ascormic Acd. Anti-Oxidant 0.1 0.1 1 0-0.2 Sodium. Anti-oxidant 0.2 0.2 0-03' 20 Metabisulrite camomer I Thener 1 .5 1 1.5 i 0-3.0 Na.H %20 Neutralizer 2.45 2.45 0.1-5 DEONtZED Camer i Whitening 93.4 94.15 85-98 Water or Agent Sovbean Milk STI or natural Whrtening Agent 1.0 0 0-15 25 extract Compound I 1 Whitenina Aaent 0 0.25 1 0-1 -52 listed in Table K. The deionized water content of these formulations could be replaced with soybean milk Table K 5 C!7k Mm i Unction %W/W EthanoI 3olvenC I1 70 40-90 PropyLene. Solvent (2) 29 1-40 Glycol Deionizea Carriec q.S. 1-40 Water STI Whtening 0 Coaspuncs wh~tening 100 1 oo To prepare this formulation Compound I was dissolved in water. The et*harol and-propylene glycol were mixed and combined with the aqueous solution 10 containing Compound I. In summary, we have demonstrated that activation of the keratinocyte receptor PAR-2 results in increased pigmentation. Preferably, such activation may be accomplished by the use of. trypsin or SLIGRL or 15 SLIGKVD or other SLIGRL or SLIGKVD derivatives. *We have also demonstrated that whitening may be accomplished by the use of serine protease inhibitors or PAR-2 antagonists, as well as by melanosome-transfer blockers. Other compounds known to those of skill in the art that 20 inhibit. melanosome transfer into keratinocytes could also be used as depigmenting agents. Compound I, - a trypsin and thrombin inhibitor, for example, inhibits melanosome transfer to keratinocytes. STI works by the same mechanism. The accumulation of 25 undelivered melanosomes in the melanocytes could induce a negative feed back , mechanism, that slows new melanosome formation. -The production 'of TRP-1, the major glycoprotein in melanocytes, is down-regulated. which leads to destabilization of tyrosinase. This 30 results in reduced melanin formation, and in a color -53 switch. to a lighter brown, as the ratio of TRP-1:TRP-2 is reduced. The melanosomes accumulation in the melanocyte after Compound I treatment, or after STI treatment, therefore, have reduced and altered melanin 5 content, which adds to the whitening effect of compound I or. STI.

Claims (40)

1. A method of effecting changes in mammalian skin pigmentation comprising administering to a mammal a pigmentation-changing effective amount of a compound which is: 5 (i) a PAR-2 antagonist which binds to or blocks but does not activate PAR-2; or (ii) a PAR-2 agonist which binds to and activates PAR-2.

2. A method according to claim 1 wherein said compound is selected from the group consisting of antagonists based on SLIGRL which bind to or block but do not activate 10 PAR-2, and antagonists based on SLIGKVD which bind to or block but do not activate PAR-2 and mixtures thereof

3. A method according to claim 2 wherein said compound is a protease inhibitor.

4. A method according to claim 2 or claim 3 wherein said compound is a serine protease inhibitor. 15

5. A method according to any one of claims 2 to 4 wherein said compound is a thrombin and/or tryptase and/or trypsin inhibitor.

6. A method according to any one of claims 2 to 5 wherein said compound is a compound of formula I: MH N E A RX R 2 (B)p 20 wherein: A is selected from the group consisting of C 8 alkyl, carboxyClualkyl, - -55 Ci-4alkoxycarbonlyCj-4alkyl, phenylCI- 4 alkyl, substituted phenylCI- 4 alkyl (where the phenyl substituents are independently selected from one or more of, Ci- 4 alkyl, perfluoroCI- 4 alkyl, CI- 4 alkoxy, hydroxy, halo, amido, 5 nitro amino, CI-<alkylamino, C 1 . 4 dialkylamino, carboxy or CI- 4 alkoxycarbonyl), formyl, Ci- 4 alkoxycarbonyl, Ci..-alkylcarbonyl, PhenylCi-alkoxycarbonyl, C3 7 cycloakylcarbonyl, phenylcarbonyl, substituted phenylcarbonyl (where the phenyl substituents are 10 independently selected from one or more of, CI- 4 alkyl, perfluoroCt. 4 alkyl, C.- 4 alkoxy, hydroxy, halo, amido, nitro, amino, CI-salkylamino, CI. 4 dialkylamino, carboxy or CI-4 alkoxycarbonyl), C.4alkylsulfonyl, Ci 4aikoxysulfonyl, perfluoroCi- 4 alkyl-sulfonyl, 15 phenyisulfonyl, substituted phenylsulfonyl (where the phenyl substituents are independently selected from one or more of, Ci- 4 alkl, perfluoroct- 4 alkyl, CI- 4 alkoxy, hydroxy, halo, amid, nitro, amino, C 1 - 4 alkylamino, Ci 4dialkylamino, carboxy or Ci-4 alkoxycarbonyl), 10 20 camphorsulfonyl, phenylC. 4 alkysulfonyl, substituted phenylCt-<alkysulfony1, CL-4alkylsulfinyl, perfluroc.. .ialkylsulfinyl, phenylsulfinyl, substituted phenylsulfinyl (where the phenyl substituents are independently selected from one or more of. Ci- 4 alkyl, 25 perfluoroc.. 4 alkyl, Ci- 4 alkoxy, hydroxy, halo, amido, nitro, amino, CL-4alkylamino, CI-4dialkylamino, carboxy or Ci-4alkoxycarbonyl), phenylCi- 4 alkylsulfinyl, substituted pennylCi..alkylsulfinyl, 1-naphthylsulfonyl, 2-naphthylsulfonyl or substituted naphthylsulfonyl 30 (where the naphthyl substituents are independently selected from one or more of, CI-qalkyl,perfluoroC 4 alkyl, CI- 4 alkoxy, hydroxy, halo, amido, nitro, amino, carboxy or Ct..4alkoxvy-carbonyl), 1-naphthylsulfinyl, 2 naphthylsulfinyl or substituted naphthylsulfinyl (where . . -56 the naphthyl substituents are independently selected from one or more of, C 1 _ 4 alkyl, perfluoroCI. 4 alkyl, C-1. 4 alkoxy, hydroxy, halo, amido, nitro, amino, Cx. 4alkylamino, CLadialkylamino, carboxy or C. 5 4alkoxycarbonyl); a D or L amino acid which is coupled as its carboxy terminus to the nitrogen depicted in formula I and is selected from the group consisting of alanine, asparagine, 2-azetidinecarboxylic acid, glycine, N-C.. 10 ealkyglycine, proline, 1-amino-!-cycloC 3 .. salkylcarboxylic acid, thiazzolidine-4-carboxylic acid, 5,5-dimethylthiazdlidine-4-carboxylic acid, oxadolidine-4-carboxylic acid, pipecolinic acid, valine, methionine, cysteine, serine, threonine, 15 norleucine, leucine, tert-leucine, isoleucine, phenylalanine, 1-naphthalanine, 2-naphthalamine, 2 thienylalanine, 3-thienylalanine, (1,2,3,4} tetrahydroisoquinoline- 1-carboxylic acid and 1,2,3,4,1-tetrahydroisoquinoline-2-caroboxylic acid 20 where the amino terminus of said amino acid is connected to a member selected form the group consisting of C- 4 alkyl,tetrazol-Syl-C,-zalkyl, carboxytCi-alkyl, CialkoxycarbonylC.4alkyl, phenylCj_ 4 alkyl, substituted phenyl C 1 4 alkyl (where the phenyl 25 substituents are independently selected from'one or more of, CI.. 4 alkyl, perfluoroc 1 4 alkyl, CI 4 alkoxy, hydroxy, halo, amido, nitro, amino, CI- 4 alkylamino, Ci 4 dialkylamino, carboy or CI 4 alkoxycarbonyl), 1,1 diphenylC.. 4 alkyl, 3-phenyl-2-hydroxypropionyl, 2,2 30 diphenyl- 1-hydroxyethylcarbonyl, (1,2,3,43 tetrahydroisoauinoline- 1-carbonyl, [1,2,3,41 tetrahydroisoauinoline-3,carbonyl, 1-methvlamino-1 cyclohexanecarbonyl, 1-hydroxy-1-cyclohexanecarbonvl, 1-hydroxy-1-pheny-lacetyl, 1-cyclohexyl-l- -57 hydroxyacetyl, 2-phenyl-2-hydroxypropionyl, 3, 3 diphenyl-2-.hycroxypropionyl, 3-cyclohexyl-2 hydroxypropionyl, fonnyl, CI-4akoxycarbonyl, CI1..2alkylcarbonyl, perfluoroC, 5 4 alkyl, CI.4alkylcarbonyl, phenylCi- 4 alkylcarbonl, substituted phenylCI- 4 alkylcarbanyl (where the phenyl substitu~ents are independently selected from one or more of, Ct.. 4 alkyl, perfluoroCX-alkyl, CI- 4 alkoxy, hydroxy, halo* amido, nitro amino, CI..4alkylamino, Cl.. 10 4dialkylamilo, carboxy or Cx. 4 alkoxycarbonyl) 1,1 diphenylCl- 4 alkylcarbonyl, substituted 1, l-diphenylC 1 ,alkylcarbonyl (where the phenyl substituents are independently selected from one or more of, CI- 4 alkyl, perfiuoro CL-.alkyl, CI..4alkoxy, hydroxy, halo, amido, 15 nitto, amino, Ct.. 4 alkylamino, Cl14dialkylamino, carboxy or CI- 4 alkoxy-carbonyl), perfluoroCt- 4 alkysulfonyl, Cj 4alkysulfonyl, CI.4alkoxysulfoxyl, phenysulfonyl, substituted phenylsulfonyl (where the phenyl substituents are Lndependently selected from one or 20 more of, C-lalkyl, perfiucro CI-4alkylamino, C 1 4dialkylamjno, carboxy or CI.4alkoxycarbonyi), 10 cxamphorsuifonyl, phenylCI- 4 alkylsulfonyl, substituted phenylCI- 4 alkylsufonyi, perfluroCI-4alkysulfinyI, C l4alkysuifinyi, phenylsulf-,nyl, substituted 25 phenysulfjinyl (where the phenyl substituents are independently selected from one or more of, CI- 4 alkyl, perfluoro C,-,alkyl, CI- 4 alkoxy, hydroxy, halo, antido, nitro, amino, CI- 4 alkylamino, CI- dialkylamino, carboxy or CL-4 alkoxycarbonyl), l-nachthysulfonl, 2 30 naohthylsulfonyIl substituted naphthylsulronyl (where the naohthyl substituents are independently selected from one or niore Of, Ct. 4 alkyl, perfluroC 1 - 4 alkyl, C 1 .. ,aikoxy, hydroxy, halo, amido, nitro, amino, Cj 4 alkyl.amino, CI..4dialkvlamino, carboy or C 1 .. -58 <alkoxycarbnyl),1-naphthysulfinyl, 2-naphthysulfinyl, and substituted naphthylsulfinyl (where the naphthyl substituents are independently selected from one or more of, CI- 4 alkyl, perfluoroci 4 alkyl, C.. 4 alkoxy, 5 hydroxy, halo amido, nitro, amino, CL- 4 alkylamino, C104dialkylamono, carboxy or C-14alkoxycarbonyl): or a poly peptide comprised of two amino acids, where the first amino acid is a D or L amino acid, bound via its carboxy terminus tot he nitrogen depicted 10 in Formula I and is selected from the group consisting of glycine, N-Cl-ealkylglycine, alanine, 2 -azetidinecarboxylic acid, praline, thiazolidine-4 carboxylic acid, 5.5-dimethylthiazolidine-4-carboxylic acid, oxazolidine-4-carboxylic acid, 1-amino-1-cycloC3-a 15 alkylcarboxylic acid, 3-hydroxypropoline, 4 hydroxyproline, 3- (CI- 4 alkoxy) praline, 4(CI 4alkoxy)proline, 3,4-dehydroprline, 2,2-dimethyl-4 thiazolidine carboxylic acid, 2.2-dimethyl-4 oxadolidine carboxylic acid, pipecolinic acid, valine, 20 methionine, cysteine, asparagine, serine, threonine, leucine, tert-leucine, isoleucine, phenylalanine, 1 naphthalanine, 2-naphthalanine, 2-thienylalanine, 3 thienylalnine, (1,2,3,41-tetrahydroisoquinoline- 2 carboxylic acid, aspartic acid-4-Cj. 4 alkyl ester and 25 glutamic acid 5-CI- 4 alkyl ester and the second D or L amino acid, is bound to the amino terminus of said first amino acid, and is selected from the group consisting of phenylalanine, 4 benzolyphenylalanine, 4-carboxyphenylalanine, 4 30 (Carboxy Cl-2alkyl)phenylalanine, substituted phen-lalanine (where the phenyl substituents are inde. adently selected from one or more of C 1 - 4 alkyl, perfluoroCi- 4 alkyl, CIaalkoxy, hydroxy, halo, amido, nitro, amino, CI-.alkylamino, CI 4 dialkvlamino, carboxy -59 or Cl.4alkoxycarbonyl), 3-benzothienylalanine, 4 biphenylalanine, homophenylalanine, octahydroindole-2 carboxylic acid, 2-pyridylalanine, 3-pyridylalanine, 4 thiazolyalanine, 2-thienylalanie, 3- (3 5 benzothienyl)alanine, 3 -thienylalanine, tryptophan, tyrosine, asparagine, 3-tri C...alkylsilylalanine, cyclohexylglycine, diphenylglycine, phenylglycine, methionine sulfoxide, methionine sulfone, 2, 2 -dicyclohexylaianine, 2-(1 10 naphthylalanine), 2 -( 2 -naphthylalanine), phenyl substituted phenylalanine (where the substituents are selected from C 1 . 4 alkyl, perfluoroC.. 4 alkyl, C.. 4 alkoxy, hydroxy, halo, amido, nitro, amino, CI- 4 alkylamino, Ci 4dialylamino, carboxy or Ci- 4 alkoxycarbonyl), aspartic 15 acid, aspartic adid-4c,.alkyl, perfluorocl- 4 alkyl, C.. 4alkoxy, hydroxy, halo, amid, nitro, amino, C 1 4alkylamino, Ci- 4 dialkylaminc, carboxy or C. 4alkoxycarbony), aspartic acid, aspartic acid-4-Cl 4 alkyl ester glutamic acid, glutamic acid-5-CI- 4 alkyl 20 ester, cycloC 3 -salkylaalanine, substituted cycIoC 3 salkylalanine (where the ring substituents are carboxy, C-4-alkyl ester, cycloC 3 -salkylalanine, substituted cycloC.qalkylalanine (where the ring substituents are carboxy, CI.4alkylcarboxy, C 1 - 4 alkoxycarbonyl or 25 aminocarbonyI), 2 , 2 -diphenylalanine and all alpha-CI salkyl of all amino acid derivatives thereof, where the amino terminus of said second amino acid is unsubstituted or monosubstituted with a member of the group consisting of formyl, Cl-12 alkyl, tetrazol-5 30 ylCl-2alkyl, carboxyCl-8 alkyl, carboalkoxyC 1 -alkyl, phenyL Ci 4 alkyl, substituted phenylc. 4 alkyl (where the phenyl substituents or independently selected from- one or more of, C. 4 alkyl, perfluoroC. 4 alkyl, Ci- 4 alkoxy, hydroxy, halo, amid, nitro, amino, C. 4 alkylamino, Cl. 4 dialkylamino, carboxy Or CL- 4 alkoxycarbonyi), 1, 1 diIpebnylC 1 - 4 alkyl, CI-6alkoxycarbonyl, pheny2.Cl 6alkoxycarbonyl, Cl-2alkylcarbonyl, perfluoroC. 4 alkylCo-4alkylcarbonyl, phenyC 1 4 ,alcylcarbonyl, 5 substituted phenyCL- 4 alkylcarbonyl (where the phenyl substituents are independently selected from one or More of*CI. 4 ai-kyl, perfluoro C; 1 . 4 alkyl, CI-4 alkoxy, hydroxy, halo, amido, nitro, amino, Cl-. 4 alkylamino, Cj 4dia3.kylamino, carboxy or C 1 . 4 alkoxycarbony.), 10 diphenylC.. 4 alkyl, perfluoroC.. 4 alkyl, Ci.. 4 alkoxycarbonyl), lO-camphorsulfonyl, phenylC 1 4 alkysulfonyl, substituted phenyC.. 4 alky].sulfonyl, Cj.. 4 alkysulfinyl, perfiucro. C 1 . 4 alkylsulfinyl, phenylsulfinyl, substituted phenylsulfinyl (where the 15 phenyl. substituents are independently selected from one or more of, CI- 4 alkyl, perfluoroC.. 4 alkyl, C 1 - 4 alkoxy, hydroxy, halo, amido, nitro, amino, CI. 4 alkylaimono, C 1 .. 4 dialkylamono, carboxy or Cl-,alkoxycarbonyl'. phenyC 1 4 aikylsulfinyl, substituted phenylC 1 - 4 alkylsulfinyl I 20 naphthylsulfonyl, 2-naphthylsulfonyl, substituted naphthylsulfonyi (where the naphthyl substituent is selected from C.-.alkyl, perfluorOCI- 4 alkyl, C 1 . 4 alkoxy, hydroxy, halo amido, nitro, amino, Ci- 4 alkylamino, Ct 4 dialkylamino, carboxy or Cl-,alkoxycarbonyll , 1 25 haphthyl-sulfinyl, 2-haphthylsulfinyl and substituted naphthyl-sulfinyl (where the naphthy. substituent is selected from CI. 4 alkyl, perfluaroC 1 4 ,alkyl, C 1 - 4 a3.koxy, hydroxy, halo, amido, nitro, amino, CI. 4 alkylautina, C 14dialkylamino, carboxy CC CI 4 alkoxycarbonyl); R, is 30 selected from the group consisting of hydrogen and alkyl; Rz is selected from the group consisting of arninoC2 salkyl, guanidiaoC?-al, CI- 4 a.LkvlguanidinoCz ',alkyl, diC 4 alkeyliouanidinoC-alkyl, amidinoC 2 -5alkyl, CI-* 4alky-IamidinioCzalkyl, diCI- 4 alky-lamidinoc 2 5 -alkyl, Ci.. 3 alkoxyC 2 ... alkyl, phenyl, substituted phenyl. (where the substituents are independently selected from one or more of amino, amidino, guanidino, C 1 .. alkylamino, CL-. 5 4 dialkylamino, halogen, perfluoro C 1 I 4 alkyl,* C 1 - 4 alkcyl, CI- 3 alkoxy or nitro), benzyl, phenyl substituted benzyl (where the substituents are independently selected from one or more of, ami-no, amidino, guanidino, C 1 -. 4 alkylamino, C 1 - 4 dialky- amino, halogen, perfluoro Ci.. 10 4 alkyl, C).-O4alkyi, CL-3alkoxy or nitro), hydroxyCz 5 alkl, C. Lsalky].aminoCz_5alkyl, Cj_5dialkylaminoC2- 5 alkyl 1 4 -amninocyclohexy1.C~raalkyl and C_alkyl.; *p is 0 or 1.: B-is 200 where n is 0-3, R, is F or CI.-Salkyl and the carbonyl moiety of B is bound to E; E is a-heterocycle selected from the group consisting 25 of oxazolin-2-yi, oxazol-2-yi, thiazol-2-yl, thiazol-5 yl, thiazol-4-yi, thiazolin-2-yl, imidazol-2-yl, 4-oxo 2-quinoxalin-2y1, Z-pyridyl, 3-pyridyl, benzo(bjthiophen-2-71, triazol-4--yl triazol-6-yl, pyrazo1-2-y., 41,5, 6, 7 -tetrahydrobe-nzothi-azol-2y1, 30 naph.thoL2-, 1-dlthiaz-7l-2-yl, naphtho(1-2-djthiazo--2-y1 cqulnoxalin- 2 -yl, isoquinolin-1-yl, isoquinolin-3-yl, benzo [bi furan-2-yl, [pyrazin.-2-yl, qruinazolin-2-yl, isothiazoi-5-yI, Lsothiazol-2-Vl, purin-Ryul and a substituted het~erocycle where the substituents are - 62 selected from Ci 4 from C-14alkyl, perfluoro Ci 4 alkyl, Ci 4 alkoxy, hydroxy, halo, amido, nitro, amino, Ci 4 alkylamino, C 14 dialkylamino, carboxy, Ci 4 alkoxycarbonyl, hydroxy or phenylCI 4 alkoxycarbonyl, hydroxy or phenylC 1Aalkylaminocarbonyl; or a pharmaceutically acceptable salt thereof 5

7. A method according to claim 6 wherein said compound contains a d phenylalanine-proline-arginine sequence.

8. A method according to claim 7 wherein said compound is (S)-N-Methyl-D phneylalanyl-N-[4-[(amino iminomethyl)amino]- I -(2-benzothiazolylcarbonyl)butyl)-L prolinamide. 10

9. A method according to any one of claims I to 3 wherein said compound is derived from one or more of the botanical families leguminosae, solanaceae, gramineae and cucurbitaceae.

10. A method according to claim 9 wherein said compound is derived from legumes.

11. A method according to claim 9 or 10 wherein said compound is selected from the 15 group consisting of: undenatured soybean extract, limabean extract, blackbean extract or mixtures thereof

12. A method according to claim I1 wherein said compound is selected from the group consisting of: fractions of undenatured soybean extract, limabean extract, blackbean extract and mixtures thereof 20

13. A method according to claim 10 wherein said compound is selected from the group consisting of soybean milk, limabean milk, blackbean milk, soybean extract, limabean extract, blackbean extract, soybean paste, limabean paste and blackbean paste and mixtures thereof

14. A method according to claim 1, wherein said compound is selected from the 25 group consisting of SLIGRL, SLIGKVD and derivatives of SLIGRL and SLIGKVD which bind to and activate PAR-2 and mixtures thereof

15. A method according to claim 14, wherein said compound is a protease which activates PAR-2. - 63

16. A method according to claim 14 or claim 15, wherein said compound is a serine protease which activates PAR-2.

17. A method according to claim 16, wherein said compound is selected from the group consisting of trypsin, tryptase, thrombin and proteases naturally-occurring in the 5 skin which activate PAR-2.

18. A method according to claim 1, wherein said compound is a melanosome transfer enhancer.

19. A method according to claim 1, wherein said compound is administered twice daily for at least eight weeks. 10

20. A method according to claim 19, wherein said compound is administered at a relatively high dosage for at least about four to about ten weeks and then applied at a relatively lower dosage on a continuous basis to maintain skin lightening effect.

21. A method according to any one of the preceding claims, wherein said compound is administered orally. 15

22. A method according to any one of claims 1 to 20, wherein said compound is administered parenterally.

23. A method according to any one of claims 1 to 20, wherein said compound is administered topically.

24. A composition when used to affect changes in mammalian skin pigmentation 20 comprising a pigmentation-changing effective amount of a compound as defined in any one of claims 1 to 23.

25. A composition according to claim 24 wherein said PAR-2 affecting compound is present in an amount of from about 0.0001% to about 15% by weight/volume of said composition. 25

26. A composition according to claim 25 wherein said compound is present in an amount from about 0.001 to about 5% of said composition. - 64

27. A composition according to claim 26 wherein said compound is present in an amount from about 0.005 to about 1% of said composition.

28. A composition according to claim 24 comprising bean milk in an amount of from about 1 to about 99% by weight. 5

29. A composition according to claim 24 comprising soybean trypsin inhibitor, limabean trypsin inhibitor or blackbean trypsin inhibitor in an amount of from about 0.01 to about 20% by weight.

30. A composition according to any one of claims 24 to 29 further comprising a cosmetically-acceptable vehicle. 10

31. A composition according to any one of claims 24 to 30, wherein said composition further comprises additional depigmenting agents.

32. A composition according to any one of claims 24 to 31, wherein said composition further comprises tyrosinase inhibitors.

33. A composition according to any one of claims 24 to 32, wherein said 15 composition further comprises liposomes.

34. A composition according to claim 33, wherein said composition comprises glycerol dilaurate, cholesterol, polyoxyethylene- 10-stearyl ether and polyoxyethylene-9 lauryl ether.

35. A composition according to any one of claims 24 to 34, wherein said 20 composition further comprises anti-oxidants.

36. A composition according to any one of claims 24 to 35, wherein said composition further comprises a sunscreen.

37. A composition according to claim 24 comprising a compound as defined in claim 13, wherein said composition comprises from about I to about 99% bean milk, from 25 about 0.1 to about 20% emulsifier and a preservative in an effective amount.

38. A composition according to claim 24, wherein said composition further comprises a compound selected from the group consisting of: anti-oxidants, sunscreens, - 65 moisturisers, bleaching agents, depigmentation agents, surfactants, foaming agents, conditioners, humectants, fragrances, viscosifiers, buffering agents, preservatives and a mixture thereof

39. Use of a compound which is: 5 (iii) a PAR-2 antagonist which binds to or blocks but does not activate PAR-2; or (iv) a PAR-2 agonist which binds to and activates PAR-2 in the manufacture of a medicament for affecting changes in the mammalian skin pigmentation. 10

40. A method according to claim 1; a composition according to claim 24; or use according to claim 39, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.