AU2006252275A1 - Methods for treating skin pigmentation - Google Patents

Description

-1- 0 o AUSTRALIA SPATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT S ORIGINAL

(N

Name of Applicant/s: Johnson Johnson Consumer Companies, Inc.

Actual Inventor/s: Stanley S Shapiro and Susan Niemiec and John Kung and Miri Seiberg Address for Service is: SHELSTON IP 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. 2002313834 dated 05 Dec 2002 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 37491AUP01 501054366 1.DOC/5844 la- O METHODS FOR TREATING SKIN PIGMENTATION The present application is a divisional application of Australian Application No.2002313834, which is incorporated in its entirety herein by reference.

1. Field of the Invention This invention is related to methods and compositions for bringing about skin t pigmentation and/or for causing skin depigmentation. More particularly, this invention

I\N

0 relates to compounds which affect melanogenesis and can be used as depigmenting agents or as agents for darkening skin.

2. Background of the Invention Skin coloring has been of concern to human beings for many years. In particular, 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 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.

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, ID precise application of all these compounds is necessary in order to achieve the desired result. Many of these Ci compounds have been found to be quite irritating to the skin and therefore undesirable for use.

The understanding of the chemical and enzymatic Cq basis of melanogenesis is heavily documented.

Melanocytes nigrate from-the embryonal neural crest into V- the skin to produce secretory granules, melanosomes, which produce melanin. Melanogenesis occurs within the C 10 melanosome, and the melanin is later distributed to CI keratinocytes via the melanocyte dendrites. The key enzyme in melanogenesis is tyrosinase, which initiates a CA cascade of reactions which convert tyrosine to the biopolymer melanin. Two tyrosinase-related proteins (TRP's) are known, TRP-l and TRP-2. These proteins share, with tyrosinase about 40% homology and have catalytic activities as well as regulatory roles in melanogenesl.s. TRP-l is the most abundant glycoprotein in melanocytes.

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 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 nvolved in the regulation of melanogenesis.

The beta isoform of the Protein Kinase C (PKC) family has been ;hown to regulate human melonogenesis through tyrosinase activation. Gene expression of tyrosinase, TRP-l and TRP-2 is coordinated. All three enyzmes are -3- O expressed in human epidermis. In melanocytes co- Scultured with keratinocytes, these transcripts are Cl expressed at a ratio of 45:45:10, respectively. In melanocytes cultured alone, only TRP-1 transcripts are Spresent, indicating that a keratinocyte-derived signal is involved in the coordinate expression of these genes.

C The regulation of keratinocyte-melanocyte interactions CI and the mechanism of melanosome transfer into O 10 keratinocytes are not yet understood.

CI 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-1, and PAR-3) in its sequence. Both receptors are activated proteolytically by an arginineserine 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.

Only PAR-2 is activated by mast cell tryptase. Both receptors could also be activated by the peptides that correspond to their new N-termini, independent of receptor cleavage. SLIGRL, the mouse PAR-2 activating peptide, is equipotent in the activation of the human receptor. While the function of the TR is well documented, the biology of the PAR-2 has not yet been fully identified. A role for PAR-2 activation in the inhibition of keratinocyte growth and differentiation has' been recently described (Derian et al., "Differential Regulation of Human Keratinocyte Growth and Differentiation by a Novel Family of Proteaseactivate Receptors", Cell Growth Differentiation, Vol.

8, pp. 743-749, July 1997).

-4- Summary of the Invention SIn accordance with this invention, we have found a method for affecting changes in mammalian skin pigmentation comprising topically applying to the skin iV of a mamma.. a compound which affects the PAR-2 pathway.

C- The compositions of this invention may contain one or n more compounds that act as trypsin, as tryptase, as IO serine protease or as PAR-2 agonists, for increase in 0 10 pigmentation. Alternatively, they may contain one or c-i 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 "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 (1986), and includes but is not limited to humans. As used herein, "receptor" shall include both intracell llar and extracellular receptors and shall mean those molecules capable of receiving and transducing a signal. The term PAR-2 refers to the protease-activated receptor-2 or a related protease 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, is a 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 melanocyres, via 0

C)

(U the PAR-2 pathway, affect melanogenesis. We have found that thrombin inhibitors, and/or tryptase inhibitors, C- and/or trypsin inhibitors and PAROD antagonists can be used as depigmenting agents without irritation of the Sskin. PAR-2 agonists and serine proteases such as trypsin and tryptase can be used as darkening agents.

l r Furthermore, PAR-2 could be useful as a target for ND whitening and darkening agents.

Brief Description of the Drawings Fig. 1A is a graph depicting the increase or decrease .n relative pigmentation of epidermal equivalents containing melanocytes treated with known pigmenting and depigmenting agents in accordance with the methods of this invention.

Fig. 1B 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.

Fig. 2 is a group of images of epidermal equivalents containing melanocytes treated with ?AR-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 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 melanocytes after Q- -6- Q exposure to ultraviolet light followed by treatment with Cq compositions of this invention.

Fig. EA is a photograph depicting gels showing the expression of TR and PAR-2 in skin, melanoma cells and e- epidermal equivalents containing melanocytes.

n Fig. 5B is a photograph depicting gels showing the expression of TR and PAR-2 by primary human melanocytes.

SFig. 6A and 6B are photographs depicting gels (C 10 showing the expression of various genes after treatment with different concentrations 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.

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 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 CL;pound I in accordance with methods of this invention at concentrations of- 0, 10 JM, 50 pM and 250 PM respectively.

Figs. 11A, 11B and 11C are photographs of electron micrograpnic views of epidermal equivalents containing melanocytes treated with compositions of this invention.

Figs. 1LE, 11F and I1H are photographs of electron micrographic views of Yucatan Swine skin treated with compositions of this invention.

c( Figs. 11D and 11G are photographs of electron U micrographic views of untreated sites of Yucatan Swine Sskin.

Figs. 12A, 12B, 12C, 12D and 12E are photographs of histolocrical F&M stained sections of Yucatan Swine I skin, as follows: 12A shows untreated skin; 12B shows (e skin treated with compositions of this invention after nq eight weeks of treatment; 12C shows skin one week after \O stopping treatment; 12D shows skin two weeks after stopping t:.eatment 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 wi-h compositions of this invention.

Fig. 14 contains ultraviolet and visible light digital photographs of human skin prior to treatment and subsequent to treatment with compositions of this invention.

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 decrease pigmentation in mammalian skin. In our opinion, some of the compounds described in U.S. Patent No. 5,52:;,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 invention. Some of these compounds are also described in Costanzo, et al., "Potent Thrombin Inhibitors That Probe the Si' Subsite: Tripeptide Transition State Analogues; Based on a Heterocycle-Activated Carbonyl -a- (1)Group", J. Med. Che., 1996, Vol. 39, pp. 3039-3043 and have the following structural formula: c-I A

R

2 p IND wherein: A iz selected from the group consisting of C..

ealkyl, carboxyCi...

4 alkyl, C1.4alkoxyc:arbonylCI- 4 alkyl, phenylCI- 4 alkyl, substituted phenylCi..

4 a.lkyl (where the phenyl substituents are independerLtly selected from one or more of, C 1 4 alkyl, perf luoroC:1i 4 alkyl, Cl-.4alkoxy, hydroxy, hl, amido, nitro am-iho, C .4alkylamjno, C1.4dialky2.mino, carboxy or CI- 4 alkoxycarbonyl), formyl, CI...4alkoxycarbonyl, CI-.2alkylcirbonyl, phenylCk-4alkoxycarbonyl, C3- 7 CYcloaky:'Lcarbonyl, phenylcarbonyl, substituted phenylcarbonyj. (where the phenyl substituents are independently selected from one or more of, Cl 1 4 alkyl, perfluoro:1 4 alkyl, CI-4.al koxy, hydr-oxy, hlo amido, nitro, arrino, C1-4alkylamino, C 14dialkylamino, carboxy or CI..

4 alkoxycarbonyl.) C14alkylsulfonl,

C

1 4alkoxysu.'ifonyl, perfluoroCl 4 alkyl-sulfonyl, Phenylsulfonyl, substituted phenlsI fonyl (hr h phenyl substituents are independently selected from one or more of, C 1 4 alkl, perfluoroCI- 4 alkyl, C1..

4 alkoxy, hydroxy, halo, amido, nitro, amino,

CI-.

4 alkylamino,

C

1 4dialkylaiuntflO, carboxy or, C 1 4 alkoxycarbonyl), camphorsulfonyi, phenylCl1 4 alkysulfonyl, substituted phenylc 1 4 alkysul fonyl, Cl-4alkylsulfinl, perfluroC..

-9- U 4alkylsu].finyl, phenylsulfinyl, substituted.

phenylsulfiiyl (where the phenyl substituents are Cl independently selected from one or more of C 1 4 alkyl, perfluoroC,..

4 alkyl, C 1 4 alkoxy, hydroxy, halo, amido, nitro, amino, Ci.

4 alkylamino, Cl.4dialkylamino, carboxy or CI.

4 alkoyxycarbonyl), phenylCi-..alkylsulfinyl, substituted pehnylCI..

4 alkylsulfinyl, l-naphthylsulfonyl, CI 2-naphthyl!;ulfonyl or substituted naphthylsulfonyl 0 10 (where the naphthyl substituents are independently CI selected f::om one or more of, CI-4alkyl,perfluoraC 1 4 alkyl, Cj_..alkoxy, hydroxy, halo, amido, nitro, amino, carboxy or Cl-4alkoxyy-carbonyl), l-naptithylsulfinyl, 2naphthylsulfinyl or substituted naphthylsulfinyl (where the naphthyl substituents are independently selected from one cr more of, C 1 4 alkyl, perfluorocl- 4 alkyl, Ct..

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

4alkylamino, CI-4dialkylamino, carboxy or Ci..

4alkoxycarbonyl); a D or L amino acid which is coupled a~s its carboxy terminus the nitrogen depicted in formula I and is selected Erom the group consisting of alanine, asparagina, 2-azetidinecarboxylic acid, glycine, N-C..

nalkyglyc:'Lne,. praline, 1- amino- 1-cycloC3ealkylcarb)oxylic acid, thiazzolidine-4-carboxylic acid, S-dimethylthiazolidine-4-carboxylic acid, oxadolidine-4-carboxylic acid, pipecolinic acid, valine, rciethionine, cysteine, serine, threonine, norleUcirLe, leucine, tert-leucine, isoleucine, phenylalzinirte, l-naphthalanine, 2-naphthalamin6, 2thienyla:lanine, 3-thienylalanine, tetrahyd::oisoau-4noli ne- 2-carboxylic acid and 1, 2, 3,4, 1 -tta-dosqio~n--aooyi acid IND CIwhere the amino terminus of said amino acid is U connected tc A member selected from the group consisting Of Cl 1 4 alkyl, tetrazol-5:y-C 1 2 alky1, carboxytCl-4Elkyl, Cl-4alkoxycarbonylC1 4alkyl, phenylC..

4 a]ikyl, substituted phenyl C 1 4 alkyl (where the phenyl substituents are independently selected from one or CImore of, CI- 4 alkyl, perfluoroC 1 4 alkyl, C 1 4 alkoxy, hycdroxy, halo, amido, nitro, amino, CI- 4 alkylamino, Ci..

IND

4 dialkylaniino, carboy or CL.4alkoxycarbonyl), lidiphenylC 1 4 alkyl, 3 -phenyl--2-hydroxypropionyl, 2,2diphenyl- 1-hydroxyethylcarbonYl, (1,2,3,41 tetrahydroisoquinoline- 1-carbonyl, [1,2,3,41 tetrahydroisoquinoline-3, carbonyl, 1-mdthylamino-lcyclohexanecarbonyl, l-hydroxy-1-cyclohexanecarbonyl, 1-hydroxy-:.-pheny-lacetyl, 1-cyclohexyl-lhydroxyace:yl, 3 -phenyl-2-hydroxypropionyl, 3,3diphenyl-2--hydroxypropionyl, 3 -cyclohexyl-2hydroxyprooionyl, formyl, CI.4alkoxycarbonyl, Cl-lzalkylcarbonyl, perfluoroCI- 4 alky1, CI...alkylcarbonyl, phenYlCI 4 alkylcarbonl, substituted phenylC 1 4 alkylcarbonyl (where the phenyl substituents are independently selected from one or more of, Ci..

4 alkyI,. perfluoroC 1 4 ,alkyl, C;, 4 alkoxy, hydroxy, halo amido, nit~ro amino, CI-.4alkylamino,

C

1 4 dialkylamino, carboxy or7 C- 4 alkoxycarbonyl) 1, l-diphenylCl- 4alkylcartionyl, substituted 1, 1-diphenylCl..

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

4 alkyl, perfluoro C 1 4 alkyI, CI- 4 alkoxy, hydroxy, halo, amido, nitro, amino, Ct- 4 alkylamino,

CI.

4 dialkylamino, carboxy or C 1 4 alkoxy-carbonyl), perfluoroCi 4 alkysulfonyl, Ci- 4alkysulfonyl,

C

1 4 alkoxysulfonyl, phenysulfonyl, substitute~d phenylsulfonyl (where the phenyl substituents are independently selected from one or IND1 more of, C-lalkyl, perfluoro CL-4alkylantino, Cj- 4dialkylamino, carboxy or. CI-4alkoxycarbonyl) cxamphorsulfonyl, phenylCl- 4 alkylsulfony1, substituted phenylC 1 4 a].kylsufonyl, perfluroCi 4 alkysulfinyl,

C-

l4alkysulfijnyl phenyisulfinyl, substituted phenysulfixtyl (where the phenyl substituents are CI independently selected from one or more of, CI- 4 alky1, CI perfuoro C1.

4 alkyl,

C~

4 alkoxy, hydroxy, halo,,amido, nitro,. amino,

C

1 4 alkylamino,

C

1 4 dialkylamino, carboxy CI or CI- 4 alkioxycarbonyl), l-rxaphthysulfonl, 2naphthylsu lfonyl,,substituted naphthylsul.fonyl (where the naphthyl Substituents are independently selected fromi one or more of, C 1 4 alkyl, perfluroC 1 4 alkyl, Ci- 4alkoxy, hydroxy, halo, azaido, nitro, amino, Ci- 4alkylamino, C1.4dialkylamino, carboy or C 1 4alkoxycar)bonyl) ,l-naphthysulfinyl, 2-naphthysulfinyl, and substituted naphthylsulfinyl (where the naphthyl substituents are independently selected from one or more of, C:1 4 alkyl, perfluoroc 1 4 -alkyl,

C

1 4 alkoxy, hydfoxy, halo amido, nitro, amino,

CI-

4 alkylamino, C1O 4 dialkylamono, carboxy or C-14alkoxycarbonyl): or a poly peotide-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 in Formula I and is selected from the group consisting of glycine, N-CI-ealkylglycine, alanine., 2azetidinecarboxylic acid, proline,. thiazolidine-4- .carboxylic acid, 5.

5 -dimethylthiazolidine.4.carboxvlic acid, oxazolidine-4-carboxylic acid, l-amino-1-cyclOC 3 8 alkylcarboxylic acid, 3 -hydroxypropoline, 4hydroxyor-yline,3-(C~I 4 alkoxy) proline, IND -12ri 4 (CL.4alkoxy)proline, 3, 4 -dehydropr line, 2, 2-dimethyl-4- U thiazoljidine carboxylic acid, 2 .2-dimethyl-4oxadolidine carboxylic acid, pipecolinic acid, valine, c-i luethionine,, cysteine, asparagine, serile, threonine, leucine, tert-leucine, isoleucine, phenylalanine, 1naphthalanmne, 2 -naphtha lanine, 2 -thienylalanine, 3thienylalnine, [1,2,3,41 -tetrahydroisoquinoline- 2carboxylic acid, *aspartic acid- 4 -Cl- 4 alkyl ester and IND glutamic acid 5-C 1 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, 4benzolyphexiylalanine, 4 -carboxyphenylalanine, 4- (Carboxy C1-2alkyl) phenylalanine, substituted phenylalanine. (where the phenyl substituents Are independently selected from one or more Of Cl- 4 alkyl, perfluoroCt..

4 alkyl, C 1 4 alkoxy, hydroxy, halo, amido, nitro, amino, CI-4alkylamino, Cl..4dialkylamino, carboxy or C1-4alkcxycarbony1) 3 -benzothienylalanine, 4biphenylaljanine, homophenylalanine, octahydroindole-2-.

carboxylic acid, 2-pyr idyl al anine, 3 -pyridylalanine, 4thiazolya:Lanine, 2-t-hienylalanie, 3- (3benzothienyl) alanine, ]-thienylalanine, tryptophan, tyrosine, asparagine, 3-tni- Ct.4alkylsilylalanine, cyclohexyiglycine, diph-enylglycine, phenylglycine, methionine sulfoxide, methionine sulfone, 2 2 -dicyclohexylalanine, 2-(1naphthylalanine), 2 -naphthylajlanine), phenyl.

sub-stituted phenylalanine *(where the substituents are selected from:CI 4 alkyl, perfluoroci, 4 alkyl, C 1 4 alkoxy, hydroxy, halo, amido, niitro, amino,

CI.

4 alkylamino,

CI-

4dialylarino, carboxy or C 1 4 alkoxycarbonyl) aspartic acid, aspartic acid-4CI..

4 alkyl, perfluoroc 1 4 alkyl Ci- 4alkoxy, hydroxy, halo, amido, nitro, amino, Cl-* NO -13- 4alkylamiflI), CI-74dialkylamino, carboxy or C 1 4alkoxycar:oony) aspartic acid, aspartic acid-4-C 4alkyl ester glutamic acid, glutamic acid-B-C 1 4 alkyl ci ester, Cyc:loC 3 -salkylaalanine, substituted cyclOC 3 aalkylalanine (where the ring substituents are carboxy,

C

1 alkyl ester, Cycloc3-salkylalanine, substituted CyCloC3.eaky]alafline (where the ring substituents are CI carboxy, Ci..4alkylcarboxy,. CL.4alkoxycarbonyl or CI amino carbo~nyl) 2, 2 -diphenylalanine and all alpha-C..

.5alkyl of all amino acid derivatives thereof, CIwhere the amino terminus of said second amino acid is Unsubstituted or monosubstituted with a member of the group consisting of formyl, C1-12 alkyl, ylCI1-2alkyl, carboxyCl-8 alkyl, carboalko xyCi..

4 alkyl, phenyl.

C

1 4 alkylt, substituted phenylC 1 4 alkyl (where the pheny. substituents or independently selected from one or more of, CI..

4 alkyl, perfluorC.-.

4 alkyl, Ct..ealkoxy, hydrcxy, halo, amido, nitro, amino, Cl-4alkylauino, Ci..

4dialkylaiuino, carboxy Or CI..4alkoxycarbonyl), 1,1dipe hnylCI- 4 alkyl, Ci-Galkoxycarbonyl, phenylCl- 6al-koxyca.rbonyl,

CI-

2 alkylcarbonyl, perfluoroC 1 4aiky'LCo-4alkylcarbonyl, phenyC;..

4 ;alkylcarbonyl, substituted o~henyCl-4alkylcarbonyl (where the phenyl substiZuents are independently selected from one or more Of c: 1 4 alkyl, perfluoro

CI-

4 alkyl,

C-

4 alkoxy, hydroxy, halo, amido, nitro, amino, Ci.4alkylamino,

C,-

4dialkvlamino, carboxy or C1.4alkoxycarbonyl),.1,1diphenyl(c-1 4 alyi perfluor6'Cltalkyl,

CI..

4alkoxy*cELrbonyl), 1O-camphorsulfonyl,. phenylC..

4 a1 kysuljionyl, substituted phenylcl-,alkylsul fonyl, Ci- 4alkysulffinyl, perfluoro CL-4alkylsulfinyl, phenyisulfinyl, substituted pheny-Isulfinyl (where the phenyl subst ituents .are independently selected from one or more Of, C'- 4 alkyl, perfluOroC-,.alkvl,

CI-

4 alkoxy, -14- CI hydroxy, halo,* amido, nitro, amino, C1.4alkylamono, Ci..

4dialkylamonci, carboxy or CI-4alkoxycarbonyl) phenyC 1 4alkylsul finrl., substituted phenylCl-4alkylsulfinyl 1!- CInahylnaphthylsulf aptylufnyl, substituted naphthylsulfonyl (where the naphthyl substituent is selected from C- 4 alkyl, perflUOroc 1 4 alkyl, CI-.

4 alkoxy, hydroxy, halo amido, nitro, amino, Cl..

4 alkylamino, Cj- 4dialkylamino, carboxy or C1..4alkoxycarbonyl),

I-

NO haphthyl-sulfjnyl, 2-haphthylsulfinyl and substituted 0 10 naphthyl-sulfinyl (where the naphthyl substituent is selected from C 1 4 alkyl, perfluoroC 1 4 ,alkyl, C- 4 alkoxy, hydroxy, halo, amido, 'nitro, amino, C 1 4 alkylamino,

C-

14dialkylam.Lno, carboxy or C1..4alkoxycarbonyl) R, is selected from the group consisting of hydrogen and alkyl; R 2 i selected from the group consisting of aminoC2-salkyl, guanidinioC 2 -alkyl, CI-.

4 alkylguanidinoC 2 salkyl, diCI-.4alkylguanidinOC 2 5alkyl, amidinoC 2 -5alkyl, C 1 4alky-lamidinoC 2 5 alkyl, diCI-.

4 alky- lamidinoC 2 5 alkyl, C 1 3alkoxyC 2 5 alkyi, phenyl, substituted phenyl (where the substituents are independently selectedi from one or m~ore 'of ami.no, amidino, guanidino, Cl- 4 alkylamino,

C

1 4dialkylamnino, halogen, perf2luoro C 1 4 alkyi,. C 1 4 alkyl,

C:

1 3 alkoxy or nitro), benzyl, phenyl substituted benzyl (where the substituents are independently selected from one or more of, amino, amidino, guanidino, Cj.

4alkylamin,, CI.4dialky-lamino, halogen, perfluoro Ci..

4alkyl, Cl--O4alkyl, CI.3alkoxy or nitro) hydrcxyC 2 ralkl, C I-ralkylauinoC 2 -5alkyl, Cl-sdialkylaminc 2 .alky1, 4 -aminocyclohextyiCo.

2 alkyl and C-alkyl; P is 0 or 1; B is cl 0 where n is 0-3, R3 is H or Ci:-Salkyl and the carbonyl mioety of B is bound to E; E is a het.erocycle selected from the group) consisting of oxazolin-2-yl, oxazol-2-yl, thiazol-2-yl, yl, thiazol-4-yl, thiazolin-2-yl, imidazol-2-yl, 4-oxo- 2 -quinoxalin-2y1, 2-pyridyl, 3-pyridyl, benzo fbithiophen-2-yl, triazol-4-yl triazol-6-yl,.

pyrazol-2-yl, 4,S, 6 7 -tetrahydrobenzothiazol-2yl, naphtho(;:', -dlthiazol-2-yl, naphthofl-2-dlthiazol-2-yl quinoxaljin- 2-yl, isoquinolin-1-yl, isoauinolin-3-yl, benzo (b .furan-2-yl, tpyrazin-2-yl, qui.-azclin-2-yI, 1sothiazol-3-yl, purin'-ayul and a substitu:ed heterocycle where the substituents are selected fr.om C 1 4 from C-l4alky, perfiucro Cj..4alkyI,C 1 4alkoxy, hydroxy, halo, amido, nitro, amino, C 1 4alkylaflLno, C, 4 dial kylamino, carboxy, Ci..

4alkoxyca3rbonyl, hydroxy or phenylCI- 4 alkylam.inocarbonyl; or pharmaceutically acceptable salts thereof.

More particularly, *in our opinion, some of the compounds of *the foregoing formula containiing a dphenyl-al.anine-pro line- .arginine motif should be effective in inhibiting the PAR-2 pathway and causing 0 -16depigmentation. One particularly preferred compound Swhich acts as a thrombin and trypsin inhibitor and is C active in depigmenting mammalian skin is (S)-N-Methyl-D- Cl phenylalanyl-N-.[4-( aminoiminomethyl) amino] (2benzothiazolylcarbonyl) butyl] -L-prolinamide (Chemical Abstracts name) (hereinafter referred to as "Compound C1 We suggest that other compounds which are analogs eg or function similarly to Compound I and are set forth in O U.S. Patent No. 5,523,308 may be active in the methods S 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 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 following plant families: Solanaceae potato, tomato, tcmatilla, and the like); Gramineae rice, buckwheat, sorghum, wheat, barley, oats and the like); Cucurbitaceae cucumbers, squash, gourd, luffa and the like); and, preferably, Leguminosae beans, 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 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 o -17eC melanosomes by melanocytes, or to receive melanosomes by keratinocytes in the skin.

SThe compounds which are active in the compositions C and methods of this invention may be delivered topically by any means known to those of skill in the art. If the ^Q delivery parameters of the topically active pharmaceutical or cosmetic agent so require, the Stopically active composition of this invention may preferably be further composed of a pharmaceutically or 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, particula:rly 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 by weight); b) compounds having the steroid backbone found in cholesterol (preferably in an amount of between about 5% and about 45% by weight); and c) one or mere fatty acid ethers having from about 12 to about 18 carbon atoms (preferably in an amount of between z:bout E. and about 70% by weight collectively), 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 choleste:ol/ polyoxyethylene ether/polyoxyethylene-9-lauryl ether (GDL liposomes) are most preferred. Preferably the liposomes are present in an amount, based upon the total volume of the composition, of from about 10 mg/mL to about 100 mg/mL, and more preferably from about 20 mg/mL to about mg/mL. A ratio of about 37.5:12.5:33.3:16.7 is most 0-18- (C preferred. Suitable liposomes may preferably be prepared in accordance with the protocol set forth in Example 1, though other methods commonly used in the C( art are also acceptable. The above described composition may be prepared by combining the desired components in a suitable container and mixing them under i ambient conditions .in any conventional high shear mixing f means well known in the art for non-ionic liposomes ND preparations, such as those disclosed in Niemiec et S 10 al., "Influence of Nonionic Liposomal Composition On Topical Delivery of Peptide Drugs Into Pilosebacious Units: An In Vivo Study Using the Hamster Ear Model," 12 Pharm. Res. 1184-88 (1995) ("Niemiec"), which is incorporated by reference herein in its entirety. We 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 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, 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 s:kill in the art may also be utilized as vehicles :.or 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- (N fragments can be chemically synthesized and provided in 0 a pure form suitable for pharmaceutical/cosmetic usage.

Products of natural extracts can be purified according (N to techniques known in the art. Recombinant sources of compounds are also available to those of ordinary skill S in the art.

c In alternative embodiments, the topically active m pharmaceutical or cosmetic composition may be D optionally combined with other ingredients such as 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 like. The compositions of this invention may also contain active amounts of retinoids 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.

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 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 l /cm2 of topically active acent is present when a change in pigmentation 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 abour 0.0001% to about 15% by weight/volume of D C<1 the composition. More preferably, it should be present Sin an amount from about 0.0005% to about 5% of the .C composition; most preferably, it should be present in C an amount of from about 0.001 to about 1% of the composition. Of course, these ranges are suggested for the foregoing components.. The lower set of ranges is C- intended to be efficacious for PAR-2 pathway Sagonists/antagonists and/or inhibitors having high

\O

therapeutic indices and which do not require C- 10 significar.tly 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 employed in the compositions of this invention in a concentration 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, 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.

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 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

\O

-21o as Compound I and related compounds; from about 20% to Sabout 99% for liquid derivatives and extracts of botanical materials; and from about 1% to about 20% for CI fractions of natural extracts and naturally-derived protease inhibitors such as STI or mixtures thereof) between ore and two times daily for a period of time C until the skin evidences a change in pigmentation.

eC This may be for from about four to about ten weeks or

\O

Smore. Thereafter, once the change in pigmentation has been achieved, a lower concentration and dose (from about 0.0)001% 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 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 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 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 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- O equivalent system was used. The epidermal equivalent system used contained melanocytes. One epidermal C equivalent system which is useful in performing this study is the MelanoDerm system, available commercially from MatTek Co. This system contains human normal t\ melanocytes, together with normal, human-derived C epidermal keratinocytes, which have been cultured to C- form a multi-layered, highly differentiated model of the O human epidermis. In the following examples, equivalents C- 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)

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 ni:rate reducing activity. Multilayered human epidermal equivalents containing melanocytes were used 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

irradiatioh and known whitening agents such as benzaldehyde and hydroquinone by increasing and reducing pigmentation, respectively. The MelanoDerm epidermal equivalents were exposed to benzaldehyde (available from Sigma of St. Louis, MO), hydroquinone (available from Sigma) and UVB irradiation. UV irradiation was performed with a UVB FS light source in an exposure chamber, with plate covers removed and Phosphatebuffered saline (PBS, from Gibco-BRL, Gaithersburg,

MD)

present in the lower chamber. UVB intensity was -23- U measured with a UVX radiometer (UVP Inc., San Gabriel, Q CA). Equivalents were treated with 0.1-0.12 J/cm No loss of viability was observed in equivalents treated

(C

N with up tc 0.3 J/cm 2 On the fourth day of exposure to the test compounds/ultraviolet irradiation, the equivalents were C-q fixed, sectioned and stained, or stained a's whole C-q without sectioning. MelanoDerm equivalents were

IO

0 formalin fixed and put in paraffin blocks, and sections Cq 10 from the MelanoDerm equivalents were stained in accordance with the following standard procedures: (1) H&E, DOPA H&E and Fontana-Mason using standard techniques known to those of skill in the art.

Alternatively, whole MelanoDerm equivalents were stained and thei: 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 molecules, which identifies primarily melanins. F&M stained sections were used for image analysis using Optomax Image Analysis Systems, from Optomax Inc., Hollis, NH. Alternatively, Empire Images database 1.1 was used on a Gateway 2000 P5-100 computer (Media Cybernetics, Silver Springs, MD) for capturing images.

Image Pro Plus version 4.0 was used for image analysis.

Parameters measured were as follows: level of pigmentation within individual melanocytes. and (2) number of pigmented melanocytes per field, for the Optomax system, or the surface area of silver deposits within melanocytes and the number of pigmen-:ed meianocytes for the Image Pro system. Using the Optomax system, surface area of silver deposits within individual melanocytes was measured in -24- C melanocytes, using multiple .sections from triplicate equivalents per treatment. The number of melanocytes c per field was calculated in these sections.

A

"pigmentation factor" was defined as the average surface area of silver deposits within an individual melanocyte, multiplied by the number of pigmented melanocytes per i field. A value of one was assigned to untreated OD controls, and values of treatment groups were normalized to their relevant controls. Using the Image Pro system, S 10 surface area of silver nitrate deposits and number of melanocytes .were measured for whole equivalents.

A

value of or.e was assigned to untreated controls and values of treatment' groups were normalized to their relevant controls.

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 (500M), hydroquinone (500M) and UVB irradiation.(0.12 J/cm z 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. Complete® Protease Inhibitor Cocktail tablets available from Boehringer Mannheim were used, containing inhibitors of chymotrypsin, thermolysin, papain, pronase, pancreatic extract and trypsin. Soybean trypsin inhibitor was available from Sigma and was dissolved in a 50 mg/ml liposome vehicle or in Ix PBS. All other protease inhibitors used in this in vitro example were dissolved in IxPBS. GDL liposbmes were prepared as set forth in Niemic, et al., above, with the exception of the following changes: the nonionic liposomal formulation contained glycerol dilaurate CEinilsynt GDL, ISP Van Dyk)/cholesterol .(Croda) /Polyoxyethylene-l 0-stearyi ether (Brij76, ICI) /polyoxy.athylene-9-lauryJ. ether, as at ratio of 37.5:12.5:33.3:16.7. Hepes buffer, 0.05M, pH 7.4 (Gibco-BRL cf 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 in Figure 113, some of the serine protease inhibitors, particularly STI (soybean trypsin inhibitor), were very effective in inhibiting melanogenesis.

TABLE A Test Ingredients FOrmtla ti on ComplJeteD Total protease inhibitor Mix-l Serine Protease inhibitors 99 g/mL Phenylmethyl-sul fonyl fluoride ("PMSF') and .g/mL L-1-Chloro-3-K4tosylamido) -4-phenyl-2but anone ("TPCK") M-ix-2 Serine protease inhibitors 0.1 g±g/mL apro tinin, ±g/mL Soybean trypsin inhibitor pLg/rnL leupeptin and 0.25 gg /rL (L-1-Chloro-3-[4tosylamido] -7-aiuino-2heptanone-HC1) ("TLCK STI Soybean trypsin inhibitor -1 mgI/mI -26- Example 2: A Protease-activated Receptor Is Involved In C Pigmentation Example 1 demonstrates that STI reduces S 5 pigmentation. STI inhibits trypsin. Because trypsin is 0C known to activate TR and PAR-2, we tested the possible Vq involvement of TR and PAR-2 in pigmentation. MelanoDerm 0D human epidermal equivalents were treated with the TR and PAR-2 agonists and antagonists set forth in Table B Cl 10 below daily for three days. On the fourth day, the samples were harvested, fixed, and DOPA, H&E or F&M staining.was performed. Histological and wholeequivalent examination revealed changes in pigmentation following the treatments. Figure 2 depicts the results 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.

Figu:re 3 shows the results of the studies set forth in this example, representing the level of pigmentation in MelanoDerm equivalents treated with TR and PAR-2 reagents. SLIGRL, a PAR-2 agonist, dramatically increased pigmentation, indicating that 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 indicates that TR is less likely to be involved in pigmentation.

i -27- TABLE B 7 Asa~ VAR-2 Dm Thrombin Activates TR Trypsin Activats TR and PPA-2 TFLLRNPIIDK TR peptlde aqonist activates T R only SLIGRL PAR-2 peprtide agonist activates eAR-2 only SFtLRN TR pept~de aqonist -activates TR, TR, cross-reacts with PAR-2 FSLLR4 Scrambled peptice Lnactive kirtzdir Specific inhibitor of thrombin Compour.d I j Thcommiin and tryps.n Lnhibitor E-xamople 3: A Dose-response Relation Between Proteaseactivated Rteceptors Signaling and Melanogenesis MelanoDerm equi valents were treated with increasing concentrations of SLIGRL, the PAR-2-peptide agonist, at 0, 10 and 50 pX in the same manner as set forth in Example 2. F&M staining was performed in the fourth day. As shown in Figure 4A, increasing concentrations of SLIGRL, the PAR-2 activator, result in increased *pigmentatihon. Trypsin, a PAR-2 activator, has the same effect. Treatment with increasing, concentrations of *Compound I, the thrombin and .t rypsin in~hibitor, .from O.lpM to 1pM resulted in decreasing pigmentation (see Figure 4;L) Pretreatment of the equivalents with UVB' irradiation increased melanogenesis, relative to untreated. controls. Compound Iwas able to reduce this UVB-induced pigmentation as well (Fig. This example demonstrates a dose-response -relation for increasing and decreasing pigmentation with the modulation of PAR-2 signaling. This example also.

demonstrates that Compound i can inhibit pigmentation and preve~nt UV-induced pigmentation.

-28- Example 4: PAR-2 is Expressed in Keratinocytes, But Not In Melanocytes PAR-2 and TR expression have been demonstrated previously in keratinocytes and fibroblasts. This example demonstrates that PAR-2 is expressed in Cq keratinocytes, but not in melanocytes. Furthermore, it Cq demonstrates that TR is expressed in both keratinocytes O and melanocytes. In order to demonstrate this, MelanoDerm human epidermal equivalents, human primary melanocyte cultures (neonatal and adult, from Clonetics of San Diego, CA) and Cloudman S91 mouse melanoma cells from ATCC of Rockville, MD were grown in culture and total RNAs were extracted using "RNA Stat-60" reagent available from "Tel-Test 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 DNase available from Promega Corporation under the tradename "RQ1 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 Corporation (May, 1995). The resulting 200ng of DNased-RNA was reverse transcribed via the procedure set forth in "Superscript II Reverse Transcriptase" a protocol published by Gibco-BRL (now Life Technologies, Incorporated) (April 1992), using 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 using about a -29unit (per 100 ul reaction) of a thermostable DNA polymerase which is commercially available from Perkin- Elmer-Cetus Corporation under the tradename "Taq polymerase" and about 0.1 umol/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-phosphatedehydrogenase (G3PDH) primers, available from Clontech 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% 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 ethanol according to well-known procedures. When primers for G3PDH were used, only 10% of the PCR reactior products were used. An RNA sample from epidermal equivalents that was not reverse-transcribed was used as a negative control for each PCR 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 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.

(N

SThe r:elative quality of each respective RT-PCR Sreaction product was then compared by analyzing the C- mRNA level of G3PDH, a "housekeeping" gene, in each respective product. As illustrated in FIG. 5 and 6, 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 S 10 are expressed in total skin and in the MelanoDerm equivalents However, S91 melanoma cells ("S91") did not express PAR-2 or TR. To investigate this further, we tested primary newborn ("mel-NB") and adult melanocytes for TR and PAR-2 expression. As 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 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, the amount of MgCl 2 required for the PCR reaction, and the length of the PCR cycle.

IND -31- Table C: DNA Primers Utilized in RT-PCR Assay Primer At. mi Cce' No.* f~ DNA c'S1 see of Imz)Cycles Seq.

att-ached MqcI e ID 1 No.

-32- AT77~GCCT

CTCCTGGGG(

TR-sense .2.5 .5 e 94 3021 CCTCTGAGTGC 1. 6 CAGArG=AC 3 72 TCrACAG Th- 2.5 .5 0 94 30 12 antizense -1 6 SS CCAAGTTAAC 3 6 72 AGCTTrGTA

ATGCTGTTAT

CAGG W3) Thromoin- 2.5 S5 094 35 13 sense I1@55 AACCTGAAGGA 3 72

GACGTGGAC

(311 Thromoin- 2.5 .5 94 35 14 antisense I 6 CAGGAGCCCAG 3 72

AATATGACTC

I0 -33- 0 Example 5: Keratinocyte-Melanocyte Contact is Required Sfor Compound I Depigmenting Effect Cl The results of Example 4 suggest that melanocytes alone might not respond to the depigmenting effect of SPAR-2 antagonists. Indeed, the level of pigmentation of human prima::y melanocytes or choleratoxin-induced S91 cells, which is reduced by hydroquinone and C benzaldehyde, was not affected by Compound I.

Since PAR-2 is not expressed in melanocytes, we tested CI the possible requirement of keratinocyte-melanocyte interactions for the depigmenting effect of Compound I.

Primary melanocyte cultures were compared to identical cultures plated under epidermal equivalents (EpiDerm, 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 Compound I, with the PAR-2 agonist SLIGRL, and with the TR agonist TFLLRNPNDK, as set forth in Table D, and DOPA stained on the fourth day. In Table D, keratinocytes are indicated by melanocyres are indicated by "M" and lack cf keratinocyte-melanocyte contact is indicated 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 -3.4- 0*results demonstrate that keratinocyte-melanocyte contact is required for the PAR-2 effect on pigmentation.

Th=L D freatowt a-cultrex Me2nolem (M 30(n K-W cmtact) VK-N amitct) InCompound I no no liqfttenLng effect effect SLZGRL no no claricening effect effect rlTFLLRNPNI)K no no no effect effect, effect Example 6: Compound I Affects Melanocyte Gene Expression MelanaDerm equivalents were treated with increasing concentrations of the thrombin and trypsin inhibitor, Compound or with increasing concentrations of the PAR-2 agonist SLIGRL. RNAs extracted from untreated and.

Compound I-treated equivalents were analyzed for gene expizession by RT-PCR in the manner set forth above in Example 4. Gene-specific primers were designed as set forth in 'Cable C above, and Clontecn primers for human G3PDH were used as .in Example 4. Melanogenic genes tested for expression level were tyrosinase, TRP-l, and TRP-2 A dose-dependent decrease in TRP-1 and a dosedependent increase in TRP-2 mRNA levels were observed in Compound I-treated samples, as shown in Figure GAL.

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 processes dopaquinone to 5, 6-dihydroxyindole carboxylic acid (DEICA) rather. than to 5. 6-dihydroxyindole

(DHI)

D This process results in brown, finely dispersed eumelanin, as opposed to insoluble black eumelanin, and Ci results in a lighter skin tone. TRP-1 stabilizes the melanogenic complex, enabling pigment production.

Q Reduced levels of TRP-1 result in reduced tyrosinase activity and reduced pigmentation. Lack of this protein results in albinism. Increasing concentrations of IND SLIGRL, however, did not affect melanogenic gene S 10 expressior (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 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 MelanoDerm 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 bt produced by the keratinocytes only. As shown in Figure 6A, treatment with Compound I resulted in increased expression of trypsin. SLIGRL, which did not affect melanogenesis gene expression (Fig. 6B) also increased trypsin expression in the equivalents. We conclude that while trypsin is a possible natural activator of PAR-2 in skin and possibly affects pigmentation, -its mRNA levels do not correlate. with pigmentation. This suggests that another, yet uniden'ified serine protease, which is inhibited by compound I, STI and the like, is the natural activator -36- Sof PAR-2 ir. the epidermis. Compounds that induce or inhibit this protease would serve as. darkening and Cl lightening agents, respectively.

Example 7: Thrombin and Trypsin Inhibitors and PAR-2 Agonists AfJfect Pigmentation In Vivo N Two cuinea pigs were treated twice daily, five D days/week for seven weeks with Compound I at 1 and S 10 PM in 70:20 ethanol:propylene glycol vehicle on one pigmented nipple.. The other nipple of each animal was treated wi-:h vehicle only and served as a control.

Chromameter measurement after seven weeks of treatment revealed a dose-dependent lightening effect of +9.6 L* 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 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.

Example 8: Thrombin 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 jM. Each compound was applied to two sites on the pig twice daily, five days per week for eight weeks. After eight weeks of treatment, chromameter measurements were taken. The application of Compound I resulted in a visible lightening effect. The PAR-2 agonist SLIGRL resulted in IN -37- U a darkening effect as measured by chromameter.

SFLLRN

C and FSLLRN had no significant effects.

Two Yucatan swine were treated for seven and a half weeks, or for ten weeks, twice daily, five days per Q week, with increasing concentrations of Compound

I.

Four concentrations of active compound were used, as follows: 0, 10, 50 and 250pM. Two sites per ND concentration were placed on opposite sides of the swine dorsum. Chromameter measurements were taken before treatment started and every two weeks thereafter.

Pictures were taken periodically and at the end of the experiment. A visible lightening effect was observed during the 4th, 5th and 6th weeks of treatment, for the 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 measuring brightness) during the treatment course of one swine are shown in Fig. 9. A saturation effect is observed, which is a time and concentration dependent. This example demonstrates a visual depigmenting effect by Compound

I,

in the animal model system most resemble pigmented human skin.

At the end of these experiments, biopsies were taken for histological and electron microscopy

(EM)

analyses. Histological samples were stained with H&E and F&M. H&E staining showed that there was no irritation, inflammatory response or changes in skin 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- S-epidermis. These results are illustrated in Figure Untreated ad vehicle-treated samples (Fig. 10A) were Cl identical and darkest. The 10 pM treatment (Fig. showed reduced pigmentation and the 50 and 250 pM j treatments (Fig. 10C, 10D, respectively) were the C< lightest.

C- The results of this example suggest that the O maximal whitening effect of Compound I could be achieved C- 10 with higher concentration over a shorter period of time or with lower concentration over a longer period of time. Thu;, at least two difference regimens may be used to achieve the desired skin whitening results.

Example 9: Ultrastructural Studies Demonstrate the Effect of Compound I On the Skin In Vitro and In Viva Ultrastructural analysis was performed on MelanoDerm equivalents and swine skin sites treated with Compound I. MelanoDerm equivalents treated with Compound I were analyzed for melanosome formation and distribution using electron rricroscopy. Treated samples contained more.

melanosomes, but less mature melanosomes, i.e., melanosome:s which evidence reduced melanin production, within the melanocytes, relative to untreated controls (Fig. 11A, 11B). Dendrites containing melanosomes were easily identified within treated keratinocytes (Fig.

11C), but. were difficult to find within control keratinocytes. This suggests abnormal melanosome formation and slow or impaired melanosome transfer into keratinocytes in the treated samples.

Skin samples from Yucatan swine treated with compound I1 for eight weeks, as described in example 8, -39- Swere also analyzed by electron microscopy. Melanosomes within keratinocytes of treated sites were smaller and C( less pigmented, compared to controls (Fig.llD, 11E and 11F). Moreover, the distribution of melanosomes within the treated skins was abnormal. Melanosomes were CI detected mainly at the epidermal-dermal border, compared to a random distribution in untreated controls (Fig.

ND 11G, 11H). While we cannot rule out other mechanisms, 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 I is revers;ible A Yucatan swine was treated with Compound I, 250 OM, 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 Figure 12B. For the following four weeks (starting at week nine of the experiment), the color of the treated sites was monitored, and two biopsies were taken each week from two :reated sites. Untreated sites were biopsied as well. The depigmenting effect could be visualizec 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 treatment, repigmentation was demonstrated histologi=ally. 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

NO

o machinery, and its effect is reversible in vivo. Figure S12A 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 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 NC treatment was stopped. Figure 12D shows sections of I sites which were treated for eight weeks with Compound 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.

Example 11: Preparation of naturally-derived products containincr

STI

Example 1 demonstrates that .the presence of 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.

To make soybean paste, soybeans were first soaked in deionized or purified water for several hours. The soybeans were ground after they were fully hydrated, with the addition of small quantities of water, if needed, t:o smoothen the paste. To make soybean milk, 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.

NO ~-41o 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.

SExample 12: Treatment With Naturally-Derived Materials that affect the PAR-2 Pathway Induces Depigmentation m Two Yucatan swine were treated for eight and ten weeks, twice a day, five days a week, with different soybean- and lima-bean-derived products. These natural products include soybean paste, soybean protein acid hydrolysate, miso, native and boiled soybean milk, and a commercia.ly available extract of soybean (Actiphyte

T

of Active Organics,. Dallas Texas), as well as purified 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.

Histological analysis of biopsies from' the treated sites following F&M staining confirmed the depigmenting effect of the soybean 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 the soy protein acid hydrolysare is explained by the denatura:ion 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 limabean trypsin inhibitor, respectively. (Example 1 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-

IO

.Example 13: An STI in liposome formulation can lighten human age spots (U An individual with three age spots on the. dorsum Ci of their hand was treated for eight weeks, twice a day, with the following: The age spot located closest to the arm was treated with placebo, containing 20 mg/ml of 1 0 liposomes. The middle age spot was not treated. The (Cq third age spot was treated with STI, in liposomes (20 mg/ml).

\O

N GDL liposomes were prepared as set forth in Niemiec, et al., above, with the exception of the following changes: the non-ionic liposomal formulation contained glycerol dilaurate (Emulsynt GDL, ISP Van Dyk)/cholesterol ether (Brij76, ICI)/polyoxyethylene-9-lauryl ether, as at ratio of 37.5:12.5:33.3:16.7. Hepes buffer, 0.05M, pH 7.4 (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.

As shown in figure 14, the age spot treated with STI becane 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 this orientation the top age spot is the placebotreated, 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 UVphotography. UV light enables the visualization of pigment deeper in the skin, demonstratihg that the STI whitening effect was not superficial. Figure 14 -43- 0 clearly demonstrates that the STI formulation was able to Slighten the lower age-spot. An increase of 15 L* units Swas. calculated for this STI-treated site, further demonstrating the ability of this treatment to lighten age spots.

V/ Example 14: Depigmenting formulations with soybean milk In making the soybean milk, it was discovered that C0 the rich enolliency of the milk would be desirable in a Sskin-care formulation. Because water is used as the C' predominan: ingredient of any oil-in-water emulsion, and in many ocher skin-care formulations we hypothesized that the soymiik 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 soybean milk. Surprisingly, we found that this substitut:on of soybean milk for water was physically stable. 'Formulations utilizing soybean milk should contain between about 71 and about 99% of soybean milk, more- preferably from about 80%. to about 95% soybean milk. Preferably, this and similar formulations should include a viscosity builder in an amount from about 0% to about 5% (more preferably, from about 0.1 to about one or more emollients in an amount up to about and/or emulsifiers in an amount from about 0.1% to about 10% (more preferably from about 3 to about and, opt:onally, a spreading agent in an amount from about 0 about 5% (more preferably from about 1 to about a preservative, a chelating agent or a humectant. The preservative should be present in an -44effective amount in order to preserve integrity of the milk and maintain the composition's activity..

Sufficient thickener should be present to impart. body to the formLulation without causing it to become so viscous that, it would hinder spreadability, from about 0 to about 10%, more preferably from about 3 to about Sunscreen, antioxidants, vitamins other depigtmentijig agents .and other skin care topical ingredients3 may also be incorporated into the compositions of this invention.

A paxticularly preferred example of a depigmenting formulation, substituting' s6ymilk for wate r is shown in table E below..

TABLE

E

Inqr I- ur-201 soybean mi k ajlunizn'jm s.tarch actenyl suc~inate cvci0!nthicone PIXG 6capric/caprylic rial cerides phenoacyethanal sucrome mo-coarte N~t,EOTA lyc erinm d i ~oparaffjin; latireth-7' Vehicle, depi omentina builder 3preading acent emollient/e J pre3ervative emuolijent/emu1 ifier chelac2.na aoent humectant t-hICXee t 84.9% 0.75% 2% 0.75% 1% 0. 11 STI, soybean paste and other trypsin inhibitorcontaining natural extracts can be incorpor-ated into such formulations to provide increasing concentrations of the serine protease inhibitor. Use levels of the added active .ingredient can range between 0.01% to in a f oritulation. Other depiginenting agents, including PAR-2 inhlbitors, tyrosinase inhibitors, hydroquinones, so~y productS,. ascorbic acid and its derivatives, as \U well as other ingredients with skin care benefits could 0 also be incorporated into this formulation.

Example 15: An Oil-in-water Emulsion depigmenting S 5 formulation Two examples of a depigmenting formulation with oilin-water emulsion are presented in Table F. A formulation with STI, where STI could be replaced with o any naturally-derived serine protease inhibitor, or V) with any naturally-derived extract or fraction thereof IND containing serine protease inhibitors, is described in Scolumn 4 of Table F. A similar formulation with Compound I i.s presented in column 5 of Table F.

Compound I :n this composition could be replaced with similar compounds, or with serine protease inhibitors or with any PAR-2 inhibitor materials having high therapeutic indices, whether derived synthetically or naturally, as the active ingredient. Suggested ranges 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- Tabnlea F Cetearyl Glucoside Surfactant 1.4 1.4 .1- 2.8 C12-15 Aikyl Surfacrant 4.0 4.0 1-6 Rentoate Octy. Emollient 1.0 1.0 Hydroxvatea rate Olmethicone Spreading 1.0 1.0 Cyclonein~one Agent Cycl~ffmhl coneSpreadinq Cetvi Alcohol Emollient 2.5 2.5 0-4 Butylaied Anti-vxidant 0.1 0.1 0-0.5 Hvdroxvtoluene Octyl Sun~screen 6.0 6.0 0-1o Methoxycinnamate Procyiparatben Preservitive 0.5 0.100.

vitamin E acetate Anti-oxidant 0.5 0.5 0o. Taccoherol Acetate Anti-oxidant I 0.-5 0.5 -(0-0.5 Glycerine +Iutectant 3.0 1 3.0 O-Pathenoi Pro-Vttamtn S O.'sr- Oisodlium EOTA Chelator. 0.1 0.01whitening

I

MetnVy1 Pacaben Preservative 0.2 0.2 Caroopser Thickener 0.35 0.35 0 Deionized Water or Carrier 76.35 77.5 50-80 Soybean Milk Whiteninq STI or natural I Whitening 1.0 0 1 0-15 extract Aaent 0.2 I Compound I Anent To prepare -rhis 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.

After mixing for ten minutes the rest of the aqueous phase ingredients were added and the mix was heated to 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 within the same formulation, in this Example and in the following examples and other embodiments of the methods and composiLtions of this invention.

-47- EXamle 16: De~iamentinar Comosjtion (Oil-in-Water Emulsion) Two a~dditional examples of an oil-in-water U emulsion dElpigmenting formulation are presented in G. A formulation with STI, where STI could be.

c-I replaced wfxth any naturally-derived serine Protease inhibitor, or with any naturally-derived extract or fraction thereof containing serine protease inhibitors, is,describ~ad in column 3 of Table G. A similar c-i 10 formulation.. with Compound I is presented in column 4 c-iof Table Compound I in this composition could be replaced with similar compounds or with ser-ine protease c-Iinhibitor or with Any *PAR-2 inhibitor materials having high therapeutic indices, whether derived synthetically IS 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.

G

Ethanoi solIvent 120 I5-20 Propylene Soivent .01-10 Glycol lyarox ye ryie Ice ThICKener/*.2 ,,.lose PO Povmn r irl es Th i cr. ene c Alltyl Polymec .U3 AcrylateI Crassocivne.t Pantnenoi i-ro-vktamin 1 0.1-3 jrca~- Fraarance 35-O lshxae7netceaokcin t 4.0 0-S VtmnEAnti-oxiclant 1.0 0-2 acet ate Sodi~um rdutraizer I U.35 0.1-0.5 GlIyce r in. Sumecxant 1.U 0-2 Water or Whlteninq Aqent Demcona Ah iei a t '1.25 60-60 L 5o oean tit Ik STI oc na~tural Whitening 0 0-15 -~rractAaent -48-

ID

To prepare this formulation, the hydroxyethylcellulose O was slowly added to the water or to the soybean milk Sand stir until completely dissolved. In a separate c- 5 .container the Acrylates/ C10-30 Alkyl Acrylate Crosspolymer was added and stir until completely dissolved. The content of the two.containers was combined anc. mixed for 20 minutes. Vitamin E acetate C( was then added and mixed, following by the addition of C- 10 Isohexadecane and Panthenol After mixing for Sfive minute:s the STI, or the natural extract, or S. 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.

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).

Example 17: Depiamenting Comosition (Water-In-Oil 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 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.

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 for the ingredients in such formulations are also -49- 0 To prepare this formulation, the hydroxyethylcellulose O was slowly added to the water or to the soybean milk Sand stir until completely dissolved. In a separate container t:he Acrylates/ C10-30 Alkyl Acrylate

C

Crosspolymer was added and stir until completely dissolved. The content of the two containers was combined a:d mixed for 20 minutes. Vitamin E acetate c was then added and mixed, following by the addition of Isohexadecane and Panthenol After'mixing for \O five minutes the STI,. or the natural extract, or 0 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.

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).

Example 17: Depiamentinc Commosition (Water-In-Oil 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 any naturally-derived serine protease inhibitor, or with any naturally-derived extract or fraction thereof containir.g 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.

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 for the ingredients in such formulations are also listed in Talle H. The deionized water content of these *formulations could be re~laced with soybean milk.

Table H Phase CTFA Name FuicUon %WW /WW Rne OIL IMinerat Oil Emoffient 2I Monooieate I Stearvi Alconal Emotient 25.0 25.0 I 20-60 I Dimetritcone I Soreamlna Anent 1 10 1.0 1 -1 I Cetvi Aicortoi Efmitent 2.0 2.0 1 0.10 Hyaracenateea Anti-oxidant 3.0 I 3.0 0-1 I Parsoi MCX Sunscreen' 3.0 3.0 I 01 I Proovioaraoen Preservative 0:5 (is 0.01-0.5 Vitamin E Anti-oxiaant 0.S 0.5 00-.

AGUEO I Glycertne Humcat 30 30 G2 Metn'ri Paramen Dreservative 0.2 0.2 10.01-0 3 Water or Soy Camrer 1 30.8 31.55 20-45 Milk Whitenmoa Anent II I STI ItWhitennAnent 1 10 0 I 0-10 I Coo I I WhitentnO Ann 1 0 i 0.25 n-1 To prepare this formulation the stearyl alcohol and mineral oi. were melted at 70 0 C. The other oil phase ingredient.,; were added and the mixtu re heated to The aqueou:: phase ingredients, which have been previously dissolved in the bulk ph ase water or Soy Milk and warmed to -70"C, were then added and the mixture was stirred until it congealed.

Example 18: Depigmentation Composition (Aqueous Gel) Two examples of a depigmenting formulation with aqueous ge~l are presented in Table J. A formulation with STI, where STI could-be replaced with any naturallIy--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 Comipound I is presented in column 4 of Table J.

Compound I in this composition could be replaced with U similar comp~ounds 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 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.

ri T-ABLE J CTFA Name I unction %WIW i %W/W I Octoxvnol-1 3 1 Surfactant f- 0.2 1 0.2 I 0.05-0.5 4-Hexadieriotc Preservative 0.1 0.1 0-.3 tienzanematihan Preservative 1.0 10j Disoctum EflTA Cheiatur 1 0.05 j0.05 0.01-0-2 Preservative Ascomic Anti-ondant 0.1 I0.1 1 0-0.2.

Sodilu M. Anti-oxidant 0.2 I0.2 I 0-0-3- -52listed in Table K. The deionized water content of c-i these formulations could be replaced with soybean milk Table K c-iC17A Ham I FunC=on %WW r R-uzW EhnlSolvent (11 70 40-90 Propylene Solvent 29 Glvrcoi Deionizea Carriec q.s. 1-40 c-i STI Whitening Compoundo Whitening 00001-1 To prepare this formulation Compound I was c-i dissolved in water. The ethanol and propylene glycol were mixed and combined with the aqueous solution containing Compound 1. En summary, we have demonstrated that activation of the keratinocyte receptor PAR-2 results in increased pigmentation. Preferably, such activation may be accompli~shed by the use of. trypsin or SLIGRL or 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- trans fer blockers.

other compounds known to those of skill in the art that 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 undeliverEtd melanosomes' in the inelanocytes could induce a. negative feed back ,mechanism, that slows new melanosom formation. 'The production 'of TRP-i, the major glycoprotein in melanocytes, is down- regulated,.

which leads to destabilization of tyrosinase. This results in reduced melanin formation, and in a color -53-

\O

switch, to a. lighter brown, as the ratio of TRP-1:TRP-2 Cl is reduced. The melanosomes accumulation in the Smelanocyte after Compound I treatment, or after STI treatment, therefore, have reduced and altered melanin S 5 content, which adds to the whitening effect of compound I or. STI.

Claims (52)

1. A method of effecting changes in mammalian skin pigmentation comprising administering to a mammal a pigmentation-changing effective amount of a compound which affects the PAR-2 pathway.

2. A method according to claim 1 wherein said compound inhibits the PAR-2 pathway.

3. A method according to claim 2 wherein said compound is an antagonist of PAR-2.

4. A method according to claim 3 wherein said compound binds to or blocks but does not activate PAR-2. A method according to claim 4 wherein said compound :Ls selected from the group consisting of antagonist:s based on SLIGRL which bind to or block but do not activate PAR-2, antagonists based on SLIGKVD which bind to or block but do not activate PAR-2 and mixtures thereof.

6. A nethod according to claim 2 wherein said compound is a protease inhibitor.

7. A method according to claim 6 wherein said compound .s a serine protease inhibitor.

8. A method according to claim 7 wherein said compound is a thrombin and/or tryptase and/or trypsin inhibitor.

9. A method according to claim a wherein said compound is a compound of formula I: S0 A BV wherein: A is selected from the group consisting of Ci-aalkyl, carboxyC,-_alkvl, Cl-4lkoycabonyCI4alylphenylC 1 4 al kyl, substituted c-iphenylC 1 4 alkyl (where the phenyl substituents are U independently selected from one or more of, C 1 4 alkyl, perf luoroC alkyl, C 1 alkoxy, .hydroxy, halo, amido, nitro aminc, CL- 4 alkylamino, CI- 4 dialkylamino, carboxy or CI- 4 alkoxyc:arbonyl), formyl, CI.4alkoxycarbonyl, CI-2alkylcar-bonyI, phenylCI. 4 alkoxycarbonyl, C3- 7 cycloakylc:arbonyl, phenylcarbonyl, substituted phenylcarbony2. (where the phenyl substituents are CI 10 independent~ly selected from one or more of, C 1 4 alkyl, perfluoraCI- 4 alkyl, CI- 4 alkoxy, hydroxy, halo, amido, CI nitro, amiao, CI-4alkylamino, CI.4dialkylamino, carboxy Or CL.. 4 alkoxycarbonyl) CL-4alkylsulfonyl, Cj- 4alkoxysulfonyl, perfluorOCI.. 4 alkyl-sulfonyl, phenylsulfonyl, substituted phenylsulfonyi. (where the phenyl substituents are independently selected from one or more of, CI- 4 alkl, perfluorOC-L 4 alkyl, C 1 4 alkoxy, hydroxy, halo, amido, nitro, amino, Cl. 4 alkylamino, Cj- 4dialkylamino, carboxy or C 1- 4 alkoxycarbonyl), camphorsul fonyl, phenylCI.. 4 alkysul fonyl, substituted phenvlCi- 4 2Llkysulfanyl, CI-4alkylsulfinyl, perfluroC.. 4 alkylsulfinyl, ohenylsulfinyl, substituted phenylsul:finyl ',where the phenyl substituents are independently selected from one or more of, C 1 4 alkyl, perffluoro: 1 4 aikyl, CI-4alkoxy, hydroxy, halo, amido, nitro, amino, CI-4alkvlamino, CI- 4 dialkylamino, carboxy or CI-4alkoxycarbonyl), phenylCt- 4 alkylsulfinyl, substituted peflnylCi, 4 alkylsulfinyl, 1-naphthylsulfonyl, 2-naphthylsulfolyl or substituted naphthylsulfonyl (where the naphthyl substituents are independently selected from one or more of, CI-.4alkyl,perfluoroC I 4 alkyl, CI- 4 alkoxv, hydroxy, halo, amido, nitro, amino, carboxy ar Cl-.alkoxyy-carbanyl) ,l-naphthylsulf inyl, 2- naphthylsuI;±inyl or substituted naphthvlsuifinyl (where IND -56- the naphthyl substituents are independently selected from one or -more of, C 1 4 alkyl, perfluoroC 1 4 alkyl, Clj. 4 alkoxy, hyd::.oxy, halo, amido, nitro, amino, C 1 4alkylamino, CI.4dialkylamino, carboxy or C 1 Cl 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, glyci ne, N-C 1 ID 10 salkyglycirxE, proline, I -amino- 1 -cycloc 3 8alkylcarbo):ylic acid, thiazzolidine-4-carboxylic acid, S-dimethylthiazolidine-4-carboxylic acid, oxadolidine-4-carboxvlic acid, pipecolinic acid, valine, Inethionine, cysteine, serine, threonine, norleucine, leucine, tert-leucine, isoleucine, phenylalanine, 1-naphthalanine, 2-naphthalamine, 2- thienylalar~ine, 3 -thienylalanine, tetrahydroisoquinoline- 1-carboxylic acid and 1,2,3,4, l-t:etrahydroisoquinoline-2-caroboxy lic ac id where the amihno terminus of said amino acid is connected a member selected form the group consisting Of CI- 4 alkyl, tetrazol-SyI-Ci--alkyl, carboxyr-C. 4 aukyl, CI-.4alkoxyarbonylC1.4alkyl, phenylC 1 4 alkyl, Substituted phenyl C 1 4 alkyl (where the phenyl substituents are independently selected from one or more of, C 1 4 alkyl, perfluoroC, 1 4 alkyl, C 1 4 alkoxy, hydroxy, l..alo, amido, nitro, amino, C 1 4 alkylamino, C 1 4dialkylanhino, carboy Or Cl-4alkoxycarbonyl), 1,1- diphenylCI- 4 alkyl, 3 -phenyl-2-hydroxyproploonyl, 2,2- diphenyl- 1-hydroxyethylcarbonyl, 4]- tetrahydroisoquinoline- 1-carbonyl, 3,4]- tetrahydroisoquinoline.3, carbonyl, 1-methylamino-1- cyclohe'xaclecarbonyl, 1 -hydroxy-l1-cyclohexanecarbonyl, l-hydroxy-l-pheny-lacetyl, 1-cyclohexyl-1- IND hydroxyacetyl, 3-phenyl-2-hydroxypropionyl, 3,3- c-Id.iphefl-l2-hydroxypropionyl, 3 -cyclohexyl- 2- U hydroxypropionyjj, formyl, Cli 4 alkoxyca rboriyl, Cl-lzalkylcarbonyl, perfluoroC,- 4 alkyl, CI.4alkylcarbonyl, phenylCi- 4 ailkylcarbonl, substitutedi phenYlCL. 4 alkylcarbonyl (where the phenyl substituents are independently selected from one or more of, CI. 4 alkyl, perfluoroCI- 4 alkyl, C1- 4 alkoxy, hyciroxy, hEilo amido, nitro amino, CL-.4alkylamino, C 1 c-I 10 4dialkylamiflo, carboxy or Cl-4alkoxycarbonyl) 1,1- diphenylC.. 4 alkylcarbonyl, substituted 1, l-diphenylC 1 c-I 4alkylcarbcnyl (where the phenyl substituents are indep-enden-:ly selected from one or more of, C 1 4 alky., perfluoro (Ci- 4 alkyl, CI-4alkoxy, hydroxy, halo, amido, nitto, ami:a-o, CI-4alkylamino, CI.- 4 dial kylamino, carboxy or CI- 4 alk:)xy-carbo nyl), perfluoroC,- 4 alkysulfonyl, Cj- 4alkysulfoziyl, C1.4akoxysulfoflyl phenysul fonyl, substituted phenylsulfanyl (where the phenyl substituents are independently selected from one or more of, C-lalkyl, perfluoro CL-4alkylamino, C 1 4dialkylamino, carboxy or Cl-4alkoxycarbonyl), cxamphorsuifonyl, phenyiCi-,alkylsulfonyl, substituted pheny1Cj-.,a~ikylsufonyli perfluroCi-,alkysulfinyl, C- l4alkysulfinyi, 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, nitro, ami.no, CI- 4 alkylamino, CI- dialkylamino, carboxy or CL.. 4 aikcoxycarbonyl), 1-naphthysulfonl, 2- naphthylsifonyl, substituted naphthylsulfonyl (where the naphthy. Substituents are independently selected fromi one or more of, C 1 4 alkyl, perfluroCI- 4 alkyl, Ci- 4alkoxy, hydroxy, halo, amido, nitro, amino, Cj- ,alkyla.-mio, CI-4dj'alkylamino, carboy or C 1 I-58 4alkoxycarbornyl) l-rahthysulfiny1, 2-naphthysulfinyl, and substituted naphthyisulfinyl (where the naphthyl substituents are independently selected from one or more of, CI- 4 alkyl, perfluoroCI- 4 alkyl, C 1 4 alkoxy, hydroxy, halo amido, nitro, amino, C 1 4 alkylamino, C104 dialkylzmono, carboxy or C-l4alkoxycarbonyl): or a poly pE!ptide comprised of two amino acids, where the f h'rst amino acid is a D or L amino acid, bound via il:s carboxy terminus tot he nitrogen depicted in Formula I and is selected from the group consisting of glycine, N-Cl-galkylglycine, alanine, 2- azetidinecarboxylic acid, proline, thiazolidine-4- carboxylic acid, 5. S-dimethylthiazolidine-4-carboxylic acid, oxazolidine-4-carboxylic acid, 1-amino-1--cycloC 3 -a alkylcarboNxylic acid, 3 -hydrox ypropo line, 4- hydroxyproline, 3- (C 1 .4alkoxy) proline, 4 (Cl-. -4alkoxy) pro line, 3, 4-dehydroprline, 2,2-dimethyl-4- thiazolidine carboxylic acid, 2.2-dimethyi-4- oxadolidine carboxylic acid, pipecolinic acid, valine, methionine, cysteine, asparagine, serine, threonine, leucine, tert-leucine, isoleucine, phenylalanine, 1- naphthalanine, 2 -naphtha lanine, 2-thienylalanine, 3- thienylalnine, 1,2,3, 4 1 -tet rahydroisoquinoiline- 2- carboxylic acid, aspartic acid- 4-CI- 4 aikyl ester and glutamic acid 5-CI. 4 alkvl 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- benzolyph~anylalanine, 4 -carboxyphen-ylalanine, 4- (Carboxy Cl-- 2 alkyl)phenylalanine, substituted phen-lalanine (where the phenyl substituents are inde- dently selected from one or more Of C 1 4 alkyl, perfluarocC 1 -alkyl, CI- 4 alkoxy, hydroxy, halo, amido, nitro, anino, C.,-alkvl amino, C I 4 dialkvl amino, carboxy Or C1.4alkoxycarbonyl), 3 -benzothienylalanine, 4- biphenylalaajne homophenylalanine, octahydroindole-2- (1 carboxylic acid, 2 -pyridylalanine, 3-pyridylalanine, 4- thiazolyalanine, 2 -thienylalanie, 3- (3- benzothienyl) alanine, 3-thienylalanine, tryptophan, tyrosine, asparagine, 3-tri- V) ~~CX.4alkylsilylalaninecloeygci, diphenylglycine, phenylglycine, methionine suif oxide, methionine sulfone, 2 2 -dicyclohexylalanine, 2-(l- IND naphthylalatnine) 2 2 -naphthylalanine) phenyl substituted phenylalanine (where the substituents are selected from CI- 4 alkyl, perfluoroC,- 4 alkyl, CI.. 4 alkoxy, hydroxy, halo, amido, nitro, amino, CI.. 4 alkylamino, Cj- 4dialylamirto, carboxy or C 1 4 alkoxycarbonyl), aspartic acid, aspa ctic acid- 4Ct.. 4 alkyl, perfluorocI- 4 alkyl, Cj- 4alkoxy, hy'droxy, halo, amido, nitro, amino, Ci- 4alkylauin(,, Cldi al kyl amino, carboxy or Cj. .4alkoxycarbony) aspartic acid, aspartic acid-4-C 1 ,alkyl estier glutamic: acid, glutamic acid-5-CI.. 4 alkyl ester, cycloC-salkylaalanine, substituted cycloC 3 8alkylalanine (where the ring substituents are carboxy, CI- 4 -alkyl ester, cycloC3-sajlkylalanine, substituted CYClOC3-gal.kylalanine (where the ring substituents are carboxy, ClI4alkylcarboxy, CI..alkoxycarbonyl or aminocarbanyl) 2 2 -diphenylalanine and all alpha-C.. salkyl of all amino acid derivatives thereof, where the amino terminus of said second amino acid is unsubstit-ited or monosubstituted with a member of the group consisting of formyl, Cl-12 alkyl, YlCl-2alkyl, carboxyCl-8 alkyl, carboalkoxyC, 1 4 alkyl, phenyl Cl.. 4 alkyl, substituted phenylC 1 4 alkyl (where the phenyl substituents or independently selected from. one or more of, CI- 4 alkyl, perfluoroC 1 4 alkyl, C 1 4 alkoxy, hydroxy, halo, arnido, nitro, amino, Cl- 4 alkylamino, Ct- CI 4 dialkylamir.o, carboxy or Cl-,alkoxycarbonyl), 1, 1- dipehnylCl 1 4 alkyl, CI-Galkoxycarbonyl, phenylCi- 6alkoxycar bonyl, C-2alkylcarbonyl, perfluoroCl- 4alkylCo-4a:.kylcarbonyl, phenyCl- 4 alkylcarbonyl, substituted phenyC 4 alkylcarbonyl (where the phenyl substituents are independently selected from one or c-imore Of* C,- 4 alkyl, perfluoro CJ-4alkyl, C1- 4 alkoxy, hydroxy, halo, amido, nitro, amino, Cl. 4 alkylamino, Ci. 4 dialkylamino, carboxy or CI- 4 alkoxycarbonyl) 1, 1- dipheny1Cl-.,alkyl, perfluorC 1 4 alkyl, Cj- 4 alkoxycarbonyl), lO-camphorsulfonyl, phenylCl- 4 alkysulfonyl, substituted phenylC 1 4 alkylsul fonyl, C 1 4 alkysulfir.yl, perfluoro CI- 4 alkylsulfinyl, phenylsulf.-Lnyl, substituted phenylsulfinyl (where the phenyl sub:stituents are independently selected from one or more of, C 1 4 alkyl, perfluoroCl- 4 alkyl, CI- 4 alkoxy, hydroxy, halo, amido, nitro, amino, C 1 4 alkylamono, C, 4 dialkylamono, carboxy or CI- 4 alkoxycarbonyl) phenyC,- 4 alkylsulf:Lnyl, substituted phenylC 1 4 alkylsulfinyl I- naphthytsulfonyl, 2-naphthyisulfonyl, substituted naphthylsulifonyl (where the naphthyl substituent is selected from C 4 alkyl, perfluoraCI- 4 alkyl, CI- 4 alkoxy, hydroxy, halo amido, nitro, amino, Ci- 4 alkylamino, Ct- 4 dialkylaiino, carboxy or CL- 4 alkoxycarbonyl), I- haphthyl-sulfinyl, 2-haphthylsulfinyl and substituted naphthyl-!sulfinyl (where the naphthyl substituent is selected f~rom CI.. 4 alkyl., perfluoroC 1 4 alkyl, CI-4alkoxy, hydroxy, >.alo, amido, nitro, amino, CI- 4 alkylamino, C- 14dialkylamino, carboxy or CI- 4 alkoxycarbonyl); R, is selected from the group consisting of hydrogen and alkyl; R 2 is selacted from the group consisting of aminoC2- salkyl, quanidinoC 2 -5alkyl, Cl- 4 alkvlguanidinoc 2 diC,-.4alkylauanidinoC,-alkyl, amidinoC 2 -5alkvl, C,- 4alkY-1axaidiixoc2-5alkyl, diCI.4alky-lamidinoCz. 5 alkyl, Ci.. 3alkoxyCz-sallkyl, phenyl, substituted phenyl (wher-e the substituents are independently selected from one or more of amino, amidino, gua nidino, CI- 4 alkylamino, Ci.. 4d.ialkylaminD, halogen, perfiucro CI. 4 alkyl,' CI- 4 alkyl, C 1 3 alkoxy or nitro), benzyl, phenyl substituted benzyl (where the !iubst-ituents are independently selected from Cl one or more of, amaino, amidino, guanidino, C 1 4alkylauino, C t -4dialky-l1amino, halogen, perfluoro Ci.. 4 alkyl, C1-C4alkyIC.3lol nto, hydroxyCz- salkl, C '.slkylaminoCz.salkyl, Cl-sdialkylaminoCz-.salkyl, 4 -aminocyclohexYlCI-, 2 alkyl and Cl-.salkyl; p, is 0 or 1; B is 200 whexe n is 0-3, R, is H! or Ci-Salkyl and the carbonyl. moiety of B is bound to E; E is a-heterocycle selected from the group consisting of oxazolin-2-yi, oxazol-.2-yl, thiazol-2-yl, 2 -quinoxa.in-2yl, 2-pyridyl, 3-pyridyl, benzo(bjthiophen-2yl, triazol-4-yl triazol-6-yl, pyrazol-2--yi, 4,5, 6 7 -tetrahydrobenzothiazol-2yl, naphthoE2, l-dlthiazol-2-yI naphtho(1-2-dlthiazol-2-yl quinoxali-a- 2-yl, isoquinolin-1-yl, isoquinolin-3-yI, benzo tb'furan-2-71, [pyrazir.-2-yl, quinazolin-2-yl, isothiazol-3-vl, purin-8yul and a substituted heterocycle where the substituents are -62- selected from CI-4 from C-14alky, perfluoro CI-4alkyl,c 1 CI 4 alkoxy, hydroxy, halo, amido, nitro, amino, Ci- U) 4alkylamino, C-.4dialkylamino, carboxy, C1- 4alkoxycarboryl, hydroxy or phenylC 1 -4 c 5 alkylaminoca::bonyl; or pharmaceutically acceptable salts thereof. A method according to claim 9 wherein said C- compound contains a d-phenylalanine-proline-arginine Ssequence. cD 10 11. A method according to claim 10 wherein said Scompound is -N-Methyl-D-phenylalanyl-N- [4- [(aminoiminomethyl)amino]-1-(2- benzothiazolylcarbonyl) butyl ]-L-prolinamide.

12. A method according to claim 6 wherein said compound is a natural product which affects the PAR-2 pathway.

13. A method according to claim 12 wherein said compound is derived from one or more of the botanical families leguminosae, solanaceae, gramineae and cucurbitaceae.

14. A method according to claim 13 wherein said compound is derived from legumes. A method. according to claim 14 wherein said compound is selected from the group consisting of: undenatured soybean extract, limabean extract, blackbean extract or mixtures thereof.

16. A method according to claim 15 wherein said compound is selected from the group consisting of: fractions of undenatured soybean extract, limabean extract, blackbean extract and mixtures thereof.

17. A method according to claim 14 wherein said compound selected from the group consisting of soybean milk, limabean milk, blackbean milk, soybean extract, limabean extract, blackbean extract, soybean S-63- paste, limabean paste and blackbean paste and mixtures thereof.

18. A method according to claim 2 wherein said Scompound is a melanosome transfer inhibitor. CI 5 19. A method according to claim 1 wherein said compound activates the PAR-2 pathway. A method according to claim 19 wherein* said ci compound is a. PAR-2 agonist which binds to and activates V' PAR-2. D 10 21. A method according to claim 20 wherein said 0 compound is selected from the group consisting of SLIGRL, SLIGKVD and derivatives of SLIGRL and SLIGKVD which bind to and activate PAR-2 and mixtures thereof.

22. A metnod according to claim 19 wherein said compdund is a protease which activates PAR-2.

23. A method according to claim 22 wherein said compound is a serine protease which activates PAR-2.

24. A method according to claim 23 wherein said compound is selected from the group consisting of trypsin, tryptase, thrombin and proteases naturally- occurring in the skin which activates PAR-2. A method according to claim 1 wherein said compound is a melanosome transfer enhancer.

26. A composition for affecting changes in mammalian skin pigmentation comprising a pigmentation-changing effective amount of a compound which affects the PAR-2 pathway.

27. A coposition according to claim 26 wherein said compound is a PAR-2 pathway inhibitor.

28. A composition according to claim 27 wherein said compound is an antagonist of PAR-2.

29. A conposition according to claim 28 wherein said compound binds to or blocks but does not activate PAR-2. -64- A composition according to claim 29 wherein said compound is selected from the group consisting of antagonists based on SLIGRL which bind to or block but do not activate PAR-2, antagonists based on SLIGKVD which bind to or block but do not activate PAR-2 and mixtures thereof.

31. A composition according to claim 26 wherein said compound is a protease inhibitor.

32. A composition- according to claim 31 wherein said compound is a serine protease inhibitor.

33. A composition according to claim 32 wherein said compound is a thrombin and/or trypsin and/or tryptase inhibitor or an inhibitor of the serine proteases naturally occurring in the skin which activate PAR-2.

34. A composition according to claim 33 wherein said compound contains a d-phenylalanine-proline-arginine motif. A composition according to claim 33 wherein said compound is (S)-N-Methyl-D-phenylalanyl-N- 4- ((aminoiminomethyl) amino (2- benzothiazo.Lylcarbonyl)butyl] -L-prolinamide.

36. A composition according to claim. 31 wherein said compound a naturally-derived product which affects the PAR-2 pathway.

37. A composition according to claim 36 wherein said compound is derived from one or more of the botanical families leguminosae, solanaceae, gramineae and cucurbitaceae.

38. A composition according to claim 37 wherein said compound is: derived from legumes.

39. A composition according to claim 38 wherein said compound is; derived from soy, lima and/or black beans. A composition according to claim 39 wherein said compound is selected from a group consisting of: <D undenatured soybean extract, Limabean extract, blackbean extract or mixtures thereof.

41. A composition according to claim 40 wherein said compound is selected from the group consisting of soybean milk, limabean milk, blackbean milk, soybean Sextract, linabean extract, blackbean extract, soybean paste, limabean paste and blackbean paste and mixtures thereof.

42. A composition according to claim 40 wherein said compound is a fraction of soybean milk, soybean extract, (c soybean paste, limabean milk, limabean extract, limabean paste, blackbean milk, blackbean extract, blackbean paste and mixtures thereof.

43. A composition according to claim 26 wherein said compound is a melanosome transfer inhibitor.

44. A composition according to claim 26 wherein said compound activates the PAR-2 pathway. A composition according to claim 44 wherein said compound is a PAR-2 agonist which binds to and activates PAR-2.

46. A conposition according to claim 45 wherein said compound is selected from the group consisting of SLIGRL, SLIGKVD and derivatives of SLIGRL and SLIGKVD which bind to and activate PAR-2 and mixtures thereof.

47. A composition according to claim 26 wherein said compound is a protease which activates PAR-2.

48. A composition according to claim 47 wherein said compound i:3 a serine protease which activates PAR-2.

49. A ccmposition according to claim 48 wherein said compound is selected from the group consisting of trypsin, tryptase, thrombin and proteases naturally- occurring in the skin which activates PAR-2. A composition according to claim 26 wherein said compound is a melanosome transfer enhancer. IO 0 -66- C

51. A composition according to claim 26 wherein said SPAR-2 affecting compound is present in an amount of from Sabout 0.00011 to about 15% by weight/volume of said C-i composition.

52. A composition according to claim 51 wherein said VI compound is present in an amount from about 0.001 to C-i about 5% of said composition. c-i V 53. A composition according to claim 52 wherein said (0 compound is present in an amount from about 0.005 to about 1% of said composition.

54. A composition according to claim 26 comprising bean milk ir. an amount of from about 1 to about 99% by weight. 'A composition according to claim 26 comprising soybean trypsin inhibitor, limabean trypsin inhibitor or blackbean t::ypsin inhibitor in an amount of from about 0.01 to about 20% by weight.

56. A method according to claim 1 wherein said composition is applied twice daily for at least eight weeks.

57. A method according to claim 56 wherein said composition is applied 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.

58. A method according to claim 1 wherein said composition is administered orally.

59. A method according to claim 1 wherein said composition is administered parenterally.

60. A cosmetic composition according to claim 26 comprising said pigmentation-affecting compound and a cosmetically-acceptable vehicle. IN -67-

61. A composition according to claim 60 wherein said composition further comprises additional depigmenting agents.

62. A composition according to claim 61 wherein said composition further comprises tyrosinase inhibitors.

63. A composition according to claim 26 wherein said composition further comprises liposomes.

64. A composition according to claim 63 wherein said composition comprises glycerol dilaurate, cholesterol, ether and polyoxyethylene-9- lauryl ether. A composition according to claim 26 wherein said composition :further comprises anti-oxidants.

66. A composition according to claim 26 wherein said composition further comprises a sunscreen.

67. A composition according to claim 41 wherein said composition comprises from about 1 to about 99% bean milk, from a:out 0.1 to about 20% emulsifier and a preservative in an effective amount.

68. A composition according to claim 26 wherein said composition further comprises a compound selected from the group consisting of: anti-oxidants, sunscreens, moisturizers, bleaching agents, depigmentation agents, surfactants, foaming agents, conditioners, humectants, fragrances, viscosifiers, buffering agents, preservatives and a mixture thereof.