MXPA99003054A - Methods for treating skin pigmentation - Google Patents

Abstract

This invention relates to methods and compositions for bringing about changes in skin pigmentation. More particularly, this invention relates to compounds which affect melanogenesis and can used as depigmentingagents or as agents for darkening skin utilizing the PAR-2 pathway.

Description

METHODS TO TREAT SKIN PIGMENTATION FIELD OF THE INVENTION This invention relates to methods and compositions for producing skin pigmentation and / or causing depigmentation of the skin. More particularly, this invention relates to compounds that affect melanogenesis, and which can be used as depigmentation agents or as agents to darken the skin.

BACKGROUND OF THE INVENTION The coloration of the skin has been of interest to humans for many years. In particular, the ability to remove hyperpigmentation, such as that found in spots by age, freckles or aging of the skin generally, is of interest to individuals who desire a uniform build. In certain areas of the world, general bleaching of the body is convenient. There are also hypopigmentation and hyperpigmentation disorders that should be treated. Similarly, the ability to generate a tanned appearance without incurring photodamage due to solar radiation, is important for many individuals. There have been many proposed methods to achieve depigmentation, as well as to achieve skin darkening. For example, kojic acid, hydroquinone, retinoids and other chemical compounds have been used to achieve depigmentation. Dihydroxyacetone and similar chemical compounds have been used for their ability to "tan" the skin without exposure to the sun.

It has been found that many of these previous solutions are not acceptable. There is often a different line of demarcation between the areas of the skin to which said prior compositions have been applied. Therefore, the precise application of all these compounds is necessary to achieve the desired result. It has been found that many of these compounds are quite irritating to the skin and, therefore, inconvenient to use., Knowledge of the chemical and enzymatic basis of melanogenesis is widely documented. Melanocytes migrate from the embryonic neural crest in the skin to produce secretory granules, melanosomes, which produce melanin. Melanogenesis occurs within the melanosome, and melanin is then distributed to the keratinocytes by the melanocyte dendrites. The key enzyme of melanogenesis is tyrosinase, which initiates a cascade of reactions that convert tyrosine into the biopolymer melanin. Two proteins related to tyrosinase (TRP's), TRP-1 and TRP-2 are known. These proteins share approximately 40% homology with tyrosinase, and have catalytic activities as well as regulatory functions in melanogenesis. TRP-1 is the most abundant glycoprotein in melanocytes. Despite 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 TRPs share structural and biological properties with the family of genes for membrane protein associated with Hsosomes (LAMP); therefore, its delivery to the melanosomal membrane could induce its activation. A phosphorylation / dephosphorylation reaction in the cytoplasmic tails of these proteins could intervene in the regulation of melanogenesis. It has been shown that the beta isoform of the protein kinase C (PCC) family regulates human melanogenesis by activating tyrosinase. The tyrosinase gene expression, TRP-1 and TRP-2, is coordinated. These three enzymes are expressed in the human epidermis. In melanocytes cocultivated with keratinocytes, these transcripts are expressed at a ratio of 45:45:10, respectively. In cultured melanocytes alone, only transcripts of TRP-1 are present, indicating that a keratinocyte-derived signal is involved in the coordinated expression of these genes. The regulation of the interactions between keratinocytes and melanocytes and the mechanism of melanosome transfer in keratinocytes have not yet been understood. Protease activated receptor 2 (PAR-2) is a receptor coupled to 7 transmembrane G proteins, that is, related to, but different from, the thrombin receptors (TR, also called PAR-1 and PAR-3) in their sequence. Both receptors are proteolytically activated by a cut of arginine-serine in the extracellular domain. The newly created N-terminal ends then activate these receptors as ligands bound. Both receptors can be activated by trypsin, but only TRs are activated by thrombin. Only PAR-2 is activated by the tryptase of mast cells. Both receptors can also be activated by the peptides corresponding to their new N-terminal ends, independent of the cutoff of the receptor. SLIGRL, the mouse PAR-2 activating peptide, is equipotent in the activation of the human receptor. Although the role of TR is well documented, the biology of PAR-2 has not yet been fully identified. Recently, a function has been described for the activation of PAR-2 in the inhibition of growth and differentiation of keratinocytes (Derian et al., "Differential Regulation of Human Ceratinocyte Growt and Differentiation by a Novel Family of Protease-active Receptors", Cell Growt &; Differentiation, Vol. 8, pp. 743-749, July 1997).

BRIEF DESCRIPTION OF THE INVENTION In accordance with this invention, a method has been found to affect changes in skin pigmentation of mammals, which comprises applying topically to the skin of a mammal a compound that affects the PAR-2 pathway. The compositions of this invention may contain one or more compounds that act as trypsin, such as tryptase, such as serine protease or as PAR-2 agonists to increase pigmentation. Alternatively, they may contain one or more compounds that act as serine protease inhibitors, trypsin inhibitors, thrombin inhibitors, tryptase inhibitors, as inhibitors of the PAR-2 pathway, or as a PAR-2 antagonist to decrease pigmentation. , or cause "depigmentation". As used herein, "mammal" means any member "of the higher vertebrate animals comprising the class" Mammalia ", as defined in Webster's medical dictionary 407 (1986) and includes, but is not limited to, humans. As used in the present "receptor" it must include both intracellular and extracellular receptors, and must mean those molecules capable of receiving and transducing a signal.The term PAR-2 refers to protease activated receptor 2 or a related receptor activated by the Protease activated receptor 2 (hereinafter referred to as "PAR-2") is a serine protease activated receptor that is expressed in numerous tissues, including keratinocytes and fibroblasts, the thrombin receptor (also called PAR-1). , hereinafter "TR"), is a receptor activated by serine protease that is expressed in numerous tissues, including keratinocytes, the biological functions of PAR-2 and TR in the skin. They do not know each other well. However, it has been found that the interactions between keratinocytes and melanocytes, through the PAR-2 pathway, affects melanogenesis. It has been found that thrombin inhibitors and / or tryptase inhibitors and / or trypsin inhibitors and PAR-2 antagonists can be used as depigmentation agents without skin irritation. PAR-2 agonists and serine proteases such as trypsin and tryptase can be used as darkening agents. In addition, PAR-2 can be useful as a target for bleaching and obscuring agents.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A is a graph showing the increase or decrease in the relative pigmentation of melanocytes containing epidermal equivalents treated with known pigmentation and depigmentation agents, in accordance with the methods of this invention. Figure 1B is a graph showing the increase or decrease in relative pigmentation in melanocytes containing epidermal equivalents, treated in accordance with the methods and compositions of this invention. Figure 2 is a group of images of melanocytes containing epidermal equivalents treated with PAR-2 agonists and compound I. Figure 3 is a graph showing the increase or decrease of relative pigmentation in melanocytes containing epidermal equivalents, and treated in accordance with the methods and compositions of this invention. Figure 4A is a graph showing the dose / response with respect to the pigmentation of melanocytes containing epidermal equivalents, when treated with compositions of this invention. Figure 4B is a graph showing the response of melanocytes containing epidermal equivalents, after being exposed to ultraviolet light, followed by treatment with the compositions of this invention. Figure 5A is a photograph showing gels showing the expression of TR and PAR-2 in the skin, melanone cells and melanocytes containing epidermal equivalents. Figure 5B is a photograph showing gels showing the expression of TR and PAR-2 by primary human melanocytes. Figures 6A and 6B are photographs that show gels showing the expression of several genes after treatment with different concentrations of compound I and SLIGRL. Figure 7 is a graph showing the effects of different compositions of this invention on the brightness of guinea pig nipple pigmentation. Figure 8 is a photograph of the skin of pigs from Yucatan that has been treated with compositions of this invention to achieve depigmentation thereof. Figure 9 is a graph showing the skin brightness of Yucatan pigs during the course of treatment in accordance with the methods and compositions of this invention.

Figures 10A, 10B, 10C and 10D are photographs of histological sections of skin of pigs from Yucatan stained with F &M and treated with compositions containing compound I according to the methods of this invention at concentrations of 0.10 μM , 50 μM and 250 μM, respectively. Figures 11 A, 11 B and 11 C are photographs of electronic micrographic views of melanocytes containing epidermal equivalents, treated with compositions of this invention. Figures 11 E, 11 F and 11 H are photographs of electronic micrographic views of the skin of pigs from Yucatan, treated with the compositions of this invention. Figures 11D and 11G are photographs of electronic micrographic views of untreated sites of the skin of pigs from Yucatan. Figures 12A, 12B, 12C, 12D and 12E are photographs of histological sections of skin of Yucatan pigs stained with F &M, as follows: 12A shows untreated skin; 12B shows skin treated with the compositions of this invention after 8 weeks of treatment; 12C shows skin a week after stopping treatment; 12D shows skin two weeks after stopping treatment, and 12E shows skin four weeks after stopping treatment. Figure 13 is a photograph of histological sections stained with F &M taken from the skin of Yucatan pigs and treated with the compositions of this invention.

Figure 14 contains digital photographs of visible and ultraviolet light of human skin before treatment and subsequent to treatment with the compositions of this invention.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES It has been 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 of the skin of mammals. In the opinion of the authors, some of the compounds described in the patent of E.U.A. No. 5,523,308, which is incorporated herein by reference, and which behave as inhibitors of thrombin and / or trypsin and / or tryptase, will be useful in the methods of this invention. Some of these compounds are also described in Costanzo et al., "Potent Trombin Inhibitors Tat Probéte S1 'Subsite: Tripeptide Transition State Analogues Based on a Heterocycle-Activated Carbonil Group", J. Med. Chem. 1996 Vol. 39, pp. . 3039-3043, and they have the following structural formula: where: A is selected from the group consisting of C?--Alkyl, C 4 -carboxyalkyl, C? -4-C alco -4 -alkylcarbonyl, C? -4 alquiloalkylalkyl, C 4 -4-substituted phenylalkyl (where the substituents of phenyl are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, C 1-4 dialkylamino 4, carboxy or C1-4 alkoxycarbonyl), formyl, C1-4 alkoxycarbonyl, C1-2 alkylcarbonyl, C1-4 phenylalkoxycarbonyl, C3-7 cycloalkylcarbonyl, phenylcarbonyl, substituted phenylcarbonyl (where the phenyl substituents are independently selected of one or more of C? -44 alkyl) perfluoroalkyl of C 1-4, alkoxy of C 1-4, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, dialkylamino of C 1-4, carboxy or C 4 alkoxycarbonyl), C 1-4 alkylsulfonyl, C 1-4 alkoxysulfonyl, C 1-4 perfluoroalkylsulfonyl phenylsulfonyl, phenylsubonyl substituted (where the phenyl substituents are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C? 4, dialkylamino of C -? -, carboxy or C 1-4 alkoxycarbonyl), 10-camphorsulfonyl, phenylalkylsulfonyl of C 1-4, phenylalkylsulfonyl of C 1-4 substituted, alkylsulfinyl of C 1-4, perfluoroalkylsulfinyl of C-T 4, phenylsulphinium , substituted phenylsulfinyl (wherein the phenyl substituents are independently selected from one or more of C 1-4 alkyl), C1-4 perfluoroalkyl, C1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C1-4 alkylamino, C1-4 dialkylamino, carboxy or C1-4 alkoxycarbonyl), phenylalkylsulfinyl Cr4, phenylalkylsulfinyl of substituted C 1-4, 1-naphthylsulfonyl, 2-naphthylsulfonyl or substituted naphthylsulfonyl (wherein the naphthyl substituents are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, carboxy or C 1-4 alkoxycarbonyl), 1-naphthylsulfinyl, 2-naphthylsulfinyl or substituted naphthylsulfinyl (where the naphthyl substituents are independently selected from one or more of C 1-4 alkyl, perfluoroalkyl of C 1-4, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, C 1-4 dialkylamino, carboxy or C 1-4 alkoxycarbonyl); an amino acid D or L which is attached at its carboxy terminus to the nitrogen shown in Figure I, and is selected from the group consisting of alanine, asparagine, 2-azetidinecarboxylic acid, glycine, N-alkylglycine of C? -8, proline , 1-amino-1-cycloalkylcarboxylic acid of C -8, thiazolidin-4-carboxylic acid, 5,5-dimethylthiazolidin-4-carboxylic acid, oxazolidin-4-carboxylic acid, pipecolinic acid, valine, methionine, cysteine, serine, threonine, norleucine, leucine, ter-leucine, soleucine, phenylalanine, 1-naphthalanine, 2-naphthalanine, 2-thienylalanine, 3-thienylalanine, acid [1, 2, 3, 4] -tetrahydroisoquinolin-1 - carboxylic acid and [1, 2, 3, 4] -tetrahydroisoquinoline-2-carboxylic acid, wherein the terminal amino group of said amino acid is attached to a member selected from the group consisting of C 1-4 alkyl, tetrazole-5 -Cl-Cr 2 alkyl, C 1-4 carboxyalkyl, C 1-4 alkoxycarbonylalkyl, C 1-4 phenylalkyl, C 1-4 phenylalkyl or (wherein the phenyl substituents are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, dialkylamino of C1-4, carboxy or C1-4 alkoxycarbonyl), 1,1-diphenylalkyl of Ci-4, 3-phene-2-hydroxypropionyl, 2,2-diphenyl-1-hydroxyethylcarbonyl, [1, 2, 3, 4 ] -tetrahydroisoquinolin-1 -carbonyl, [1, 2, 3, 4] -tetrahydroisoquinoline-3-carbonyl, 1-methylamino-1-cyclohexanecarbonyl, 1-hydroxy-1-cyclohexanecarbonyl, 1-hydroxy-1-phenylacetyl, -cyclohexyl-1-hydroxyacetyl, 3-phenyl-2-hydroxypropionyl, 3,3-diphenyl-2-hydroxypropionyl, 3-cyclohexyl-2-hydroxypropionyl, formyl, C1-4 alkoxycarbonyl, C1-12 alkylcarbonyl, perfluoroalkyl of C1-4, C1-4 alkylcarbonyl, C1-4 phenylalkylcarbonyl, substituted C1-4 phenylalkylcarbonyl (wherein the phenyl substituents are independently selected from one or more of C1-4 alkyl, perfluoroalkyl of C1-4, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, C 1-4 dialkylamino, carboxy or C 1-4 alkoxycarbonyl), 1,1-diphenylalkylcarbonyl of C1-4, substituted C1-4-1-diphenylalkylcarbonyl (where the phenyl substituents are independently selected from one or more of C1-4 alkyl, C1-4 perfluoroalkyl, C1-4 alkoxy, hydroxy , halogen, amido, nitro, amino, C 1-4 alkylamino, dialkylamino of C 1-4, carboxy or alkoxycarbonyl of C 1-4), perfluoroalkylsulfonyl of C 1-4, alkylsulfonyl of C 1-4, alkoxysulfonyl of C 1-4, phenylsulfonyl substituted (wherein the phenyl substituents are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, dialkylamino C1-4. carboxy or C 1-4 alkoxycarbonyl), 10-camphorsulfonyl, C 1-4 phenylalkylsulfonyl, substituted C 1-4 phenylalkylsulfonyl, C 1-4 perfluoroalkylsulfinyl, C 1-4 alkylsulfinyl, phenylsulphinyl, substituted phenylsulfinyl (where the phenyl substituents are selected independently of one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, C 1-4 dialkylamino, carboxy or C 1-4 alkoxycarbonyl), 1-naphthylsulfonyl, 2-naphthylsulfonyl, substituted naphthylsulfonyl (where the naphthyl substituents are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy) -4, hydroxy, halogen, amido, nitro, amino, C1-4 alkylamino, dialkylamino of C1-4, carboxy or alkoxycarbonyl of C4), 1-naphthylsulfinyl, 2-naphthylsulfinyl, and substituted naphthylsulfinyl (where the substituents of naphtyl are independently selected from one or more alkyl of C 1-4, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, C 1-4 dialkylamino, carboxy or C 1-4 alkoxycarbonyl); or a polypeptide formed by two amino acids, wherein the first amino acid is an amino acid D or L, attached via its carboxy terminal end, to the nitrogen shown in formula I, and is selected from the group consisting of glycine, N-alkylglycine C1-8, alanine, -zetidinecarboxylic acid, proline, thiazolidin-4-carboxylic acid, 5,5-dimethylthiazolidin-4-carboxylic acid, oxazolidin-4-carboxylic acid, C3-8 1-amino-1-cycloalkylcarboxylic acid, 3-hydroxyproline, -hydroxyproline, 3- (C) alkoxy proline, 4- (C 1-4 alkoxy) proline, 3,4-dehydroxyproline, 2,2-dimethyl-4-thiazolidine carboxylic acid, 2,2-dimethyl-4-acid carboxylic oxazolidin, pipecolinic acid, valine, methionine, cysteine, asparagine, serine, threonine, leucine, ter-leucine, isoleucine, phenylalanine, 1-naphthalanine, 2-naphthalanine, 2-thienylalanine, 3-thienylalanine, acid [1, 2, 3,4] -tetrahydroisoquinoline-2-carboxylic ester, 4-alkyl ester of aspartic acid and 5-alkyl ester of C -? - 4 glutamic acid, and the second or amino acid D or L is attached to the terminal amino group of said first amino acid, and is selected from the group consisting of phenylalanine, 4-benzoylphenylalanine, 4-carboxyphenylalanine, 4- (carboxyl-C1-2 -aryloxy) phenylalanine, substituted phenylalanine (wherein the Phenyl substituents are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, C 1-4 dialkylamino. , carboxy or C 1-4 alkoxycarbonyl), 3-benzothienylalanine, 4-biphenylalanine, homophenylallanine, octahydroindol-2-carboxylic acid, 2-pyridylalanine, 3-pyridylalanine, 4-thiazothialanine, 2-thienylalanine, 3- (3-benzothienyl) alanine, 3-thienylalanine, tryptophan, tyrosine, asparagine, 3-trialkylsilylalanine of C? -4, cyclohexylglycine, diphenylglycine, phenylglycine, methionine sulfoxide, methionine sulfone, 2,2-dicyclohexylalanine, 2- (1-naphthylalanine), 2- (2-naphthylalanine), phenylalanine substituted with phenyl (don of the phenyl substituents are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, dialkylamino of C 1. -4, carboxy or C1-4 alkoxycarbonyl), aspartic acid, 4-C-alkyl aspartic acid, C1-4 perfluoroalkyl, C- alkoxy, hydroxy, halogen, amido, nitro, amino, C1- alkylamino , dialkylamino of CM, carboxy or alkoxycarbonyl of C1-4, aspartic acid, 4-alkyl ester of C1-4 of aspartic acid, glutamic acid, 5-alkyl ester of C1-4 of glutamic acid, C3-8 cycloalkylalanine, cycloalkylalanine of substituted C3-8 (where the ring substituents are carboxy, C-alkyl ester, C3.8 cycloalkylanine, substituted C3.8 cycloalkylanine (where the ring substituents are carboxy, C1-4 alkylcarboxy, alkoxycarbonyl or aminocarbonyl of C-, 2,2-diphenylalanine and C -? - 5 alkyl all alpha of all s amino acid derivatives thereof, wherein the amino terminal group of said second amino acid is unsubstituted or monosubstituted with a member of the group consisting of formyl, C-M2 alkyl, tetrazol-5-ylalkyl of C-2, carboxyalkyl of C-β-8, carboalkoxyalkyl of C 1-4, phenylalkyl of C-1,4, phenylalkyl of C-α-4 substituted (where the phenyl substituents are independently selected from one or more of C 1-4 alkyl, perfluoroalkyl of C1-4, C1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C1-4 alkylamino, dialkylamino of C1-4, carboxy or alkoxycarbonyl of C1-4), 1, 1-diphenylalkyl of C-, alkoxycarbonyl of C-uß, phenylalkoxycarbonyl of CIT, alkylcarbonyl of C? -2, perfluoroalkyl of de C 1-4, phenylalkylcarbonyl of C 1-4, phenylalkylcarbonyl of C? -4 substituted (where the phenyl substituents are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nit ro, amino, C1-4 alkylamino, C1-4 dialkylamino, carboxy or C1-4 alkoxycarbonyl), 1, 1-diphenylalkyl of C-, perfluoroalkyl of C? -4, alkoxycarbonyl of C-1-4, 10-camphorsulfonyl, phenylalkylsulfonyl of d-4, phenylalkylsulfonyl of substituted C-? -4, alkylsulfinyl of C 1-4, C 1-4 perfluoroalkylsulfinyl, phenylsulphinyl, substituted phenylsulfinyl (wherein the phenyl substituents are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, C 1-4 dialkylamino, carboxy or C 1-4 alkoxycarbonyl), phenylalkylsulfinyl Ci.

, Fenilalquilsulfinilo C- substituted, 1-naftilsuIfonilo, 2-naphthylsulfonyl, substituted naphthylsulfonyl I (wherein the substituents of naphthyl independently of one or more of C1-4alkyl is selected, C1-4 perfluoroalkyl, C1-4 alkoxy , hydroxy, halogen, amido, nitro, amino, alkylamino C1-4 dialkylamino C1-4 carboxy or C1-4 alkoxycarbonyl), 1-haftilsulfinilo, 2-naphthylsulfinyl and substituted haftilsulfinilo (where the phenyl substituents are selected independently of one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C? - alkoxy, hydroxy, halogen, amido, nitro, amino, C? - alkylamino, C 1-4 dialkylamino, carboxy or C 4 alkoxycarbonyl); R1 is selected from the group consisting of hydrogen and alkyl; R2 is selected from the group consisting of C2.8 aminoalkyl, guanidinoalkyl of C2-5 alkylguanidino C- C2-5 alkyl, -dialkylguanidino C? -4-alkyl C2-5, C2-5 amidinoalquilo, C 1-4 alkylamidino-C 2-5 alkyl, C 2-4 dialkylamidoino C 2-5 alkyl, C 2-5 alkoxy C 2-5 alkyl, phenyl, substituted phenyl (where the substituents are independently selected of one or more of amino, amidino, guanidino, C- alkylamino, C 1-4 dialkylamino, halogen, C 1-4 perfluoroalkyl, C 1-4 alkyl, C 1-13 alkoxy or nitro), benzyl, benzyl substituted with phenyl (wherein the substituents are independently selected from one or more of amino, amidino, guanidino, C? -4 alkylamino, C 1-4 dialkylamino, halogen, C 1-4 perfluoroalkyl, C 1-4 alkyl, C -? - 3 alkoxy or nitro), C2.5 hydroxyalkyl, C 2-5 alkylamino of C 2-5, dialkylamino of C? -5-C 2-5 alkyl, 4-aminocyclohexylalkyl of C 0 -2 and C 1-5 alkyl; p is 0 or 1; B is wherein n is from 0 to 3, R3 is H or C -? - alkyl, and the carbonyl portion of B is attached to E; E is a heterocycle selected from the group consisting of oxazolin-2-yl, oxazol-2-yl, thiazol-2-yl, thiazol-5-yl, thiazol-4-yl, thiazolin-2-yl, imidazole-2 yl, 4-yl oxo-2-quinoxalin-2-2-pyridyl, 3-pyridyl, benzo [b] thiophen-2-yl, thiazol-4-yl yl pyrazol-2-thiazole-6-yl, 4,5,6,7-tetrahydrobenzothiazol-2-yl, naphtho [2,1-d] thiazol-2-yl, naphtho [1-2-d] thiazol-2-yl, quinoxalin-2-yl, isoquinolin-1- yl, isoquinolin-3-yl, benzo [b] furan-2-yl, pyrazin-2-yl, quinazolin-2yl, isothiazol-5-yl, isothiazol-3-yl, purin-8-yl and a heterocycle substituted, wherein the substituyent.es are selected from C-1-4 alkyl, C perfluoroalkyl, C- alkoxy, hydroxy, halogen, amido, nitro, amino, C -? - 4 alkylamino, dialkylamino C1 -4, carboxy, C 1-4 alkoxycarbonyl, hydroxy or phenylalkylcarbonyl of C-; Or pharmaceutically acceptable salts thereof. More particuy, in the opinion of the authors, some of the compounds of the above formula containing a d-phenylalanine-proline-arginine motif must be effective in inhibiting the PAR-2 pathway and causing depigmentation. A particuy preferred compound that acts as an inhibitor of thrombin and trypsin and is active in depigmentation of mammalian skin is (S) -N-methyl-D-phenylalanyl-N- [4 - [(aminoiminomethyl) amine ] -1- (2-benzothiazolylcarbonyl) butyl] -L-prolinemia (Chemical Abstracts name) (hereinafter referred to as "Compound I"). It is suggested that other compounds that are analogous or that function in the same manner as compound I and are described in the U.S.A. No. 5,523,308, may be active in the methods and compositions of this invention. Other compounds that inhibit trypsin, such as serine protease inhibitors, and in particu soybean trypsin inhibitor (STI), will also be useful in the methods of this invention. Extracts of soy, bean and black beans, and other natural products made from these bean plants such as, but not limited to, bean milk, bean paste, miso and the like, also serve to reduce pigmentation by this mechanism. Other sources of serine protease inhibitors can be extracted from the species belonging to the following plant families: Solanaceae (eg, potato, tomato, tomatillo, and the like); Gramineae (for example, rice, buckwheat, sorghum, wheat, barley, oats, and the like); Cucurbitaceae (for example, cucumbers, squash, zucchini, scourer and the like); and, preferably, Leguminosae (e.g., beans, peas, lentils, peanuts, and the like). Although it is not desired to be limited by the following theory, it is speculated that the compounds capable of affecting the pigmentation of the skin interact directly or indirectly with the PAR-2 of keratinocytes or its activating protease, and thus affect melanogenesis, directly or indirectly. Possibly, the compounds of this invention induce, in the case of increased pigmentation, or reduce, in the case of reduced pigmentation, the signal to transport melanosomes by melanocytes, or to receive melanosomes by keratinocytes in the skin. The compounds that are active in the compositions and methods of this invention can be released topically by any means known to the person skilled in the art. If the release parameters of the topically active pharmaceutical or cosmetic agent so require, the topically active composition of this invention can be further preferably composed of a pharmaceutical or cosmetically acceptable carrier capable of functioning as a delivery system to allow agent penetration. Topically active on the skin. An acceptable vehicle for topical release of some of the compositions of this invention, particularly proteins such as trypsin and STI, may contain liposomes. Liposomes are most preferably nonionic and contain a) glycerol dilaurate (preferably in an amount of between about 5% and about 70% by weight, b) compounds having the steroid skeleton found in cholesterol (preferably in an amount of between about 5% and about 45% by weight); and c) one or more fatty acid ethers having from about 12 to about 18 carbon atoms (preferably in an amount between about 5% and about 70% by weight collectively), wherein the constituent compounds of the liposomes are preferably a ratio of approximately 37.5: 12.5: 33.3: 16.7. Most preferred are liposomes comprised of glycerol dilaurate / cholesterol / polyoxythylene 10-stearyl ether / polyoxyethylene 9-lauryl ether (GDL liposomes). Preferably, the liposomes are present in an amount, based on the total volume of the composition, of from about 10 mg / ml to about 100 mg / ml, and preferably from about 20 mg / ml to about 50 mg / ml. A ratio of 37.5: 12.5: 33.3: 16.7 is preferred. Suitable liposomes can preferably be prepared according to the protocol indicated in Example 1, although other methods commonly used in the art are also acceptable. The composition described above can be prepared by combining the desired components in a suitable container and mixing them under ambient conditions in any conventional high shear mixing medium well known in the art for non-ionic liposome preparations, such as those described in Niemiec. and others, "Influence of Nonionic Liposomal Composition On Topical Delivey of Petid Drugs Into Pilosebacious Units: An In Vivo Study Using the Hamster Ear Model," 12 Pharm. Res. 1184-88 (1995) ("Niemiec"), which is incorporated herein by reference in its entirety. We have found that the presence of these liposomes in the compositions of this invention can increase the depigmentation capabilities of some of the compositions of this invention.

Other preferable formulations may contain, for example, soy milk or other liquid formulations derived directly from legumes or other suitable vegetable. For example, said formulation may contain a large proportion of soy milk, an emulsifier that maintains the physical stability of the soy 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 skilled in the art can also be used as carriers for the release of the compositions of this invention. The source of active compound to be formulated will generally depend on the particular form of the compound. The small organic molecules and the peptide fragments can be chemically synthesized and provided in a pure form suitable for pharmaceutical / cosmetic use. The products of natural extracts can be purified according to known techniques. Recombinant sources of the compounds are also available to the person skilled in the art. In alternative embodiments, the topically active pharmaceutical or cosmetic composition can be optionally combined with other ingredients such as humectants, cosmetic adjuvants, antioxidants, bleaching agents, tyrosinase inhibitors and other known pigmentation agents, surfactants, foaming agents, conditioners, humectants, fragrances, viscosity enhancers, buffering agents, preservatives, sunscreens and the like. The compositions of this invention may also contain active amounts of retinoids (ie, compounds that bind to any member of the retinoid receptor family), and include for example, tretinoin, retinol, tretinoin esters or retinol, and the like. The topically active pharmaceutical or cosmetic composition should be applied in an amount effective to effect changes in the pigmentation of mammalian skin. As used herein, "effective amount" means a quantity sufficient to cover the region of the skin surface on which a change in pigmentation is desired. Preferably, the composition is applied in free form to the surface of the skin, such that based on 1 cm 2 of skin surface, it is present from about 2 μl / cm 2 to about 200 μl / cm 2 of topically active agent when a pigmentation change is desired. When a trypsin and thrombin inhibitor is used, such as compound I or its analogues, a synthetic or naturally occurring formulation such as an active compound should be present in the amount of from about 0.0001% to about 15% weight / volume of the composition. Preferably, it should be present in an amount from about 0.0005% to about 5% of the composition; more preferably, it should be present in an amount from about 0.001 to about 1% of the composition. Of course, these scales are suggested for the previous components. It is intended that the lower group of scales be effective for agonists / antagonists of the PAR-2 pathway, and / or inhibitors that have high therapeutic indices and do not require significantly larger concentrations or doses to be effective in the methods of this invention. These compounds can be of natural or synthetic origin. The liquid derivatives and natural extracts obtained directly from plant or botanical sources can be used in the compositions of this invention in a concentration (w / v) of about 1 to about 99%. Fractions of natural extracts and protease inhibitors of natural origin such as STI may have a different preferred scale, from about 0.01% to about 20%, and preferably from about 1% to about 10% of the composition. Of course, mixtures of the active agents of this invention can be prepared and used together in the same formulation, or in serial applications of different formulations. The present inventors have unexpectedly found that when topically active agents such as agonists and / or inhibitors of PAR-2 and trypsin and / or thrombin and / or tryptase and / or their inhibitors, are applied topically to the skin of an animal, a significant change in pigmentation is achieved. Preferably, depigmenting agents (as well as other agents of this invention that affect pigmentation) 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 as compound I and related compounds; from about 20% to about 99% for liquid derivatives and extracts of botanical materials; and from about 1% to about 20% for factions of natural extracts and protease inhibitors of natural origin, such as STI or mixtures thereof) between one and two times a day for a period until the skin exhibits a change in pigmentation . This may be about 4 to about 10 weeks or more. Then, once the pigmentation change is achieved, it can be applied at a less frequent time, for example about once a day to about twice a week, a lower concentration and doses (from about 0.00001% to about 0.005%). for compounds having high therapeutic indices, such as compound I and related compounds, from about 10% to about 90% for liquid derivatives and botanical extracts, and from about 0.01% to about 5% for fractions of natural and inbreeding extracts of protease of natural origin, such as STI or mixtures thereof) of active ingredient. The effects of the active agents of this invention are reversible; therefore, to maintain these effects, continuous application or administration must be carried out. The invention described illustratively herein may conveniently be practiced in the absence of any component, ingredient, or step not specifically described herein.

Several examples are given below to better illustrate the nature of the invention and the manner of carrying it out, although they do not serve to limit the scope of the methods and compositions thereof.

EXAMPLE 1 Protease inhibitors affect pigmentation To study the possible functions of the PAR-2 pathway in pigmentation, it was used. an equivalent in vitro epidermal system. The epidermal equivalent system used contained melanocytes. An epidermal equivalent system that is useful for developing this study is the MelanoDerm system, commercially available from MatTek Co .. This system contains normal human melanocytes, together with normal human-derived epidermal keratinocytes, which have been cultured to form a highly differentiated, multi-stratified model. of the human epidermis. In the following examples, the equivalents were treated with the test compounds for 3 days, and the samples were cultured on the fourth day after starting treatment. The harvested equivalents were stained with DOPA (a substrate for tyrosinase) and H & E (a standard histological stain) or as a Fontana-Mason stain (F &M), another stain known to the person skilled in the art. F &M staining is a silver staining technique that markedly and neatly cleans melanins that have high silver nitrate reducing activity. Human multiple-layer epidermal equivalents containing melanocytes were used as an in vitro model system to study the effect of protease inhibitors on melanogenesis. The epidermal equivalents used were commercially available as MelanoDerm from MatTek of Ashland, MA .. These equivalents are known to respond to ultraviolet light irradiation ("UVB"), and bleaching agents such as benzaldehyde and hydroquinone are known to increase and reduce the pigmentation, respectively. The epidermal equivalents of MelanoDerm were exposed to benzaldehyde (available from Sigma of St. Louis, Mo), hydroquinone (available from Sigma) and UVB irradiation. The UV irradiation was carried out with a UVB FS light source in an exposure chamber, with the plate covers removed and phosphate buffered saline (PBS, from Gibco-BRL, Gaithersburg, MD) present in the lower chamber . The intensity of the UVB light was measured with a UVX radiometer (UVP Inc., San Gabriel, CA). The equivalents were treated with 0.1-0.12 J / cm2. No loss of viability was observed in treated equivalents up to 0.3 J / cm2. On the fourth day of exposure to the test compounds / ultraviolet irradiation, the equivalents were fixed, sectioned and stained, or stained as a whole without sectioning. The MelanoDerm equivalents were fixed in formalin and placed in paraffin blocks, and sections of the MelanoDerm equivalents were stained in accordance with the following standard procedures: (1) H &E, (2) DOPA + H &E and (3) ) Fontana-Mason ("F &M") using standard techniques known to those skilled in the art. Alternatively, complete equivalents of MelanoDerm were stained, and their images were captured for image analysis. Three sections were processed per equivalent, three equivalents per experiment. Each experiment was repeated three times. DOPA is a substrate for tyrosinase. F &M identifies silver nitrate reducing molecules, which mainly identifies melanins. Sections stained with F &M were used for image analysis using Optomax image analysis systems, Optomax Inc., Hollis, NH. Alternatively, the Empire image database 1.1 was used in a P5-100 Gateway 2000 computer (Media Cybernetics, Silver Springs, MD) for image capture. Image Pro Plus version 4.0 was used for image analysis. The parameters measured were the following: (1) level of pigmentation within individual melanocytes, and (2) number of pigmented melanocytes per field, for the Optomax system, or (1) the surface area of silver deposits within melanocytes, and (2) the number of pigmented melanocytes for the Image Pro system. Using the Optomax system, the surface area of deposits was measured of silver within individual melanocytes in 60 melanocytes, using multiple sections of equivalents in triplicate per treatment. The number of melanocytes per field was calculated in these sections. The "pigmentation factor" was defined as the average surface area of silver deposits within an individual melanocyte, multiplied by the number of pigmented melanocytes per field. A value of 1 was assigned to untreated controls, and the values of the treatment groups were normalized to their relevant controls. Using the Image Pro system, the surface area of silver nitrate deposits and the number of melanocytes were measured by complete equivalents. A value of 1 was assigned to the untreated controls, and the values of the treatment groups were normalized to their relevant controls. Figure 1A is a graph showing the increase or decrease in relative pigmentation, measured and calculated by the full equivalent / Image Pro system, as described above, by exposure to benzaldehyde (50μM), hydroquinone (50μM) and UVB irradiation (0.12 J / cm2). Equivalents of human epidermis were also exposed to protease inhibitor mixtures, which are described in Table A below. Protease inhibitors are available from Boehringer Mannheim of Indianapolis, IN. Tablets from a cocktail of complete protease inhibitor available from Boehringer Mannheim, containing inhibitors of chymotrypsin, thermolysin, papain, pronase, pancreatic extract and trypsin were used. The soybean trypsin inhibitor ("STI") was obtained from Sigma, and was dissolved in 50 mg / ml of liposome vehicle or in 1xPBS. All other protease inhibitors used in this example in vitro were dissolved in 1xPBS. GDL liposomes were prepared as described in Niemic et al., Cited above, except for the following variants: the non-ionic liposomal formulation contained glycerol dilaurate (Emulsynt GDL, ISP Van Dyk) / cholesterol (Croda) / ether 10- polyoxyethylene stearyl (Brij76, ICI) / polyoxyethylene 9-lauryl ether, at a ratio of 37.5: 12.5: 33.3: 16.7. Hepes pH buffer, 0.05M, pH 7.4 (Gibco-BRL from 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 meanogenesis. As described in Figure 1B, some of the serine protease inhibitors, in particular STI (soybean trypsin inhibitor), were very effective in inhibiting melanogenesis.

TABLE A EXAMPLE 2 A protease-activated receptor intervenes in pigmentation Example 1 shows that STI reduces pigmentation. STI inhibits trypsin. Because it is known that trypsin activates TR and PAR-2, the possible partition of TR and PAR-2 in pigmentation was tested. MelanDerm epidermal equivalents of human were treated with the TR and PAR-2 agonists and antagonists described in Table B below, daily for three days. On the fourth day, the samples were harvested, fixed and stained with DOPA, H &E or F &M. Histological examination and complete equivalents revealed changes in pigmentation after treatments. Figure 2 shows the results of this example. As shown herein, the PAR-2 peptide agonist, SLIGRL, induced pigmentation in individual melanocytes. Treatment with compound I, a thrombin and trypsin inhibitor, resulted in reduced pigmentation. Figure 3 shows the results of the studies described in this example, which represent 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 could intervene in pigmentation. Hirudin, a specific thrombin inhibitor, and TFLLRNPNDK, a selective TR agonist, had no effect on pigmentation. However, SFLLRN, a less specific TR agonist, showed a tendency to clear or reduce pigmentation. This indicates that TR is less likely to intervene in pigmentation.

TABLE B EXAMPLE 3 Dose response relationship between protease-activated receptor signaling and melanogenesis Equivalents of MelanoDerm were treated with increasing concentrations of SLIGRL, the peptide agonist of PAR-2, at 0, 10 and 50 μM, in the same manner as described in Example 2. Staining with F &M was carried out on the fourth day. As shown in Figure 4A, increasing concentrations of SLIGRL, the activator of PAR-2, results in increased pigmentation. Trypsin, an activator of PAR-2, has the same effect. Treatment with increasing concentrations of compound I, the thrombin and trypsin inhibitor, from 0.1 μM to 1 μM resulted in a decreasing pigmentation (see Figure 4A). Pretreatment of the equivalents with UVB irradiation increased the melanogenesis compared to the untreated controls. Compound I also reduced this pigmentation induced by UVB light (Fig. 4B). This example demonstrates a dose response relationship to increase and decrease pigmentation, with modulation of PAR-2 signaling. This example also demonstrates that compound I can inhibit pigmentation and prevent pigmentation induced by UV light.

EXAMPLE 4 PAR-2 is expressed in keratinocytes, but not in meanocytes The expression of PAR-2 and TR has been previously demonstrated in keratinocytes and fibroblasts. This example demonstrates that PAR-2 is expressed in keratinocytes, but not in melanocytes. In addition, it demonstrates that TR is expressed in both keratinocytes and melanocytes. To demonstrate this, epidermal equivalents of MelanoDerm from human, cultures of primary melanocytes from human (neonatal and adult, from Clonetics of San Diego, CA) and mouse melanoma cells from Cloudman S91 of ATCC from Rockville, MD, were developed in culture. , and the total number of RNA molecules was extracted using reagent "RNA Stat-60", available from "Tel-Test B" Incorporated as described in Chomczymski, "Single Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-phenol-chloroform extraction, "162 Anal. Biochem. 156-69 (1987). A sufficient amount of RNase-free DNase, available from Promega Corporation under the trade name of "RQ1 Rnase-free DNase", was then added to the RNA extracted from each sample, so that each respective product gave 200 ng of RNA treated with DNase. using the procedures described in "" RNase-free DNase ", protocol published by Promega Corporation (May 1995). The resulting 220 ng of RNAse treated with DNase were reverse transcribed ("RT") by the procedure described in "Superscript II Reverse Transcriptase", a protocol published by Gibco-BRL (now Life Technologies, Incorporated) (April 1992) , using random hexamers such as random initiators that are commercially available from Life Technologies, Incorporated.

• The resulting RT products were then amplified by polymerase chain reaction ("PCR") using approximately 0.5 units (per 100 μl reaction) of a thermostable DNA polymerase that is commercially available from Perkin-Elmer-Cetus Corporation under the trade name "Taq polymerase", and approximately 0.1 μmoles / reaction of specific initiators of TR and PAR- 2, as described in Table C and in Marthinuss et al. 1995, incorporated herein by reference, or by initiators of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), available from Clontech Laboratories, Inc., of Palo Alto, CA in accordance with the procedures described in Marthinuss et al. 1995, or in the protocol accompanying the Clontech primers Laboratories. The PCR products were then analyzed using bromide gels % ethidium / 2% agarose according to well-known methods in the • technique, to compare the level of expression of certain genes in keratinocytes and melanocytes. When it was necessary to achieve better visualization, the resulting PCR products were precipitated with ethanol in accordance with well-known procedures. When initiators were used for G3PDH, only % of the PCR reaction products were used. A sample of RNA 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 in the relevant bands in the gels. As a positive control, a human skin RNA sample was used which was reverse transcribed when there were no commercial positive controls. The migration of the RT-PCR products on the gels was always identical to that of the positive controls, and those of the amplifier sizes reported. The relative quality of each respective RT-PCR reaction product was then compared by analyzing the level of G3PDH mRNA, a "housekeeping" gene, in each respective product. As illustrated in Figures 5 and 6, it was found that gene expression for G3PDH is similar at all time points examined, which thus allowed the comparison of the relative levels of gene expression for the desired genes. Figure 5A shows that, as expected, TR and PAR-2 are expressed in total skin and MelanoDerm equivalents ("DM"). However, S91 melanoma cells ("S91") did not express at PAR-2 or TR. To better investigate this, primary melanocytes of newborn ("mel-NB") and adult ("mel-A") were tested for the expression of TR and PAR-2. As shown in Figure 5B, the primary melanocytes of human express TR, but not PAR-2. Therefore, it is suggested that PAR-2 agonists and antagonists can interact with keratinocytes, but not with melanocytes, in MelanoDerm equivalents, and that TR agonists and antagonists can interact with both keratinocytes and melanocytes. Therefore, an interaction between keratinocytes and melanocytes is suggested, during which the PAR-2 signal in keratinocytes is converted into a pigmentation endpoint. 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.

TABLE C DNA initiators used in the RT-PCR test EXAMPLE 5 -Se i-p -m. -I "e -re f? Mntapfn p ~ .iierat.nnnitO-melanO t • nn for the -l depigmenting effect of compound I The results of Example 4 suggest that melanocytes alone may not respond to the depigmenting effect of PAR-2 antagonists. In fact, the level of pigmentation of human primary melanocytes or S91 cells induced by choleratoxin, which is reduced by hydroquinone and benzaldehyde, was not affected by compound I. Since PAR-2 is not expressed in melanocytes, the authors of the present proved the possible requirement of keratinocyte-melanocyte interactions for the depigmenting effect of compound I. Primary cultures of melanocyte were compared with identical cultures plated in epidermal equivalents (EpiDerm, lacking melanocytes) to create a culture Associated without contact between keratinocytes and melanocytes. These were also compared with the MelanoDerm equivalents, where the melanocytes are present in the basal layer of the equivalent. The cultures were treated for 3 days with compound I, with the SLIGRL agonist of PAR-2, and with the TFLLRNPNDK agonist of TR as indicated in Table D, and stained with DOPA on the fourth day. In Table D, the keratinocytes are indicated with "K", the melanocytes are indicated with "M" and the lack of keratinocyte-melanocyte contact is indicated as "non-contact K-M". As shown in Table D, no pigmentation effect was observed between primary melanocytes and in associated cultures treated with these agents. In the MeianoDerm equivalents, compound I was reduced and SLIGRL induced pigmentation, while TFLLRNPNDK had no effect. These results show that keratinocyte-melanocyte contact is required for the effect of PAR-2 on pigmentation.

TABLE D EXAMPLE 6 Compound I affects the expression of the melanocyte gene Equivalents of MelanoDerm were treated with increasing concentrations of the thrombin inhibitor and trypsin, compound I, or with increasing concentrations of the PAR-2 agonist SLIGRL. RNAs were extracted from the untreated ones, and equivalents treated with compound I were analyzed to determine the expression of the gene by means of RT-PCR in the manner indicated above in example 4. Gene-specific primers were designed as indicated in Table C above, and Clontech primers for human G3PDH were used as in example 4. The melanogenic genes tested for expression level were tyrosinase, TRP-1 and TRP-2. A dose-dependent reduction in TRP-1 mRNA levels and a dose-dependent increase in TRP-2 mRNA levels were observed in the samples treated with compound I, as shown in Figure 6A . However, tyrosinase expression was not affected. These changes correlate with the dose-dependent whitening effect of this inhibitor. Both patterns of gene expression resulted in a clearance effect. The enzyme TRP-2 processes the dopaquinone in 5,6-dihydroxindolcarboxylic acid (DHICA), instead of 5,6-dihydroxyindole (DHI). This process results in finely dispersed coffee eumelanin, unlike insoluble black eumelanin, and results in a lighter skin tone. TRP-1 stabilizes the melanogenic complex allowing the production of pigment. Reduced levels of TRP-1 result in reduced tyrosinase activity and reduced pigmentation. The lack of this protein results in albinism. However, the increasing concentrations of SLIGRL do not affect the expression of the melanogenic gene (Figure 6B). TRP-1 and TRP-2 are specific for melanocytes. Compound I inhibits trypsin and thrombin. Hirudin, a specific thrombin inhibitor, has no effect on pigmentation, as was observed previously in Example 2. In this way, the authors of the present decided to test whether trypsin and thrombin are expressed in the skin. A probe designed to detect both brain and gastric trypsins, as described in Table C, detected the expression of both mRNAs in a sample of total skin mRNA available from Invitrogen of Carlsbad, California, USA, as well as in equivalents of MelanoDerm The same expression pattern was detected for thrombin. Neither trypsin nor thrombin were expressed in normal melanocytes (Figures 5A, B). These data suggest that if trypsin activates PAR-2, it could be produced by keratinocytes only. As shown in Figure 6A, treatment with compound I resulted in increased trypsin expression. SLIGRL, which does not affect the genetic expression of melanogenesis (Figure 6B) also increased the expression of trypsin in the equivalents. The authors of the present study concluded that while trypsin is a possible natural activator of PAR-2 in the skin, and possibly affects pigmentation, its mRNA levels do not correlate with pigmentation. This suggests that another yet unidentified serine protease, which is inhibited by compound I, STI and the like, is the natural activator of PAR-2 in the epidermis. The compounds that induce or inhibit this protease could serve as darkening and clarifying agents respectively.

EXAMPLE 7 Inhibitors of thrombin and trypsin and PAR-2 agonists affect pigmentation In vivo Two guinea pigs were treated twice daily, five days a week for seven weeks, with compound I in a concentration of 1 and 10 μM in ethanol vehicle: propylene glycol, 70:30 on a pigmented nipple. The other nipple of each animal was treated with vehicles only and served as a control. The measurement in Cromámetro after seven weeks of treatment, revealed a dose-dependent clearance effect of +9.6 L * and almost 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 a concentration of 10 μM, twice daily five days a week for eight weeks. The measurement of the chromameter 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 indicated in figure 7.

EXAMPLE 8 Inhibitors of thrombin and trypsin and PAR-2 agonists affect In Vivo pigmentation.

A dwarf Yucatan pig was treated with compound I, SFLLRN, FSLLRN and SLIGRL, in a concentration of 10 μM. Each compound was applied in two places on the pig twice daily, five days a 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, caused a darkening effect as measured by the chromameter. SFLLRN and FSLLRN had no significant effects. Two Yucatan pigs were treated for seven and a half weeks, or for ten weeks, twice daily five days a week, with increasing concentrations of compound I. Four concentrations of active compound were used, as follows: 0, 10, 50 and 250 μM. Two sites were placed by concentration on opposite sides of the back of the pig. Chromameter measurements were taken before starting the treatment and then every two weeks. Photographs were taken periodically and at the end of the experiment. A visible lightening effect was observed during the fourth, fifth and sixth weeks of treatment, for the treatments of 250, 50 and 10 μM, respectively. By the eighth week, the whitening effect of the two highest doses were similar. These results are illustrated in Figure 8. The readings of the chromimeter (L *, which measure brightness) during the course of treatment of a pig, are shown in Figure 9. A saturation effect was observed, which is time dependent and . concentration. This example demonstrates a visual depigmentation effect by compound I, in the animal model system that most resembles pigmented human skin. At the end of these experiments, biopsies were taken for histological analysis and electron microscopy (EM). Histological samples were stained with H &E and F &M. Staining with H &E showed that there was no irritation, inflammatory response or changes in skin architecture, demonstrating the safety of the use of compound I for long periods. Staining with F &M showed that there is reduced pigmentation in the treated samples, both in the basal layer and in the entire epidermis. These results are illustrated in Figure 10. The untreated and vehicle treated sample (Figure 10A) were identical and darker. Treatment with 10 μM (Figure 10B) showed reduced pigmentation and the 50 and 250 μM treatments (Figure 10C, 10D, respectively) were the clearest. The results of this example suggest that the maximum whitening effect of compound I could be achieved with a higher concentration for a shorter period or with a lower concentration for a longer period. In this way, at least two different regimens can be used to achieve the desired skin whitening results.

EXAMPLE 9 Ultrastructural studies demonstrate the effect of compound I on skin in vitro and in vivo An ultrastructural analysis was performed on MelanoDerm equivalents and pigskin sites treated with compound I. The MelanoDerm equivalents treated with compound I were analyzed to determine the formation and distribution of melanosome using electron microscopy. The treated samples contained more melanosomes, but less mature melanosomes, that is, melanosomes that have reduced melanin production within the melanocytes, relative to the untreated controls (Figures 11A, 11B). Melanosomes containing dendrites were easily identified within the treated keratinocytes (Figure 11C), but it was difficult to find them within the control keratinocytes. This suggests abnormal melanosome formation and delayed or impaired transfer of melanosome to the keratinocytes in the treated samples. Also analyzed by skin microscopy were skin samples from Yucatan pigs treated with compound I for 8 weeks as described in example 8. The melanosomes within the keratinocytes of treated sites were smaller and less pigmented, compared to the controls (figure 11 D, 11E and 11F). In addition, the distribution of melanosomes within the treated skins was abnormal. The melanosomes were detected mainly in the epidermis-dermis boundary, in comparison with a random distribution in the untreated controls (figure 11G, 11 H). Although the present authors can not rule out other mechanisms, they suggest that keratinocytes treated with compound I were unable to actively take or receive melanosomes from the dendrites present.

EXAMPLE 10 The depigmenting effect of compound I in vivo is reversible A Yucatan pig was treated with compound I, 250 μM, during 8 weeks, 2 times a day, 5 times a week on eight sites. All the sites showed visible depigmentation at the end of the treatment period, as indicated in Figure 12B. During the next 4 weeks (beginning week 9 of the experiment), the color of the treated sites was monitored, and two biopsies were taken each week from the two treated sites. Biopsy of the untreated sites was also taken. The depigmenting effect could be visualized in one and two weeks after treatment, and a complete reversal was observed in the fourth week. Histological examination of sections of skin stained with F &M confirmed the visually observed repigmentation (as indicated in figure 12). As soon as one week after the treatment, repigmentation was demonstrated histologically. The visual observations correlate with the histological demonstration of pigmentation of the stratum corneum. This example demonstrates that compound I does not induce permanent damage to the pigmentation machinery, and that its effect is reversible in vivo. Figure 12A shows two histological sections stained with F &M from sites that were not treated with compound I. Figure 12B shows two histological sections stained with F &M from sites that were treated with compound I for 8 weeks. Figure 12C shows sections of sites that were treated for 8 weeks with compound I, one week after stopping treatment. Figure 12D shows sections of sites that were treated for 8 weeks with compound I, two weeks after stopping treatment. Figure 12E shows sections of sites that were treated for 8 weeks with compound I, four weeks after stopping treatment. As indicated in Figure 12E, the sections were completely repigmented four weeks after the end of the treatment.

EXAMPLE 11 Preparation of products of natural origin that contain STI Example 1 demonstrates that the presence of soy trypsin inhibitor in any lightening formulation is convenient for its depigmenting activity. Based on the analytical test, it has been determined that soy milk and soybean meal are rich sources of soybean trypsin inhibitor. To make the soybean paste, soy beans were first soaked in deionized or purified water for several hours. The soybeans were ground after being completely hydrated with the addition of small amounts of water if necessary, to soften the pasta. To make soy milk, the same procedure was developed with the addition of more water (the milling process allows the extraction of soy milk). After harvesting, the soy milk was filtered to remove any residual part of the soybean husk. Soy milk, soybean paste and millet were prepared to be used as naturally occurring materials that contain STI and are capable of lightening skin color.

EXAMPLE 12 Treatment with materials of natural origin that affect the PAR-2 pathway induces depigmentation Two pigs from Yucatan were treated for 8 and 10 weeks, twice a day, five times a week, with different products derived from soybeans and beans. These natural products include soybean paste, acid hydrolyzate from soy protein, millet, natural and boiled soy milk, and a commercially available soy extract (Actiphyte ™ from Active Organic, Dallas Texas), as well as purified STI and different preparations of trypsin inhibitors from soybeans and beans. At seven weeks of treatment, all sites were visually clearer than the surrounding skin, except for the sites treated with boiled soya milk and acid hydrolyzate of soy protein. Histological analysis of biopsies of sites treated after staining with F & amp; amp; amp; amp;; M, confirmed the depigmentation effect of soybean and bean products. An example of such histological data is given in Figure 13. The lack of depigmenting activity in boiled soya milk and in the acid hydrolyzate of soy protein, is explained by the denaturation or degradation of the soy proteins in these preparations. , respectively. The authors of the present theoretically consider that the depigmentation agents active in soybean and bean products are soybean trypsin (STI) inhibitor and bean trypsin inhibitor, respectively. (Example 1 shows the depigmenting effect of STI in vitro). This example demonstrates that natural extracts containing trypsin inhibiting activity can be used as bleaching agents that affect the PAR-2 pathway.

EXAMPLE 13 An STI in liposome formulation can clear age spots in humans An individual with three age spots was treated on the back of his or her hand for eight weeks, twice a day, with the following: the nearest localized age spot on the arm was treated with placebo containing 20 mg / ml of liposomes. The middle part of the age spot was not treated. The third age spot was treated with STI, 1%, in liposomes (20 mg / ml). GDL liposomes were prepared as indicated in Niemiec, et al., Cited above, with the exception of the following changes: the non-ionic liposomal formulation contained glycerol dilaurate (Emulsynt GDL, ISP Van Dyk / cholesterol (Croda) / polyoxyethylene ether) 10-stearyl in a 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 Digital photographs were taken in UV and light visible at time 0, 4 and 8 weeks of treatment L * values (brightness) were calculated from the images using Adobe Photoshop As shown in figure 14, the age spot treated with STI became clearer after 8 weeks of treatment Figure 14 is a combination of four photographs The left box is the visible light photograph of the hand, before (upper) and after (lower) 8 weeks of treatment. , the manc has superior is treated with placebo, the middle spot is without treatment, and the lower spot is treated with STI. The right frame shows the same hand at the same time points, using UV photography. UV light allows the visualization of the deepest pigment in the skin, demonstrating that the whitening effect of STI was not superficial. Figure 14 clearly demonstrates that the STI formulation was able to clear the lower spot. An increment of 15 L * units was calculated for this site treated with STI, demonstrating in addition the capacity of this treatment to lighten age spots.

EXAMPLE 14 Depigmentation formulations with soy milk In the preparation of soy milk, it was discovered that the rich emolliency of the milk would be convenient in a skin care formulation. Since water is used as the predominant ingredient of any oil-in-water emulsion and in many other skin care formulations, the present inventors consider that soy milk can be used to replace deionized water in such formulations. However, it would be expected that this type of formulation was not physically stable due to the immiscibility of the oil and water components of soy milk. Surprisingly, they found that this substitution of soy milk for water is physically stable. The formulations using soy milk should contain between about 1% and about 99% soy milk, preferably about 80% to about 95% soy milk. Preferably, this formulation and similar formulations should include a viscosity increaser in an amount from about 0% to about 5% (preferably 0.1 to about 2%), one or more emollients in an amount of up to about 20% and / or emulsifiers in an amount of from about 0.1% to about 10% (preferably from about 3 to about 5%), and optionally an extender in an amount of from about 0 to about 5% (preferably from about 1 to about 2%) , a preservative, a chelating or moisturizing agent. The preservative must be present in an effective amount to preserve the integrity of the milk and maintain the activity of the composition. Sufficient thickener must be present to impart body to the formulation without causing it to become too viscous to prevent its spreading, for example about 0 to about 10%, preferably about 3 to about 5%. Sunscreens, antioxidants, vitamins, other depigmenting agents and other topical ingredients for skin care can also be incorporated into the compositions of this invention. A particularly preferred example of the depigmenting formulation that replaces water with soy milk is shown in Table E below.

TABLE E STI, soybean paste and other natural extracts containing trypsin inhibitor can be incorporated into such formulations to provide increasing concentrations of the serine protease inhibitor. The levels of use of the added active ingredient can vary between 0.01% to 15% in a formulation. Other depigmenting agents may also be incorporated into this formulation, including inhibitors of PAR-2, tyrosinase inhibitors, hydroquinones, soy products, ascorbic acid and its derivatives, as well as other ingredients with skin care benefits.

EXAMPLE 15 A depigmenting formulation of oil in water emulsion Table F shows two examples of a depigmenting formulation with oil in water emulsion. In column 4 of table F, a formulation with STI is described, in which the STI could be replaced with any serine protease inhibitor of natural origin, or with any naturally occurring extract or fraction thereof containing serine protease inhibitors. In column 5 of table F a similar formulation with compound I is presented. Compound I in this composition could be replaced with similar compounds, or with serine protease inhibitors or with any PAR-2 inhibitor material having high therapeutic indices , whether of synthetic or natural origin, as the active ingredient. The suggested scales for the ingredients in these formulations are also presented in Table F. The deionized water content of these formulations can be replaced with soy milk.

TABLE F To prepare this formulation, the ingredients of the liquid phase were combined and mixed at 85 ° C, and then cooled to 60 ° C. In a separate container, carbopol was added slowly to water or soy milk. After mixing for 10 minutes, the rest of the ingredients in the aqueous phase were added and the mixture was heated to 60 ° C. The two phases were then combined, mixed for 10 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 compositions of this invention.

EXAMPLE 16 Depigmentation composition (oil in water emulsion) In Table G, two additional examples of a depigmenting formulation in oil-in-water emulsion are presented. Column 3 of Table G describes a formulation with STI, where the STI could be replaced with any serine protease inhibitor of natural origin or with any extract of natural origin or fraction thereof containing serine protease inhibitors. In column 4 of table G a similar formulation with compound I is presented. Compound I in this composition could be replaced with similar compounds or with serine protease inhibitor or with any PAR-2 inhibitor material having high therapeutic indices, either of natural or synthetic origin, as the active ingredient. The suggested scales for the ingredients in these formulations are also indicated in Table G. The deionized water content of these formulations could be replaced with soy milk. TABLE G To prepare this formulation, the hydroxyethylcellulose was slowly added to water or soy milk and stirred until complete dissolution. In a separate container, the interlaced polymer of acrylates / C10-30 alkyl acrylate was added and stirred until completely dissolved. The contents of the two containers were combined and mixed for 20 minutes. Vitamin E acetate was then added and mixed, followed by the addition of isohexadecane and panthenol (98%). After mixing for 5 minutes, the STI, or the natural extract, or the compound I, together with propylene glycol, was added and stirred for 5 minutes. Then glycerin was added and the formulation was stirred for 20 minutes. Finally, the pH was adjusted with sodium hydroxide to 8 for STI (the scale is 6-8.5) or to 7 for compound I (the scale is 5.5-8.5).

EXAMPLE 17 Depigmentation composition (water-in-oil emulsion) Table H shows an example of a depigmenting formulation with water-in-oil emulsion. Column 4 of Table H describes a formulation with STI, where the STI could be replaced with any serine protease inhibitor of natural origin, or with any extract of natural origin or fraction thereof containing serine protease inhibitors. In column 5 of table H a similar formulation with compound I is presented. Compound I in this composition could be replaced with similar compounds or with serine protease inhibitor or with any PAR-2 inhibitor material having high therapeutic indices, either of natural or synthetic origin, as the active ingredient. The suggested scales for the ingredients in these formulations are also presented in Table H. The deionized water content of these formulations could be replaced with soy milk. TABLE H To prepare this formulation, stearyl alcohol and mineral oil were melted at 70 ° C. The other ingredients of the oil phase were added and the mixture was heated to 75 ° C. The ingredients of the aqueous phase, which had previously been dissolved in most of the aqueous phase or soy milk and then heated to 70 ° C, were then added, and the mixture was stirred until frozen.

EXAMPLE 18 Depigmentation composition (aqueous gel) Table J shows two examples of a depigmenting formulation with aqueous gel. Column 3 of Table J describes a formulation with STI, where the STI could be replaced with any serine protease inhibitor of natural origin, or any naturally occurring extract or fraction thereof containing serine protease inhibitors. In column 4 of table J a similar formulation with compound I is presented. Compound I in this composition could be replaced as the active ingredient with similar compounds or with serine protease inhibitor or with any PAR-2 inhibitor material having high therapeutic indices, either of natural or synthetic origin. The suggested scales for the ingredients in these formulations are also presented in Table J. The deionized water content of these formulations could be replaced with soy milk.

BOX J Solvent based depigmenting composition Table K presents an example of a depigmenting formulation containing solvent. Column 3 of Table K describes a formulation with STI, where the STI could be replaced with any serine protease inhibitor of natural origin, or any naturally occurring extract or fraction thereof containing serine protease inhibitors. In column 4 of table K a similar formulation with compound I is presented. Compound I in this composition could be replaced as the active ingredient with similar compounds or with serine protease inhibitor or with any PAR-2 inhibitor material having high therapeutic indices, either of natural or synthetic origin. The suggested scales for the ingredients in these formulations are also presented in Table K. The deionized water content of these formulations could be replaced with soy milk.

TABLE K To prepare this formulation, compound I was dissolved in water. The ethanol and propylene glycol were mixed and combined with the aqueous solution containing the compound I. In summary, the present inventors have shown that the activation of the keratinocyte receptor PAR-2 results in increased pigmentation. Preferably, said activation can be achieved by the use of trypsin or SLIGRL or SLIGKVD, or other derivatives of SLIGRL or SLIGKVD. They have also shown that whitening can be achieved by the use of serine protease inhibitors or PAR-2 antagonists, as well as by means of melanosome transfer blockers. Other compounds known to those skilled in the art that inhibit the transfer of melanosome to keratinocytes could also be used as depigmenting agents. The compound I, an inhibitor of trypsin and thrombin, for example, inhibits the transfer of melanosome to keratinocytes. The STI works through the same mechanism. The accumulation of melanosomes not released in the melanocytes could induce a negative feedback mechanism that retards the formation of new melanosome. The production of TRP-1, the largest lycopene in the melanocytes, is negatively regulated which leads to the destabilization of tyrosinase. This results in reduced melanin formation, and a lighter coffee color change, reducing the ratio of TRP-1.; TRP-2. The accumulation of melanosomes in the melanocyte after treatment with compound I, or after treatment with STI, has therefore reduced and altered the melanin content, which adds to the whitening effect of compound I or STI.

Claims (68)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for effecting changes in mammalian skin pigmentation, comprising administering to a mammal an effective amount to change the pigmentation of a compound that affects the PAR-2 pathway.

2. A method according to claim 1, characterized in that said compound inhibits the PAR-2 pathway.

3. A method according to claim 2, characterized in that said compound is a PAR-2 antagonist.

4. A method according to claim 3, characterized in that said compound binds to PAR-2, or blocks it, but does not activate it.

5. A method according to claim 4, characterized in that said compound is selected from the group consisting of antagonists based on SLIGRL, which bind to PAR-2 or block it, but do not activate it, antagonists based on SLIGKVD that are they bind PAR-2 or block it, but they do not activate it, and mix it.

6. A method according to claim 2, characterized in that said compound is a protease inhibitor.

7. A method according to claim 6, characterized in that said compound is a serine protease inhibitor.

8. - A method according to claim 7, characterized in that said compound is an inhibitor of thrombin and / or tryptase and / or trypsin.

9. A method according to claim 8, characterized in that said compound is a compound of formula I: Wherein: A is selected from the group consisting of C? .s alkyl,? or C-? - carboxylalkyl, C 1-4 dCalkyl alkoxycarbonyl, C 1-4 phenylalkyl, substituted C 1-4 phenylalkyl ( wherein the phenyl substituents are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, dialkylamino of C 1. -4, carboxy or C1-4 alkoxycarbonyl), formyl, alkoxycarbonyl 15 ^^ C1-4 alkylcarbonyl Cr2, phenylalkoxycarbonyl C1-4 cycloalkylcarbonyl C3-7, phenylcarbonyl, substituted phenylcarbonyl (where the phenyl substituents independently of one or more of C1-4alkyl is selected , C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, C 1-4 dialkylamino, carboxy or C 1-4 alkoxycarbonyl), 20 alkylsulfonyl C1-4 alkoxysulfonyl C1-4 perfluoroalquilsulfonilo C1-4 phenylsulfonyl, substituted fenilsufonilo (where the phenyl substituents independently of one or more of C1-4alkyl is selected, perfluoroalkyl C1- 4, C1-4alkoxy, hydroxy, halo, amido, nitro, amino, alkylamino C1-4 dialkylamino C1-4 carboxy or C1-4 alkoxycarbonyl), 10-camphorsulfonyl, C1-4 fenilalquilsulfonilo , fenilalquilsulfonilo C1-4 substituted, alkylsulfinyl C1-4 perfluoroquilsulfinilo C1-4 phenylsulfinyl, substituted phenylsulfinyl (where the phenyl substituents independently of one or more of C1-4alkyl is selected, C1-4 perfluoroalkyl , C1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, alkylamino of 1-4. C1-4 dialkylamino, carboxy or C1-4 alkoxycarbonyl), Ci-4 fenilalquilsulfinilo, fenilalquilsulfinilo of C1-4 substituted, 1-naphthylsulfonyl, 2-naphthylsulfonyl or naphthylsulfonyl sub substituted (wherein the naphthyl substituents are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, carboxy or C1-4 alkoxycarbonyl), 1-naphthylsulfinyl, 2-naphthylsulfinyl or substituted naphthylsulfinyl (where the substituents are independently naphthyl one or more of C1-4alkyl, perfluoro C1-4alkyl, C1-4alkoxy selected, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, C 1-4 dialkylamino, carboxy or C 1-4 alkoxycarbonyl); an amino acid D or L which is attached at its carboxyl terminal end to the nitrogen shown in Figure I, and is selected from the group consisting of alanine, asparagine, 2-azetidinecarboxylic acid, glycine, C? -8 N-alkylglycine, proline, C3-8 1-amino-1-cycloalkylcarboxylic acid, thiazolidin-4 acid -carboxylic acid, 5,5-dimethylthiazolidin-4-carboxylic acid, oxazolidin-4-carboxylic acid, pipecolinic acid, valine, methionine, cysteine, serine, threonine, norleucine, leucine, ter-leucine, isoleucine, phenylalanine, 1-naphthalanine, 2- naphthalanine, 2-thienylalanine, 3-thienylalanine, [1, 2, 3, 4] -tetrahydroisoquinoline-1-carboxylic acid and [1, 2, 3, 4] -tetrahydroisoquinoline-2-carboxylic acid, where the group Amino terminal of said amino acid is attached to a member selected from the group consisting of C 1-4 alkyl, tetrazol-5-yl-d-2 alkyl, C 1-4 carboxyalkyl, C 1-4 alkoxycarbonylalkyl, phenylalkyl of C- 1-4, C 1-4 substituted phenylalkyl (wherein the phenyl substituents are independently selected from one or more of C 1-4 alkyl, perfluoroalkyl of C? -4, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, dialkylamino of C 1-4, carboxy or C 1-4 alkoxycarbonyl), 1,1-diphenylalkyl of C-1-4, 3-phenyl-2-hydroxypropionyl, 2,2-diphenyl-1-hydroxyethylcarbonyl, [1,2,3,4] -tetrahydroisoquinolin-1-carbonyl, [1, 2, 3, 4] - tetrahydroisoquinoline-3-carbonyl, 1-methylamino-1-cyclohexanecarbonyl, 1-hydroxy-1-cyclohexanecarbonyl, 1-hydroxy-1-phenylacetyl, 1-cyclohexyl-1-hydroxyacetyl, 3-phenyl-2-hydroxypropionyl, 3,3- diphenyl-2-hydroxypropionyl, 3-cyclohexyl-2-hydroxypropionyl, formyl, C 1-4 alkoxycarbonyl, C 1-12 alkylcarbonyl, C 1-4 perfluoroalkyl, C 1-4 alkylcarbonyl, C 1-4 phenylalkylcarbonyl, phenylalkylcarbonyl C1-4 substituted (wherein the phenyl substituents are independently selected from one or more of C1-4 alkyl, C1-4 perfluoroalkyl, C1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C1 alkylamino) -4, C1-4 dialkylamino, c arboxi or C1-4 alkoxycarbonyl), 1, 1-diphenylalkylcarbonyl C1-4, 1, 1-diphenylalkylcarbonyl substituted C1-4 (wherein the phenyl substituents are independently selected from one or more C1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, C 1-4 dialkylamino, carboxy or C 1-4 alkoxycarbonyl), C 1-4 perfluoroalkylsulfonyl , C 1-4 alkylsulfonyl, C 1-4 alkoxysulfonyl, substituted phenylsuiphenyl (wherein the phenyl substituents are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, dialkylamino of C 1-4, carboxy or alkoxycarbonyl of C 1-4), 10-camphorsulfonyl, phenylalkylsulfonyl of C 1-4, phenylalkylsulfonyl of C 1-4 substituted, C1-4 perfluoroalkylsulfinyl, C1-4 alkylsulfinyl, phenylsulfinyl, substituted phenylsulfinyl (wherein the substituents of phenyl are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, dialkylamino C1-4, carboxy or C1-4 alkoxycarbonyl), 1-naphthysulfonyl, 2-naphthylsulfonyl, substituted naphthylsulfonyl (wherein the naphthyl substituents are independently selected from one or more of C1-4 alkyl, C1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, C 1-4 dialkylamino, carboxy or C 1-4 alkoxycarbonyl), 1-naphthylsulfinyl, 2-naphthylsulphinyl, and substituted naphthylsulfinyl (wherein the naphthyl substituents are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino , C 1-4 dialkylamino, carboxy or C 1-4 alkoxycarbonyl); or a polypeptide formed by two amino acids, wherein the first amino acid is an amino acid D or L, attached via its terminal carboxyl terminus to the nitrogen shown in formula I, and is selected from the group consisting of glycine, N-alkylglycine of C? -8, alanine, 2-azetidinecarboxylic acid, proline, thiazolidin-4-carboxylic acid, 5,5-dimethylthiazolidin-4-carboxylic acid, oxazolidin-4-carboxylic acid, 1-amino-1-cycloalkylcarboxylic acid of C3-8, 3-hydroxyproline, 4-hydroxyproline, 3- (C 1-4 alkoxy) proline, 4- (C 1-4 alkoxy) proline, 3,4-dehydroxyproline, 2,2-dimethyl-4-thiazolidine carboxylic acid, 2-hydroxyproline. , 2-dimethyl-4-oxazolidin carboxylic acid, pipecolinic acid, valine, methionine, cysteine, asparagine, serine, threonine, leucine, ter-leucine, isoleucine, phenylalanine, 1-naphthalanine, 2-naphthalanine, 2-thienylalanine, 3-thienylalanine , [1, 2,3,4] -tetrahydroisoquinoline-2-carboxylic acid, 4-alkyl ester of C? -4 aspartic acid and 5-alkyl ester C 1-4 acid of glutamic acid, and the second amino acid D or L is attached to the terminal amino group of said first amino acid, and is selected from the group consisting of phenylalanine, 4-benzoylphenylalanine, 4-carboxyphenylalanine, 4- (carboxyalkyl) of C1-2) phenylalanine, substituted phenylalanine (wherein the phenyl substituents are independently selected from one or more of C1-4 alkyl, C1-4 perfluoroalkyl, C1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, C 1-4 dialkylamino, carboxy or C 1-4 alkoxycarbonyl), 3-benzothienylalanine, 4-biphenylalanine, homophenylalanine, octahydroindol-2-carboxylic acid, 2-pyridylalanine, 3- pyridylalanine, 4-thiazothialanine, 2-thienylalanine, 3- (3-benzothienyl) alanine, 3-thienylalanine, tryptophan, tyrosine, asparagine, 3-trialkylsilylalanine C1-4, cyclohexylglycine, diphenylglycine, phenylglycine, methionine sulfoxide, methionine sulfone, 2,2-dicyclohexylalanine, 2- (1-naphthylalanine), 2- (2-naphthi) lalanin), phenyl substituted phenylalanine (wherein the substituents are independently selected from C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, C1-4 dialkylamino, carboxy or C1-4 alkoxycarbonyl), aspartic acid, 4-C4 alkyl of aspartic acid, CM perfluoroalkyl, CM alkoxy, hydroxy, halogen, amido, nitro, amino, alkylamino of CM , CM dialkylamine, carboxy or alkoxycarbonyl of CM, aspartic acid, 4-alkyl ester of aspartic acid CM, glutamic acid, 5-alkyl ester of glutamic acid CM, C3-8 cycloalkylanine, substituted C3-8 cycloalkylanine ( wherein the ring substituents are carboxy, CM alkyl ester, C3.8 cycloalkylanine, substituted C3-8 cycloalkylanine (wherein the ring substituents are C, -C 4, C 4 alkylcarboxyl, alkoxycarbonyl, or CM 2 aminocarbonyl). , 2-diphenylalanine and alkyl of C -? - 5 all alpha of all the amino acid derivatives thereof, wherein the amino terminal group of said second amino acid is unsubstituted or monosubstituted with a member of the group consisting of formyl, C1-12 alkyl, tetrazole -5-C1-6alkyl, carboxyalkyl of C -? 8 > CM-carboalkoxyalkyl, CM phenylalkyl, substituted C1-4 phenylalkyl (wherein the phenyl substituents are independently selected from one or more of C1-4 alkyl, C1-4 perfluoroalkyl, C1-4 alkoxy, hydroxy , halogen, amido, nitro, amino, alkylamino of O1-4, dialkylamino of C 1-4, carboxy or alkoxycarbonyl of C 1-4), 1,1-diphenylalkyl of CM, alkoxycarbonyl of C? -6, phenylalkoxycarbonyl of d-? , C 1 -C 2 alkylcarbonyl, C 1-4 perfluoroalkyl C-alkylcarbonyl, CM phenylalkylcarbonyl, substituted CM phenylalkylcarbonyl (wherein the phenyl substituents are independently selected from one or more of d-4 alkyl, C1 perfluoroalkyl 4, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, C 1-4 dialkylamino, C 1-4 carboxy or alkoxycarbonyl), 1,1-diphenylalkyl of C? -4, perfluoroalkyl of CM, alkoxycarbonyl of CM, 10-camphorsulfonyl, phenylalkylsulfonyl of CM, phenylalkylsulfonyl of substituted CM, alkylsulfinyl of CM, CM perfluoroalkylsulfinyl, phenylsulfinyl, substituted phenylsulfinyl (wherein the phenyl substituents are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy , halogen, amido, nitro, amino, C 1-4 alkylamino, dialkylamino of C 1-4, carboxy or alkoxycarbonyl of C 1-4), phenylalkylsulfinyl of C? -4, phenylalkylsulfinyl of substituted CM, 1-naphthylsulfonyl, 2- naphthylsulfonyl, substituted naphthylsulfonyl (where the naphthyl substituents are independently selected from one or more of C 1-4 alkyl, C 1-4 perfluoroalkyl, C 1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino) 4, C1-4 dialkylamino, carboxy or alkoxycarbonyl C1-4), 1 -haftiisulfinyl, 2-haftiisulfinyl and substituted naphthylsulfinyl (wherein the phenyl substituents are independently selected from one or more of C1-4 alkyl, C1-4 perfluoroalkyl, C1-4 alkoxy, hydroxy, halogen, amido, nitro, amino, C 1-4 alkylamino, dialkylamino of C -? - 4, carboxy or C 1-4 alkoxycarbonyl); R-i is selected from the group consisting of hydrogen and alkyl; R2 is selected from the group consisting of C2.8 aminoalkyl, C2-5 guanidinoalkyl, C2-5 alkyl CM-guanidino, CM2-C2-5 alkyl dialkylguanidino, C2-5 amidinoalkyl, C? Alkylamidino? -4-C2-5 alkyl, C2-4 dialkylamidoino C2-5 alkyl, C2-5 alkoxy C2-5 alkyl, phenyl, substituted phenyl (wherein the substituents are independently selected from one or more than amino, amidino, guanidino, C -4 alkylamino, dialkylamino of CM, halogen, perfluoroalkyl of C, alkyl of CM, alkoxy of C-? 3 or nitro), benzyl, benzyl substituted with phenyl (wherein the substituents are independently selected from one or more of amino, amidino, guanidino, CM alkylamino, CM dialkylamino, halogen, CM perfluoroalkyl, CM alkyl, C-? 3 alkoxy or nitro), C2.5 hydroxyalkyl, C 2-5 alkylamino of C 2-5 alkyl, dialkylamino of Ct-5-C 2-5 alkylo, 4-aminocyclohexylalkyl of Co-2 and C 1-5 alkyl; p is 0 or 1; B is wherein n is from 0 to 3, R3 is H or C1-5 alkyl, and the carbonium portion of B is attached to E; E is a heterocycle selected from the group consisting of oxazolin-2-yl, oxazol-2-yl, thiazol-2-yl, thiazol-5-yl, thiazol-4-yl, thiazolin-2-yl, imidazole- 2-yl, 4-oxo-2-quinoxalin-2-yl, 2-pyridyl, 3-pyridyl, benzo [b] thiophen-2-yl, thiazol-4-yl, thiazol-6-yl, pyrazole-2- ilo, 4,5,6,7-tetrahydrobenzothiazol-2-yl, naphtho [2,1-d] thiazol-2-yl, naphtho [1-2-d] thiazol-2-yl, quinoxalin-2-yl, isoquinoline- 1-yl, isoquinolin-3-yl, benzo [b] furan-2-yl, pyrazin-2-yl, quinazolin-2-yl, isothiazol-5-yl, isothiazol-3-yl, purin-8-yl and a substituted heterocycle, wherein the substituents are selected from CM alkyl, CM perfluoroalkyl, CM alkoxy, hydroxy, halogen, amido, nitro, amino, CM alkylamino, CM dialkylamino, carboxy, CM alkoxycarbonyl, hydroxy or phenylalkylcarbonyl of CM; Or pharmaceutically acceptable salts thereof.

10. A method according to claim 9, characterized in that said compound contains a d-phenylalanine-proline-arginine sequence.

11. A method according to claim 10, characterized in that said compound is (S) -N-methyl-D-phenylalanyl-N- [4 - [(aminoiminomethyl) amino] -1- (2-benzothiazolylcarbonyl) butyl] -L-prolinamide.

12. A method according to claim 6, characterized in that said compound is a natural product that affects the PAR-2 pathway.

13. A method according to claim 12, characterized in that said compound is derived from one or more of the botanical families of legumes, Solanaceae, Graminaceae and Cucurbitaceae.

14. A method according to claim 13, characterized in that said compound is derived from legumes.

15. A method according to claim 14, characterized in that said compound is selected from the group consisting of: denatured soy extract, bean extract, black bean extract or mixtures thereof.

16. - A method according to claim 15, characterized in that said compound is selected from the group consisting of: fractions of denatured soy extract, bean extract, black bean extract and mixtures thereof.

17. A method according to claim 14, characterized in that said compound is selected from the group consisting of soy milk, bean milk, black bean milk, soybean extract, bean extract, black bean extract, pasta of soybean, bean paste and black bean paste, and mixtures thereof.

18. A method according to claim 2, characterized in that said compound is an inhibitor of melanosome transfer.

19. A method according to claim 1, characterized in that said compound activates the PAR-2 pathway.

20. A method according to claim 19, characterized in that said compound is a PAR-2 agonist that binds PAR-2 and activates it.

21. A method according to claim 20, characterized in that 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. .

22. A method according to claim 19, characterized in that said compound is a protease that activates PAR-2.

23. - A method according to claim 22, characterized in that said compound is a serine protease that activates PAR-2.

24. A method according to claim 23, characterized in that said compound is selected from the group consisting of trypsin, tryptase, thrombin and proteases of natural origin in the skin that activate PAR-2.

25. A method according to claim 1, characterized in that said compound is an enhancer of the melanosome transfer.

26.- A composition to effect changes in the pigmentation of mammalian skin, comprising an effective amount to change the pigmentation of a compound that affects the PAR-2 pathway.

27. A composition according to claim 26, characterized in that said compound is an inhibitor of the PAR-2 pathway.

28. A composition according to claim 27, characterized in that said compound is an antagonist of PAR-2.

29. A composition according to claim 28, characterized in that said compound binds PAR-2 or blocks it, but does not activate it.

30. A composition according to claim 29, characterized in that said compound is selected from the group consisting of antagonists based on SLIGRL, which bind PAR-2 or block it, but do not activate, antagonists based on SLIGKVD that bind to PAR-2 or block it, but do not activate it, and mix it.

31. A composition according to claim 26, characterized in that said compound is a protease inhibitor.

32. A composition according to claim 31, characterized in that said compound is a serine protease inhibitor.

33.- A composition according to claim 32, characterized in that said compound is an inhibitor of thrombin and / or trypsin and / or tryptase, or an inhibitor of serine proteases of natural origin in the skin, which activates PAR-2 .

34. A composition according to claim 33, characterized in that said compound contains a d-phenylalanine-proline-arginine motif.

35.- A composition according to claim 33, characterized in that said compound is (S) -N-methyl-D-phenylalanyl-N- [4 - [(aminoiminomethyl) amino] -1- (2-benzothiazolylcarbonyl) but l] -L-prolynamide.

36.- A composition according to claim 31, characterized in that said compound is a product of natural origin that affects the PAR-2 route.

37.- A composition according to claim 36, characterized in that said compound is derived from one or more of the botanical families of legumes, Solanaceae, Graminaceae and Cucurbitaceae.

38. - A composition according to claim 37, characterized in that said compound is derived from legumes.

39.- A composition according to claim 38, characterized in that said compound is derived from soybean, bean and / or black bean.

40.- A composition according to claim 39, characterized in that said compound is selected from a group consisting of denatured soy extract, bean extract, black bean extract or mixtures thereof.

41. A composition according to claim 40, characterized in that said compound is selected from the group consisting of soy milk, bean milk, black bean milk, soybean extract, bean extract, black bean extract, pasta of soybean, bean paste and black bean paste and mixtures thereof.

42.- A composition according to claim 40, characterized in that said compound is a fraction of soy milk, soybean extract, soybean paste, bean milk, bean extract, bean paste, black bean milk, extract of black beans, black bean paste and mixtures thereof.

43.- A composition according to claim 26, characterized in that said compound is an inhibitor of melanosome transfer.

44. A composition according to claim 26, characterized in that said compound activates the PAR-2 pathway.

45. - A composition according to claim 44, characterized in that said compound is a PAR-2 agonist that binds PAR-2 and activates it.

46. A composition according to claim 45, characterized in that 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, characterized in that said compound is a protease that activates PAR-2.

48. A composition according to claim 47, characterized in that said compound is a serine protease that activates PAR-2.

49.- A composition according to claim 48, characterized in that said compound is selected from the group consisting of trypsin, tryptase, thrombin. and proteases of natural origin in the skin, which activate PAR-2.

50.- A composition according to claim 26, characterized in that said compound is an enhancer of the melanosome transfer.

51. A composition according to claim 26, characterized in that said compound that affects PAR-2 is present in an amount of approximately 0.0001% to approximately 15% by weight / volume of said composition.

52. - A composition according to claim 51, characterized in that said compound is present in an amount of about 0.001 to about 5% of said composition.

53. A composition according to claim 52, characterized in that said compound is present in an amount of about 0.005 to about 1% of said composition.

54.- A composition according to claim 26, characterized in that it comprises soy milk in an amount of approximately 1 to approximately 99% by weight.

55.- A composition according to claim 26, characterized in that it comprises soybean trypsin inhibitor, bean trypsin inhibitor or black bean trypsin inhibitor in an amount of about 0.01 to about 20% by weight.

56.- A method according to claim 1, characterized in that said composition is applied twice a day for at least 8 weeks.

57. A method according to claim 56, characterized in that said composition is applied in a relatively high dosage for at least about 4 to about 10 weeks and then applied to a relatively lower dosage in a continuous base to maintain the skin lightening effect.

58.- A method according to claim 1, characterized in that said composition is administered orally.

59. - A method according to claim 1, characterized in that said composition is administered parenterally.

60. A cosmetic composition according to claim 26, characterized in that it comprises said pigmentation-affecting compound, and a cosmetically acceptable vehicle.

61.- A composition according to claim 60, further characterized in that said composition comprises additional depigmenting agents.

62. A composition according to claim 61, further characterized in that said composition comprises tyrosinase inhibitors. 63.- A composition according to claim 26, further characterized in that said composition comprises liposomes. 64.- A composition according to claim 63, characterized in that said composition comprises glycerol dilaurate, cholesterol, polyoxyethylene-10-stearyl ether and polyoxyethylene-9-lauryl ether. 65.- A composition according to claim 26, further characterized in that said composition comprises antioxidants. 66.- A composition according to claim 26, further characterized in that said composition comprises a sunscreen. 67.- A composition according to claim 41, characterized in that said composition comprises about 1 to about 99% soy milk, about 0.1 to about 20% emulsifier and a preservative in an effective amount. 68.- A composition according to claim 26, further characterized in that said composition comprises a compound selected from the group consisting of: antioxidants, sunscreens, humectants, bleaching agents, depigmentation agents, surfactants, foaming agents, conditioners, humectants , fragrances, viscosifying agents, buffering agents, preservatives and a mixture thereof.