MXPA97005433A - Antagonists of alpha-melanocyte stimulating hormone and methods based on myself - Google Patents

Abstract

Alpha-melanocyte stimulating hormone antagonist peptides are described along with methods for inhibiting the effects of the alpha-melanocyte stimulating hormone on hormone-sensitive cells or tissues. In particular, methods to clarify skin pigmentation and to treat malignant melanoma, as well as diagnostic kits for practicing the invention are described.

Description

ANTAGONISTS OF THE ALPHA-MELANOCYTE STIMULATING HORMONE AND METHODS BASED ON THEM i. introduction The present invention provides the peptide antagonists of the alpha-melanocyte stimulating hormone and the methods for inhibiting the effects of the alpha-melanocyte stimulating hormone on cells or tissues sensitive to this hormone, including the clearance of the iodine. dt; Id leS UeSt íiiiuuiie and til tiatdiulelito malignant melanoma. 2. Antecedent.es of the invention Pigmentation and skin staining are related to the amount and distribution of melanin in the mole a rin c r m m e s. Or * »i ri" rm i pn c I ^ r ^^ V > I ap nn io r 1 1 p 1 acro 1 ^ epidermis includes keratmocytes and the me- nocytes that supply meiosomes, ie, melanoma, to the keratomocytes It contains pigment-like grains, through dendritic processes.Melanin is a dark pigment that is produced by oxidation of the tyrosma to dopa and dopaqumona by means of the above tyrosmase, to produce compounds that are poiimerized to form meianin. Localized procedures that currently lack some safe and effective method of cosmetic treatment, these disorders include pigmented spots, such as freckles (freckles), solar lentigines (also known as liver spots or chloasma), acanthosis nigricans (a disorder hypermelanotic), café-au-lait spots, nevi (moles) and elasma (localized darkening of the skin after delivery). need for a method to regulate the pigmentation tone for the total surface of the skin, either for cosmetic purposes, for example, to lighten the color, or to block the damaging effects on appearance caused by certain endogenous disorders. Until now, no safe and effective method to achieve control of epidermal pigment tone has been available. The endogenous hormone that is involved in the treatment is the stimulating hormone 0 ° "'aifa- eocyte (?, A-MSH") (Lerner et al., 1961, Nature , 169, 176-179). The a-MSH also participates in the regulation of the central nervous system and in the functions of the immune system (De eid, 1993, Ann JSIY Acad. Sci., 680, 20-28, xatro, Í990, Brain Res., 536, Í24-Í32; Luger, et al., 1993, Ann. N.? Acad. Sci., 660, 567-570 / Cannon, et al., 1966, J. lmmunoi., 137, 2232-2236; Murphy et al. ai., 963, Science 22l, 1992-193), growth (Strand, f. i., et al., 1993, Ann. N Acad. sci., 660, 29-49), mitogenesis (Halaban et al., 1993, Ann. IMY Acad. Sci., 689, 290-300); and melanoma (Varga et al., 1974, Proc. Nati, Acad.Sci.U.S.A. 71, 1590-1593). The indirect sustenance of the hypothesis that a-MSH is an endogenous regulator of the basal tone of the skin arises from experiments in which the elimination of the pituitary gland induces the clearance of the pigmentation of fish, amphibians and lower mammals (Chavín, 1956 , Exp. Zool., 133, i-36; Smith, 191, Science, 44, 75-758; Alien, 1916, Science, 44, 755-758; Kust, CC, 1965, Gen Comp. Endocrinol., 5 , 222-231). In addition, anecdotal clinical reports claim that humans become abnormally pale as a result of the loss of pituitary function caused by disease or hypophysectomy (Eeiig et al., 1967, Endocrinology and Metaboiism, McGra-Hili Book Company, New York, 1 -26). In humans, MSH is released not only from the pituitary but also apparently by means of dermal keratmocytes after exposure to ultraviolet light (Luger, et al., 1993, Ann. NY Acad. Sci., Bdu, jü. - 7ü). Although it is well known that MHS injection in animals or humans causes an increase in skin tone beyond the Pásales levels, the contribution of endogenous a-MSH to tonic pigmentation in the Oasai line is unknown. .

It has been shown that MSH effects an increase in the activity of tyrosinase in the cultured cells of the mouse eianoma.He found that the synchronized cells respond to the MHS only in the ü2 phase of the cell cycle. The binding of the MSH marked with "" I occurs predominantly in ü2. Thus, it is proposed that MSH can activate the adenylate cyclase of the melanoma cell by binding to an MSH receptor in the ü2 phase (varga et al., 1974, Proc. Nati. Acad. Sci. US-a ., li (5) 1590-3). In this manner, a practical antagonist of a-MSH activity will provide it with a useful pharmacological agent to confirm the function of endogenous a-MSH and provide methods to regulate all biological functions mediated by a-MSH, including the tone of pigmentation of the skin and malignant meianoma cancer cells. Recently Jayawickreme et ai., November of 1 W4. of Bioi. Cnem, 269 (47) 29846-29854, identified a series of poiypeptide antagonists of a-MSH, for example, octapeptides and nonapeptides. however, the larger peptide molecules have limited the ability to penetrate the tissues of interest. Therefore, there is a need for an a-MSH antagonist that is a small molecule, eg, less than 500 Da, more able to diffuse easily into tissue structures, to regulate tissues responsive to a-MSH. . 3. SUMMARY OF THE INVENTION The present invention provides the peptide antagonists of a-MSH and furthermore provides methods for regulating the function of cells and tissues that respond to the hormone a-MSH. The invention provides the peptide antagonists according to the active peptides of a-MSH of Table 1, infra. In this manner, the invention provides the peptide antagonists of a-MSH comprising an amino acid sequence of RST, wherein R, S and T are amino acid residues and R is selected from the group consisting of D-Trp, D- Phc, D-Tyr,? CD-'i'rp and D-Hie, except that, when S is Arg and T is Nle or an amide of these then R is * -Phe D- and. Ac-D-Trn or D-His * when S is Lys, D-Arg, Leu, Nie, Ala, Met or Abu and T is Nie or an amide of these, then R is D-TG?; V when s is A p v 'i is Leu, Nie, Nva, Met, D-Nie, Lie, Abu, Val, Arg or D-Arg or an amide of these, then R is D-Trp; S is selected from the group consisting of Arg, Lys, D-Arg, Leu, Nie, Ala, Met, and Abu; and T is selected from the group consisting of Leu Nie, Nva, Met, D-Nie, Lie, Abu, Val, Arg, D-Arg and these amides. The present invention also provides molecules having structures analogous to the described peptide antagonists.

The invention also provides a method for inhibiting the activity of a-MSH in a cell or tissue responsive to a-MSH by contacting a cell or tissue with a peptide antagonist of a-MSH according to the invention. The invention further provides a method of clarifying the skin color of an animal by administering an effective amount of a peptide antagonist of the a-MSH according to the invention, to an animal, for example, a mammal such as Being a human, who needs this treatment, treatment methods are provided for pigmented spots, nevi, freckles, meiasms and for a local and systemic cosmetic lightening of the skin color. The administration can be topical or systemic. The invention further provides a method of treating malignant melanoma by administering an effective amount of a α-MSH peptide antagonist according to the invention, in an animal, for example, a mammal such as a human in need of this treatment. The invention further provides a method of modulating the immune system by administering an effective amount of a peptide antagonist of a-MSH, according to the invention, to an animal such as a human in need of this treatment. In yet another embodiment, the invention provides a pharmaceutical composition of a peptide antagonist in accordance with the active a-MSH antagonist peptides of Table 1, intra. Thus, the invention provides the peptide antagonists of a-MSH comprising an amino acid sequence of? -st, wherein R, S, and T are amino acid residues and R is selected from the group consisting of u-trp ,? -Phe, D-tyr, Ac-D-trp, and D-HIS, except that, when S is Arg and T is Nie or an amide of these, then R is u-Phe,? -tyr, Ac- D-trp or D-HÍS; when s is Lys, u-Arg, Leu, Nie, Ala, Met, or Abu, and T is Nie or an amide of these, then R is D-trp; and when S is Arg and T is Leu, Nie, Nva, Met, D-Nle, lie, Abu, val, Arg or? -Arg or an amide of these, then R is u-trp; s is selected from the group consisting of Arg, Lys, D-Arg, Leu, Nie, Ala, Met and Abu; and T is selected from the group consisting of Leu, Nie, Nva, Met, u-Nie, lie, Abu, val, Arg,? -Arg and these amides, together with a pharmaceutically acceptable excipient. In an alternative embodiment, the invention provides a tripeptide antagonist of the a-megnocyte-stimulating hormone which is identified by the method of preparing a combinatorial screening library comprising a library of tripeptide molecules of random structure. The combinatorial randomization library is then contacted with a test system for the activity of the a-melanocyte-stimulating hormone, followed by the identification of the tripeptide molecules that antagonize the activity of the a-melanocyte-stimulating hormone.

. DESCRIPTION OF THE DRAWINGS Figure 1 exemplifies the organization of the combinatorial library composed of 96 sub-libraries which is used for the identification of the tripeptide antagonist. ABBREVIATIONS: Abu, acid 2-am? Nobut? R? Co; ? -Abu, 4-aminobutyric acid; e-Ahx, 6-aminohexanoic acid; Aib, 2-am? No? Sobutir? Co; ß-Ala, acid 3-app nn rnpi om no; Orn, nrnifina; Hyp, trans hirir xiprnl ína; Nie, norieucin; Nva, norvaline. Figures 2A and ZB show visual images of ia-MSH antagonist responses, produced by they contain D-trp in position i (ie the su-bioiioteca D-trp). The image on the left, (Figure 2A), shows a 6 cm culture plate (Paicon) with tno I anp nrpc yann pc rntrii oylrnn? Rrarpca nrotra aria r eiatonin (i Nm during 3? in) and a-MSH (15 Nm during rain), just after the application of approximately 6UU globules of the D-trp sub-biololiate. At 60 min, (Fig. 2t), white circular patterns had appeared in response to the local release of nearby blood cells. Figure 3 provides a dose-response curve for the candidate antagonists that were tested in their ability to inhibit the pigment dispersion induced by a-MSH in cultured Xenopus melanofora cells. The graph shows an example of the inhibition curves for three Aβ peptides. (picture), A, (dark triangles) and A, - (clear circles) (see Table 1) identified from the diffusion tests. Two other peptides that are not found in library screenings, A?, Are included for comparison. (dark circle) An (light triangle). The responses to melamine dispersion were quantified by measuring the transmittance through a monolayer of meianophores cultured pretreated with meiatonin i nM (Potenza, et al., 1992, Anal Biochem., 206, 315-22) and the curve was adjusted with the logistic equation (De? .ean et ai.,? 7 ?, / mi u. fñysioi., 235, ¿Í9 -ÍÍG2; . Each point represents the average and standard deviation of the sample (SSD) of four independent measurements taken two years after the addition of a-MSH (i5 Nm) plus the previous peptides at the indicated concentrations. The results are expressed as a percentage of relative treatment with a-MSH alone.

Figure 4 illustrates the competitive inhibition of a-MSH by D-Trp-Arg-Leu-NH; (dWRL) which is demonstrated using the Schild regression analysis (Arunlakshana, et al., 1959, Br. J. Pharm., 14, 48-58). The equilibrium dissociation constant (pK,) is 7.2 ± 0.1 M and the slope of the regression is 1.6 ± Ü.Ü3. Dashed lines indicate a confidence level of 99?. Figure 5 illustrates the dose ratios (rd) for Figure 4 that were obtained from the concetration-response curves for a-MSH taken in the absence (clear circles) and the presence of i μM (dark diamonds), μM (light triangles) and 100 μM (dark circles) of DWRL. (The EC50 for a-MSH alone is 2.5 ± 0.3 Nm, and for a-MSH + DWRL, it is 4.6 ± 0.6 μM). each point represents the average and the SSD of 4 independent transmittance measurements. Ti = initial transmittance (2 mm). Tf = final transmittance (60 min). The DWRL does not cause changes in the EC values for the vasoactive intestinal peptide ("VIP") or Arg0] - vasotosine ("AVT") (data not shown). Figure 6 shows that D-trp-Arg-Nie-NH (OW-R-Nie), a 4U μM, block the stimulation of the second cAMP messenger mediated by the a-MSH (ln Nm), but not the stimulation of the AMPC evoked by AVT (6 nM). The ÜVT oxytocin antagonist ([d (CH,). ,, Tyr (Me) ", Orn"] - vasotocma, from Peninsula), at 20 μM is used as a control to block the responses evoked by 6 Nm of AVT . Measurements of intracellular cAMP were taken from confluent xenopus melanophores developed on tissue tissue plates with 24 wells (Ealcon). Each bar represents the average and the SSD of 4 independent measurements. * (Test T; P <0.001) for all groups except those with asterisks. Figure 7 shows the functional antagonism of a human MSH receptor by dWRL in xenopus fibroblasts (Daniols, et al., 1990, Pigment Cell Res, 3, 38-43) transfected with "Vector alone" (pcDNAI / NEO; Invitrogen ) or with "HMelMSHR" (pcDNAI / NEO) containing a human meianoma MSH receptor inserted. Control = without additional medications, MSH = 5 nM a-MSH. The concentration of the dWRL was 10 μM. Forskolm = lüü μM forskolm (/ ß-desacetyl- / ß- [g- (N-metiipiperazmo) -butyryl, from Caibiochem). The figure is a photograph demonstrating that the topical application of the DWRL tppeptide (1 Mm in H; 0) on the skin of Xenopus l eavi s causes a local lightening of the pigmentation at coloring levels "similar to aiOino".

Figure 9 is a photograph showing that the systematic injection of the DWRL tpppptide in Xenopus l eavis causes an aciation of the skin pigmentation on the body surface. 3 animals were injected with DWRL (40 μmoles / kg) or with D-Trp-Abu-Arg-NH; (control), and at 20 min they took the albino appearance as shown. The three control animals are dark. Figure 10 exemplifies the structure-function comparisons in the third position for the 6 most potent L molecules identified by random screening from their mixture containing D-Trp in position 1 and Arg in position 2. Three additional substitutions in the position 3, not found in the randomization, are included as a comparison. The antagonist activity correlates with the hydrophobicity and charge characteristics of the R group found in position 3. The length of the chain and the spherical impediment due to substitution in the β-carbon are also important.

. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the peptide antagonists, according to the active peptides of α-MSH of Table 1, see. In this manner, the invention provides the a-MSH peptide antagonists comprising an RS-1i sequence of amino acids, wherein K, sy 'i are amino acid residues and R is selected from the group consisting of D-irp, D-pne, D-'iyr, Ac-D-trp and D-HIS, except that, when S is Arg and i is Nie or an amide of i z these, then R is D-pne, u-tyr, Ac-D-trp or D-HIS; when s is Lys, D-Arg, Leu, Nie, Ala, Met or Abu and T is Nie or an amide of this then R is D-trp; and when S is Arg and T is Leu, Nie, Nva, Met, D-Nle, Lie, Abu, Val, Arg or D-Arg or an amide of these, then R is D-trp; s is selected from the group consisting of Arg, Lys, D-Arg, Leu, Nie, Ala, Met and Abu; and T is selected from the group consisting of Leu Nie, Nva, Met, D-Nle, Lie, Abu, Val, Arg, D-Arg and amides thereof. In a preferred embodiment, the peptide consists of more than 10 amino acids, including but not limited to 15 amino acids, kin other preferred embodiments, the peptide consists of no more than 8 or no more than 12 amino acid residues (including amino acid residue derivatives) or amino acid analogues). In yet another preferred embodiment, the peptide consists of three contiguous amino acid residues, such as a tripeptide, derived from the amino acid analogue residues, where the three residues of my orases are the antagonists. assets of aa MSii as shown in Table 1, look. The present invention also provides the analogs and derivatives of the peptides. The term "amino acid" or "amino acid residue" as ULxi? D in Id piesenLe be xeíiexe iut > These are amino acids that occur naturally, that occur in a non-natural form or their derivatives, or at a l? molecule that has the conformation of an amino acid. The term "peptide" refers to any molecule that contains a peptide bond and consists of naturally occurring amino acids residues, found in non-natural form or derivatives thereof, or to a molecule having the conformation of an amino acid, A peptide bond, as used herein, refers to an amide bond that binds to two adjacent amino acid residues. In a particular embodiment, the a-MSH antagonist is one of the following tpeptides: D-T'rp-Arg-Leu; D-Trp-Arg-Nie; D-Trp-Arg-Nva; D-Trp-Arg-Met; D-Trp-Arg-D-Nie; D-Trp-Arg-Iie; D-Trp-Arg-Abu; D-Trp-Arg-Vai; D-Trp-Arg-Arg; D-Trp-Arg-D-Arg; D-Trp-Lys-Nie; D-Trp-D-Arg-Nle; D-Trp-Leu-Nie; D-Trp-Nie-Nle; D-Trp-Ala-Nie; D-Trp-Met-Nle; D-Trp-Abu-Nie; D-Phe-Arg-Nle; D-Trp-Arg-Nie; D-Ac-Trp-Arg-Nie; Trp-Arg-Nie and D-His-Arg-Nie. Compositions containing one or more of the aforementioned tripeptides are also provided. In another embodiment, the peptide has a molecular weight of less than D? Gives. In still another embodiment, the peptide can be an antagonist of tripeptide α-MSH which is identified by screening a combinatorial and synthetic random library for the α-MSH antagonist activity. Simply by way of example, the combinatorial library can be prepared according to the methods of Houghten et al., 1991, Nature 354, 84, Houghten et al., 1992, Biotechniques 13, 412 or Jaya ickreme, et al., 1994, Proc. Nati Acad. Sci. U.S.A. 91, 1614-1618, in which the peptides are synthesized in a randomly structured manner, without inclination toward homology with the a-MSH. Randomness is defined so that the size and / or structure of a resulting peptide can not be predicted at any position. It is also contemplated that other random libraries known for the art can be used to identify the α-MSH antagonist peptides. The screening can be carried out by any of the methods known in the art, and in a preferred aspect, it is carried out as described in the following examples. The peptides and amino acids comprising the peptides are not limited to the 20 natural amino acids. Non-traditional amino acids include, but are not limited to, the D isomers of the common amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, nidroxiproiin, sarcosine, citrulline, cysteic acid, t-butyigiycin, t-outiiaianin, phenylemycin, cyclohexyllanine , ß-alanine, amino acids designers as It can be the ß-methylamino acids, Ca-methylammocids, Na-methylammocids and analogous amino acids in general. In addition, amino acids may include Abu, 2-ammobutyl acid; ? -ADu, acid 4-ammoout? R? Co; e-Añx, 6-ammo hexanoic acid; Aib, acid 2-ammo? Sobut? R? Co; β-Ala, 3-ammopropionic acid; Orn, ornithma; Hyp, trans hydroxyprolma; Nie, norleucine; Nva, norvaline. In addition, the amino acid can be D (dextrorrotatope) or L (ievorrotatory). In addition, the peptides and / or the amino acids can be obtained by bio-blocking groups, including but not limited to, acetylation or carboxylation in the terminal amino group and amidation in the carboxyl terminal to provide the protected derivatives. The α-MSH antagonist peptides identified by comomatory screening can be prepared by methods well known in the art. For example, in torma oreve, peptide synthesis in solid phase consists in the coupling of the carooxiio group of the amino acid c-termmai to a ream and the addition in successive form of protected amino acids in the N-affame position. The protecting groups can be any of those known in the art. Before adding each new amino acid to the chain, the protective group of the previously added amino acid is removed from the chain. The coupling of the amino acids to the appropriate reams is described in Kivier et al., U.S. Patent No. 4,244,956. These solid phase syntheses have been described, for example, by Merrifield, 1964, j. Am. Chem. Soc, 85: 2149; vale et al. 1981, Science 213: 1394-1397; Marki et al., 1981 j. Am. Chem. soc. 103: 3178 and in the United States Patents or. 4, 305, 872 and 4,316,891. In a preferred aspect, an automated peptide synthesizer is employed. The purification of the synthesized peptides can be carried out by conventional methods including chromatography (for example, ion exchange, affinity and sizing column chromatography), centrifugation, differential solubility or by any other standard technique for the purification of proteins. In a preferred embodiment, the HPLC (reversed phase high resolution liquid chromatography) is used. L = structure-function relationship determined to the tripeptide antagonists described herein, as described infra as shown in the Pleura l and > z * ra ~? = > position of the third amino acid, it can also be used na ra n c a n a r a r a r a r r r r r r r r r r a r a r a r p a l i n c a n c e r an a I n a c r-m o also have inhibitory properties for a-MSH. Of et > Secondly, it was concluded that the invention of molecules, in addition to those expressly described, that share the structure, hydrophobicity, characteristics of l / loading and sidechain properties of the peptide antagonists that are exemplified herein. . i α-MSH antagonists having therapeutic and cosmetic utility The peptide antagonists of α-MSH have therapeutic and cosmetic utility in the modulation of a-MSH-mediated functions, eg, clearance of skin pigmentation , the modulation of the immune system and the treatment of melanoma. By "clearance" is meant the reduction of the pigment tone provided by the melanin present in the skin to obtain a lighter skin color. Although it is not intended to adhere to any particular mechanism of action, peptide antagonists are considered to be competitive inhibitors of α-MSH in the α-MSH receptors, resulting in a blockage of the effects that maintain pigmentation of α-MSH. Endogenous MSH. In this way, the meianma particles present in ia-MSH sensitive cells are dispersed in the presence of the α-MSH antagonist peptides due to blockade of the α-MSI α-halogen receptor. This is due to the elimination of the energetic tonic of the MSH on the aggregation of meiacin. When the peptides are administered topically, the skin ib clarifies where the peptide is applied. When the peptide is administered systemically, the entire surface of the skin becomes significantly clearer in the color of the pigmentation. The a-MSH antagonist peptides can be used to treat the following localized conditions including, but not limited to, spots or pigmented areas such as nevi or moles, freckles, melasma and post-inflammatory hyperpigmentation. It is also possible to clarify the complexion for cosmetic purposes both locally and systemically, as desired. Antagonistic peptides of α-MSH provide methods for the treatment of malignant melanoma by administering to an individual an effective amount of a peptide antagonist according to the invention. The invention also provides methods for modulating the immune response of an animal or person by administering an effective amount of a peptide antagonist of α-MSH to an animal or person in need of this treatment. In addition, other diseases and clinical correlations of undesirable a-MSH activity can be treated with the α-MSH antagonist peptides according to the invention. and 5.2 identification of the peptide antagonists of a-MSH Traditional methods for the development of drugs for peptide receptors are usually based on the screening of modified natural agonist peptide structures and usually give rise to the identification of large molecules. In contrast, as described in the examples, the small molecule peptide antagonists of a-MSH, according to the invention, were identified by an alternative screening strategy of large quantities of random small tripeptide molecules prepared on surfaces of beads in a multipurpose combinatorial peptide library ("MUPL") according to the diffusion assay of jayawicicreme, et al., 1994,. mol, cnem., 2óym 29 «4b-29854 (v> jayaw? c? reme i") and Jayawicicreme et al., 1994 proc. Nati. Acad. Sci (U..), 91, 16i -? 6 6 ("jayawic reme n"), which are incorporated herein by reference in their entirety, as described in the specific examples, the method used allowed the screening of a library of beads carrying random tripeptidic molecules. of the bioassay consisted of meianophores on agarose gel pretreated with meiatonin. -_? in contact with the agarose gel. The controlled release of the peptides from the beads was performed by a gas phase release procedure according to Jayawickreme i and 11, supra. The dispersion of the pigment induced by the diffusion of the released peptides was observed after 5 or 10 minutes and moni oreated by subtraction of visual images. . 3 Treatment and Compositions The invention provides methods of treatment by administering, to an individual, an effective amount of an α-MSH antagonist peptide according to the invention. In a preferred aspect, the peptide is purified. Preferably, the individual is an animal, for example, a mammal and more preferably a human. The various delivery systems are known and can be used to administer the a-MSH antagonist peptide of the invention, for example, in aqueous solution, encapsulation in iiposomes, microparticles, microcapsules, receptor-mediated endocytosis (see, for example, Wu and Wu, 1967, Biol. Chem., 262, 4429-4432). Methods of administration include, but are not limited to, direct application to the skin, intradermal, intramuscular, intravenous, intranasal, epidural and oral routes. The peptides, according to the invention, can be administered z by any convenient route, for example, by infusion or injection of the bolus, by means of absorption through the epithelial or mucocutaneous linings (e.g., the oral mucosa, the rectal and intestinal mucosa). In a specific embodiment, it may be desirable to administer the peptide, according to the invention, locally in the area that it is desired to treat by any of the aforementioned methods. The present invention also provides the pharmaceutical compositions. These compositions comprise a therapeutically or cosmetically effective amount of a peptide according to the invention and a pharmaceutically acceptable carrier or excipient. This carrier includes, but is not limited to, water, saline, for example, physiological saline, buffered saline, dextrose, glycerol, ethanoi, and combinations thereof. Is the formulation correct or adequate for? C?? R? I? . The composition, if desired, may also contain minor amounts of wetting or suspending agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, cream, gel or powder. The composition can be formulated as a suppository with the binders and additional carriers such as the glycerides. The oral formulation may include standard carriers such as the pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Commonly, compositions for intravenous administration are solutions in aqueous, isotonic, and sterile buffer solution, when necessary, the composition may also include a solubilizing agent and a local anesthetic to relieve pain at the site of injection. In general, the ingredients are supplied separately or mixed in a unit dosage form, for example, as a freeze-dried dry powder or water-free concentrate in a hermetically sealed container as may be known in the art. I l o r a r c a c o r r o r I-Y? If the active agent is to be administered, when the composition is to be administered by infusion, it can be dosed with an infusion bottle containing water or sterile pharmaceutical grade saline. When the compositions are administered by injection, a sterile water vial for injection or saline may be provided so that the ingredients can be mixed prior to administration. The a-MSH peptide antagonists of the invention can be formulated as neutral forms or salts. Pharmaceutically acceptable salts include those which are formed with amino groups, such as those obtained from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc. and those that are formed with free carboxyl groups such as those obtained from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-etiiamino ethanol, histidine, procaine, and so on. The amount of the a-MSH peptide antagonist of the invention that will be effective in the treatment of a particular disorder or cosmetic condition will depend on the nature of the antagonist, disorder or condition and can be determined by standard clinical techniques. In addition, the in vi tro or m vi ve tests can optionally be used to help identify the optimal dosage ranges. The precise dose that should be used in the formulation will also depend on the route of administration and the seriousness of the disease or cosmetic condition and should be decided according to the judgment of the physician and the circumstances of each patient. The effective dose can be extrapolated from the dose-response curves obtained from the test systems with animal or animal models as provided herein. an elective amount of the peptide antagonists of z a-MSH is easily determined by administering graded doses of peptide antagonists according to the invention and observing the desired effect. The data provided in Table I and Figures 2-and, intra, will aid in the determination of the effective amounts necessary for the purpose of lightening the skin by providing ICÜO concentrations, dose-response curves for the representative peptides and the examples in vi vo. In one embodiment, the effective concentration for dWRL (D-Trp-Arg-Leu) in a conventional formulation for, for example, skin lightening, is in the range of 1 μM to 10 mM. In a preferred embodiment, the effective concentration of the dWRL for a conventional formulation is in the range of 1 μm to 5 mM. In a more preferred embodiment, the effective concentration for a topical formulation is in the range of 500 μM to 2 mM. In a particular embodiment, the effective concentration for a topical formulation is close to i mM. In another embodiment, an effective dose of OWRL for systemic administration for, e.g., skin ailment, is in the range of 1 to 4000 μmol / kg body weight. In a more preferred embodiment, the effective dose of the dWRL for systemic administration for, for example, skin ailment, is in the range of 30 to 100.

Z 5 μmoi / kg body weight. In another embodiment, the effective dose of dWRL for systemic administration is in the range of 30 to 100 μmol / kg body weight. The concentrations and effective doses for each of the other exemplified tripeptide antagonists can, for example, be easily determined by reference to Table 1 of the IC, which shows the relative potencies of the peptide antagonists for the tripeptide dWRL. In another alternative embodiment, the invention comprises kits containing an effective amount of an α-MSH antagonist peptide according to the invention. In this way, it is contemplated that the kit contains one or more containers with at least one ia-MSH antagonist peptide according to the invention, simply by way of example, the kit will contain a peptide or antagonist peptides of ia a- MSH formulated for application in the skin or for administration by the routes of administration mtradermia, intramuscular, intravenous, tranasal, epidural and oral. The kits may contain a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained-release formulation, cream, gel or powder formulation of the α-MSH antagonist peptide in pre-mixed form or as separate ingredients easy to mix or be Z? Formulated in a peptide formulation or a pharmaceutical composition containing an effective amount of an α-MSH antagonist peptide according to the invention. The invention is further described in the following examples which in no sense are proposed to limit the scope of the invention. 6. EXAMPLES 6.1 Design and construction of the tripeptide combinatorial library A synthetic tripeptide random combinatorial library was prepared using Emoc (fluorenylmethoxycarbonyl) chemistry on resin bound to MBHA (4-methyl-benzhydrylamine) (substitution level 0.95 mmol / g ) as described in jayawickreme iyn, supra, in combination with simultaneous multiple peptide synthesis (La et al., 1991, N'ature, 354, 63-84 and Houghten et al., Nature, 354, 84-86). A process of division, copulation and recombination was used to synthesize the tppeptides on the sub-libraries of pearls. The sub-library contained 2304 (i? 46? 46J possible tripeptides or combinations of similar sequences dl? t; LupepLidob.It is uL lizu i (see id riyuid i) i represent the molecule in position i that comprises each sub -bibiiotheca.Place 1 contains one of the 48 Z I molecules listed for the other two positions, or one of the 48 acetylated equivalents (Ac). AA, up to AA¡, in position 2 and 3 are the 20 normal levorotatory (non-predetermined) amino acids, minus ys, plus 18 equivalent dextrorotatory isoforms (D). 11 additional molecules are listed to carry a total of 48. During the preparation for the screening, approximately 4-t, of the total molecules attached to the bead were released using the controlled procedure of splitting of trifluoroacetic acid (TEA) in gas phase , followed by treatment with NH <; / H? gaseous to return non-toxic pearls for direct use in biological assays. The separated molecules remained non-covalently associated with their origin beads until they were released for the assay.

The skin meianoforos of Xenopus l U V.? J Cii _- 1-4 were maintained. -L O- V < V- fc. . J.? J_ C ^ / ilLW C C-JV-l liJ U Cll _L O above (Daniolos et al., 1994, Pigment Ceil Res. 3, 38- 3; Uz- -t- et ai; 1993 J. Bioi. chem., 268, Í9i26-? 9? 33). they sowed 1 ^ my nnoc io rol n'l ac on t? "I s / -" a c io r-n I t i G 1 ~ I c n "t a r r r (Paicon) in 5 ml of conditioned medium with fibroblasts and incubated at 27 ° C for at least 48 h. For each After testing, the medium was removed from the plate and replaced with 3.5 ml of Sea Plaque Agarose at 0.9; The cells were pretreated with melatonin (1 nM for 30 in) and a-MSH (15 nM for 2 min), and approximately 600 beads were applied from each sub-library. The beads remained separated from the melanophore cell layer by a 2 mm thick interposed agarose, allowing only the dissociated free molecules to interact on the cell surface. The translocation of the pigment (dispersion) induced by the tppeptide was onitored by means of visual image subtraction as described in McClintock et al., 1993, Anal. Biochem., 209, 198-305. 6. 3 Determination of the peptide sequence The peptide beads were selected from the positive areas, washed with N-methylpyriidoire ("NMP"), dichloromethane, ("DCM") and methane! (v, MeOH ") and were re-prepared for a second round of screening to select single sensitive pearls for positive signals by poorly distributing the pearls on a polystyrene sheet The selected pearls of the first or second scent voivisrop to unfold by means of the procedure tpcloroacét? co / NHi, / H; 0 gaseous The individual pearls sensitive to positive signals Then they were collected, washed with NMP, DCM and MeOH, placed on a glass fiber filter and their sequence was determined using an Applied Biosystems 476A sequencer. 6. 4 Dose-response studies For the dose-response studies, all tripeptides were synthesized in a Rink Amide MBHA 0.25 mmoi resin using the normal t oc chemistry. After the synthesis, the peptides were purified using LAP and reverse phase chromatography on a column of c. The dose-response curves were obtained by plaque microtitre assays (Potenza et al, 1992, Pigment Ce11 Res., 5, 3 / 2- / «). Melanophores cells (1 «,? / well) were plated on 96-well tissue culture plates (Paicon). Before an assay, the medium was supplemented with medium conditioned with an ibrobiset containing i nni of c-MSH, 1 nm of melatomine and different concentrations of tripeptide. For control experiments, 1 nm of a-MSH was replaced with bombesin fragment ([8-14], oxytocin or without a-MSH.) Dose-response curves or competitive inhibition curves for a-MSH a obtained by varying the concentration of a-MSH at different concentrations of the tripeptides. or? 6.5 Results The screening of the multipurpose peptide libraries as described in the above was carried out at the level of release of 10 picomoles per bead [A i-, of the total) so that the responses of the molecules with potencies could be observed. as low as 100 μM. The library of 221,184 components was organized as 96 sub-libraries with 2304 different peptide combinations per mixture. Each one defined by the chemical structure of the amino terminal position. The sub-libraries that start with D-Trp, D-Phe, D-T'yr and Ac-D-Trp produced response patterns indicating the presence of multiple molecules similar to antagonists, with the most numerous and powerful signals. developed by the pearls of the D-Trp library (figure 4). No similar signals were observed from any of these sub-libraries when the pigment dispersion was activated 6 nM AVT ([Arg "] - Sigma vasotocin; EC; <2 nM or 4 nM VIP (Vasoactive intestinal eptide from Sigma; EC ^ * i nMj instead of i5 nM a-MSH (Peninsula; EC ~ Z nM) After identification by the diffusion test, the candidate antagonists were re-synthesized, purified (Jaya ickreme 1 and 11, Supra) and were tested (Figures 2-9) .The results were organized and tabulated in Table I, below, and THE two antagonists Jl potent, D-trp-Arg-Leu-NH (DWRL) and D-trp-Arg-Nle-NH < have 1C5¿ values of 620 ± 150 nM (mean ± SE) and 930 ± 220 nM respectively, against 15 nM of a-MSH. These antagonists are competitive and specifically block the a-MSH-induced activation of transfected human and endogenous amphibian MSH receptors (Figures 2-9). The DWRL tppeptide had a dissociation constant in equilibrium (Ke) of 63 ± 15 nM (figure 2c), in line with a 1C;, - predicted of approximately 96 nM that a-MSH had been applied at its Cso- value TABLE 1 Pepetidic sequence JQ, (μM) A D-Trp -Arg -X -NH A 'Leu o.62 ± 0.15 A: Nie 0.93 ± 0.22 A, Nva 3.3 ± ii A: Met 5.6 ± 2.6 A D-Nie 9.9 ± 1.8 A, - lie 49 ± 9 A Abu 82 ± 41 A: - Val 237 ± 100 A Arg 261 ± 66 A.- D-Arg 664 ± 397 A--? Abs inactive A. eAhx inactive A, j Ala inactive A, 4 ßAla inactive B D- -Trp -X -Nie- NH- Bi Lys 15 ± 1 B D-Arg 30 ± 11 B3 Leu 48 ± 2.2 B4 Nie 59 ± 12 B5 Wing 65 ± 18 B, Met 121 ± 27 B-, Abu 405 ± 69 tí. Asp inactive C X -Arg -Nie -NH. C i D- -Phe 4.4 ± 1.2 C; D- -Tyr 28 ± 2 C, Ac-D- 43 ± 8 Trp c * Trp 100 ± 2 or D- -HlS 318 ± 105 6. b Structure-function relationships In addition to identifying tripeptide antagonists with molecular weights of less than 500 daltons that are better able to provide systemic and topical methods of blocking the endogenous activity of α-MSH, simultaneous detection of multiple signals from The antagonists that arise from different sub-libraries provided an opportunity to compare similarities and differences in the structure of the antagonist. This allowed the determinations of the components that contribute to receptor interaction and blockage and the information is potentially useful for another development of non-peptidic antagonists. The active sequences were deduced iteratively by screening sub-mixtures of the D-trp sub-library. For example, response patterns in the diffusion assay indicated that the most potent molecules contain Arg in position 2, while other sub-mixtures with this position 2 (ie Lys, D-Arg, Met, Leu and Nie) had less activity similar to the MSH receptor antagonist. Table 1, from Al to Ao, show the 6 most potent molecules that were identified by screening the sub-mixture of D-trp-Arg-x. After identification by the diffusion assay, the candidate antagonists turned to (Figures 2-9). The two most potent antagonists, D-Trp-Arg-Leu-NH (dwKL) and D-trp-Arg-Nie-NH have c values of 620 ± 150 nM (mean ± SE) and 930 ± 220 nM respectively, against 15 nM of the a-MSH. These antagonists are competitive and specifically block the activation induced by the α-MSH of the MSH receptors in the transfected and endogenous amphibian human meianoma (Figures 7-9). The DWRL peptide had an equilibrium dissociation constant (Ke) of 63 ± 15 nM (Figure 5), in line with a C, or predicted of approximately 96 nM that had a-MSH been applied at its EC value ", . The multiple MSH receptor antagonist molecules identified from the sub-mixture D-Trp-Arg-x comprise a series of related peptides in structural form (Figure 10) that form a series of permissible substitutions, wherein the activity is not abolished by differences in the structure at position 3. Within this group, measurements of 1C (inhibitory concentration) show that the potency of the antagonist correlates positively with the length of the hydrocarbon group of the side chain. Nle > Nva > Abu > Ala, and they correlate negatively with the presence of a β-methyl group, ie Nva > Ile and Abu > Val. The elimination of the group -methium of the Leu results in a decrease of 50 c in the antagonistic potency as manifested by the comparison with the Nva. When Met is located in position 3, there is a 6-fold reduction in power compared to Nie, which has a similar group but does not contain sulfur. The strongest hydrophilic substitution, with proven charge, Arg, causes a 300-fold decrease in power compared to Nie (see Labia i A). The sub-mixtures of isomer D also contain positive signals, but have reduced potency compared to the sub-mixtures of the L-isomer. The D-Nle, which was included for the comparison, was 10 times more potent than the L isomer in the third position . The vast majority of the tripeptides selected with the diffusion assay showed no activity similar to the α-MSH antagonists. Although this is expected given the high efficiency of receptor-ligand interactions, the negative results are informative. The absence of activity in most library mixtures tested suggests that the changes made in position 1 are not well tolerated in terms of the interaction with the receiver. In addition to the D-Trp, it was found that only the D-Phe clearly related and to a lesser extent the D-Tyr in the non-acetylated mixtures, show a significant activity similar to the α-MSH antagonist. Confirmation of this information arises from the finding that these are also the two most powerful and permissible substitutions in position i in a general structure x-Arg-Nie-NH (see ia 'labia 1, cl and C2), where x represents all the non-acetylated combinations described in Figure 1. In this way, the positive signals observed in the screening sub-mixes probably arise as a result of the structural similarity for positives in the D-trp library and do not represent additional unrelated structures. A seemingly similar image 3b is conserved for the positives observed within the sub-position mixes of the D-trp library. Substitutions of x in the structure D-trp-x-Nle-NH with Lys D-Arg, Met, Leu and Nie result in peptides showing antagonist activity (see Table 1, cell and B5), and, as expected, each has a lower potency than with Arg in position 2. Therefore, it is evident that the peptides selected from the random sieves, because they give stronger signals, are in effect the most potent MSH receptor antagonists in the library. Variations in the size of the combinatorial test bead, for example, contribute less to differences in signal strength than a difference of 1 or 2 orders in power. 6. 7 quantification of the cyclic AMP With 24-well tissue culture plates (Paicon) 2 days before the assay, immediately before a test, the cells were washed with medium L-15 ai 70-σ (sigma) containing 0.05 of bovine serum albumin and then treated with i nm of meiatonin in the same medium. Then, the cells were washed for 5 min in L-5 to 70; more 0.05. of bovine serum albumin plus 0.05 mm of 3-isobutyl-i-methyixanthin (Aidrich) plus i nm of meiatonin, followed by 30 min of 3 / treatment with the test drug in the same medium. After treatment with the drug, the cells were washed twice with phosphate-buffered saline solution. As shown in figure 6, the D-trp-Arg-Nle-NH, (dW-R-Nle), at 40 μM, blocks the stimulation of the second AMPC messenger mediated by a-MSH (10 nM), but does not block the stimulation of the AMPC evoked by the AVT (6 nM). The ÜVT oxytocin antagonist ([d (CH)., Try (Me) ", orn '-'] - Vasotocin, from Peninsula), at 20 μM is used as a control to block the responses evoked by 8 nM of AVT. 6. 8 Functional Antagonism of a Human MSH Receptor by dWRL in xenopus fibroblasts To demonstrate that a human MSH receptor can be inhibited by tripeptide antagonists, the xenopus fibroblasts were transfected with a vector expressing a human MSH receptor and the response of cAMP was determined with and without the tripeptide as illustrated in Figure 7. The xenopus fibroblasts (Daniolos, et al., 1990, Pigment Ceii Res, 3, 36-43) were transfected with "vector alone" (pcDNAI / NEO, Invitroqen) or with "HMelMSHR" (pcDNAI / NEO) containing an insert of the MSH receptor of the human meianoma. (Mountjoy et al., 1992 Science, 257, 248-1225, by Roger Cone). Control = without additional medications. MSH = 5 to you nM μM a-MSH. The concentration of the dWRL was 10 μM. Forskolin = 100 μM forskolin (7β-desacetyl-7β- [g- (N-methylpiperazino) -butyryl, from Calbichem). Transfections were performed by eiectroporation ("5 x 10" 'cells per 400 μL in phosphate-buffered saline at 70 °, pH 7.0, plus 10 μg of NAC (using 0.2 cm cells on a BTX ECM-600 (475 v, 720 ohm and 400 μt.) 46 hours after transfection, the confluent cells, plated in 12-well tissue culture plates (Paicon) were rinsed for one hour with 70 * L-15 medium (Sigma) which contained 0.5 *. of bovine serum albumin (thiSA) (Sigma) and again for 5 minutes with bovine serum albumin (BSA) Sigma added to 5i, and another P7 riuran p 5 min with I HMX (3- i snbnt i 1 - I tnet i 1 xanf i na of Aidrich) 0.5 mM added The drugs under test were then added with 1BMX present for 45 mm and the MDMA was extracted with 1 ml of ethanoi ai 0 per well. the protein bound to cAMP (21, Amersham Kit) Each bar represents the average and the SSD of 3 independent measurements, except the control group. HMeiMSHR were n = 6. * (Test T; p < 0.üüb) for all groups except for other groups that carry a single asterisk. ** (test T-P < ü. ?? 6) for the other groups. As shown in Figure 7, the tripeptide dWRL Significantly, formation of AMPC in cells transfected with HMelMSHR (human melanoma MSH receptor) was significantly associated with cells transfected with HMelMSHR treated with MSH alone. 6. 9 Topical efficacy for skin lightening To demonstrate that pigment clearance is induced locally at the skin level, topical dWRT (1 mM in n, ü) was applied to the skin surface of Xenopus laevie. (figure 8). It turned out a clarified skin tone similar to albino in the place of application. These results indicate that the peptide acts transdermally and that, under normal conditions of adaptation to darkness, the tonic coloration is mediated by the endogenous melanotropm and that the elimination of this influence causes the frog to adopt a clarified state "pdleClu? dxbino". 6. 10 Stica st_em? Ca for clearing the ?? e-. To confirm the effect of a-MSH on the skin tonic color Xenopus l eavi s adapted to the dark was injected with dWRL (40 μM / kg) or with D-Trp-Abu-Arg-NH (control). The dWRL caused complete aciaramiento of each frog tested (n = ó) at 20 minutes, while no change was observed in the control group (n = 6) (figure 9).

The present invention should not be limited in scope by the specific embodiments described herein. In fact, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the aforementioned description and the accompanying data, it is proposed that these modifications fall within the scope of the claims. . The various publications are mentioned in the present, the descriptions of which are incorporated herein by reference in all their strengths.

Claims (2)

1. RE1 INDICATIONS A peptide antagonist of the a-megnocyte stimulating hormone having an amino acid sequence comprising RST, wherein R, S and T are amino acid residues and R is selected from the group consisting of D-Trp, D-Phe, D -T'yr, Ac-D-Trp and D-His, except that, when S is Arg and T is Nie or an amide of this, then R is D-Phe, D-Tyr, Ac-D-Trp or D -His; when S is Lys, D-Arg, Leu, Nie, Ala, Met or Abu and T is Nie or an amide of this, then R is D-Trp; and when S is Arg and T is Leu, Nie, Nva, Met, D-Nle, Lie, Abu, Val, Arg or D-Arg, or an amide of these, then R is D-Trp; S is selected from the group consisting of Arg, Lys, D-Arg, Leu, Nie, Ala, Met and Abu; and T is selected from the group consisting of Leu, Nie, Nva, Met, D-Nle, Lie, Abu, Val, Arg, D-Arg and amides thereof. The antagonist of the a-melanocyte-stimulating hormone, according to Claim I, which is a tripeptide. The antagonist of the a-meiocyte stimulating hormone, according to claim 1, having an amino acid sequence D-Trp-Arg-Leu. The antagonist of normo stimulating the a-meianocyte, according to claim I, which 4 does it have an amino acid sequence D-trp-Arg-Nie. 5. . The α-melanocyte stimulating hormone antagonist, according to claim 1, having an amino acid sequence D-Trp-Arg-Nva. 6. The α-melanocyte stimulating hormone antagonist, according to claim 1, having an amino acid sequence D-trp-Arg-Met. 7. The antagonist of the alpha-melanocyte stimulating hormone according to claim 1 having an amino acid sequence of D-trp-Arg-D-Nle. 8. The α-melanocyte stimulating hormone antagonist according to claim 1, having an amino acid sequence D-T'rp-Lys-Nle. 9. The antagonist of the amino-stimulating hormone, according to claim 1, having an amino acid sequence D-'irp-D-Arg-Nie. lü The antagonist of the amino acid stimulating hormone, according to claim 1, having an amino acid sequence D-Trp-Leu-Nie. 11. The antagonist of the normonae stimulating agent of the aegiocyte, according to claim 1, which has a? sącue-nciß de? minoacidos ü- ro-Nie- i. 12. The antagonist of the stimulating hormone of the amino acid, in accordance with the claim, which has an amino acid sequence of D-Phe-Arg-Nle 13. The antagonist of the amine-stimulating hormone, in accordance with claim i, which has an amino acid sequence Tyr-Arg-Nle. 14. The α-melanocyte stimulating hormone antagonist, according to claim 1, having an amino acid sequence Ac-D-trp-Arg-Nle. 15. A method for inhibiting the activity of the a-melanocyte-stimulating hormone in a cell or tissue responsive to the a-melanocyte-stimulating hormone, comprising the step of contacting the cells or tissue with an effective dose of a peptide antagonist of the a-melanocyte-stimulating hormone, wherein the pppfiico antagonist cop.st.a of a .SRr.nenr.i of RST amino acids, wherein R, S and T are amino acid residues and R is selected from the group consisting of D-Trp, D-Phe, D-'Tyr, Ac-D-Trp and D-His, except that, when S is Arg and T is Nie or an amide of this, then R is D- Phe, D-Tyr, Ac-D-Trp or D-His; when S is Lys, D-Arg, Leu, Nie, Ala, Met or Abu and T is Nie, or an amide of this, then R is D-Trp; and when S is Arg and T is Leu, Nie, Nva, Met, D-Nie, Lie, Abu, Val, Arg or D-Arg or an amide of these, then R is D-Trp; S is selected from the group consisting of Arg, Lys, D-Arg, Leu, Nie, Ala, Met and Abu; and T is selected from the group consisting of Leu, Nie, Nva, Met, D-Nle, Lie, Abu, Val, Arg, D-Arg and amides of these. 16. a method for lightening the color of an animal, the method comprising the step of administering to an animal an effective amount of a peptide antagonist of the a-melanocyte-stimulating hormone, wherein the peptide antagonist comprises a sequence of RST amino acids, where R, S and T are amino acid residues and R is selected from the group consisting of D-Trp, D-Phe, D-Tyr, Ac-D-Trp and D-His, except that, when S is Arg and T is Nie or an amide of this, then R is D-Phe, D-Tyr, Ac-D-Trp or D-His; when S is Lys, D-Arg, Leu, Nie, Ala, Met or Abu and T is Nie or an amide of this, then R is D-Trp; and when S is Arg and T is Leu, Nie, Nva, Met, D-Nle, Lie, Abu, Val, Arg or D-Arg or an amide of these, then R is D-Trp; S is selected from the group consisting of Arg, Lys, D-Arg, Leu, Nie, Ala, Met and Abu; and T is selected from the group consisting of Leu, Nie, Nva, Met, u-Nie, Lie, Abu, Val, Arg, D-Arg and these amides. i /. Everything, according to the claim ib, where the animal is a human. J. u. J_I p "i u? _ T ?, &ct do with claim v, where peptide antagonist is administered topically. i > 19. The method, according to claim 16, wherein the antagonist is administered by means of a method selected from the group consisting of topical administration, systemic administration by injection and oral administration. The method, according to claim 16, wherein the peptide antagonist is a tripeptide. 21. The method according to claim 20, wherein the tripeptide has an amino acid sequence of D-trp-Arg-Leu. 22. The method according to claim 20, wherein the tripeptide has an amino acid sequence of D-rp-Arg-Nle. 23. The method according to claim 20, wherein the tripeptide has an amino acid sequence of D-Trp-Arg-Nva.
2. 2 . The method according to embodiment 20 wherein the tripeptide has an amino acid sequence of D-trp-Arg-Met. 25. The method, according to claim 20, wherein the tripeptide has an amino acid sequence of D-trp-Arg-D-Nie. 26. The method, according to claim 20, wherein the tripeptide has an amino acid sequence of D-trp-Lys-Nie. 4b 27. The method according to claim 20, wherein the tripeptide has an amino acid sequence of D-trp-D-Arg-Nle. The method, according to claim 20, wherein the tripeptide has an amino acid sequence of D-trp-Leu-Nle. 29. The method according to claim 20, wherein the tripeptide has an amino acid sequence of D-trp-Nle-Nie. 30. The method according to claim 20, wherein the tripeptide has an amino acid sequence of D-Phe-Arg-Nle. 31. The method according to claim 20, wherein the tripeptide has an amino acid sequence of tyr-Arg-Nie. 32. The method according to claim 20, wherein the tripeptide has a amino acid sequence of Ac-D-trp-Arg-Nie-NH .. 33. A method of treating the malignant meianoma comprising the step of administering to an animal an effective amount of a peptide wherein the peptide has an amino acid sequence consisting of RST, wherein R, S and 'i are amino acid residues and R is selected from the group consisting of D- trp, D-Phe, D-Tyr, Ac-D-trp and D-HIS, except that, when S is Arq and T 4 / is Nie or an amide of it, then R is D-Phe, D-Tyr, Ac-D-tr? or D-HÍS; when S is Lys, D-Arg, Leu, Nie, Ala, Met or Abu and T is Nie or an amide thereof, then R is D-trp; and when S is Arg and T is Leu, Nie, Nva, Met, D-Nle, He, Abu, val, Arg or D-Arg or an amide of these, then R is D-tr ?; s is selected from the group consisting of Arg, Lys, D-Arg, Leu, Nie, Ala, Met and Abu; and T is selected from the group consisting of Leu Nie, Nva, Met, o-Nie, ile, Abu, val, Arg, D-Arg and amides thereof. a pharmaceutical composition containing a peptide with an amino acid sequence consisting of RST, wherein R, S and T are amino acid residues and R is selected from the group consisting of D-Txp, D-Phe, D-Tyr, Ac -D-trp and D-HIS, except that, when S is Arg and is Nie or an amide of it, then R is D-Phe, D-'i'yr, A c- i i- 'l' rn M- H i c • r-i i a nrJ? < -: or c I. c M- fl rrt 1, 011 M "l o Ala, Met or Abu and T is Nie or an amide of it, then P is D-trp; and when S is Aro and T is Leu, Nie, Nva, Met, D-Nle, Lie, Abu, Val, Arg or D-Arg or an amide of these, then R is D-trD; s is selected from the apD consisting of Arg, Lys, D-Arg, Leu, Nie, Ala, Met and Abu; and T is selected from the group consisting of Leu Nie, Nva, Met, D-Nle, Lie, Abu, Val, Arg, D-Arg and these amides. 4tí 35. The pharmaceutical composition according to claim 32, wherein the pharmaceutically acceptable carrier is selected from the group consisting of a solution, cream or lotion suitable for topical application, a physiological salt or buffer suitable for systemic injection, a slow release carrier and a composition suitable for oral administration. 36. An α-melanocyte stimulating hormone antagonist peptide that is identified by a method comprising the steps of a. prepare a combinatorial library consisting of a library of random sequence tpeptide molecules; b. the screening of the combinatorial library to detect activity that antagonizes the melanocyte-stimulating hormone; c. the identification of tripeptidic molecules that have activity that antagonizes the a-meanocyte-stimulating hormone. 37. a kit containing, in one or more containers, a peptide antagonist of the ameriancyte-stimulating hormone, which is selected from the group consisting of D-Trp-Arg-Leu; D-Trp-Arg-Nie; D-Trp-Arg-Nva; D-Trp-Arg-Met; D-Trp-Arg-D-Nie; D-T'rp-Argile; D-Trp-Arg-Abu; D- and Trp-Arg-vai; D-trp-Arg-Arg; D-trp-Arg-D-Arg; D-Trp-Lys-Nie; D-trp-D-Arg-Nle; D-trp-Leu-Nle; D-trp-NLe-Nle; D-trp-Aia-Nie; D-trp-Met-Nie; D-trp-Abu-Nle; D-Phe-Arg-Nle; D-trp-Arg-Nle; D-Ac-trp-Arg-Nle; Trp-Arg-Nle and D-His-Arg-Nle and their amides. a kit, according to claim 37, further containing, in one or more containers, a pharmaceutically acceptable carrier, suitable for the administration of the peptide antagonist of the a-melanocyte-stimulating hormone.