CN1179105A - Antagonists of alpha-melanocyte stimulating hormone and methods based thereon - Google Patents

Abstract

Peptide antagonists of alpha-melanocyte stimulating hormone are disclosed, together with methods of inhibiting the effects of alpha-melanocyte stimulating hormone on cells or tissues sensitive to that hormone. In particular, methods for lightening the pigmentation of skin and for treating malignant melanoma, as well as kits for practicing the invention are also disclosed.

Description

α-melanotropin antagonist and correlation technique thereof

1. preface

The invention provides the peptide agonist of α-melanotropin, and the method for inhibition α-melanotropin to the effect of its sensitive cells or tissue is provided, comprise skin whitening, regulate immunne response and treatment malignant melanoma.

2. background of invention

The pigmentation of skin with darken with the epidermis melanosome in melanic content and distribute relevant.The epidermis cell group comprises keratinocyte and melanocyte, and the latter provides melanosome by dendrite for keratinocyte, promptly contains melanic pigment granule.Melanin is black pigment, and it is to produce like this: under the catalysis of tryrosinase, tyrosine is oxidized to DOPA and DOPA quinone, the chemical compound repolymerization that it generated becomes melanin.

The local excessive pigmentation has many kinds, also lacks cosmetic treatment safely and effectively at present.These pigmentations comprise mottle, as freckle, sun nevus (also claiming the liver nevus), acanthosis nigricans (the excessive sexual maladjustment of a kind of melanin), coffee with milk speckle, nevus, chloasma (local skin deepening in puerperal).The method of regulating the whole skin tone is that people pay close attention to and need always, its objective is that in order to improve looks for example skin whitening perhaps stops the outward appearance that is caused by the endocrine disturbance to be damaged.Up to the present, also there is not method in full force and effect to be used for the tone of reconciliation statement chromatosis.

The endogenous hormones relevant with Pigmented adjusting be α-melanotropin (" α-MSH ") (Lerner etc., 1961, Nature 189,176-179).α-MSH is also relevant with following factors: the adjusting of central nervous system and function of immune system (De Weid, 1993, Ann.NY Acad.Sci.680,20-28; Tatro, 1990, Brain Res.536,124-132; Luger etc., 1993, Ann.NY Acad.Sci.680,567-570; Cannon etc., 1986, J. Immunol.137,2232-2236; Murphy etc., 1983, Science221,192-193), growth (Strand, F.l. etc., 1993, Ann.NY Acad.Sci.680,29-49), cell division (Halaban etc., 1993, J.Ann.NY Acad.Sci.689,290-300), and melanoma (Varga etc., 1974, Proc.Natl.Acad.Sci.U.S.A.71,1590-1593).

The indirect support of hypothesis that to α-melanotropin is the endogenous regulatory factor of skin key colour is tested from some, in these experiments, extract Fish, amphibian and mammiferous hypophysis such as low can induce its pigmentation alleviate (Chavin, 1956, J.Exp.Zool.133,1-36; Smith, 1916, Science44,75-758; Allen, 1916, Science44,755-758; Rust, C.C., 1965, Gen. Comp.Endocrinol.5,222-231).

In addition, some clinical examples report, pale unusually (Felig etc., 1987. can become when the people causes the pituitary function forfeiture owing to disease or pituitectomy Endocrinology and MetabolismMcGraw-Hill Book Company, New York, 1-26).To the mankind, not only hypophysis discharges MSH, and the Keratoderma cell behind irradiation under ultraviolet ray, also obviously discharge MSH (Luger etc., 1993, Ann.N.Y.Acad.Sci.680,567-570).As everyone knows, MSH is injected to the animal or human and can make skin color exceed the key colour level.Endogenous α-MSH still belongs to unknown to the Pigmented contribution of key colour so far.

Shown that MSH can make the tyrosinase activity of the mouse melanin tumor cell of cultivation strengthen.Find that also synchronization cell only has response in the G2 phase of cell cycle to MSH. ¹²⁵The combination of the MSH of I labelling mainly occurs in the G2 phase.Therefore, infer MSH may by activate on the MSH receptor that is attached to G2 phase cell the melanoma cell adenyl cyclase (Varga etc., 1974, Proc.Natl.Acad.Sci.U.S.A., 71 (5), 1590-3).

Therefore, the active antagonist of practical α-MSH can provide the medicament of confirming endogenous α-MSH effect, and the method for all biological function (comprising cutaneous pigmentation color harmony malignant melanoma cell) of regulating α-MSH mediation is provided.

Recently, people such as Jayawickreme is in November, 94 J.of Biol.Chem.269 (47), identified a series of polypeptide class α-MSH antagonist among the 29846-29854, for example octapeptide and nonapeptide antagonist.Yet the larger peptide molecule sees through and makes the ability of the interested tissue of people is limited.Therefore need micromolecule α-MSH antagonist, for example less than 500 daltonian antagonisies, this antagonist preferably is easy to be diffused in the organizational structure to regulate α-MSH sensitive organization.

3. summary of the invention

The invention provides the peptide agonist of α-MSH, and then have the cell of response and the function of tissue that method is provided to α-MSH for regulating.The present invention provides peptide agonist according to the active alpha-MSH peptide of following table 1.Therefore, the invention provides such peptide agonist of α-MSH, it comprises the R-S-T sequence, wherein R, S and T are amino acid residues, R is selected from D-Trp, D-Phe, D-Tyr, Ac-D-Trp, Trp and D-His, just when S be Arg and T when being Nle or its amidate, R is D-Phe, D-Tyr, Ac-D-Trp, Trp or D-His; When S is Lys, D-Arg, Leu, Nle, Ala, Met or Abu, and T is when being Nle or its amidate, and R is D-Trp; When S is Arg, and T is when being Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg or D-Arg or its amidate, and R is D-Trp; S is selected from Arg, Lys, D-Arg, Leu, Nle, Ala, Met and Abu; T is selected from Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg, D-Arg and amidate thereof.The present invention also provides the molecule that similar structures is arranged with disclosed peptide agonist.

The present invention also provides a kind of method, promptly by α of the present invention-MSH peptide agonist is contacted and suppresses α-MSH activity with α-MSH response cell or tissue.

The present invention also provides the method that the animal skin outward appearance is brightened, and this method is the animal that needs this type of treatment by giving, and for example mammal such as people use the α of the present invention-MSH peptide agonist of effective dose and finish.Provide Several Methods to be used for the treatment of pigmented spots, nevus, freckle, chloasma, and the outward appearance that can be used for part or whole body brighten beauty treatment.

The present invention also provides a kind of method for the treatment of malignant melanoma, and this method is the mammal that needs this type of treatment by giving, and for example the people uses the α of the present invention-MSH peptide agonist of effective dose and finishes.Using can be partial or general.

The present invention also provides a kind of adjusting immune method, and this method is the mammal that needs this treatment by giving, and for example the people uses the α of the present invention-MSH peptide agonist of effective dose and finishes.

In another embodiment, the invention provides the pharmaceutical composition of the peptide agonist of the active alpha-MSH antagonism peptide that provides in the table 1 below a kind of basis.Therefore the invention provides α-MSH antagonist with R-S-T aminoacid sequence.Wherein R, S and T are amino acid residues, and R is selected from D-Trp, D-Phe, D-Tyr, Ac-D-Trp, Trp and D-His, just when S be Arg and T when being Nle or its amidate, R is D-Phe, D-Tyr, Ac-D-Trp, Trp or D-His; When S is Lys, D-Arg, Leu, Nle, Ala, Met or Abu, and T is when being Nle or its amidate, and R is D-Trp; When S is Arg, and T is when being Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg or D-Arg or its amidate, and R is D-Trp; S is selected from Arg, Lys, D-Arg, Leu, Nle, Ala, Met and Abu; T is selected from Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg, D-Arg and amidate thereof.And pharmaceutical carrier.

In an optional embodiment, the invention provides a kind of three peptide antagonists of α-melanotropin, identify this three peptide antagonists by preparation combined sorting library, contain the tripeptides molecular library of random structure in the library.This contacts with α-melanotropin active testing system in combined sorting library at random again, identifies active three peptide molecules of antagonism α-melanotropin subsequently.

4. accompanying drawing is described

Fig. 1 shows constituting of the combined sorting library that formed by 96 sublibraries that are used to identify three peptide antagonists.Abbreviation: Abu, 2-aminobutyric acid; γ-Abu, the 4-aminobutyric acid; ε-Abx, 6-aminocaprolc acid; Aib, the 2-aminoisobutyric acid; β-Ala, the 3-alanine; Orn, ornithine; Hyp, trans hydroxyproline; Nle, nor-leucine; Nva, norvaline.

Fig. 2 A and 2B show the video image of α-MSH antagonist reaction, produce on the microballon of these images in first combination sublibrary (being the D-Trp sublibrary) that comprises D-Trp.Left figure (Fig. 2 A) shows the 6 centimetres of agarose culture dishs (Falcon product) that are coated with the Xenopus laevis melanocyte, these cells are after the microballon of just using about 600 D-Trp sublibraries, with melatonin (1Nm, 30 minutes) and α-MSH (15Nm, 30 minutes) pretreatment.After 60 minutes (Fig. 2 B),, the part that is close to microballon demonstrates white circular pattern owing to discharging.

Fig. 3 provides the dose response curve of antagonist to be determined, and these antagonisies are used to test its inhibition ability to pigment diffusion in the Xenopus laevis melanocyte of α-MSH inducing culture.Curve chart shows 3 peptide A ₁(square), A ₄(black triangle) and A ₆The example of the inhibition curve of (hollow circle) (seeing Table 1), these three peptides are identified by diffusion experiment.Two other peptide A that in library screening, does not find ₉(filled circles) and A ₁₁In (black triangle) also is included in as a comparison.The light transmission rate of the cultivation melanocyte by measure seeing through 1nM melatonin and processing (Potenza etc., 1992, Anal.Biochem206,315-22) can carry out quantitatively melanin reactions with corresponding logical equation curve (De Lean etc., 1978, Am.J.Physiol.235, E97-E102).Every bit is represented the average and the sample standard deviation (SSD) of 4 separate measured values, and these measured values are to obtain carry out 2 hours after the free peptide that adds α-MSH (15Nm) and desired concn after.The gained result represents with accounting for corresponding independent percent with α-MSH processing gained result.

Fig. 4 shows D-Trp-Arg-Leu-NH ₂(dWRL) to the competitive inhibitory effect of α-MSH.This inhibitory action by the Schild regression analysis be confirmed (Arunlakshana etc., 1959, Br.J.Pharm.14,48-58).Equilibrium dissociation constant (pKc) is 7.2 ± 0.1M, and regression slope is 1.6 ± 0.03.Dotted line shows that confidence level is 99%.

The dosage rate of Fig. 5 displayed map 4 (dr), these ratios add 1 μ M (solid diamond) from not adding DWRL (hollow circle) among α-MSH, the concentration-response curve the during DWRL of 10 μ M (hollow triangle) and 100 μ M (solid circles) concentration.(EC during α-MSH individualism ₅₀Be 2.5 ± 0.3Nm, α-MSH and DWRL and EC when depositing ₅₀Be 4.8 ± 0.6 μ M).Each point is represented four the independently average and the SSD of light transmittance test.The initial light transmittance of Ti=(2 minutes).The final light transmittance of Tf=(60 minutes).DWRL does not cause the EC of vasoactive intestinal peptide (" VIP ") and 8 arginine vasotocins (" AVT ") ₅₀The variation of value (these data are not listed).

Fig. 6 shows D-Trp-Arg-Nle-NH ₂(dW-R-Nle) the cAMP second message,second messenger stimulation of blocking-up α-MSH mediation when 40 μ M, but do not block the activated cAMP stimulation of AVT (8nM).Oxytocin antagonist GVT ([d (CH ₂) ₅, Tyr (Me) ², Orn ⁸]-vasotocin; The Peninsula product) when 20 μ M, is used as the contrast of blocking-up by the activated response of 8Nm AV.The measurement of CAMP is that the Xenopus laevis melanocyte that converges that is used in growth in the 24 hole tissue culture plate (Falcon) carries out in the cell.Each bar chart is represented the average and the SSD of 4 independent measurement values. ^*(T-test; P＜0.001), is with except the asterisk person for whole test groups.

Fig. 7 show people MSH receptor be subjected to dWRL in the Xenopus laevis fibroblast functional antagonism (Daniolos etc., 1990, Pigment Cell Res.3,38-43).These Xenopus laevis fibroblasts are subjected to " Vector alone " (pcDNAI/NEO, Invitrogen produces) or contain Humanmachine tumour MSH receptor inserting segmental " HMelMSHR " transfection (pcDNAI/NEO).Contrast=no medication, MSH=5nM α-MSH, dWRL concentration=10 μ M, forskolin=100 μ M forskolin (7 β-deacetylate-7 β-[g-(N methyl piperazine)-butyryl); Calbiochem produces).

Fig. 8 is a photos, is used for showing that local application tripeptides DWRL (1mM aqueous solution) makes the skin whitening of calm pigment to Xenopus laevis skin and forms the colour of skin of " albinism sample ".

Fig. 9 is a photos, is used for showing the whitening effect that causes whole body pigmentation skin to Xenopus laevis as systemic injection.Injected (40 μ M/kg) DWRL or D-Trp-Abu-Arg-NH for three Xenopus laevis ₂(contrast) in 20 minutes, presents the body surface feature of albinism sample, as shown in the figure.Three contrast Xenopus laevis are still dark skin.

Figure 10 shows the comparison of the strongest L-type molecule of 6 activity in the structure function of the 3rd position, these molecules be by random screening way from the 1st be D-Trp, identify in the 2nd the arginic sublibrary.Other 3 molecules the 3rd replacement of not finding in random screening are also listed in this as a comparison.The activity of antagonist is relevant with the hydrophobicity and the charge characteristic of the 3rd R group.

5. detailed Description Of The Invention

The present invention relates to the peptide agonist of active alpha in the following table 1-MSH antagonistic peptide. Therefore, originally Invention provides the α of the governing response α-cell of MSH hormone and the function of tissue-MSH antagonist And method. Specifically, the invention provides contain the R-S-T sequence α-MSH peptide class is short of money Anti-dose, wherein R, S and T are amino acid residues, and R is selected from D-Trp, D-Phe, D-Tyr, Ac-D-Trp, Trp and D-His, just working as S is that Arg and T are Nle Or during its amidate, R is D-Phe, D-Tyr, Ac-D-Trp, Trp or D-His; When S is Lys, D-Arg, Leu, Nle, Ala, Met or Abu, and T When being Nle or its amidate, R is D-Trp; When S is Arg, and T be Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg or D-Arg or its amidatioon During thing, R is D-Trp; S be selected from Arg, Lys, D-Arg, Leu, Nle, Ala, Met and Abu; T be selected from Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg, D-Arg and amidate thereof. In preferred embodiments, this polypeptide is by 10 Individual, include but not limited to 15 amino acid. In another embodiment, this polypeptide comprises and is no more than 8 Individual or be no more than 12 amino acid residues (comprising amino acid residue derivative or amino acid analogue).

In a further preferred embodiment, this type of peptide molecule contains as 3 of tripeptides continuous amino Acid residue (derivative or the amino acid analogue that comprise amino acid residue), wherein these three amino acid Residue is the active alpha that provides in the following table 1-MSH antagonist. The present invention also provides the class of these peptides Like thing and derivative.

Term used herein " amino acid " or " amino acid residue " refer to that naturally occurring, non-natural deposits Or the amino acid residue of deriving or the molecule with Amino acid conformation.

Term " peptide " refers to any molecule that contains peptide bond, and it is existed by naturally occurring, non-natural Or the amino acid residue of deriving or have the molecular composition of Amino acid conformation. Peptide bond refers to connect two phases The amido link of adjacent amino acid residue.

In specific embodiments, α-MSH antagonist is one of following three peptide molecules: D-Trp-Arg-Leu; D-Trp-Arg-Nle; D-Trp-Arg-Nva; D-Trp-Arg-Met; D-Trp-Arg-D-Nle; D-Trp-Arg-Ile; D-Trp-Arg-Abu; D-Trp-Arg-Val; D-Trp-Arg-Arg; D-Trp-Arg-D-Arg; D-Trp-Lys-Nle; D-Trp-D-Arg-Nle; D-Trp-Leu-Nle; D-Trp-Nle-Nle; D-Trp-Ala-Nle; D-Trp-Met-Nle; D-Trp-Abu-Nle; D-Phe-Arg-Nle; D-Tyr-Arg-Nle; D-Ac-Trp-Arg-Nle; Trp-Arg-Nle and D-His-Arg-Nle. Also provide comprise one or more on State the composition of tripeptides.

In another embodiment, the molecular weight of peptide is less than 500 dalton.

In another embodiment, peptide can be from the combinatorial libraries of random synthesis by the screening α-The tripeptides α that the MSH antagonistic activity obtains-MSH antagonist. As example, combinatorial libraries can be pressed Houghten etc., 1991,Nature354,84, Houghten etc., 1992,Biotechniques13,412 or Jayawickreme etc., 1994,Proc.Natl.Acad.Sci.U.S.A.91,1614-1618 method, in the library synthetic peptide class have random structure and with the homology of α-MSH Irrelevant. Limiting " at random " is not have any predictable size and any position for the peptide that makes gained The structure of putting. Other random libraries known in the art also can be used for identifying α-MSH antagonistic peptide. Sieve Choosing can be undertaken by known method in any this area, and preferably sieves described in following embodiment Choosing.

The aminoacid of these peptides and formation peptide is not limited to naturally occurring 20 seed amino acids.Non-classical aminoacid includes but not limited to following molecule: the D type isomer of common amino acid, α-An Jiyidingsuan, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, tert-butyl group glycine, tert-butyl group alanine, phenylglycine, Cyclohexylalanine, Beta-alanine, designer's (designer) aminoacid such as Beta-methyl aminoacid, the Ca-methylamino acid, Na-methylamino acid, and general amino acid analogue.In addition, aminoacid also comprises: Abu, 2-aminobutyric acid; γ-Abu, the 4-aminobutyric acid; ε-Abx, 6-aminocaprolc acid; Aib, the 2-aminoisobutyric acid; β-Ala, the 3-alanine; Orn, ornithine; Hyp, trans hydroxyproline; Nle, nor-leucine; Nva, norvaline.And aminoacid can be D type (dextrorotation) or L type (left-handed).And these peptides and/or aminoacid can form derivant by the sealing to group, include but not limited to acetylation, at amino terminal carboxylated and in the carboxyl terminal amidatioon so that protected derivant to be provided.

α-MSH antagonism peptide of identifying by combined sorting can prepare with method as known in the art.For example, briefly, solid-phase synthesis is arranged, comprise the aminoacid that is coupled to the amino acid whose carboxyl of C-terminal on the resin and constantly adds N-α protection.Blocking group can be as known in the art any.Before the growing chain, the previous aminoacid that adds was wanted deprotection at new aminoacid of every adding.The method that aminoacid is coupled to suitable resin is seen Rivier etc., United States Patent (USP) 4,244, No. 946.This type of solid phase synthesis is seen for example Merrifield, 1964, J.Am.Chem.Soc.85:2149; Vale etc., 1981, Science213:1394-1397; Marki etc., 1981, J.Am.Chem. Soc.103:3178 and United States Patent (USP) 4,305,872 and 4,316, No. 891.In aspect preferred, use the automatic peptide synthesizer.

The purification of synthetic peptide is undertaken by standard method, comprises chromatography (for example ion exchange, affinity chromatograph, volume column chromatography), and is centrifugal, the standard technique of differential solubility method or any other purifying protein.In preferred embodiments, can use reversed-phase HPLC (high performance liquid chroma-tography).

The structure and the function relationship of three peptide antagonists disclosed herein see accompanying drawing for details.Its 3rd amino acids as shown in figure 10.Can prepare with reference to this relation has α-the similar molecular structure of MSH antagonist activities.Therefore, except structure, hydrophobicity, charge characteristic and the side chain character of peptide agonist described herein, the present invention also attempts to comprise these similar molecules.

5.1 α-MSH antagonist has treatment and beautifying use

α-MSH peptide agonist has effect to have the purposes of treatment and beauty treatment aspect because of its function to α-MSH mediation.For example it can alleviate cutaneous pigmentation, regulates immune system and treatment melanoma.

" brighten (lightening) " and mean and alleviate the tone that melanin causes in the skin, make whiteness of skin.Do not wish to be limited, it is believed that peptide agonist is the competitive inhibitor of α-MSH to α-MSH receptor, the pigmentation of endogenous α-MSH capable of blocking by any concrete mechanism of action.Therefore, the melanin granule that is present in α-MSH response cell can be owing to the sealing to endogenous α-MSH receptor scatter in the presence of α-MSH antagonism peptide.This can remove the enhancement effect of MSH to the melanin accumulation.When this peptide was used in the part, the skin at the position of administration for peptides will bleach.When this peptide of systemic administration, the tone on whole skin surface will shoal.

α-MSH antagonism peptide can be used for treating following local disease, includes but not limited to hyperpigmentation after mottle, color lump such as nevus, birthmark, freckle, chloasma, the inflammation.If need, also can be that purpose is carried out the part and the whole body colour of skin brightens with the beauty treatment, as previously mentioned.

By the peptide agonist of the present invention of effective dose is used to patient, α-MSH antagonism peptide provides method for the treatment malignant melanoma.

By α-MSH peptide agonist of using effective dose for the animal or human who needs this treatment, the present invention also provides the method for the immunne response of regulating the animal or human.

In addition, active diseases associated of other and bad α-MSH and clinical symptoms can be treated with α of the present invention-MSH antagonism peptide.

5.2 the evaluation of α-MSH peptide agonist

The conventional method of the drug development of peptide receptor is usually based on the screening to the natural agonist peptide structure that changes, and general what identify is macromole.On the contrary, as be shown in the examples, the small-molecule peptide antagonist of α-MSH of the present invention is according to improving one's methods to people's such as Jayawickreme diffusion method of testing, a large amount of micromolecule tripeptides at random identify by screening, these micromolecule tripeptides were in the surface preparation of the microballon of multipurpose combined peptide library (" MUPL "), as people such as Jayawickreme 1994 J.Biol.Chem.269,29846-29854 (" Jayawickreme I ") and 1974, Proc.Natl.Acad.Sci. (U.S.A.)91,1614-1618 (" Jayawickreme II ") is described, and these two pieces of documents intactly are hereby incorporated by.

Described in specific embodiment, employed method can be screened being loaded with at random the library of the microballon of three peptide molecules.The biological detection culture medium is by being made up of the agarose gel of melanocyte through the melatonin processing is also long thereon.Microballon is contacted with agarose gel.Carry out the controlled release of peptide molecule from the microballon with the gas phase method for releasing of Jayawickreme I and II (the same).Observe in 5 or 10 minutes by the peptide molecule that discharges and spread inductive pigment dispersion, and monitor with the video image relief method.

5.3 treatment and compositions

The invention provides the treatment that α of the present invention-MSH antagonism peptide carries out by use effective dose to the experimenter.Peptide is purification preferably.The experimenter is animal preferably, and for example mammal most preferably is the people.

Known have a multiple induction system, can be used for using α of the present invention-MSH antagonism peptide, for example aqueous solution, liposome methods, microgranule, microcapsule, the effect of receptor-mediated cell phasmid (see Wu and Wu, 1987, J.Biol.Chem.262,4429-4432).Application process includes but not limited to be directly used in skin, Intradermal, intramuscular, intravenous, nasal cavity, epidural and oral cavity route.Peptide class of the present invention can be used by any suitable approach.For example infusion or single fast injection are by epithelium or mucosa lining (as oral mucosa, rectum and intestinal mucosa).

In specific embodiment, use peptide of the present invention at the regional area of any said method treatment of needs, the result also may be ideal.

The present invention also provides pharmaceutical composition.Said composition comprises peptide of the present invention and the pharmaceutical carrier or the excipient of treatment or cosmetic effective dose.Carrier includes but not limited to water, saline solution for example normal saline, buffer, glucose, glycerol, ethanol and their conjugate.Said preparation should be suitable for the method used.

If need, said composition also can comprise a small amount of wetting agent or emulsifying agent, perhaps the pH buffer agent.It can be solution, suspension agent, Emulsion, tablet, pill, capsule, slow-releasing agent, unguentum, gel or powder agent.It also can be made into suppository, uses conventional junction mixture and carrier such as triglyceride.Oral medicine can contain standard vector such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate.

In specific embodiments, said composition can be made the pharmaceutical composition that is suitable for to people's used for intravenous injection according to a conventional method.Typical intravenous injection compositions is the sterile isotonic buffer.In case of necessity, also can comprise cosolvent and local anesthetic in this compositions, to eliminate the pain of injection site.Usually, each composition provides with independent or blended unit dosage form, such as the freeze-dried powder in the container that the is contained in sealing ampoule or the pouch of bright active drug dosage (as show) or there is not aqueous concentrate.When being used for infusion, it can divide to install to and fills in pharmaceutical grade sterilized water or the brinish infusion bottle.During as injection, need the Injectable sterile water of an ampoule or saline before injecting, to mix with ingredient.

α of the present invention-MSH antagonism peptide can be made into the form of neutrality or salt.Pharmaceutical salts comprises the salt that those and free amine group form, the salt that example hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid etc. form, with the salt that forms with free carboxy, the salt that forms as sodium, potassium, ammonium, calcium, hydrated ferric oxide., 2-aminopropane., triethylamine, 2-ethyl amido alcohol, histidine, procaine etc.

Deficiency disorder that α of the present invention-MSH peptide agonist treatment is concrete or the effective dose when using as beauty treatment depend on the antagonist properties that is suitable for this deficiency disorder or cosmetic conditions, and are determined by standard clinical techniques.In addition, external or body build-in test can have randomly with helping determine the optimal dose scope.Used exact dose also depends on the order of severity of route of administration, disease in the preparation, cosmetic conditions also should be according to professional's judgement and patient's state of an illness decision.Effective dose can be inferred according to dose response curve external or that the animal model test macro is obtained provided herein.

According to the present invention and to the observation of Expected Results, be easy to determine the effective dose of the peptide agonist of α-MSH by the dosage grade of peptide agonist.This part data provides among table 1 and the accompanying drawing 2-9 below.The IC that is provided is provided ₅₀Example in the concentration, the dose response curve of representative peptide class and body, these data will help to determine to make the colour of skin to brighten needed effective dose.

In one embodiment, in brightening to the purpose topical formulations with for example colour of skin, the valid density scope of dWRL (D-Try-Arg-Leu) at 1 μ M between the 10mM.In preferred embodiments, effective dWRL concentration range of topical formulations at 100 μ M between the 5mM.In a more preferred embodiment, the valid density scope of topical formulations at 500 μ M between the 2mM.In one embodiment, the valid density of topical formulations is about 1mM.

In another embodiment, dWRL is used for the effective dosage ranges of systemic administration (for example skin whitening) between per kilogram of body weight 1-4000 μ M.In preferred embodiments, to be used for the effective dosage ranges of systemic administration (for example skin whitening) be per kilogram of body weight 20-200 μ M to dWRL.In a more preferred embodiment, dWRL is used for the effective dosage ranges of systemic administration (for example skin whitening) between per kilogram of body weight 30-100 μ M.Effective dosage ranges when in another embodiment, dwRL makes systemic administration is 30-100 μ M.

The IC of the peptide agonist relative potency of the demonstration dWRL tripeptides that provides by for example reference table 1 ₅₀Value, the just valid density and the dosage of easily definite various other exemplary three peptide antagonists.

In another optional embodiment, the present invention includes the test kit of the α of the present invention-MSH antagonism peptide that contains effective dose.This test kit will contain one or more containers that at least a α of the present invention-MSH antagonism peptide is housed.As example, this test kit can contain α-MSH antagonism peptide or be used for skin, or the peptide class by subcutaneous, intramuscular, intravenous, intranasal, epidural administration or oral administration.This test kit can contain the α-MSH antagonism peptide of solution, suspension agent, Emulsion, tablet, pill, capsule, slow releasing agent, unguentum, gel or powder agent form.This α-MSH antagonism peptide can be pre-mixed or separately deposit is easy to blended composition.It also can be mixed with a kind of form or pharmaceutical composition of peptide, wherein contains the antagonism peptide of the present invention of effective dose.

The present invention will be further described in the following embodiments, and these embodiment to the scope of the invention without limits.

6. embodiment

6.1 the design of tripeptides combinatorial library and structure

The tripeptides of synthetic combinatorial library at random is with Fmoc (fluorenylmethyloxycarbonyl) and the chemical crosslinking resin of MBHA (4-methyl-.alpha.-aminodiphenylmethane .) (replacement rate=9.5mM/g), as described in JayawickremeI and II, the same, and carry out the synthetic (Lam etc. of a plurality of peptides simultaneously, 1991 Nature354,83-84 and Houghten etc., Nature354,84-86) prepare.To use separation, coupling and recombination method when synthesizing the tripeptides in the microballon sublibrary.

Each sublibrary may contain 2304 (1 * 48 * 48) plants tripeptides or the combination of class tripeptide sequence.First amino acid position (see figure 1) in the molecule of each sublibrary of Z96 representative formation.This locational molecule also belongs in addition in 2 positions a kind of in 48 kinds of molecules, or equivalent is changed in corresponding acetylation (Ac).AA in the position 2 and 3 ₁And AA ₃₇Represent 20 kinds of left-handed (not pre-fixing) aminoacid, remove cysteine, add the isomer of 18 kinds of corresponding dextrorotation (D) aminoacid.Add other 11 kinds of molecules, sum total is 48.

In the preparation that is used for screening, about 4% extension pearl molecule is separated under the effect of controlled gas phase trifluoroacetic acid (TFA) cracking process.Through gasification NH ₃/ H ₂O handles the back as the nontoxic microballon that is directly used in biological detection.Separated molecule still remains on the microballon with non-covalent bonded state, just is released when detecting.

6.2 functional living being detects

By preceding method Xenopus laevis dermal melanin cell is kept in the culture fluid (Daniolos etc., 1990, Pigment Cell Res.3,38-43; Potenza etc., 1991, Pigment Cell Res.4,186-192 and Karne etc., 1993, J.Biol.Chem.268,19126-19133).With 1,500,000 cell inoculations in the 6 cm of tissue culture plates that contain 5 milliliters of fibroblast culture medium (Falcon product), 27 ℃ of cultivations at least 48 hours.When each the detection, remove this culture medium, change 3.5 milliliter 0.9% Sea Plaque agarose.Cell will be used melatonin (1nM, 30 minutes) and α-MSH (15mM, 2 minutes) pretreatment.Use 600 microballons in each sublibrary.These microballons are separated by the agarose and the melanocyte of one deck 2 millimeters thick.Only allow complete free molecule and cell surface to have an effect.

The inductive pigment of tripeptides shifts (diffusion) video image subduction technical monitoring, this technology such as McClintock etc., and 1993, Anal.Biochem.209, described in the 298-305.

6.3 peptide sequencing

With the microballon N-crassitude (" NMP ") of hot spot, dichloromethane (" DCM ") and methanol (" MeOH ") clean the back and take turns the microballon that screening has the positive reaction signal as second.These microballons sporadicly can be dispersed in the enterprising row filter of polyethylene film therebetween.Screen the microballon gas phase trifluoroacetic acid/NH that obtains from the first round ₃/ H ₂The O method is cracked into single microballon.These positive marks' single microballon is collected on the glass fiber filter, washes with NMP, DCM and MeOH.And check order with Applied Biosystems 476A sequenator.

6.4 dose response research

For carrying out dose response research, with standard Fmoc chemical method synthetic whole tripeptides on the Rink of 0.25mmol AmideMBHA resin.After synthetic, with HPLC and these peptides of C18 post reversed phase chromatography method purification.With microtitration plate measure dose response curve (Potenza etc., 1992, Pigment Cell Res.5,372-378).Melanocyte (18,000/ hole) is put into 96 hole tissue culturing plates (Falcon product).After the detection, culture medium is with containing 1nM α-MSH, and the tripeptides of 1nM melatonin and variable concentrations replenishes this culture medium.Measure in the matched group, use Bombesin-[8-14] fragment, oxytocin replaces 1nM α-MSH or α-MSH is changed to blank.Measure α-MSH dose response curve or competition inhibition curve by the α-MSH concentration that changes in the variable concentrations tripeptides.

6.5 result

Carry out multipurpose peptide library screening method as stated above and carry out (accounting for total amount 4%) with every microballon 10pM emission levels.So that the molecular reaction that is low to moderate 100 μ M is tired also can be observed.This has the library of 221,186 kinds of components to be made up of 96 sublibraries, and each sublibrary has 2,304 kinds of peptide molecule combinations.These sublibraries are distinguished by the amino terminal of its peptide.With D-tryptophan, dexamphetamine propylhomoserin, L-tyrosine and acetyl dextrorotation tryptophan is that response curve that the sublibrary of N-terminal produces shows and has multiple antagonist-like molecules, wherein has signal that the sublibrary microballon of dextrorotation tryptophan end produces at most also the strongest (Fig. 4).When adding 8nM AVT ([arginine ⁸]-oxytocin, Sigma produces; EC ₅₀≈ 2nM) or 4nM VIP (blood vessel intestinal peptide, Sigma produces; EC ₅₀≈ 1nM) rather than 15nM α-MSH (Peninsula produces; EC ₅₀≈ 2nM) time, from these sublibraries, do not observe similar reaction signal.

After the diffusion Test Identification, antagonist to be selected is carried out resynthesis and purification (JayawickremeI and II, the same), and measure (Fig. 2-9).The result provides in table 1.Concerning α-MSH, two antagonist D-Trp-Arg-Leu-NH the strongest ₂(DWRL) and D-Trp-Arg-Nle-NH ₂IC ₅₀Value is respectively 620 ± 150nM (mean value SE) and 930 ± 220Nm.These antagonisies belong to the induced activity of competitive inhibition and the endogenous α-MSH of single-minded blocking-up amphibian and human MSH receptor are played sealing process (Fig. 2-9).The equilibrium dissociation constant of tripeptides DWRL (Ke) is 63 ± 15Nm (Fig. 2 C), if press the IC of desired α-MSH ₅₀Value is 96Nm approximately, and the α-MSH in the test has reached its EC ₅₀Value.

Table 1

Peptide sequence IC ₅₀(μ M) A D-Trp-Arg-X-NH ₂A ₁Leu 0.62 ± 0.15A ₂Nle 0.93 ± 0.22A ₃Nva 3.3 ± 1.1A ₄Met 5.6 ± 2.6A ₅D-Nle 9.9 ± 1.8A ₆Ile 49 ± 9A ₇Abu 82 ± 41A ₈Val 237 ± 100A ₉Arg 261 ± 68A ₁₀D-Arg 664 ± 397A ₁₁γ Abu non-activity A ₁₂ε Ahx non-activity A ₁₃Ala non-activity A ₁₄β Ala non-activity B D-Trp-X-Nle-NH ₂B ₁Lys 15 ± 1B ₂D-Arg 30 ± 11B ₃Leu 48 ± 2.2B ₄Nle 59 ± 12B ₅Ala 65 ± 18B ₆Met 121 ± 27B ₇Abu 405 ± 69B ₈Asp non-activity C X-Arg-Nle-NH ₂C ₁D-Phe 4.4 ± 1.2C ₂D-Tyr 28 ± 2C ₃Ac-D-Trp 43 ± 8C ₄Trp 100 ± 2C ₅D-His 318 ± 105

6.6 structure-functional dependency

Except identifying that those are easy to whole body or partial α-MSH activity are risen three peptide antagonists of molecular weight less than 500Da of blocking effect, the various type antagonist signal that also each sublibrary is produced is tested simultaneously.Thereby might the similarities and differences of antagonist structure be compared.Can determine the composition relevant by comparing, and the data of this respect has potential using value to further exploitation non-peptide antagonist with acceptor interaction and sealing process.Screening to the secondary storehouse of dextrorotation tryptophan sublibrary can be derived bioactive sequence.

For example, diffusion test reaction curve shows that the 2nd amino acids of the molecule that reaction is the strongest is an arginine, is that (as lysine, dextrorotation arginine, methionine, leucine, nor-leucine) then shows lower MSH receptor antagonist-like activity in other amino acid whose secondary sublibraries at the 2nd.In the table 1, A ₁To A ₆6 the strongest molecules of reaction that screening D-Trp-Arg-X word bank obtains have been shown.

After the evaluation through the diffusion test, antagonist to be selected is carried out resynthesis and purification (Jayawickreme I and II, the same) and measures (Fig. 2-9).With respect to 15nM α-MSH, two antagonist D-Trp-Arg-Leu-NH the strongest ₂(dWRL) and D-Trp-Arg-Nle-NH ₂IC ₅₀Value is respectively 620 ± 150nM (mean value SE) and 930 ± 220Nm.These antagonisies belong to the induced activity of competitive inhibition and the endogenous α-MSH of single-minded blocking-up amphibian and human MSH receptor are played sealing process (Fig. 7-9).The equilibrium dissociation constant of peptide dWRL (Ke) is 63 ± 15nM (Fig. 5), if press the IC of desired α-MSH ₅₀Value is 96 approximately, and the α-MSH in the test has reached its EC ₅₀Value.

Various type MSH receptor antagonist agent molecule filters out from D-Trp-Arg-X level word bank, and the latter forms peptide molecule (Figure 10) relevant on a series of structures.They have constituted interchangeable structure, and its activity can be owing to the architectural difference of the 3rd amino acids is degenerated.In this group peptide, IC (inhibition concentration) measured value reflects that antagonist tires and hydrocarbon side chain lengths positive correlation, i.e. nor-leucine＞norvaline＞2-aminobutyric acid＞alanine, and with the negative correlation that exists of Beta-methyl, as norvaline＞isoleucine; 2-aminobutyric acid＞valine.From leucine, remove γ-methyl and make antagonist potency ratio norvaline low 50%.But tire than the nor-leucine that similar R group unvulcanised is arranged during when methionine and to reduce 6 times at the 3rd.Replace in the test at electric charge, what hydrophilic was the strongest is arginine, and its potency ratio nor-leucine (sees Table 1, A) for low 300 times.The reaction of dextroisomer word bank also is positive, but compares with the laevoisomer word bank, and it is tired and has reduced.The dextrorotation nor-leucine is than high 10 times tiring of the 3rd laevoisomer.

Most of tripeptides that screens from the diffusion test does not show similar α-MSH antagonistic activity.Though estimating has the height specificity between receptor and the part, what major part obtained is negative findings.The library of most of test lacks the change of active explanation position 1 can not obey the receptor acting category well.Except that the dextrorotation tryptophan, just have only in non-acetylation library that dextrorotation tyrosine demonstrates similar α-MSH antagonist activities on dextrorotation alanine and the littler degree.Finding that they also are X-Arg-Nle-NH ₂After 2 of position 1 replaceable aminoacid the most effective, this observed result obtains confirming (seeing Table 1, C1 and C2) in the general formula.Whole 48 kinds of non-acetylation compositionss in the X representative graph 1 herein.

Therefore, the increase of the time observing positive signal in screening may be owing to be similar to positive reaction molecule rather than other irrelevant structures in the dextrorotation tryptophan library on its structure.In the sublibrary in dextrorotation tryptophan library, also obviously observe the similar positive phenomenon that increases.At D-Trp-X-Nle-NH ₂Change X into lysine in the structure, dextrorotation arginine, methionine, leucine and nor-leucine can produce the polypeptide (seeing Table 1, B1 and B5) of antagonist activities and as desired, it is tired than low with arginine.Therefore, the resulting peptide of random screening is because it has the strongest signal, and becomes in fact the most virtuous MSH receptor antagonist in this library.Various changing factors in the combined sorting, for example, the size of microballon is littler than the tire influence of amino acid whose order modification of difference to the influence of signal intensity difference.

6.7 cyclic adenosine monophosphate is quantitative

Tested preceding 2 days, melanocyte is taped against in the 24 hole tissue culturing plates (Falcon product).Before facing test, clean cell with the 70%L-15 culture medium (Sigma product) that contains 0.05% bSA.The 1nM melatonin that is used in then in the same culture medium is handled cell.The mixture of reuse 70%L-15+0.05% bSA+0.5mM 3-isobutyl-1-methylxanthine (Aldrich product)+1nm melatonin was washed 5 minutes, and reuse is furnished with the same culture medium of test medicament and handled 30 minutes.Cell after chemicals treatment is washed 2 times with the salt of 70% phosphate-buffered.

As shown in Figure 6, D-Trp-Arg-Nle-NH ₂(dW-R-Nle), under 40 μ M, the cAMP second message,second messenger stimulation of sealing α-MSH (10nM) mediation, but do not seal activated cAMP stimulation by AVT (8nM).Oxytocin antagonist GVT ([d (CH ₂) ₅, Tyr (Me) ², Orn ₈]-vasotocin; Derive from Peninsula), under 20 μ M, be used as the contrast that the activated sealing of 8nM AVT is replied.

6.8 dWRL is to the functional antagonism of people MSH receptor in the Xenopus laevis fibroblast

For reference MSH receptor is subjected to the inhibition of three peptide antagonists,, when tripeptides exists and do not exist, measure the cAMP reaction, as shown in Figure 7 with of the carrier transfection of Xenopus laevis fibroblast with expressing human MSH receptor.

The Xenopus laevis fibroblast (Daniolos etc., 1990, Pigment Cell Res.3,38-43) with " Vector alone " (pcDNAI/NEO, Invitrogen produce) transfection or with transfection " HMelMSHR " (pcDNAI/NEO contains the Humanmachine tumour receptor of insertion) (Mountjoy etc., 1992, Science257,1248-1251; Roger Cone gives).Contrast=no additional medicaments.MSH＝5nM?μM?α-MSH。DWRL concentration is 10 μ M.Forskolin=100 μ M forskolin (7 β-deacetylation-7 β-[g-(N methyl piperazine subbase)-butyryl; Calbiochem produces).

Transfection carries out with electroporation that (≈ 5 * 10 ^-6In the saline of cell/400 μ l, 70% phosphate-buffered, pH7.0 adds 10 μ g cDNA to be measured) use 0.2 centimetre of test tube of BTX ECM-600 (475V, 720 ohm, 400 μ F).After the transfection 48 hours, the fused cell that is tiled in 12 hole tissue culturing plates (Falcon product) was washed 5 minutes with the 70%L-15 culture medium that contains 0.5% bSA BSA (Sigma product).Reuse is added with 0.5mM IBMX (3-isobutyl-1-methylxanthine, Aldrich produces) and washed 5 minutes.When IBMX exists, add the test medicament, every hole adds 1 milliliter of 60% ethanol extracting cAMP.CAMP is from cAMP conjugated protein (21; Amersham produces test kit).Each bar line is represented independent average and the SSD that measures three times, and contrasts-HMelMSHR test group n=6.Except that other groups that have single asterisk, all groups are ^*(T-Test; P＜0.006), other groups is ^*(T-Test; P＜0.006).

As shown in Figure 7, and only handle hMel MSHR cells transfected with MSH and compare, tripeptides is formed with more obvious inhibitory action to cAMP in hMel MSHR (the Humanmachine tumour MSH receptor) cells transfected.

6.9 the partial result of skin whitening

For showing that pigment brightens is inductive by the part in the skin level, and dWRL (1mM aqueous solution) is by the local skin surface (Fig. 8) that is applied to Xenopus laevis.Applying the lighter albinism sample skin color of position appearance.These results show that peptide of the present invention can transdermal effect, and under scotopic normal condition, mediate enhanced pigmentation by endogenous melanotropin in vivo, and the removal of this influence gives Rana nigromaculata lighter " albinism sample " state.

6.10 the general effect of skin whitening

Be the effect of conclusive evidence α-MSH, give scotopic Xenopus laevis injection dWRL (40 μ M/Kg) or D-Trp-Abu-Arg-NH the enhancing skin color ₂(matched group).DWRL causes that in 20 minutes every is tried Xenopus laevis (n=8) whole body whitening effect, but at matched group (n=8) no change (Fig. 9) then.

The present invention is not subjected to the restriction of specific embodiments scope described herein.In fact, except that described herein, according to the front describe and related data to make various change of the present invention also be conspicuous to those skilled in the art.These change the scope that also belongs to claim.This paper has quoted from different publications, and its disclosure intactly is incorporated herein by reference at this.

Claims (38)

1. the peptide agonist of α-melanotropin, it has the aminoacid sequence that comprises R-S-T, wherein R, S and T are amino acid residues, R is selected from D-Trp, D-Phe, D-Tyr, Ac-D-Trp, Trp and D-His, just when S be Arg and T when being Nle or its amidate, R is D-Phe, D-Tyr, Ac-D-Trp, Trp or D-His; When S is Lys, D-Arg, Leu, Nle, Ala, Met or Abu, and T is when being Nle or its amidate, and R is D-Trp; When S is Arg, and T is when being Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg or D-Arg or its amidate, and R is D-Trp; S is selected from Arg, Lys, D-Arg, Leu, Nle, Ala, Met and Abu; T is selected from Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg, D-Arg and amidate thereof.

2. the antagonist of the α-melanotropin of claim 1, it is a kind of tripeptides.

3. the antagonist of the α-melanotropin of claim 1, it has aminoacid sequence D-Trp-Arg-Leu.

4. the antagonist of the α-melanotropin of claim 1, it has aminoacid sequence D-Trp-Arg-Nle.

5. the antagonist of the α-melanotropin of claim 1, it has aminoacid sequence D-Trp-Arg-Nva.

6. the antagonist of the α-melanotropin of claim 1, it has aminoacid sequence D-Trp-Arg-Met.

7. the antagonist of the α-melanotropin of claim 1, it has aminoacid sequence D-Trp-Arg-D-Nle.

8. the antagonist of the α-melanotropin of claim 1, it has aminoacid sequence D-Trp-Lys-Nle.

9. the antagonist of the α-melanotropin of claim 1, it has aminoacid sequence D-Trp-D-Arg-Nle.

10. the antagonist of the α-melanotropin of claim 1, it has aminoacid sequence D-Trp-Leu-Nle.

11. the antagonist of the α-melanotropin of claim 1, it has aminoacid sequence D-Trp-Nle-Nle.

12. the antagonist of the α-melanotropin of claim 1, it has aminoacid sequence D-Phe-Arg-Nle.

13. the antagonist of the α-melanotropin of claim 1, it has aminoacid sequence Tyr-Arg-Nle.

14. the antagonist of the α-melanotropin of claim 1, it has aminoacid sequence Ac-D-Trp-Arg-Nle.

15. one kind is suppressed the active method of α-melanotropin in α-melanotropin response cell or tissue, it comprises the peptide agonist of described cell or tissue with the α-melanotropin of effective dose is contacted, wherein said peptide agonist comprises aminoacid sequence R-S-T, wherein R, S and T are amino acid residues, R is selected from D-Trp, D-Phe, D-Tyr, Ac-D-Trp, Trp and D-His, just when S be Arg and T when being Nle or its amidate, R is D-Phe, D-Tyr, Ac-D-Trp, Trp or D-His; When S is Lys, D-Arg, Leu, Nle, Ala, Met or Abu, and T is when being Nle or its amidate, and R is D-Trp; When S is Arg, and T is when being Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg or D-Arg or its amidate, and R is D-Trp; S is selected from Arg, Lys, D-Arg, Leu, Nle, Ala, Met and Abu; T is selected from Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg, D-Arg and amidate thereof.

16. method that the animal colour of skin is brightened, it comprises the peptide agonist of using the α-melanotropin of effective dose to animal, wherein said peptide agonist comprises aminoacid sequence R-S-T, wherein R, S and T are amino acid residues, R is selected from D-Trp, D-Phe, D-Tyr, Ac-D-Trp, Trp and D-His, just when S be Arg and T when being Nle or its amidate, R is D-Phe, D-Tyr, Ac-D-Trp, Trp or D-His; When S is Lys, D-Arg, Leu, Nle, Ala, Met or Abu, and T is when being Nle or its amidate, and R is D-Trp; When S is Arg, and T is when being Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg or D-Arg or its amidate, and R is D-Trp; S is selected from Arg, Lys, D-Arg, Leu, Nle, Ala, Met and Abu; T is selected from Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg, D-Arg and amidate thereof.

17. the method for claim 16, wherein said animal is the people.

18. the method for claim 16, wherein said peptide agonist is local application.

19. the method for claim 16, wherein said antagonist are to use by being selected from local application, general injection and Orally administered method.

20. the method for claim 16, wherein said peptide agonist is a tripeptides.

21. the method for claim 20, wherein said tripeptides has aminoacid sequence D-Trp-Arg-Leu.

22. the method for claim 20, wherein said tripeptides has aminoacid sequence D-Trp-Arg-Nle.

23. the method for claim 20, wherein said tripeptides has aminoacid sequence D-Trp-Arg-Nva.

24. the method for claim 20, wherein said tripeptides has aminoacid sequence D-Trp-Arg-Met.

25. the method for claim 20, wherein said tripeptides has aminoacid sequence D-Trp-Arg-D-Nle.

26. the method for claim 20, wherein said tripeptides has aminoacid sequence D-Trp-Lys-Nle.

27. the method for claim 20, wherein said tripeptides has aminoacid sequence D-Trp-D-Arg-Nle.

28. the method for claim 20, wherein said tripeptides has aminoacid sequence D-Trp-Leu-Nle.

29. the method for claim 20, wherein said tripeptides has aminoacid sequence D-Trp-Nle-Nle.

30. the method for claim 20, wherein said tripeptides has aminoacid sequence D-Phe-Arg-Nle.

31. the method for claim 20, wherein said tripeptides has aminoacid sequence Tyr-Arg-Nle.

32. the method for claim 20, wherein said tripeptides has aminoacid sequence Ac-D-Trp-Arg-Nle-NH ₂

33. method for the treatment of malignant melanoma, it comprises the peptide of using effective dose to animal, described peptide has the aminoacid sequence that comprises R-S-T, wherein R, S and T are amino acid residues, R is selected from D-Trp, D-Phe, D-Tyr, Ac-D-Trp, Trp and D-His, just when S be Arg and T when being Nle or its amidate, R is D-Phe, D-Tyr, Ac-D-Trp, Trp or D-His; When S is Lys, D-Arg, Leu, Nle, Ala, Met or Abu, and T is when being Nle or its amidate, and R is D-Trp; When S is Arg, and T is when being Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg or D-Arg or its amidate, and R is D-Trp; S is selected from Arg, Lys, D-Arg, Leu, Nle, Ala, Met and Abu; T is selected from Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg, D-Arg and amidate thereof.

34. pharmaceutical composition that comprises peptide, described peptide has the aminoacid sequence that comprises R-S-T, wherein R, S and T are amino acid residues, R is selected from D-Trp, D-Phe, D-Tyr, Ac-D-Trp, Trp and D-His, just when S be Arg and T when being Nle or its amidate, R is D-Phe, D-Tyr, Ac-D-Trp, Trp or D-His; When S is Lys, D-Arg, Leu, Nle, Ala, Met or Abu, and T is when being Nle or its amidate, and R is D-Trp; When S is Arg, and T is when being Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg or D-Arg or its amidate, and R is D-Trp; S is selected from Arg, Lys, D-Arg, Leu, Nle, Ala, Met and Abu; T is selected from Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg, D-Arg and amidate thereof.

35. the pharmaceutical composition of claim 32, wherein said pharmaceutical carrier are selected from solution, cream or the washing liquid that is suitable for topical application, are suitable for the physiology salt or the buffer agent of general injection, are suitable for Orally administered slow-released carrier and compositions.

36. the peptide agonist of α-melanotropin, it is to identify with the method that comprises the steps:

A. prepare a kind of combinatorial library, this library comprises the library of random sequence three peptide molecules;

B. screen described combinatorial library, to detect the activity of antagonism melanotropin;

C. identify active three peptide molecules with described antagonism α-melanotropin.

37. test kit, it comprises the peptide agonist of α-melanotropin in one or more containers, described peptide agonist is selected from D-Trp-Arg-Leu, D-Trp-Arg-Nle, D-Trp-Arg-Nva, D-Trp-Arg-Met, D-Trp-Arg-D-Nle, D-Trp-Arg-Ile, D-Trp-Arg-Abu, D-Trp-Arg-Val, D-Trp-Arg-Arg, D-Trp-Arg-D-Arg, D-Trp-Lys-Nle, D-Trp-D-Arg-Nle, D-Trp-Leu-Nle, D-Trp-Nle-Nle, D-Trp-Ala-Nle, D-Trp-Met-Nle, D-Trp-Abu-Nle, D-Phe-Arg-Nle, D-Tyr-Arg-Nle, D-Ac-Trp-Arg-Nle, Trp-Arg-Nle and D-His-Arg-Nle or its amidate.

38. the test kit of claim 37, it also comprises the pharmaceutical carrier of the peptide agonist that is suitable for using described α-melanotropin in one or more containers.

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