CN103497237A

CN103497237A - Alpha-MSH analogue with therapeutic activity

Info

Publication number: CN103497237A
Application number: CN201310198558.0A
Authority: CN
Inventors: 托马斯·恩格尔布雷希特·诺克尔德·约纳森; 瑟伦·尼尔森; 约尔延·弗洛基尔; 比亚内迪尤·拉尔森
Original assignee: AbbVie Inc
Current assignee: AbbVie Inc
Priority date: 2005-08-26
Filing date: 2005-08-26
Publication date: 2014-01-08
Anticipated expiration: 2025-08-26
Also published as: CN103497237B

Abstract

The invention relates to an alpha-MSH analogue with therapeutic activity. The invention describes a peptide analogue of alpha-melanocyte stimulating hormone (alpha-MSH), which has improved efficacy when compared with natural alpha-MSH. Compared with alpha-MSH, the alpha-MSH analogue shows improved anti-inflammatory effect and improved capability of preventing ischaemia. The invention further discloses an application of the peptide to the preparation of pharmaceutical compositions, and the pharmaceutical compositions which are used for treatment or prevention of tissue diseases of one or more organs of mammal.

Description

The alpha-MSH analogue of therapeutic activity

The application is to be on August 26th, 2005 applying date of original application, and application number is 200580051680.9, and what denomination of invention was the Chinese patent application of " alpha-MSH analogue of therapeutic activity " divides an application.

Technical field

The present invention relates to the peptide analogs of alpha-Melanocyte stimulating hormone (α-MSH), described peptide analogs is compared the effect with raising with natural α-MSH peptide.With α-MSH, compare, alpha-MSH analogue shows the ability that the treatment of the antiphlogistic effects of raising and raising or prevention and local asphyxia or back are whole body damage, organ damage or cell injury that the blood vessel local asphyxia of pouring into again is relevant.

Background technology

Native peptides alpha-Melanocyte stimulating hormone (α-MSH) is known for people as the natural agonist of 1 type, 3 types, 4 types and 5 type melanocortin (MC) acceptors.The MC acceptor belongs to the type of G-protein linked receptor.All receptor subtypes all are coupled to activated form G-stimulatory protein(SP) (G-stimulatory protein), this means that receptor activation relates to the cAMP content of raising.ACTH is the natural aglucon of 2 receptors (MC2).

Carried out a large amount of research about the MC acceptor in many tissues.Know, 1 receptor (MC1) that α-MSH is bonded thereto with great avidity is for example expressed in brain at some tissues and cell, comprises stellate cell, testis, ovary, scavenger cell and neutrophilic granulocyte.Yet MC1 may express in the tissue of wide region more, although this also needs to set up.The selectivity of MC receptors bind different MS H peptide changes.MC1 with great avidity in conjunction with α-MSH, also with lower avidity in conjunction with β-MSH, γ-MSH and ACTH.According to reports, MC2 is only in conjunction with ACTH, and not in conjunction with any MSH peptide.High-affinity to the aglucon of other acceptors comprises γ-MSH(MC3-acceptor) and β-MSH(MC4-acceptor).On the contrary, MC5 in the mode identical with MC1 with low-down avidity in conjunction with MSH peptide (to α-MSH the highest avidity).

There are various functions by activating the MSH-peptide that the MC-acceptor works, comprise that immunity, anti-inflammatory, thermoregulation, pain, aldosterone are synthetic, blood pressure regulation, heart rate, vascular tone, cerebral blood flow (CBF), nerve growth, placenta are grown, many hormones (for example aldosterone, thyroxine, prolactin, FSH) synthetic/discharge.ACTH generates on (steroidoneogenesis) and has Main Function at the activation ketosteroid.α-MSH also induces skin pigment to form.

Need Special attention will be given to, the many effects that related to about which acceptor of MSH peptide, particularly α-MSH are not also set up fully.The anti-inflammatory action of α-MSH relates to various processes and comprises that interference NO production, endothelin-1 effect, IL-10 form by inference, and this is associated with again the MC1 acceptor of expressing in scavenger cell and monocyte.

Confirmed that the MC acceptor is important (Lipton and Catania1997) to the activation of α-MSH in various inflammatory processes: 1) suppress the chemotaxis migration (Catania1996) of neutrophilic granulocyte.2) α-MSH comprises that its analogue suppresses the release (Goninard1996) to the cytokine (IL-1, TNF-α) of LPS processing response.3) suppress the TNF-α (Wong, K.Y. etc., 1997) to the bacterial endotoxin response.4) the TNF-α that ICV or IP administration α-MSH suppress to give by part the cental system that LPS causes produces.5) confirmed that α-MSH alleviates the inflammation of the acute renal failure (Star, R.A. etc., 1995) that experimental inflammatory bowel diseases (Rajora, N. etc., 1997) and local asphyxia induce.6) by suppressing inducing and exciting of Contact hyper sensitization, α-MSH also has certain preventive effect and induces the haptens tolerance, and people infer that α-MSH may mediate skin inflammation and the dermopathic important negative regulator of high hyperplasia (Luger, T.A., 1997).Finally, α-MSH causes discharging (Hartmeyer, M., 1997) from the IL-8 of the raising of the endotheliocyte of the micro-vasculature of skin.

Hypoxia (local asphyxia) and reperfusion injury are all important factors in people's physiopathology.The example of tissue hypoxia disease (anticipating in refilling process as damage) comprises recycle system shock, myocardial ischaemia, apoplexy, the property crossed a kidney local asphyxia, large surgical operation and organ transplantation.Because be extremely common M & M reason owing to ischemic disease, and because organ transplantation is more and more frequent, in order to improve the public's health level, be starved of the therapeutic strategy with restriction reperfusion injury ability.The basic pathology physiology of ischemia reperfusion damage is complicated, not only relate to the typical inflammation of neutrophilic granulocyte-infiltration is poured into to reaction again, and relating to again the genetic expression of perfused tissue/organ inner cell factor, described cytokine comprises tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1 β, IL-6, IL-8, interferon-γ and ICAM-1 (ICAM-1).In addition, it is believed that, by direct inhibition shrinkability and cell death inducing, the local TNF-α produced facilitates as caused organ dysfunction after local asphyxia in heart after infarct.Due to local asphyxia and/or pour into again complicated character; verified simple anti-inflammatory treatment concept is invalid: therefore; most of experimental studies point to such fact, in order to protect, avoid reperfusion injury, and the interaction capable with the companion of an above activation pathway need to be arranged.Verified, α-MSH has the ability of anti-inflammatory, oxidation resistant and anti-apoptotic simultaneously, this for this compound in order to protect the effect of avoiding reperfusion injury to provide good explanation.

The receptor affinity (to for example MC4 acceptor) that the specific modification of the amino-acid residue of known α-MSH aminoacid sequence can cause peptide to improve, the biological activity extended or more receptor-specific in conjunction with profile (receptor-specific binding profile) ( deng, 1997; Hruby etc., 1995; Sawyer etc., 1980; Hiltz etc. 1991; Scarden ì ngs etc., 2000).Yet, when being intended to produce the peptide medicine, these peptides also have the problem to the low stability of enzyme liberating.

As mentioned above, the problem of the medicine of exploitation peptide therapeutic activity be peptide fast and degraded by enzymes very efficiently, usually there is the transformation period in the several minutes scope.Proteolytic enzyme and other proteolytic ferments are immanent, particularly all the more so in gi tract, therefore when oral peptide usually in a plurality of sites to the sensitivity of degrading, and in blood, liver, kidney and blood vessel endothelium to a certain extent also to the sensitivity of degrading.In addition, particular peptide usually on an above key in main chain to the sensitivity of degrading; Each hydrolysis position is mediated by specific proteases.Even overcome these obstacles, particularly for neuropeptide, run into the difficulty aspect they leap blood brain barrier transportations.

In order to improve the metabolic stability of peptide, the 1999(WO99/46283 such as Larsen) developed a kind of SIP(of being called as structure and induced probe) technology.Sip technique is induced the use of probe based on structure, this structure induces probe to take short peptide sequence as representative, appends to C-end or the N-end of parent's peptide or appends to (Lys) that C-holds and N-holds simultaneously ₆.Based on intramolecular hydrogen bond, structure induces probe to force parent's peptide to become more orderly conformation, whereby, contrary with the peptide in random-coil conformation, and peptide mosaic (being connected to the peptide of probe) is low to the proteolytic enzyme susceptibility.As structurized result, the peptide mosaic more is difficult to by proteasome degradation.Additional SIP usually causes the raising of enzyme stability of peptide and while biological activity remain unchanged (Rizzi etc. 2002) to biologically active peptides.

Summary of the invention

Surprisingly, the present invention is verified, with natural α-MSH peptide, compares, and α-MSH and alpha-MSH analogue modify at the SIP-of the N-of peptide end the maximum effect that has improved peptide.With natural α-MSH peptide, compare, peptide of the present invention has demonstrated the ability of the prevention ischemic conditions of the antiphlogistic effects that improves and raising.

Therefore, the present invention relates to specific peptide, described peptide is included in that the SIP N-end of peptide divided modifies and α-MSH of dividing in the C-end of peptide or the aminoacid sequence of α-MSH variant.

First aspect, the invention provides the peptide that adds up to 12～19 amino-acid residues, and described peptide comprises following aminoacid sequence:

X-Aa ₁-Aa ₂-Aa ₃-Aa ₄-Aa ₅-Y-Aa ₆-Aa ₇-Z

Wherein X comprises 6 amino-acid residue R1-R2-R3-R4-R5-R6, and wherein R1, R2, R3, R4, R5 and R6 can be Lys or Glu independently, and

Wherein Y comprise be selected from His-Phe-Arg, His-(D-Phe)-Arg, His-Nal-Arg and His-(D-Nal)-aminoacid sequence of Arg, and

Wherein Z comprises the aminoacid sequence that is selected from Lys-Pro-Val and Lys-Pro-(D-Val), and

Aa wherein ₁, Aa ₂, Aa ₃, Aa ₄, Aa ₅, Aa ₆and Aa ₇can be amino-acid residue or the disappearance that any naturally occurring or non-natural exists independently, and

The carboxyl terminal of wherein said peptide is-C (=O)-B1 that wherein B1 is selected from OH, NH ₂, NHB2, N (B2) (B3), OB2 and B2, wherein B2 and B3 are independently selected from can substituted C _1-6alkyl, can substituted C _2-6alkenyl, can substituted C _6-10aryl, can substituted C _7-16aralkyl and can substituted C _7-16alkylaryl; With

The aminoterminal of wherein said peptide is (B4) HN-, (B4) (B5) N-or (B6) HN-, wherein B4 and B5 independently selected from H, can substituted C _1-6alkyl, can substituted C _2-6alkenyl, can substituted C _6-10aryl, can substituted C _7-16aralkyl and can substituted C _7-16alkylaryl; B6 be B4-C (=O)-.

The invention still further relates to described peptide for the preparation of the purposes of pharmaceutical composition of the patient's condition that is used for the treatment of or prevents the tissue of mammiferous one or more organs.In addition, the present invention relates to for example pharmaceutical composition of composition, peptide according to the present invention is in purposes medically and the method for the patient's condition that is used for the treatment of the tissue of mammiferous one or more organs, wherein said pharmaceutical composition comprises that one or more are according to peptide of the present invention and pharmaceutically acceptable carrier, described method comprise give effective dose according to peptide of the present invention.Particularly, the present invention pays close attention to the method that is used for the treatment of the patient's condition that the toxic effect by local asphyxia, inflammation and/or poisoning or pharmacological agent causes.

Summary of the invention

The present invention relates to the peptide of therapeutic activity, described peptide has the effect of the organ dysfunction that alleviates or prevent from being caused by local asphyxia, inflammation and/or poison toxic effect poisoning or pharmacological agent.

As herein defined, if it can be used in treatment, alleviates or weaken morbid state, the physiology patient's condition, symptom or etiology indication or estimates or its diagnosis, peptide sequence is " therapeutic activity " so.Prevent morbid state, the physiology patient's condition, symptom or etiology indication if it can be used in, peptide sequence is " prophylactic activity " so.The preparation of pharmacological activity is also physiology and/or bioactive.Pharmacological activity is measured in vitro, body or the effect of material (peptide) in external physiological system or biosystem, and can use well known in the art, for particular peptide or have in peptide, in vitro, body standard of similar physiological function or the analyzed in vitro method is analyzed.

Peptide of the present invention

The present invention relates to a kind of peptide, described peptide comprises that α-MSH that the C-end that is included in peptide is divided or the aminoacid sequence of α-MSH variant and the structure of dividing in the N-end of peptide induce probe (SIP).Peptide of the present invention is called as alpha-MSH analogue.In this specification sheets and claim, these terms are used by synonym.

α-MSH variant is defined as a kind of aminoacid sequence, described aminoacid sequence and natural α-MSH(Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val, SEQ ID NO:101) compare, be modified to and there is at least one amino-acid residue disappearance, replacement, stack or modify in sequence.α-MSH variant preferably has following structure: Aa ₁-Aa ₂-Aa ₃-Aa ₄-Aa ₅-Y-Aa ₆-Aa ₇-Z, wherein Y comprises the aminoacid sequence that is selected from His-Phe-Arg, His-(D-Phe)-Arg, His-Nal-Arg and His-(D-Nal)-Arg, and wherein Z comprises and is selected from Lys-Pro-Val and Lys-Pro-(D-Val), and Aa wherein ₁, Aa ₂, Aa ₃, Aa ₄, Aa ₅, Aa ₆and Aa ₇can be amino-acid residue or the disappearance that any naturally occurring or non-natural exists independently.

In this article, term " amino-acid residue " refers to the amino-acid residue (non-natural amino-acid residue) that any naturally occurring amino-acid residue (natural amino-acid residue) or non-natural exist.

Natural amino-acid residue is defined in the amino-acid residue that nature occurs, for example Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Tyr, Thr, Trp, Val.

As for α-MSH variant structure, the example of preferred natural amino-acid residue is Ser, Tyr, Met, Glu, Ile, Trp and Gly.

That non-natural amino-acid residue is defined as not occurring at nature but amino-acid residue that experiment is created.Non-natural amino-acid residue comprises synthetic α, the amino acid that β or gamma-amino acid residue (no matter L-configuration or D-form) and side chain are modified, the tyrosine of for example modifying, wherein aromatic ring also replaces for example one or more halogens, sulfonic group, nitro etc., and/or phenylol is converted into ester group etc., described non-natural amino-acid residue comprises the amino acid of side chain protected, the method of wherein knowing according to the those of ordinary skill in chemistry of peptides field is protected amino acid side chain, such as being recorded in the method with J.Jones and Jones1991 such as Bodanszky etc. 1994.The example of preferred non-natural amino-acid residue is nor-leucine (Nle), Nal(β-2-naphthyl-L-Ala), D-Nal(β-2-naphthyl-D-alanine), D-phenylalanine (D-Phe) and D-Val (D-Val).

Aspect the widest, the present invention relates to add up to the peptide of 12～19 amino-acid residues, described peptide comprises following aminoacid sequence:

X-Aa ₁-Aa ₂-Aa ₃-Aa ₄-Aa ₅-Y-Aa ₆-Aa ₇-Z

Wherein Y comprises the aminoacid sequence that is selected from His-Phe-Arg, His-(D-Phe)-Arg, His-Nal-Arg and His-(D-Nal)-Arg, and

In the context of the present invention, term " can be substituted " be used to refer to described group can be by one or more C of being selected from _1-8alkyl, C _1-8alkoxyl group, oxo (can be expressed as the enol form of isomery), carboxyl, amino, hydroxyl (can be expressed as the keto-acid of isomery when being present in the enol system), nitro, cyano group, dihalo-C _1-8alkyl, three halos-C _1-8the group of alkyl, halogen replaces once or several times, and for example 1～5 time, preferably 1～3 time, more preferably 1～2 time.Usually, above-mentioned replacement can be carried out other optional replacement.

In this article, term " C _1-6alkyl " be to be used to refer to saturated hydrocarbon chain straight chain or side chain; and wherein the longest chain has 1～6 carbon atom, for example methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, sec-butyl, the tertiary butyl, amyl group, isopentyl, neo-pentyl, hexyl, heptyl and octyl group.The side chain hydrocarbon chain is to be used to refer to the C replaced by hydrocarbon chain at any one carbon atom _1-6alkyl.

In this article, term " C _2-6alkenyl " be the straight or branched alkyl that is used to refer to 2～6 carbon atoms and contains one or more pairs of keys.C _2-6the illustrative examples of alkenyl comprises allyl group, high allyl, vinyl, crot(on)yl, butenyl, pentenyl and hexenyl.C with two keys more than _2-6the illustrative examples of alkenyl comprises butadienyl, pentadienyl, hexadienyl and hexatriene base and their branching form.The position of unsaturated link(age) (two key) can be any one position in carbochain.

Term " C in this article _3-8-cycloalkyl " be for covering three, four, five, six, seven and octatomic ring of a carbon atoms, and term " heterocycle " is to be used to refer to three, four, five, six, seven and the octatomic ring that carbon atom forms together with 1～3 heteroatoms.Heteroatoms is independently selected from oxygen, sulphur and nitrogen.

C _3-8-cycloalkyl and heterocycle can optionally contain one or more unsaturated link(age)s, but described unsaturated link(age) is not in forming the situation of aromatics π-electron system.

Preferred " C _3-8-cycloalkyl " illustrative examples be cyclopropane, tetramethylene, pentamethylene, cyclopentadiene, hexanaphthene, tetrahydrobenzene, 1，3-cyclohexadiene, 1; 4-cyclohexadiene, suberane, suberene, 1,2-cycloheptadiene, 1,3-cycloheptadiene, 1; 4-cycloheptadiene and 1,3,5-cycloheptatriene.

The illustrative examples of " heterocycle " is the 2H-thiapyran, the 3H-thiapyran, the 4H-thiapyran, tetrahydric thiapyran, the 2H-pyrans, tetrahydropyrans, piperidines, 1, 2-dithia tetrahydrobenzene, 1, the 2-dithian, 1, 3-dithia tetrahydrobenzene, 1, the 3-dithian, 1, 4-dithia tetrahydrobenzene, 1, the 4-dithian, 1, the 2-dioxine, 1, the 2-dioxane, 1, the 3-dioxine, 1, the 3-dioxane, 1, the 4-dioxine, 1, the 4-dioxane, piperazine, 1, the 2-oxathiene, 1, the 2-oxathiane, 4H-1, the 3-oxathiene, 1, the 3-oxathiane, 1, the 4-oxathiene, 1, the 4-oxathiane, 2H-1, the 2-thiazine, tetrahydrochysene-1, the 2-thiazine, 2H-1, the 3-thiazine, 4H-1, the 3-thiazine, 5, 6-dihydro-4H-thiazine, 4H-1, the 4-thiazine, tetrahydrochysene-1, the 4-thiazine, 2H-1, 2-

piperazine, 4H-1,2-

piperazine, 6H-1,2-

piperazine, 2H-1,3-

piperazine, 4H-1,3-

piperazine, 4H-1,4-

piperazine, maleimide, succinimide, imidazoles, pyrazoles, pyrroles,

azoles, furazan, barbituric acid, thiobarbituricacidα-, dioxopiperazine, different

azoles, glycolylurea, dihydrouracil, morpholine, trioxane, 4H-1, 2, 3-trithio heterocycle hexene, 1, 2, the 3-trithian, 1, 3, the 5-trithian, six hydrogen-1, 3, the 5-triazine, tetramethylene sulfide, tetrahydrofuran (THF), pyrroline, tetramethyleneimine, pyrrolidone, pyrrolidine-diones, pyrazoline, pyrazolidine, tetrahydroglyoxaline, imidazolidine, 1, the 2-dioxole, 1, the 2-dioxolane, 1, the 3-dioxole, 1, the 3-dioxolane, 3H-1, the 2-dithiole, 1, the 2-dithiolane, 1, the 3-dithiole, 1, the 3-dithiolane, different

azoles quinoline, different azoles alkane,

the azoles quinoline, azoles alkane, thiazoline, thiazolidine, 3H-1,2-oxygen thia cyclopentenes, 1,2-oxathiolane, 5H-1,2-oxygen thia cyclopentenes, 1,3-oxygen thia cyclopentenes, 1,3-oxathiolane, 1,2,3-trithio heterocyclic pentene, 1,2,3-trithio heterocycle pentane, 1,2,4-trithio heterocycle pentane, 1,2,3-trioxa cyclopentenes, 1,2,3-trioxa pentamethylene, 1,2,4-trioxa pentamethylene, 1,2,3-triazoline and 1,2,3-triazoles alkane.Combination to heterocycle can be in heteroatomic position or by the carbon atom of heterocycle.

Term " aryl " is to be used to refer to aromatic carbocyclic or member ring systems in this article.In addition, term " aryl " comprises wherein at least two aromatic rings of condensed ring system, or at least one aryl and at least one C _3-8-cycloalkyl, or at least one aryl and shared at least one chemical bond of at least one heterocycle.The illustrative examples of " aryl " ring comprises can substituted phenyl, naphthyl, phenanthryl, anthryl, acenaphthenyl, 1,2,3,4-tetrahydro naphthyl, fluorenyl, indenyl, indyl, coumaran base, tonka bean camphor base, chromanyl, isochroman base

and base (azulenyl).Preferred aryl is phenyl.

Term " C in this article _7-16aralkyl " be to be used to refer to C _1-6the C that alkyl replaces _6-10aryl.

Term " C in this article _7-16alkylaryl " be to be used to refer to C _6-10the C that aryl replaces _1-6alkyl.

In one embodiment, the present invention relates to add up to the peptide of 12～19 amino-acid residues, described peptide comprises the aminoacid sequence in the choosing group that freely following material forms:

X-Y-Z，

X-Aa ₁-Y-Z，

X-Aa ₁-Aa ₂-Y-Z，

X-Aa ₁-Aa ₂-Aa ₃-Y-Z，

X-Aa ₁-Aa ₂-Aa ₃-Aa ₄-Y-Z，

X-Aa ₁-Aa ₂-Aa ₃-Aa ₄-Aa ₅-Y-Z，

X-Aa ₁-Y-Aa ₆-Z，

X-Aa ₁-Aa ₂-Y-Aa ₆-Z，

X-Aa ₁-Aa ₂-Aa ₃-Y-Aa ₆-Z，

X-Aa ₁-Aa ₂-Aa ₃-Aa ₄-Y-Aa ₆-Z，

X-Aa ₁-Aa ₂-Aa ₃-Aa ₄-Aa ₅-Y-Aa ₆-Z，

X-Aa ₁-Y-Aa ₆-Aa ₇-Z，

X-Aa ₁-Aa ₂-Y-Aa ₆-Aa ₇-Z，

X-Aa ₁-Aa ₂-Aa ₃-Y-Aa ₆-Aa ₇-Z，

X-Aa ₁-Aa ₂-Aa ₃-Aa ₄-Y-Aa ₆-Aa ₇-Z, and

X-Aa ₁-Aa ₂-Aa ₃-Aa ₄-Aa ₅-Y-Aa ₆-Aa ₇-Z。

Aa wherein ₁, Aa ₂, Aa ₃, Aa ₄, Aa ₅, Aa ₆and Aa ₇can be any natural or non-natural amino-acid residue or disappearance independently, and

In a preferred embodiment, the present invention relates to a kind of peptide, wherein peptide comprises following aminoacid sequence:

X-Aa ₁-Aa ₂-Aa ₃-Aa ₄-Aa ₅-Y-Aa ₆-Aa ₇-Z。

Aa wherein ₁, Aa ₂, Aa ₃, Aa ₄, Aa ₅, Aa ₆and Aa ₇can be any natural or alpha-non-natural amino acid independently.Thereby, Aa ₁, Aa ₂, Aa ₃, Aa ₄, Aa ₅, Aa ₆and Aa ₇all be present in peptide of the present invention.

In one embodiment, the present invention relates to according to peptide of the present invention, wherein aminoterminal is (B4) HN-, wherein B4=H.

In another embodiment, the present invention relates to according to peptide of the present invention, the carboxyl terminal of wherein said peptide is-C (=O)-B1, wherein B1=OH.

Having several method can be used for stabilized peptide makes its anti-degraded and reduces the ability that peptide for example, is reacted with other compounds, reagent and/or peptide/protein (in blood plasma).The invention still further relates to the peptide of being modified by those methods well known in the art according to the present invention.In a preferred embodiment, the present invention relates to according to peptide of the present invention the wherein aminoterminal of the peptide modification that is acetylation.Therefore, in a preferred embodiment, the present invention relates to according to peptide of the present invention, wherein aminoterminal is (B6) HN-, wherein B6=B4-C (=O)-, and B4=CH ₃.Another preferred embodiment in, the present invention relates to according to peptide of the present invention, wherein the carboxyl terminal of peptide is amidated modification.Therefore, the present invention relates to according to peptide of the present invention, the carboxyl terminal of wherein said peptide is-C (=O)-B1, wherein B1=NH ₂.

Aspect the widest in the present invention, X is selected from Lys-Lys-Lys-Lys-Lys-Lys(sequence table sequence 37(SEQ ID No:37)), Glu-Lys-Lys-Lys-Lys-Lys(sequence table sequence 38), Lys-Glu-Lys-Lys-Lys-Lys(sequence table sequence 39), Lys-Lys-Glu-Lys-Lys-Lys(sequence table sequence 40), Lys-Lys-Lys-Glu-Lys-Lys(sequence table sequence 41), Lys-Lys-Lys-Lys-Glu-Lys(sequence table sequence 42), Lys-Lys-Lys-Lys-Lys-Glu(sequence table sequence 43), Glu-Glu-Lys-Lys-Lys-Lys(sequence table sequence 44), Glu-Lys-Glu-Lys-Lys-Lys(sequence table sequence 45), Glu-Lys-Lys-Glu-Lys-Lys(sequence table sequence 46), Glu-Lys-Lys-Lys-Glu-Lys(sequence table sequence 47), Glu-Lys-Lys-Lys-Lys-Glu(sequence table sequence 48), Lys-Glu-Glu-Lys-Lys-Lys(sequence table sequence 49), Lys-Glu-Lys-Glu-Lys-Lys(sequence table sequence 50), Lys-Glu-Lys-Lys-Glu-Lys(sequence table sequence 51), Lys-Glu-Lys-Lys-Lys-Glu(sequence table sequence 52), Lys-Lys-Glu-Glu-Lys-Lys(sequence table sequence 53), Lys-Lys-Glu-Lys-Glu-Lys(sequence table sequence 54), Lys-Lys-Glu-Lys-Lys-Glu(sequence table sequence 55), Lys-Lys-Lys-Glu-Glu-Lys(sequence table sequence 56), Lys-Lys-Lys-Glu-Lys-Glu(sequence table sequence 57), Lys-Lys-Lys-Lys-Glu-Glu(sequence table sequence 58), Glu-Glu-Glu-Lys-Lys-Lys(sequence table sequence 59), Glu-Glu-Lys-Glu-Lys-Lys(sequence table sequence 60), Glu-Glu-Lys-Lys-Glu-Lys(sequence table sequence 61), Glu-Glu-Lys-Lys-Lys-Glu(sequence table sequence 62), Glu-Lys-Glu-Glu-Lys-Lys(sequence table sequence 63), Glu-Lys-Glu-Lys-Glu-Lys(sequence table sequence 64), Glu-Lys-Glu-Lys-Lys-Glu(sequence table sequence 65), Glu-Lys-Lys-Glu-Glu-Lys(sequence table sequence 66), Glu-Lys-Lys-Glu-Lys-Glu(sequence table sequence 67), Glu-Lys-Lys-Lys-Glu-Glu(sequence table sequence 68), Lys-Lys-Lys-Glu-Glu-Glu(sequence table sequence 69), Lys-Lys-Glu-Lys-Glu-Glu(sequence table sequence 70), Lys-Lys-Glu-Glu-Lys-Glu(sequence table sequence 71), Lys-Lys-Glu-Glu-Glu-Lys(sequence table sequence 72), Lys-Glu-Lys-Lys-Glu-Glu(sequence table sequence 73), Lys-Glu-Lys-Glu-Lys-Glu(sequence table sequence 74), Lys-Glu-Lys-Glu-Glu-Lys(sequence table sequence 75), Lys-Glu-Glu-Lys-Lys-Glu(sequence table sequence 76), Lys-Glu-Glu-Lys-Glu-Lys(sequence table sequence 77), Lys-Glu-Glu-Glu-Lys-Lys(sequence table sequence 78), Lys-Lys-Glu-Glu-Glu-Glu(sequence table sequence 79), Lys-Glu-Lys-Glu-Glu-Glu(sequence table sequence 80), Lys-Glu-Glu-Lys-Glu-Glu(sequence table sequence 81), Lys-Glu-Glu-Glu-Lys-Glu(sequence table sequence 82), Lys-Glu-Glu-Glu-Glu-Lys(sequence table sequence 83), Glu-Lys-Lys-Glu-Glu-Glu(sequence table sequence 84), Glu-Lys-Glu-Lys-Glu-Glu(sequence table sequence 85), Glu-Lys-Glu-Glu-Lys-Glu(sequence table sequence 86), Glu-Lys-Glu-Glu-Glu-Lys(sequence table sequence 87), Glu-Glu-Lys-Lys-Glu-Glu(sequence table sequence 88), Glu-Glu-Lys-Glu-Lys-Glu(sequence table sequence 89), Glu-Glu-Lys-Glu-Glu-Lys(sequence table sequence 90), Glu-Glu-Glu-Lys-Lys-Glu(sequence table sequence 91), Glu-Glu-Glu-Lys-Glu-Lys(sequence table sequence 92), Glu-Glu-Glu-Glu-Lys-Lys(sequence table sequence 93), Lys-Glu-Glu-Glu-Glu-Glu(sequence table sequence 94), Lys-Glu-Glu-Glu-Glu-Glu(sequence table sequence 94), Glu-Lys-Glu-Glu-Glu-Glu(sequence table sequence 95), Glu-Glu-Lys-Glu-Glu-Glu(sequence table sequence 96), Glu-Glu-Glu-Lys-Glu-Glu(sequence table sequence 97), Glu-Glu-Glu-Glu-Lys-Glu(sequence table sequence 98), Glu-Glu-Glu-Glu-Glu-Lys(sequence table sequence 99), Glu-Glu-Glu-Glu-Glu-Glu(sequence table sequence 100).

The preferred peptide of the present invention is the stable compound of following peptide sequence at present:

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 1)

Glu-Glu-Glu-Glu-Glu-Glu-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 2)

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 3)

Glu-Glu-Glu-Glu-Glu-Glu-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 4)

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Nle-Glu-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 5)

Glu-Glu-Glu-Glu-Glu-Glu-Ser-Tyr-Ser-Nle-Glu-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 6)

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Nle-Glu-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 7)

Glu-Glu-Glu-Glu-Glu-Glu-Ser-Tyr-Ser-Nle-Glu-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 8)

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Nle-Glu-His-D-Nal-Ar g-Trp-Gly-Lys-Pro-Val(sequence table sequence 9)

Glu-Glu-Glu-Glu-Glu-Glu-Ser-Tyr-Ser-Nle-Glu-His-D-Nal-Ar g-Trp-Gly-Lys-Pro-Val(sequence table sequence 10)

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Nle-Glu-His-D-Nal-Ar g-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 11)

Glu-Glu-Glu-Glu-Glu-Glu-Ser-Tyr-Ser-Nle-Glu-His-D-Nal-Ar g-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 12)

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 13)

Glu-Glu-Glu-Glu-Glu-Glu-Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 14)

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 15)

Glu-Glu-Glu-Glu-Glu-Glu-Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 16)

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Ser-Ile-Ile-Ser-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 17)

Glu-Glu-Glu-Glu-Glu-Glu-Ser-Ser-Ile-Ile-Ser-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 18)

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Ser-Ile-Ile-Ser-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 19)

Glu-Glu-Glu-Glu-Glu-Glu-Ser-Ser-Ile-Ile-Ser-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 20)

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Ser-Ile-Ile-Ser-His-D-Nal-Ar g-Trp-Gly-Lys-Pro-Val(sequence table sequence 21)

Glu-Glu-Glu-Glu-Glu-Glu-Ser-Ser-Ile-Ile-Ser-His-D-Nal-Ar g-Trp-Gly-Lys-Pro-Val(sequence table sequence 22)

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Ser-Ile-Ile-Ser-His-D-Nal-Ar g-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 23)

Glu-Glu-Glu-Glu-Glu-Glu-Ser-Ser-Ile-Ile-Ser-His-D-Nal-Ar g-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 24)

Lys-Lys-Lys-Lys-Lys-Lys-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 25)

Glu-Glu-Glu-Glu-Glu-Glu-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 26)

Lys-Lys-Lys-Lys-Lys-Lys-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 27)

Glu-Glu-Glu-Glu-Glu-Glu-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 28)

Lys-Lys-Lys-Lys-Lys-Lys-Nle-Glu-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 29)

Glu-Glu-Glu-Glu-Glu-Glu-Nle-Glu-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 30)

Lys-Lys-Lys-Lys-Lys-Lys-Nle-Glu-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 31)

Glu-Glu-Glu-Glu-Glu-Glu-Nle-Glu-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 32)

Lys-Lys-Lys-Lys-Lys-Lys-Nle-Glu-His-D-Nal-Arg-Trp-Gly-Ly s-Pro-Val(sequence table sequence 33)

Glu-Glu-Glu-Glu-Glu-Glu-Nle-Glu-His-D-Nal-Arg-Trp-Gly-Ly s-Pro-Val(sequence table sequence 34)

Lys-Lys-Lys-Lys-Lys-Lys-Nle-Glu-His-D-Nal-Arg-Trp-Gly-Ly s-Pro-(D-Val) (sequence table sequence 35) and

Glu-Glu-Glu-Glu-Glu-Glu-Nle-Glu-His-D-Nal-Arg-Trp-Gly-Ly s-Pro-(D-Val) (sequence table sequence 36)

Can carry out stabilization by N-end and/or the C-end of modifying above-mentioned peptide, for example the C-end of the N-of acetylize peptide of the present invention end and/or amidation peptide of the present invention.

For natural amino acid, with the three-letter code of knowing, provide aminoacid sequence.The modification of natural amino acid residue and replacement abbreviation are as follows: Nle is the abbreviation for nor-leucine.D-Nal is the abbreviation for β-2-naphthyl-d-L-Ala.The D-Val(D-α-amino-isovaleric acid) be the abbreviation for the D-form α-amino-isovaleric acid.The D-Phe(D-phenylalanine) be the abbreviation for the D-form phenylalanine.

In a preferred embodiment, the present invention relates to a kind of peptide, described peptide is the compound that the N-end is acetylation and the C-end is amidated of following aminoacid sequence:

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 1).

Another preferred embodiment in, the present invention relates to according to peptide of the present invention, described peptide is the compound that the N-end of following aminoacid sequence is acetylation and the C-end is amidated:

Glu-Glu-Glu-Glu-Glu-Glu-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 2).

Another preferred embodiment in, the present invention relates to according to peptide of the present invention, described peptide is that the acetylize of N-end and the C-of following aminoacid sequence holds amidated compound:

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-(D-Val) (sequence table sequence 3).

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Nle-Glu-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 5).

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Nle-Glu-His-(D-Nal)-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 9).

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 13).

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Ser-Ile-Ile-Ser-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 17).

As mentioned above, with naturally occurring peptide α-MSH, compare, peptide of the present invention has result for the treatment of and/or the peak response of raising and/or the maximum effect of raising of raising.

The contriver checked the biology effect of some peptides of the present invention:

Ac-(Lys) ₆-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH ₂(sequence table sequence 1* is acetylation and is amidated at the C-end at the N-end),

Ac-(Glu) ₆-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH ₂(sequence table sequence 2* is acetylation and is amidated at the C-end at the N-end),

Ac-(Lys) ₆-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-(D-Val)-NH ₂(sequence table sequence 3* is acetylation and is amidated at the C-end at the N-end),

Ac-(Lys) ₆-Ser-Tyr-Ser-Nle-Glu-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-Val-NH ₂(sequence table sequence 5* is acetylation and is amidated at the C-end at the N-end),

Ac-(Lys) ₆-Ser-Tyr-Ser-Nle-Glu-His-(D-Nal)-Arg-Trp-Gly-Lys-Pro-Val-NH ₂(sequence table sequence 9* is acetylation and is amidated at the C-end at the N-end),

Ac-(Lys) ₆-Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH ₂(sequence table sequence 13* is acetylation and is amidated at the C-end at the N-end),

Ac-(Lys) ₆-Ser-Ser-Ile-Ile-Ser-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-Val-NH ₂(sequence table sequence 17* is acetylation and is amidated at the C-end at the N-end).

Usually, " Ac-" illustrates that peptide of the present invention is acetylation at the N-end, " NH ₂" illustrate that peptide of the present invention is amidated at the C-end.

In the human leukocyte suspension (experimental program 1), all seven peptide dosage rely on ground and suppress the TNF-α accumulation (embodiment 1-7) that LPS induces.Surprisingly, we find to compare with natural melanotropin α-MSH, all seven peptides are more effective and available energy more, the former is defined by the maximum suppression effect that TNF-α is produced, and the latter is defined (embodiment 1-7) by providing maximum TNF-α accumulation to suppress required compound concentration.

Induce the scheme (experimental program 2) of systemic inflammatory rat injecting LPS by vein; the contriver has also studied the effect (sequence table sequence 1*, sequence table sequence 2*, sequence table sequence 3*, sequence table sequence 5*, sequence table sequence 9*, sequence table sequence 13*, sequence table sequence 17*, all * are acetylation and are amidated at the C-end at the N-end) of above listed seven peptides.We confirm, described peptide has greatly suppressed the TNF-α accumulation that in the circulating, LPS induces.Surprisingly; all seven peptides (sequence table sequence 1*, sequence table sequence 2*, sequence table sequence 3*, sequence table sequence 5*, sequence table sequence 9*, sequence table sequence 13*, sequence table sequence 17*, all * are acetylation and are amidated at the C-end at N-end) can suppress circulating in the TNF-α that induces of LPS be accumulated to the degree higher than natural melanotropin α-MSH (embodiment 1-7).

In suck the scheme that LPS induces inflammation by rat (experimental program 3), the contriver has also studied the effect of following peptide:

Verified these two peptides (sequence table sequence 1* and 5*) greatly suppress the eosinophil accumulation (embodiment 1 and embodiment 2) that in alveolar, LPS induces.Surprisingly, peptide (sequence table sequence 5*), except this effect to eosinophil, also suppresses significantly neutrophilic granulocyte and infiltrates than the high a lot of degree of the degree of finding in the rat processing with natural melanotropin α-MSH.

One property crossed local asphyxia (temporal ischemia) of kidney usually is regarded as the result of ypotension, hypovolemia, surgical operation intervention, and described result relates to the reduction of kidney and/or Aorta volume of blood flow, or relevant with septicemia.The acute renal failure that this causes local asphyxia to be induced, for most of patient, it will worsen as chronic renal failure.At present also do not occur that effective methods for the treatment of prevents the development of renal failure.In the common discovery of local asphyxia latter stage, be the development of the concentrated defect of urine, the concentrated defect of described urine has the formation without solute urine (solute free urine) output of raising.

People know, the EARF (ARF) of being induced with the local asphyxia of the rat that perfusion is induced again by local asphyxia causes the characteristic structural modification of the renal cells relevant to the infringement of urinary concentrating mechanism.The ARF model that this local asphyxia is induced provides suitable model to estimate the effect of MSH analogue in the damage of inducing in local asphyxia.In the Serious acute renal failure of being induced by the temporary transient two-way obstruction Renal artery exhausts, the inventor after deliberation peptide Ac-(Lys) ₆-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH ₂the effect of (sequence table sequence 1* is acetylation and is amidated at the C-end at N-end), and the effect of itself and native peptides α-MSH is compared to (experimental program 6).While within 5 days, being estimated after the temporary transient obstruction Renal artery, with the rat of vehicle treated, develop into polyuria (being defined by the diuresis amount than contrast rat high 101%), described control rats is carried out false renal artery obstruction.Surprisingly; the described compound (sequence table sequence 1* is acetylation and is amidated at the C-end at the N-end) of taking the molar weight identical with native peptides α-MSH makes diuresis amount normalizing fully; this illustrates that described peptide has the ability that the ARF that local asphyxia induces is avoided in protection, and processes and can not make to urinate production normalizing with native peptides in this model.

Acute Myocardial Infarction in developed country (AMI) is one of modal cause of death.Due to unexpected coronary thrombosis, AMI almost always occurs in and has in patient coronarius.At present, fibrinolytic therapy or initial stage percutaneous tranluminal coronary angioplasty (PTCA) are the methods for the treatment of of standard, can in the patient of 50-70%, obtain and pour in early days (spontaneous filling rate again is less than 30%) again.The purpose of perfusion is the size that reduces infraction again, thereby reduces the development of impaired myocardial function.Whole effects of fibrinolysis/PTCA are that short-term and long-term mortality descend 20%.Yet AMI is attended by inflammatory reaction, this is prerequisite for healing and cicatrization.Coronary occlusion has seriously reduced to the volume of blood flow of cardiac muscle part, and this has damaged energy metabolism significantly.Local asphyxia continues considerable time (> 20min) cause infraction and cause inflammatory reaction, when pouring into again the local asphyxia cardiac muscle, both can accelerate and enlarge.

Ischemic/reperfusion (MIR) not only activates the typical inflammation of neutrophilic granulocyte-infiltration is poured into to reaction again, and the genetic expression of the activation myocardial cell factor, described cytokine comprises tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1 β, IL-6, IL-8, interferon-γ and ICAM-1 (ICAM-1).This local myocardial of cytokine is crossed expression and is not only played an important role in the adjusting of infarct size but also in the myocardial dysfunction development of (comprising vessel wall recast, heart failure and cardiac hypertrophy).In addition, it is believed that, by direct inhibition shrinkability and cell death inducing, the local TNF-α generated facilitates organ dysfunction after ischemic.

Increasing experimental study has confirmed that anti-inflammatory/anti-oxidant/anti-apoptotic strategy has the ability of the infarct size that reduces the MIR animal model.Yet, there is no the unusual effect of clinical research confirmation in the mankind.

In the Ischemic/reperfusion model of rat, in described model, left front coronary artery gets clogged 60 minutes, just before removing coronary occlusion, uses the processing according to peptide of the present invention, and then follows the tracks of rat 3 hours.Then estimate the ability that described peptide reduces infarct size, and the effect of itself and native peptides α-MSH is compared to (experimental program 5).

Surprisingly, all following three peptides:

Ac-(Lys) ₆-Ser-Tyr-Ser-Nle-Glu-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-Val-NH ₂(sequence table sequence 5* is acetylation and is amidated at the C-end at the N-end), and

Ac-(Lys) ₆-Ser-Tyr-Ser-Nle-Glu-His-D-Nal-Arg-Trp-Gly-Lys-Pro-Val-NH ₂(sequence table sequence 9* is acetylation and is amidated at the C-end at the N-end) reduces infarct size to the degree (embodiment 1,3-4) higher than native peptides α-MSH.

According to functional performance top and the described peptide of embodiment, the peptide that the present invention relates to have at least one following characteristic:

A) generation of the TNF-α that inhibition LPS induces, wherein TNF-α is produced by human leukocyte

B) suppress the eosinophil infiltration of inflammation-induced in lung

C) suppress the neutrophilic granulocyte infiltration of inflammation-induced in lung

D) suppress the TNF-α accumulation of inflammation-induced in circulating

E) alleviate the acute renal failure that local asphyxia is induced

F) reduce myocardial infarction area

G) alleviate the degree of heart failure after myocardial infarction

H) alleviate lung blood vessel hypertension

I) alleviate the renal failure of cisplatin induction

Peptide can have these characteristics more than, and for example 2,3,4,5,6,7,8 or above-mentioned all characteristics.These characteristics can be enough be tested as the method for summarizing in embodiment.

As mentioned above, being characterized as of alpha-MSH analogue of the present invention has the effect improved than natural α-MSH.

In this specification sheets and claim, term " effect " is defined as the obtainable peak response of compound.In the various tests of embodiment record, alpha-MSH analogue of the present invention can produce the peak response higher than natural α-MSH.

Preferably, with α-MSH, compare, it is minimum 10% that the TNF-α that the LPS that alpha-MSH analogue of the present invention suppresses to be produced by human leukocyte induces produces, and more preferably 25% and most preferably 40%.

Further, the eosinophil that alpha-MSH analogue of the present invention can suppress inflammation-induced in lung infiltrates, and by the ability of eosinophil quantity in the liquid that reduces bronchoalveolar lavage or similar approach collection, measures.When with α-MSH relatively the time, we find that the minimum expected effect is that eosinophil reduces 10%, more preferably 25% and most preferably 50%.

In addition, the neutrophilic granulocyte that alpha-MSH analogue of the present invention can suppress inflammation-induced in lung infiltrates, and by the ability of neutrophilic granulocyte quantity in the liquid that reduces bronchoalveolar lavage or similar approach collection, measures.When with α-MSH, relatively the time, finding that the minimum expected effect is that neutrophilic granulocyte reduces 10%, more preferably 20% and most preferably 40%.

With α-MSH, compare, it is minimum 10% that the TNF-α that alpha-MSH analogue of the present invention can also suppress inflammation-induced in circulating produces, and more preferably 25% and most preferably 40%.

In addition, alpha-MSH analogue of the present invention can alleviate the acute renal failure that local asphyxia is induced, and this measures by the ability that alleviates polyuria degree after local asphyxia.When with α-MSH, relatively the time, finding that the minimum expected effect is that polyuria reduces 10%, more preferably 30% and most preferably 50%.

Further, alpha-MSH analogue of the present invention can reduce myocardial infarction area, by the ability that reduces the size of necrosis area in the local asphyxia cardiac muscle, proves.When with α-MSH, relatively the time, finding that the minimum expected effect is that infarct size reduces 10%, more preferably 20% and most preferably 30%.

On the other hand, alpha-MSH analogue of the present invention can alleviate the degree of heart failure after myocardial infarction, and the performance by direct measurement left ventricle end diastolic pressure or similar quantivative approach assess cardiac proves.When with α-MSH, relatively the time, finding that the minimum expected effect is that degree in heart failure reduces 10%, more preferably 20% and most preferably 25%.

On the other hand, alpha-MSH analogue of the present invention can alleviate lung blood vessel vascular hypertension.When with α-MSH, relatively the time, finding that the minimum expected effect is that lung blood vessel vascular hypertension reduces 10%, more preferably 20% and most preferably 30%.

On the other hand, alpha-MSH analogue of the present invention can alleviate the renal failure of cisplatin induction.When with α-MSH, relatively the time, finding that the minimum expected effect is that hypomagnesemia (hypomagnesia) and/or renal glomerulus infiltration rate reduce 10%, more preferably 20% and most preferably 30%.

As previously mentioned, native peptides α-melanotropin (α-MSH) is as the natural agonist of 1 type, 3 types, 4 types and 5 type melanocortin (MC) acceptors for people know, and ACTH is the natural aglucon of 2 receptors (MC2).Because described peptide comprises the aminoacid sequence of α-MSH or its analogue, described peptide has the ability that activates one or more melanocortin receptors (i.e. 1 type, 3 types, 4 types or 5 type melanocortin receptors).

The preparation method of peptide of the present invention

The method that peptide of the present invention can be known by this area itself is prepared.Thereby α-MSH, α-MSH variant, alpha-MSH analogue and X primitive (motif) can for example, by peptide technology of preparing (solution synthetic method or the Merrifield type solid-phase synthesis) preparations of standard.

In a possible synthesis strategy; can prepare by solid-phase synthesis by peptide of the present invention: the peptide sequence (α-MSH, α-MSH variant, alpha-MSH analogue) that at first uses protection, coupling and the deprotection steps structure pharmacologically active of known standard; after this use the aminoacid sequence that continues coupling primitive X with the mode like bioactive peptide of building on bioactive peptide, finally from the whole peptide of carrier excision.This strategy has obtained a kind of peptide, and wherein peptide sequence X is covalently bound on the peptide of this pharmacologically active on the N-of peptide end nitrogen-atoms.

Another possible strategy is to prepare respectively the sequence of α-MSH peptide/analogue and X-primitive (or its part) by solution synthetic method, solid-phase synthesis, recombinant technology or enzymic synthesis method, then in solution or use solid phase technique or its combination by two sequences of known fragment condensation step coupling.In one embodiment, can prepare by recombinant DNA method by α-MSH peptide/analogue, can prepare by solid-phase synthesis by the X primitive.α-MSH peptide/analogue can be undertaken by using the chemical bond method with engaging of X primitive.This technology allows assembling complete unshielded peptide fragment (Liu etc., 1996) under the high specific mode.Can also form and carry out described joint by the enzymatic peptide bond of albumen, its technology that a kind of high special is provided be to pass through the complete unshielded peptide fragment of peptide keyed engagement (Kullmann, 1987).

The example of suitable solid support material (SSM) be such as the resin of functionalization as polystyrene, the latex of polystyrene, polyacrylamide, polydimethylacrylamiin, polyoxyethylene glycol, Mierocrystalline cellulose, polyethylene, polyoxyethylene glycol grafting, wear promise immunomagnetic beads (dynabeads) etc.

Should be understood that, by the C-terminal amino acid of the peptide sequence of X-primitive or α-MSH, α-MSH variant, it is necessary or needs that the C-terminal amino acid of alpha-MSH analogue is connected on the solid support material by connexon commonly used, connexon commonly used is for example 2, the 4-dimethoxy-4 ' '-hydroxyl-benzophenone, 4-(4-hydroxy-methyl-3-methoxyphenoxy)-butyric acid, 4-hydroxy-methyl phenylformic acid, 4-methylol-phenylium, 3-(4-hydroxymethyl phenoxy group) propionic acid, and p-[(R, S)-a[l-(9H-fluorenes-9-yl) methoxymethylamide base]-2, the 4-Dimethoxyphenyl]-phenoxy group-acetic acid.

Optionally with one or more, be suitable for the method for " scavenging agent " (such as dithioglycol, tri isopropyl silane, phenol, thioanisole etc.) combination of this purpose by acid (such as trifluoroacetic acid, trifluoromethanesulfonic acid, hydrogen bromide, hydrogenchloride, hydrogen fluoride etc.), peptide of the present invention can be excised from the solid support material, perhaps, method such as ammonia by alkali, hydrazine, alkoxide such as sodium ethylate, oxyhydroxide such as sodium hydroxide etc., excise peptide joiner of the present invention from the solid support material.

Peptide of the present invention also can use general method well known to those skilled in the art and principle to be prepared by recombinant DNA technology.By established standard method phosphoramidite method (phosphoamidite method) for example, can prepare synthetically the nucleotide sequence of the peptide of the present invention of encoding.According to the phosphoramidite method, for example, in automatic dna synthesizer, synthetic on suitable carrier, purifying, annealing, connection and clone's oligonucleotide.

Then the nucleotide sequence of peptide of the present invention of encoding is inserted in recombinant expression vector, and described recombinant expression vector can be any carrier that can carry out easily the recombinant DNA step.The selection of carrier depends on its host cell that will be proceeded to usually.Therefore, described carrier can be the spontaneous carrier copied, and as the outer individual existence of karyomit(e) and its, copies the carrier that does not rely on chromosome duplication, for example plasmid.In addition, described carrier can be a kind of carrier, and, when being transferred to host cell, carrier is integrated in the host cell gene group and together with the karyomit(e) be integrated into it and copies.

In carrier, the nucleotide sequence of coding peptide of the present invention need to be operably connected on suitable promotor.Promotor can be the nucleotide sequence arbitrarily that demonstrates transcriptional activity and can derive from the gene of coding and the albumen of host cell homology or allos in selected host cell.Being used for instructing the nucleotide sequence example that transcribe, suitable promotor of the peptide of code book invention at mammalian cell is SV40 promotor, MT-1(metallothionein gene) promotor or adenovirus 2 major late promoters, Rous sarcoma virus (RSV) promotor, cytomegalovirus (CMV) promotor and bovine papilloma virus (BPV) promotor.The suitable promotor that is used in insect cell is the polyhedrin promotor.

Suitable promotor (transcribing of its nucleotide sequence for the peptide that instructs code book invention, especially in bacterial host cell) example be the promotor obtained from following source: the intestinal bacteria lactose operon, streptomyces coelicolor gelase gene (dagA), subtilis type froctosan saccharase gene (sacB), bacillus licheniformis alpha-amylase gene 5(amyL), bacstearothermophilus maltogenic amylase gene (amyM), bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), subtilis xylA and xylB gene, with protokaryon β-lactamase gene, and tac promotor.More promotor is recorded in " Useful proteins from recombinant bacteria " Scientific American, 1980,242:74-94; With Sambrook etc., 1989(is the same).

Be used for instructing the nucleotide sequence of the peptide of code book invention to transcribe at filamentous fungal host cell, the example of suitable promotor is the promotor from the gene acquisition of the following enzyme of encoding: aspergillus oryzae TAKA amylase, rhizomucor miehei (Rhizomucor miehei) aspartate protease, the aspergillus niger neutral alpha-amylase, the aspergillus niger acid-resistant alpha-amylase, aspergillus niger or Aspergillus awamori glucoamylase (glaA), rhizomucor miehei lipase, the aspergillus oryzae Sumizyme MP, the aspergillus oryzae triosephosphate isomerase, the Aspergillus nidulans acetamidase, Fusarium oxysporum (Fusarium oxysporum) tryptase lytic enzyme (is recorded in United States Patent (USP) 4, 288, 627, be hereby incorporated by), and their hybrid.For filamentous fungal host cell, particularly preferred promotor is the hybrid of the promotor of TAKA amylase, the NA2-tpi(gene that comes own coding aspergillus niger neutral alpha-amylase and aspergillus oryzae triosephosphate isomerase) and glaA promotor.

In yeast host, useful promotor can obtain from following gene: yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) Hydratase, phosphoenolpyruvate (ENO-I) gene, yeast saccharomyces cerevisiae galactokinase gene (GALl), Ethanol in Saccharomyces cerevisiae desaturase/glyceraldehyde-3-phosphate dehydrogenase gene (ADH2/GAP) and yeast saccharomyces cerevisiae glycerol 3-phosphate kinase gene.Be recorded in Romanos etc. 1992, yeast (Yeast) 8:423-488 for other useful promotors of yeast host cell.

The nucleotide sequence of peptide of the present invention of encoding can also be operably connected to suitable terminator for example on the human growth hormone terminator.Preferred terminator for filamentous fungal host cell obtains from the gene of the following enzyme of encoding: aspergillus oryzae TAKA amylase, aspergillus niger glucoamylase, Aspergillus nidulans aminobenzoate synthetic enzyme, aspergillus niger alpha-glucosidase and Fusarium oxysporum tryptase lytic enzyme.

For the preferred terminator of yeast host cell, from the gene of the following enzyme of encoding, obtain: yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) Hydratase, phosphoenolpyruvate, brewing yeast cell pigment C(CYC1), Ethanol in Saccharomyces cerevisiae desaturase/glyceraldehyde-3-phosphate dehydrogenase.Be recorded in the 1992(such as Romanos for other useful terminators of yeast host cell the same).

Described carrier can also comprise element for example polyadenylation signal (for example,, from SV40 or adenovirus 5Elb district), transcriptional enhancer sequence (for example, SV40 enhanser) and translational enhancer (for example, the enhanser of encoding adenovirus VA RNAs).In addition, the preferred polyadenylation signal for filamentous fungal host cell obtains from the gene of the following enzyme of encoding: aspergillus oryzae TAKA amylase, aspergillus niger glucoamylase, Aspergillus nidulans aminobenzoate synthetic enzyme.Useful polyadenylation signal for yeast host cell is recorded in Guo and Sherman, and 1995, molecular cytobiology (Molecular Cellular Biology) 15:5983-5990.

Recombinant expression vector can also comprise the DNA sequence dna that can make described carrier copy in described host cell.The example of this sequence (when host cell is mammalian cell) is the replication orgin of SV40 or polyomavirus.The example of the replication orgin of bacterium is the replication orgin of plasmid pBR322, pUC19, pACYC177, pACYC184, pUBHO, pE194, pTA1060 and pAM β l.The example that is used for the replication orgin of yeast host cell is the combination of combination, CEN3 and the ARS1 of 2 microns replication orgin (2micron origin of replication), CEN6 and ARS4.Replication orgin can be have sudden change replication orgin so that in host cell its function be temperature sensitive (referring to Ehrlich, 1978, Proc.Natl.Acad.Sci.USA75:1433).

Described carrier can also comprise selected marker, for example its product is supplied the gene of the defect of host cell, for example, for the gene of the Tetrahydrofolate dehydrogenase of encoding (DHFR) or give the gene to drug resistance, described medicine is for example Liu Suanyan NEOMYCIN SULPHATE, Geneticin (geneticin), penbritin, Totomycin (hygromycin).Suitable mark for yeast host cell is ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.Can be selected from the equivalent of following group and other species of originating for the selected marker of filamentous fungal host cell, described group includes but not limited to the amdS(acetamidase), the argB(ornithine transcarbamylase), bar(grass fourth phosphinothricin acetyl transferring enzyme), hygB (hygromix phosphotransferase), niaD(nitrate reductase), the pyrG(Orotidine-5 '-'-the phosphoric acid salt decarboxylase), sC(vitriol adenosyl transferase), trpC(aminobenzoate synthetic enzyme) and careless ammonium phosphine resistance marker.Preferred selected marker for the aspergillus cell is the amdS of Aspergillus nidulans or aspergillus oryzae and the bar mark of pyrG mark and streptomyces hygroscopicus.In addition, selectivity can complete by cotransformation, for example as WO91/17243, is put down in writing, and selected marker is on independent carrier.

For connecting the nucleotide sequence of encode respectively peptide of the present invention, promotor and terminator, and they are inserted into to the step contained in the suitable carrier that copies necessary information, known (reference, such as Sambrook etc., is enumerated in quoted passage) to those skilled in the art.

The host cell that expression vector proceeds to can be any cell that can produce peptide of the present invention, it can be eukaryotic cell, invertebrates (insect) cell or vertebrate cells (for example xenopus leavis oocytes or mammalian cell), especially insect cell and mammalian cell for example.The example of suitable mammal cell line be COS (for example, ATCC CRL1650), BHK (for example., ATCC CRL1632, ATCC CCL10) or CHO (e.g., ATCC CCL61) clone.

For transfection mammalian cell and the method for expressing the DNA sequence dna be transferred to cell can be any method well known in the art (for example, MANIATIS, T., E.F.FRITSCH and J.SAMBROOK, press of 1982Molecular Cloning:A Laboratory Manual. cold spring harbor laboratory, the cold spring port, New York).

Host cell can also be unicellular pathogenic agent (for example prokaryotic cell prokaryocyte) or non-unicellular pathogenic agent (eukaryotic cell).Useful unicellular cell be bacterial cell for example gram-positive microorganism (include but not limited to bacillus cell, Alkaliphilic bacillus (Bacillus alkalophilus) for example, bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus brevis (Bacillus brevis), bacillus circulans (Bacillus circulans), bacillus coagulans (Bacillus coagulans), Bacillus lautus (Bacillus lautus), Bacillus licheniformis (Bacillus licheniformis), Bacillus megatherium (Bacillus megaterium), bacstearothermophilus (Bacillus stearothermophilus), subtilis (Bacillus subtilis) and bacillus thuringiensis (Bacillus thuringiensis)), perhaps streptomyces cell (for example, shallow Streptomyces glaucoviolaceus (Streptomyces lividans) or mouse ash streptomycete (Streptomyces murinus)), perhaps Gram-negative bacteria (for example intestinal bacteria (E.Coli) and pseudomonas (Pseudomonas sp)).The conversion of bacterial host cell can be for example by protoplast transformation, by using competent cell, carrying out by electroporation or by fusion method.

Host cell can be the fungal cell." fungi " used herein comprises Ascomycota (phyla Ascomycota), Basidiomycota (Basidiomycota), chytrid door (Chytridiomycota) and in conjunction with bacterium door (Zygomycota) and oomycetes door (Oomycota) and all mitospore fungies (mitosporic fungi).Representative group of Ascomycota comprises, as, Neurospora (Neurospora), penicillium Pseudomonas (Eupenicillium) (=Penicillium (Penicillium)), Emericella (Emericella) (=aspergillus (Aspergillus)), loose capsule bacterium (Eurotium) (=aspergillus (Aspergillus)) and the true yeasts of listing below.Fungal host cells can be also yeast cell.Here " yeast " used comprises the yeast that ascosporogenous yeast (ascosporogenous yeast) (Endomycetaceae (Endomycetales)), basidiospore form yeast (basidiosporogenous yeast) and belong to imperfect fungi door (Fungi Imperfecti) (gemma guiding principle (Blastomycetes)).For the substratum of culturing cell, can be the conventional medium (for example contain suitable additive containing blood serum medium or serum free medium) of any suitable growth mammalian cell, or for the insect cell of growing, yeast cell or fungal cell's suitable culture medium.Suitable medium can obtain or prepare (for example, the catalogue of U.S.'s representative microbial DSMZ) according to published formula from commercial suppliers.

Then reclaim the peptide of the present invention by cells produce from substratum by ordinary method, described method comprises by centrifugal or filtration separates host cell from substratum, with the salt protein ingredient of ammonium sulfate precipitation supernatant liquor or filtrate for example, by purifying such as ion-exchange chromatography, affinity chromatography of various chromatographys.

Therefore, the present invention relates to for the preparation of the method according to peptide of the present invention, described method recombinant DNA technology, it comprises the steps: that (a) is transferred to the nucleotide sequence of coding for said peptides in host cell, (b) cultivate described host cell, separate described peptide with (c) from described culture; Perhaps, (a) under the condition that allows described peptide to produce, cultivate the recombinant host cell of the nucleotide sequence that contains coding for said peptides, separate described peptide with (b) from described culture.

Purposes

The invention still further relates to the purposes of peptide according to the present invention at field of medicaments, particularly with above one or more or the relevant purposes of the patient's condition cited below, discomfort or disease.

In one embodiment, the present invention relates to the purposes of one or more peptides according to the present invention for the preparation of pharmaceutical composition, described pharmaceutical composition is used for the treatment of or prevents the patient's condition of the tissue of mammiferous one or more organs.Described organ is selected from but is not limited in following formed group: kidney, liver, brain, heart, muscle, marrow, skin, bone, lung, respiratory tract, spleen, exocrine gland, bladder, incretory gland, comprise oviducal reproductive organ, eye, ear, vascular system, the gi tract that comprise small intestine, colon, rectum and anal tube and prostate gland.

As mentioned above, with α-MSH, compare, peptide of the present invention demonstrates the antiphlogistic effects of raising and the ability that prevents the local asphyxia patient's condition of raising.

Therefore, the present invention relates to the purposes of one or more peptides according to the present invention for the preparation of pharmaceutical composition, described pharmaceutical composition is used for the treatment of or prevents the patient's condition of the tissue of mammiferous one or more organs, and the wherein said patient's condition is local asphyxia or the inflammation patient's condition.The described patient's condition can also be the exhaustion owing to toxin or drug-induced cell, tissue or organ.

In this specification sheets and claim, if this explanation do not got rid of especially in context, term " treatment " generally includes the treatment of the existing patient's condition and the prevention (prophylactic treatment) of this patient's condition.

Aspect the widest, the present invention relates to any patient's condition wherein the normal function of organ or tissue due to local asphyxia or inflammation, change.Damage can comprise acute and/or chronic injury.Chronic injury comprises the situation of the repeated trauma between decubation wholly or in part in organ or tissue.

Local asphyxia

In this specification sheets and claim, local asphyxia is defined as the volume of blood flow that one or more organs are reduced, and causes the oxygen that tissue reduces to carry and/or utilize.Local asphyxia can occur in one or more organs, and described organ comprises (non-complete list (non-listing list)): brain, heart, four limbs, kidney,spleen,liver, intestines, stomach, lung, eye, skin, muscle, pancreas, endocrine organ and other.

By reduce/stop completely arterial blood to be supplied with, local asphyxia induces many tissue reactions to comprise neutrophilic granulocyte accumulation, other inflammatory reactions and necrocytosis.Local asphyxia relates to numerous disease, and it is relevant to large surgery (major surgery) and be secondary to other seriously diseases.It is very useful that evaluation can suppress or prevent (completely or partially) many cell/tissue/organ damages that occur as ischemic result or the compound of destruction.

The patient's condition of being treated can be produced owing to the local asphyxia of tissue or by the local asphyxia of tissue, for example stricture of artery disease or any other completely or the blood supply of part limit.Local asphyxia can be acute or chronic, and this depends on the severity of disease, and in addition, the described patient's condition can be reversible or irreversible.The example of the reversible patient's condition can be owing to blood pressure drops in operation or other interventional therapy processes.Therefore, the patient's condition of being treated can be the reduction ypotension for example of any system volume of blood flow, and it will affect the system volume of blood flow to intestines, heart, kidney or any other organ.

In one embodiment, the present invention relates to peptide according to the present invention for the preparation of the purposes that is used for the treatment of ischemic pharmaceutical composition, the wherein said patient's condition is to be caused by acute, subacute or chronic local asphyxia.

Acute, the subacute or chronic local asphyxia of organ or four limbs or tissue can be caused by numerous disease.It comprises (non-complete list) there is thrombotic atheromatous disease, from heart or from blood vessel or from the aortic aneurysm of the embolism of any organ, spasm, other organs or aneurysma, chest or abdomen or dissecting aneurysm, owing to cardiopathic ypotension, owing to the systemic disease ypotension of (comprising transmissible disease or anaphylaxis), owing to the ypotension of one or more toxic chemicals or poison or medicine.

In the second embodiment, the present invention relates to peptide according to the present invention for the preparation of the purposes that is used for the treatment of ischemic pharmaceutical composition, the wherein said patient's condition is caused by the Secondary cases local asphyxia.

The local asphyxia that is secondary to disease or the patient's condition can be selected from following disease and the patient's condition and observe at one or more: diabetes, hyperlipidaemia, Buerger's disease (Buerger's disease), Takayasu's syndrome, the property a crossed arteritis, mucocutaneous lymphnode syndrome (mucocutaneous lymphnode syndrome), cardiovascular syphilis, the connective tissue disease as Raynaud disease, blue phlebitis, blood vessel wound (comprising Iatrogenic injury for example cannulation or operation or organ transplantation).In addition, this list comprises the local asphyxia caused by one or more organ surgeries, one or more organ transplantation, operation insertion transplanting, device, graft, artificial limb or other biological medical cpds or equipment.

In the 3rd embodiment, the present invention relates to the purposes according to peptide of the present invention, the wherein said patient's condition is to be caused by the local asphyxia owing to septic shock or the patient's condition relevant to systemic ypotension.

Inflammatory symptom

Term " inflammatory symptom " in this article refers to a kind of patient's condition, and in the described patient's condition, for example T lymphocyte specific reaction or antibody cause raising of inflammatory cell and Nei Sheng medium chemical substance with the mechanism of reacting of antigen.In some cases, the normal function of organ or tissue changes due to the increase of vessel invasion and/or the contraction of visceral smooth muscle.This inflammation patient's condition can produce inflammatory disease.

In one embodiment, the present invention relates to the purposes of one or more peptides according to the present invention for the preparation of pharmaceutical composition, described pharmaceutical composition is used for the treatment of or prevents the patient's condition of the tissue of mammiferous one or more organs, and the wherein said patient's condition is the inflammation patient's condition.

The inflammation patient's condition can cause by inflammatory disease, and it comprises (non-complete list): sacroiliitis (comprising the disease relevant with sacroiliitis), osteoarthritis, rheumatoid arthritis; SpA (for example ankylosing spondylitis), reactive arthritis (comprising rheumatic fever sacroiliitis afterwards), anaphylactoid purpura and Reiter disease.In addition, inflammatory diseases comprises connective tissue disease for example vasculitis, crystallization deposition disease (comprising gout), tetra-sodium joint disease, the acute Calcified periarthritis of systemic lupus erythematous, polymyositis/dermatomyositis, systemic sclerosis, MCTD, sarcoidosis and primary Sjogren syndrome (comprising keratoconjunctivitis sicca), polymyalgia rheumatica and other types.In addition, inflammatory diseases comprises juvenile arthritis (still's disease), psoriasis, osteoarthritis, be secondary to the osteoarthritis of excessive mobile disease, congenital dysplasia, the femoral head epiphysis gets loose, Perthes disease (Perthes ' disease), intra-articular fracture, meniscectomy, obesity, the recurrent dislocation, repetitive, crystallization deposition disease and cartilage metabolism abnormality disease (comprising the tetra-sodium joint disease), ochronosis, hemochromatosis, ischemic necrosis (comprising the sickle cytopathy), therapeutics with adrenocortical hormone or other drug, decompression sickness, septic or infective arthritis (comprise tuberculous arthritis, meningococcal arthritis, gonococcus property sacroiliitis, Salmonellas property sacroiliitis), infective endocarditis (comprises by viridans streptococci, enterococcus faecalis, streptococcus aureus, staphylococcus epidermidis, Histoplasma, Brucella, Candida, the endocarditis that Aspergillus and shellfish Na Shi cock phase body (Coxiella burnetii) cause), viral arthritis (comprises infecting rubella is arranged, mumps, hepatitis B, HIV or parvovirus), or the recidivity hemarthrosis.In addition, inflammatory disease comprises that vasculitis, for example owing to infecting germy infectious vasculitis, comprises that the spirochete disease is as Lyme disease, syphilis, Rickettsiae and mycobacterial infections, fungi, virus or protozoan infection.In addition, inflammatory disease comprises non-infectious vasculitis, and the vasculitis that comprise polyarteritis, wegener granulomatosis, allergic granulomatous vasculitis (Churg-Strauss syndrome) under Takayasu's disease pulseless disease, giant cell arteritis (crosses property arteritis and polymyalgia rheumatica), Buerger's disease, polyarteritis nodosa, microscope, is secondary to connective tissue disease comprises systemic lupus erythematous, polymyositis/dermatomyositis, systemic sclerosis, MCTD, sarcoidosis and primary Sjogren syndrome.In addition, inflammatory disease comprises the vasculitis that is secondary to rheumatic arthritis.

In addition, inflammatory disease comprises and is secondary to vasculitis, inflammatory bowel, primary biliary cirrhosis, the Goodpastures syndrome (Goodpastures syndrome Goodpasture syndrome) that hypersensitive non-infectious vasculitis and leukocytosis vasculitis comprise serum sickness, anaphylactoid purpura, drug-induced vasculitis, essential mixed cryoglobulinemia, hypocomplementemia, send out with other malignant tumours connection.

In addition, inflammatory disease comprises all types of children's sacroiliitis, and for example juvenile arthritis comprises still's disease, juvenile form rheumatoid arthrosis, Juvenile ankylosing spondylitis.

In addition, inflammatory disease comprises that air flue disease and downtake case are as chronic obstructive pulmonary disease (COPD), allergic asthma and anallergic asthma, rhinallergosis, supersensitivity and nonallergic conjunctivitis.In addition, inflammatory diseases also comprises supersensitivity or nonallergic dermatitis.

Inflammatory disease comprises all types of storage disorderses in addition, as gout, tetra-sodium joint disease, acute Calcified periarthritis.

The inflammation patient's condition of all types of generation backaches of inflammatory disease for example comprises, between infection, septic diskitis, tuberculosis, malignant tumour (metastatic hepatic neoplasm, myelomatosis and other), tumor of spine, ankylosing spondylitis, acute dish disease/osteoarthritis, osteoporosis, richets between outstanding, chronic dish in addition.It also comprises scleromalacia, hyperparathyroidism, renal osteodystrophy, spondylolisthesis, Taper Pipe stenosis birth defect and fibromyalgia.

Inflammatory disease comprises that all types of soft tissue rheumatism comprise bursitis, tenosynovitis or aponeurositis, tendon end inflammation, Crushed nerve disease, periarthritis or capsulitis, muscular tone and muscle function imbalance in addition.

Inflammatory disease comprises that the inflammatory disease of gastrointestinal system (comprises all types of gastritis in addition, pemphigus, large blister quasi-Pemphigus, optimum mucous membrane pemphigoid), disease of salivary gland (sarcoidosis for example, saliva blockage and Sjogren syndrome), (for example gastro oesophageal reflux disease (GORD) or infection have the Candida species to the esophagus inflammation, herpes simplex and cytomegalovirus), the inflammatory disease of stomach (comprises acute and chronic gastritis, helicobacter pylori infection, menetrier's disease), the inflammation of small intestine (comprises coeliac disease, seitan supersensitivity enteropathy, dermatitis herpetiformis, tropical diarrhoea, Whipple disease, radiation enteritis, the systemic amyloidosis disease, connective tissue disease comprises systemic lupus erythematous, polymyositis/dermatomyositis, systemic sclerosis, MCTD and sarcoidosis), Eosinophilic Gastroenteritis, Intestinal lymphangiectasia, inflammatory bowel (comprising Crohn's disease and ulcerative colitis), diverticulosis of colon, and irritable bowel syndrome.

Preferred embodiment, the present invention relates to the purposes of one or more peptides according to the present invention for the preparation of pharmaceutical composition, described pharmaceutical composition is used for the treatment of or prevents the patient's condition of the tissue of mammiferous one or more organs, and the wherein said patient's condition is the inflammation patient's condition that is selected from pneumonia, sacroiliitis, dermatitis, pancreatitis and inflammatory bowel.

Drug-induced cell, tissue and organ failure

At an embodiment, the present invention relates to the purposes of one or more peptides according to the present invention for the preparation of pharmaceutical composition, described pharmaceutical composition is used for the treatment of or pre-Ozoban or drug-induced cell, tissue or organ failure.

In this specification sheets and claim, " drug-induced cell, tissue or organ failure " is defined as cell or tissue function and/or the modal variation of being induced by the pharmacology compound.Described pharmacology compound includes but not limited to that the cancer chemotherapy cartridge bag draws together cis-platinum, carboplatin, Dacarbazine (dacarbezine), Procarbazine, altretamine, semustine, lomustine, carmustine, busulfan, thio-tepa, melphalan, endoxan, Chlorambucil, dichloromethyldiethylamine, azacitidine, carat Qu Bin, cytosine arabinoside, fludarabine, mercaptopurine, methotrexate, Tioguanine, Zyloric, bleomycin, gengshengmeisu, zhengdingmeisu, Docetaxel, Zorubicin (inferior Baudrillard enzyme element), etoposide, idarubicin, irinotecan, mitomycin, taxol, Plicamycin, topotecan, vincaleucoblastine, vincristine(VCR), Vinorelbine, A Masa crith grace (amasacrine), asparaginase, hydroxyurea, miaow is for his grace (mititane), mitoxantrone, as the microbiotic of aminoglycoside comprises Streptomycin sulphate, Liu Suanyan NEOMYCIN SULPHATE, kantlex, Amikacin Sulphate, gentamicin, tobramycin, sisomicin, netilmicin, immunosuppressive compounds is as ciclosporin, tricyclic antidepressant, lithium salts, prenylamine and phenothiazines.

Wherein the normal function of cell, tissue or organ the reformed patient's condition comprise the patient's condition with following disease-related: toxic reaction (comprising drug-induced toxicity) and the acute and chronic injury of local asphyxia, acute and/or chronic inflammatory diseases, anaphylaxis, rheumatosis, infection (comprising that virus, fungi, bacterium infect), prion and other microorganisms and other infectious preparations well known in the art, form of ownership.Chronic injury comprises the situation with the repeated injury of the time of restoring the wholly or in part variation of organ or tissue's function.The patient's condition that wherein normal function of cell, tissue or organ has changed comprises damage, itself and one or more organs or relevant for the implantation of other devices of transplanting, and can expect that peptide of the present invention is useful in also or preventing in the treatment of the described patient's condition.Described organ can derive from individuality self, animal body self or derive from other individualities or animal.This comprises: organ transplantation, bone are transplanted, soft tissue is implanted (silicone implantations), metal and plastics implantation, or other transplantable devices medically.Described individual representative and other Mammalss.

The patient's condition of being treated can be also, by cancer or by having, organ (is for example comprised to lung, bronchiole, upper air flue to respiratory system, and/or on heart and/or on kidney and/or on gastro-intestinal system) precancerous lesion of impact causes, comprises the transitivity malignant tumour of acute leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, lymphosarcoma, myelomatosis, any cause of disease.Can expect that peptide of the present invention also will be useful in the treatment of the described patient's condition or prevention.

In addition, the patient's condition of being treated can be also to be selected from following disease and to be caused by any: diabetes, the patient's condition with empty stomach LDL-cholesterol levels of raising, there is the empty stomach LDL-cholesterol of combination raising and the patient's condition of triglyceride levels, the patient's condition with Serum Triglyceride level of raising, the patient's condition with empty stomach HDL-cholesterol levels of raising, retroperitoneal fibrosis, lupus erythematosus, many knot property arteritis, scleroderma, polymyositis, dermatomyositis, rheumatic arthritis, allergy, serum sickness, hemolytic anemia and supersensitivity granulopenia.Can expect that peptide of the present invention also will be useful in the treatment of the described patient's condition or prevention.

Many infection that cause performance to reduce may have the influence of tissue and disturb normal function, and it can be improved by the peptide of the present invention that gives effective dose.These infection comprise protozoon, virus, bacterium and fungi infestation, and comprise for example AIDS of the patient's condition, bacterial septicemia, systemic fungal infection, rickettsiosis, toxic shock syndrome, infectious mononucleosis, chlamydia trachomatis, chlamydia psittaci, cytomegalovirus infection, the arc bacillus, Salmonellas, influenza, poliomyelitis, toxoplasmosis, the lassa fever disease, yellow jack, schistosomicide, intestinal bacteria, faecalis, Bacillus proteus (preteus), Klebsiella, pseudomonas, streptococcus aureus, staphylococcus epidermidis, Candida albicans, tuberculosis, parotitis, infectious mononucleosis, hepatitis and Coxsackie virus.

The patient's condition of being treated can be relevant with the chemical wound that relates to one or more toxicants and/or medicine.These medicines comprise tricyclic antidepressant, lithium salts, prenylamine and phenothiazines, the cancer chemotherapy cartridge bag is drawn together cis-platinum, carboplatin, Dacarbazine (dacarbezine), Procarbazine, altretamine, semustine, lomustine, carmustine, busulfan, thio-tepa, melphalan, endoxan, Chlorambucil, dichloromethyldiethylamine, azacitidine, carat Qu Bin, cytosine arabinoside, fludarabine, mercaptopurine, methotrexate, Tioguanine, Zyloric, bleomycin, gengshengmeisu, zhengdingmeisu, Docetaxel, Zorubicin ((inferior Baudrillard enzyme element), etoposide, idarubicin, irinotecan, mitomycin, taxol, Plicamycin, topotecan, vincaleucoblastine, vincristine(VCR), Vinorelbine, amasacrine, asparaginase, hydroxyurea, mititane, mitoxantrone, as the microbiotic of aminoglycoside comprises Streptomycin sulphate, Liu Suanyan NEOMYCIN SULPHATE, kantlex, Amikacin Sulphate, gentamicin, tobramycin, sisomicin, netilmicin, immunosuppressive compounds is as ciclosporin.The physics wound comprises that electromagnetic radiation also can cause damage, can it be alleviated by the alpha-MSH analogue according to the present invention of using effective dose.

The patient's condition of being treated according to the present invention can also comprise connective tissue disease for example scleroderma, systemic lupus erythematous, perhaps neuromyopathy, for example Du Xing (Duchenne) type progressive muscular dystrophy, Freed rely uncommon (Friedreich) ataxia, myotonia atrophica.The described patient's condition can for example relate to the tissue of mammal intestine.

The present invention relates to the purposes according to peptide of the present invention, in free following the formed group of wherein said patient's condition choosing: myocardial ischemia, stenocardia, pericarditis, myocardial infarction, myocardial ischemia, myocarditis, myxedema (myxodemia), endocarditis.

In one embodiment, the present invention relates to the purposes according to peptide of the present invention, the wherein said patient's condition is relevant to irregular pulse.

Methods for the treatment of

The invention still further relates to be used for the treatment of or prevent the method in the patient's condition of the tissue of one or more organs of the mammalian subject of its needs, described method to comprise to give effective dose one or more according to peptide of the present invention.The described patient's condition can be ischemic conditions or the inflammation patient's condition and/or from the result of the toxic action of poisoning or pharmacological agent.

Prevent that to transplanting that produce or the associated patient's condition by any organ or vascular, comprising the reaction of graft to the host, methods for the treatment of of the present invention from being special effective.In these patient's condition, whole organ is extremely responsive to all changes about nutrition, metabolism, perfusion etc., and we believe, according to treatment of the present invention, can stablize the described patient's condition and make described tissue, to the situation of the function of any compressing organ, resistance more be arranged.The method according to this invention also is included in the process of being transplanted to acceptor the peptide of the present invention that gives the organ graft effective dose, comprises that the peptide of the present invention that adds effective dose is in grafting matrix.

In addition, the application provides evidence, for example, in severe disease (myocardial ischemia), uses alpha-MSH analogue treatment according to the present invention to prevent significantly death and dysfunction of organ.

One of modal heart patient's condition is intermittent stenocardia or pectoralgia, and wherein treatment according to the present invention is special effective.Relate to the anginal patient's condition and comprise Unstable angina, stable angina pectoris and Prinzmetal variant angina pectoris.

On the other hand, can in the situation caused by pericarditis, myocardial infarction, myocardial ischemia, myocarditis, myxodemia, endocarditis, utilize described prevention and treatment.

The patient's condition of being treated can be relevant to irregular pulse, itself or primary disease or be secondary to another individual patient's condition.The example of ARR a variety of causes comprise acute infection (particularly those affect the acute infection of lung) thereby, pulmonary infarction, ypotension, apoplexy, hypoxemia or can accelerate myocardial ischemia and cause ARR anemia.Thereby irregular pulse can worsen cycle penalty and set up wrong, self-perpetuating circulation.

We believe, will increase for developing ARR threshold value according to treatment of the present invention, thereby prevent ARR development.By directly to the delivery system effect or indirectly by guiding or the patient's condition effect of ARR reason, realizing described effect.

The syndromes that can alleviate according to the inventive method or irregular pulse can be idiopathic or insecondary, can be selected from: ventricular tachyarrhythmias or upper ventricular tachyarrhythmias, atrioventricular block, sinus node disease, Wolff-Parkinson-White syndromes, Len é gres disease, Lev disease and any syndromes that relates to undesired cardiac muscle connection between atrium and ventricle.

Be intended to suppress ARR antiarrhythmic therapy method and usually be attended by the new ARR danger of generation.Irregular pulse can be used as owing to the overdose drug toxicity reaction and occurs.Yet especially in the process of the medicine group treatment with being called as IA class medicine, irregular pulse can be used as the side effect that dose relies on (idiosyncratic reaction-in the situation that drug level develop in the active drug concentration range) and occurs.According to another embodiment, the patient's condition can be caused by one or more antiarrhythmic drugs, comprise purple foxglove, Quinidine, disopyramide, adenosine, aprindine, flecainide, amiodarone, sotalol, mexiletine, beta-Blocking agent, verapamil.

Can expect that using alpha-MSH analogue according to the present invention to treat will reduce the ARR danger of development, irregular pulse is because the treatment of the association with other antiarrhythmic drugs produces.

In another aspect of this invention, the patient's condition can be to be characterized as one or more measure by ecg scanning method (ECG) abnormal.Extremely can relate on ECG, be selected from one or more variations that following configuration changes that are selected from: P ripple, ST fragment, T ripple, QRS complex wave, Q ripple, Δ ripple and U ripple.

Other patient's condition that can alleviate by the peptide according to the present invention that gives effective dose are that organ (for example heart) ionogen is chaotic and self is chaotic, comprise relative concentration abnormal of the another kind of ion of a kind of ion pair.This patient's condition comprises one or more electrolytical Abnormal Serum concentration, and described ionogen is selected from the group formed as follows: potassium, calcium, sodium and magnesium.

According to the present invention, the tissue that can be affected comprises the one or more cell types that are present in organ, can be selected from: epithelial cell, scavenger cell, reticuloendothelial system monocyte, neutrophilic granulocyte, eosinophilic granulocyte, basophilic granulocyte, T-cell, B-cell, mastocyte, dendritic cell.Especially, T-cell, B-cell and mastocyte have special meaning on the one hand at this.

Preferred aspect of the present invention relates to prevention or treatment, wherein prophylactically gives the development for any sign of preventing the patient's condition or the patient's condition according to the dosage of alpha-MSH analogue of the present invention.

Prevention or preventative treatment can be ongoing treatment or for the patient's that prevents from suffering from coronary stricture heart attack in surgical procedure for example.Prophylactic treatment can also be for short duration.Those skilled in the art can be concrete based on the emergency situation evaluation treatment procedure.Preferred embodiment, described treatment or prevention can reduce local asphyxia infarct size coronarius.With untreated individuality, compare, this infarct size can reduce 20%, and for example at least 30%, preferably at least 50%.

Therefore, prophylactically give the foundation for any sign of preventing the patient's condition or the patient's condition according to the dosage of alpha-MSH analogue of the present invention.

Can be used as single dose, rule or continuous administration according to the dosage of alpha-MSH analogue of the present invention, or given as administration sequentially.

Administration can be application, conduit and implantation, oral administration, the administered parenterally that general administration, topical comprise the medicine target system, and for example subcutaneous administration, intramuscularly, intravenous injection, abdominal injection, intrathecal injection, pulmonary administration are for example by suction, external application, mucosa delivery, percutaneous dosing.

Therefore, administration comprises the general administration; Be expelled to tissue or comprise joint in body cavity; Be implanted to tissue or in body cavity; Use skin outward or arrive any stomach and intestine surface, or comprising the body cavity lining to mucomembranous surface.

Because can clearly find out from above, the present invention relates to the purposes of peptide according to the present invention for the preparation of medicine, described medicine is used for the treatment of by any relevant route of administration or prevents any patient's condition disclosed herein.

Pharmaceutical preparation and composition

The invention still further relates to and comprise one or more pharmaceutical compositions according to peptide of the present invention.Described pharmaceutical composition can also comprise one or more pharmaceutical carriers.Further, described pharmaceutical composition can also comprise the acceptable vehicle of one or more pharmacy.

Pharmaceutical composition according to the present invention may be, but not limited to, parenteral composition, oral compositions, topical composition, transmucosal composition or transdermal composition.

In following examples, provided and contained one or more suitable compositions according to peptide of the present invention.For giving individual (animal or human) administration, material is preferably made pharmaceutical composition, and it contains described material and the optional acceptable vehicle of one or more pharmacy.

Composition can be with for example solid-state, the form of semi-solid state or fluid composition exist such as, such as but not limited to, but bio-absorbable sheet, wet tissue paper, dressing, aerogel dressing, hydrocolloid dressing, film, foam, thin slice, bandage, plaster, e Foerderanlage, implant, pulvis, particle, small-particle, capsule, agarose or chitosan pearl, tablet, pill, bead, microcapsule, microballoon, nano particle, spray agent, aerosol, suction apparatus, gel, hydrogel, paste, ointment, emulsifiable paste, soap, suppository, Wei Jituorui (vagitorie) toothpaste, solution, dispersion liquid, suspension, milk sap, mixture, washing lotion, collutory, shampoo, enema, test kit, described test kit comprises for example two containers that separate, first container is containing with good grounds peptide of the present invention, second container contains suitable medium in order to joining first container to obtain stand-by composition before use, and some other suitable form, for example implant or implant coating or a kind of form of using under implantation and transplanting condition that is suitable for.

Composition can be prepared by conventional pharmaceutical methods; referring to for example " Remington: the science of pharmaceutics and put into practice (The science and practice of pharmacy) " second edition .Mack Publishing; Easton PA; 2000ISBN0-912734-04-3 " and " pharmaceutical technology encyclopedia (Encyclopedia of Pharmaceutical Technology) "; Swarbrick, J.& J.C edits .Boylan, Marcel Dekker, Inc., New York, 1988ISBN0-8247-2800-9.

The pharmaceutical composition that contains active substance is as delivery system.Term " delivery system " means a kind of pharmaceutical composition (pharmaceutical preparation or formulation) in this article, and it is the active substance to human body or animal body at the administration interval scale.Therefore, term " delivery system " comprises common pharmaceutical composition such as emulsifiable paste, ointment, liquid, powder, tablet etc., and more complicated formulation such as sprays, plaster, bandage, dressing, device etc.

As mentioned above, can comprise pharmacy or the acceptable vehicle of cosmetology for the pharmaceutical composition according to purposes of the present invention.

At the composition for according to purposes of the present invention, the selection of the acceptable vehicle of pharmacy and optimum concn thereof generally can not be predicted, and need to be determined based on its measuring.Whether the acceptable vehicle of a kind of pharmacy is suitable for generally also depending in pharmaceutical composition the type of selected formulation.Yet the those of ordinary skill of pharmacy field can be at " Remington:The science and practice of pharmacy " second edition .Mack Publishing, Easton PA, 2000ISBN0-912734-04-3 " find guidance.

The acceptable vehicle of pharmacy is the substantially harmless material of individuality that will give composition.This vehicle will meet the requirement that drug control department promulgates usually.Official's pharmacopeia for example British Pharmacopoeia, American Pharmacopeia and European Pharmacopoeia has all been stipulated the standard of the acceptable vehicle of known pharmacy.

Below provided the summary for the relevant pharmaceutical composition according to purposes of the present invention.This summary is based on specific route of administration.Yet, it should be understood that formulation that can be different at the acceptable vehicle of those pharmacy or the situation that is used in combination under, the application of the specific acceptable vehicle of pharmacy is not limited to the specific function of particular dosage form or this vehicle.

Parenteral composition

For the general application, can contain conventional avirulent pharmaceutically acceptable carrier and vehicle according to composition of the present invention, comprise microballoon and liposome.

Composition used according to the invention comprises solid-state, semi-solid state and the fluid composition of form of ownership.The composition of certain relevant is, for example solution, night, emulsion, gel, implant and implant suspend.

The acceptable vehicle of pharmacy can comprise, solvent, buffer reagent, sanitas, wetting agent, intercalating agent, antioxidant, stablizer, emulsifying agent, suspension agent, jelling agent, thinner, disintegrating agent, tackiness agent, lubricant and wetting agent.The example reference of different reagent is as follows.

Topical composition, transmucosal composition and transdermal composition

For the application to mucous membrane or skin.Nontoxic pharmaceutically acceptable carrier and vehicle usually be can contain for the composition according to purposes of the present invention, microballoon and liposome comprised.

For solid-state, semi-solid state and the fluid composition that comprises form of ownership according to the composition of purposes of the present invention.The composition of certain relevant is, for example, paste, ointment, hydrophilic ointment, emulsifiable paste, gel, hydrogel, solution, emulsion, suspension night, lotion, liniment, Li Suorui Bright (resoriblet), suppository, enema, vaginal suppository, molded vaginal suppository, vaginal capsule, vaginal tablet, shampoo, person mile, soap agent, paste, sprays, pulvis, film, foam, bedding and padding, sponge (for example collagen sponge), pad, dressing (for example, as, absorbent wound dressing), wetting agent, bandage, plaster and transdermal delivery system.

The acceptable vehicle of pharmacy can comprise solvent, buffer reagent, sanitas, wetting agent, intercalating agent, antioxidant, stablizer, emulsifying agent, suspension agent, jelling agent, ointment base, suppository base, infiltration toughener, spices, Derma-Guard, thinner, disintegrating agent, tackiness agent, lubricant and wetting agent.The example reference of different reagent is as follows.

Oral compositions

For comprise all types of solid-state, semi-solid states and fluid composition according to the composition of purposes of the present invention.The composition of certain relevant be for example solution, suspension, emulsion, nude film, controlled release tablet, enteric coated tablet, mouthful dispersible tablet, effervescent tablet, chewable tablet, soft capsule, hard capsule, controlled release capsule, enteric coated capsule, naked particle, effervescent granule, the particle for the preparation of the liquid orally used, coated granule, enteric coated particles, controlled release granule, for the pulvis of oral administration and for the preparation of the pulvis of the liquid orally used.

The acceptable vehicle of pharmacy can comprise solvent, buffer reagent, sanitas, wetting agent, intercalating agent, antioxidant, stablizer, emulsifying agent, suspension agent, jelling agent, thinner, disintegrating agent, tackiness agent, lubricant and wetting agent.The example reference of different reagent is as follows.

The example of all ingredients

The example of solvent is but is not limited to water, alcohol, vegetables oil or fish oil, (for example, edible oil, as Prunus amygdalus oil, Viscotrol C, theobroma oil, Oleum Cocois, Semen Maydis oil, Oleum Gossypii semen, Toenol 1140, sweet oil, plam oil, peanut oil, seed of Papaver somniferum L. powder, rapeseed oil, sesame oil, soybean oil, sunflower oil and tea-seed oil), the poly-alkylsiloxane of mineral oil, fatty oil, whiteruss, polyoxyethylene glycol, propylene glycol, glycerine, liquid, and its mixture.

The example of buffer reagent is but is not limited to citric acid, acetic acid, tartrate, lactic acid, phosphoric acid, diethylamine etc.

Be but be not limited to parabens (methyl p-hydroxybenzoate for example for the example of the sanitas of composition, ethyl p-hydroxybenzoate, propylparaben, butyl p-hydroxybenzoate, p-Hydroxybenzoic acid isobutyl ester, p-Hydroxybenzoic acid isopropyl ester), potassium sorbate, Sorbic Acid, phenylformic acid, methyl benzoate, Phenoxyethanol, bronopol, Bu Luonidaokesi (bronidox), MDM glycolylurea (MDM hydantoin), IPBC, ethylenediamine tetraacetic acid (EDTA) (EDTA), benzalkonium chloride and phenylcarbinol, perhaps preservative blends.

The example of wetting agent is but is not limited to glycerine, propylene glycol, sorbyl alcohol, lactic acid, urea, and corresponding mixture.

The example of intercalating agent is but is not limited to EDETATE SODIUM salt and citric acid.

The example of antioxidant is but is not limited to butylated hydroxy anisole (BHA), xitix and derivative, tocopherol and derivative thereof, halfcystine, with and composition thereof.

The example of emulsifying agent is but is not limited to naturally occurring natural gum, for example Sudan Gum-arabic or tragacanth, the natural for example soybean lecithin of phosphatide that exists; The sorbitan monooleate derivative; Lanolin; Wool wax alcohol; The sorb sugar ester; Direactive glyceride; Fatty alcohol; Fatty acid ester (for example tri-glyceride of lipid acid); And composition thereof.

The example of suspension agent is but is not limited to Mierocrystalline cellulose and derivatived cellulose, for example carboxymethyl cellulose, Natvosol, hydroxypropylcellulose, Vltra tears, carrageenin, Sudan Gum-arabic, gum arabic, tragacanth, and their mixture.

The example of gel matrix and viscosity-increasing agent is but is not limited to whiteruss, polyethylene, fatty oil, colloid silica or colloidal aluminum, zinc soap, glycerine, propylene glycol, tragacanth, carboxy vinyl polymer, neusilin, carboxyvinyl polymer

hydrophilic polymer, for example carboxymethyl cellulose, Natvosol of starch or derivatived cellulose for example, and other derivatived celluloses, water-swellable hydrocolloid, carrageenin, hyaluronate (hyaluronic acid derivatives that for example optionally contains sodium-chlor), and alginates (comprising the propylene glycol alginates).

The example of ointment base is but is not limited to the condensation product of beeswax, paraffin, hexadecanol, cetyl palmitate, vegetables oil, sorbitan fatty(acid)ester (this Pan Span), polyoxyethylene glycol and sorbitan fatty(acid)ester and oxyethane, for example polyethylene oxide sorbitan monooleate (tween Tween).

The example of hydrophobic ointment matrix is but is not limited to the poly-alkylsiloxane of paraffin, vegetables oil, animal tallow, synthetic glyceride, wax, lanolin and liquid.

The example of hydrophilic ointment matrix is but is not limited to solid-state macrogols(polyoxyethylene glycol).

The example of powdery components is but is not limited to alginates, collagen, lactose, can form the powder (absorbing liquid/wound fluid) of gel when being applied to wound.

The example of thinner and disintegrating agent is but is not limited to lactose, sucrose, Emdex (emdex), calcium phosphate, calcium carbonate, calcium sulfate, N.F,USP MANNITOL, starch and Microcrystalline Cellulose.

The example of tackiness agent is but is not limited to sucrose, sorbyl alcohol, Sudan Gum-arabic, sodium alginate, gelatin, starch, Mierocrystalline cellulose, Xylo-Mucine, methylcellulose gum, hydroxypropylcellulose, polyvinylpyrrolidone and polyoxyethylene glycol (Polyetyleneglycol).

The example of wetting agent is but is not limited to sodium lauryl sulphate and Polysorbate 80.

The example of lubricant is but is not limited to talcum, Magnesium Stearate, calcium stearate, silicon oxide, palmityl stearyl glyceryl ester (precirol) and polyoxyethylene glycol.

The example of coating agent is but is not limited to hydroxypropylcellulose, Vltra tears, polyvinylpyrrolidone, ethyl cellulose and polymethacrylate.

The example of suppository base is but is not limited to oleum theobromatis, mixed fatty glycerides and polyoxyethylene glycol.

Alpha-MSH analogue can be present in medicament with the amount of the 0.001-99% of medicament weight, is preferably 0.01-75%, more preferably 0.1-20%, especially 1-15% 1-10% for example.

Dosage depends on the patient's condition of being treated.Various medicines can be used under dosage well known in the art.Can be expected that, one or more dosage according to peptide of the present invention, in the scope of 1ng-100mg/kg body weight, are preferably 1 μ g-10mg/kg body weight, more preferably 10 μ g-1mg/kg body weight, for example 50-500 μ g/kg body weight.

On the other hand, the present invention relates to pharmaceutical composition as above, it comprises that one or more are according to peptide of the present invention and optional pharmaceutically acceptable carrier.

Can be by preparing according to pharmaceutical composition of the present invention by routine techniques well known in the art and conventional pharmaceutical carriers.In addition, pharmaceutical composition can be the arbitrary form that is suitable for arbitrary purposes described herein.

This paper record and claimed invention are not limited to the scope of embodiment disclosed herein, because these embodiments are used for illustrating several aspect of the present invention.Anyly be equal to embodiment all within the scope of the invention.In fact, except this paper shown and describe those revise, from the various modifications of the present invention of above-mentioned specification sheets, will become apparent to those skilled in the art.These revise the scope that also falls into accompanying claim.

This paper has quoted various reference, and its disclosed content is incorporated to this paper as a reference in the mode of their integral body.

Run through this specification sheets, word " comprises ", or its variation contains as " comprising " or " comprising " should be understood to finger element, integer or the step of pointing out, the perhaps group of element, integer or step, but element, integer or the step of not getting rid of any other, or the group of element, integer or step.

About the above-mentioned specification sheets of various aspects of the present invention and the embodiment of these aspects, it should be understood that foregoing description or arbitrary feature that mention, relevant to an embodiment of one aspect of the present invention and/or an aspect and characteristic also are suitable for any or all other aspects and/or embodiment described in the invention by analogizing.

Below will the present invention be described by the drawings and Examples of following unrestricted effect.

Embodiment

Following general description for testing the method for peptide of the present invention.The result of proof peptide provides in embodiment 1-7.The target of method is test peptide of the present invention for antiphlogistic effects and inhibition or prevents cell/tissue/organ damage of occurring as the result of the toxic action of local asphyxia, inflammation or medicine or the ability of destruction.

The feature of the deterioration of inflammatory reaction or chronic inflammatory diseases is the production of cell source medium (for example tumor necrosis factor-alpha (TNF-α), interleukin (IL-1 β, IL-8), nitrogen oxide (NO) and oxyradical), this finally can induce widely distributed endothelial injury, described endothelial injury has the angiosthenia loss of whole body vascular, the capillary wettability improved, lasting ypotension and dysfunction of organ, and it is attended by the white corpuscle accumulation (comprising neutrophilic granulocyte and eosinophilic granulocyte) in alveolar space in lung.By inducing the inflammatory mediator that comprises TNF-α, the lipopolysaccharides discharged from infectious agent (LPS) is playing an important role aspect the inflammatory reaction to infecting.Therefore, there is the methods for the treatment of that suppresses TNF-α throughput and believe to there is significant antiphlogistic effects.The contriver stimulates to produce inflammatory reaction (referring to experimental program 1-3) with LPS in many schemes, for the main mark of the antiphlogistic effects according to peptide of the present invention, is to suppress the ability that TNF-α produces.

By reduce/arterial blood is supplied with and is stoped the local asphyxia of inducing to induce many tissue reactions completely, comprises neutrophilic granulocyte accumulation, other inflammatory reactions and necrocytosis.Most cell/tissue/organ damages that evaluation can suppress or prevent (completely or partially) to occur as ischemic result or the compound of destruction are very useful.The contriver uses two one property crossed local asphyxia models: 1) the myocardial ischemia-reperfusion model of rat, it has simulated back is the development of the Acute Myocardial Infarction of blood supply recovery, because it obtains (experimental program 4) by fibrinolytic therapy or coronary angioplasty; 2) two-way renal artery obstruction, it induces acute renal failure (ARF), that it is similar to seen in the patient who carries out large surgery interventional therapy, by the instantaneous ARF(that reduces to induce of kidney blood supply due to the abdominal aortic aneurysm embodiment interventional therapy of can being performed the operation) (experimental program 5).

Nephrotoxicity is the known side effect of plus cisplatin in treatment.Although the dosage of restriction renal toxicity not necessarily still affects Most patients, observe the obvious reduction of renal glomerulus infiltration rate in therapeutic process.The renal toxicity of cis-platinum can be regarded as the direct cytotoxin damage of the nephron of the thick section of ascending branch of medulla externa layer (the especially S3 fragment of proximal tubule) and Henle ring.Therefore, plus cisplatin in treatment usually causes the heavy native defect of uriniferous tubules, comprises the ability of the dilute urine of damage.Observe hypomagnesemia in about 50% the patient who crosses with plus cisplatin in treatment, it is likely the re-absorbed defect of magnesium (Mg) due to kidney.It is the important factor that protection avoids the Nephrotoxicity of cyclosporin A that nearest research shows to supplement Mg, and has pointed out recently the possible relation between the nephrotoxicity of Mg loss and cisplatin induction.Therefore, be intended to prevent that the treatment of hypomagnesemia has useful effect, not only in order to reduce the needs that supplement Mg but also in order to reduce the renal toxicity of cis-platinum.Peptide according to the present invention is checked in experimental program 6 for the renal toxicity of cisplatin induction.

Method and material

In the method be described below, peptide of the present invention is experimental compound.

Experimental program 1

Suppress the generation of the external TNF-α induced by the LPS of human leukocyte generation

Collect the 20mL human blood in containing the vacuum blood collecting tube of EDTA (vacutainer tube).Use peace agate West Asia specification sheets (Amersham ' s Instruction) 71-7167-00AD, the Ficoll-Paque of 2002-06+(Ficoll-Paque Plus) carrys out separating periphery blood monocytic cell (PBMC).Use platform to expect that blue solution (Sigma (Sigma)) is to the PBMC counting, and with 5 * 10 ⁵the concentration of cell/mL by the PBMC incubation at RPMI1640(Ai Puli chemical products (Applichem)) in, RPMI1640 be added with 10mM hydroxyethyl piperazine ethanesulfonic acid (Hepes) (Sigma), 2mM L-glutaminate (Sigma), 0.1% bovine serum albumin (BSA) (Sigma) and 50U/50 μ g/mL penicillin/streptomycin (Sigma).At 5%CO ₂, 95% air atmosphere in, under 37 ℃, the PBMC incubation of separating is had to substratum, 10ng LPS/mL(Sigma) and the 24 hole flat undersides (Corning Incorporated (Corning Incorporated)) of experimental compound in.After 18 hours, sample is centrifugal, use the TNF-α that detects supernatant liquor from the tumor necrosis factor alpha (Tumour Necrosis Factor Alpha) [(h) TNF-α] of the mankind's hundred vertical gram (Human Biotrak) enzyme-linked immunosorbent assay (ELISA) systems (Amersham).

Each donor adopts following mode by the sample incubation:

PBMC is in RPMI (time contrast)

PBMC is at 10ng LPS/mL (carrier)

PBMC is in 10ng LPS/mL, 10 ^-17m α-MSH or alpha-MSH analogue

PBMC is in 10ng LPS/mL, 10 ^-15m α-MSH or alpha-MSH analogue

PBMC is in 10ng LPS/mL, 10 ^-13m α-MSH or alpha-MSH analogue

PBMC is in 10ng LPS/mL, 10 ^-11m α-MSH or alpha-MSH analogue

PBMC is in 10ng LPS/mL, 10 ^-9m α-MSH or alpha-MSH analogue

PBMC is in 10ng LPS/mL, 10 ^-7m α-MSH or alpha-MSH analogue

All samples are diluted to 1.4 * 10 from initial liquid storage ^-4m and 1.8 * 10 ^-3between M.

Process and avoid compound to be bonded on the surface of bottle with protection in the bottle that all solution was coated with at BSA.

Data mean with mean number ± SE.The impact that the TNF-α that experimental compound is induced LPS discharges is expressed as the percentage ratio of TNF-α accumulation in the LPS-vehicle group.With the not all contrasts of t-check analysis in pairs of student.Think under confidence level 0.05 (p) that difference is significant.

Experimental program 2

Suppress the TNF-α production that the LPS in the rat body induces

Laboratory animal: female Wistar rats (220-240g) is from Charlie river (Charles River), Sulzfeld, Germany, and make its live in that temperature (22-24 ℃) and humidity (40-70%) are controlled, have 12 hours the bright-dark circulation (carrying out illumination from 6:00A.M. to 6:00P.M.) room in.The rodents feed that rat is maintained the standard (thering is 140mmol/kg sodium, 275mmol/kg potassium and 23% protein, A Ertuomin international corporation (Altromin International), Lage, Germany), and there is the freedom that approaches water source.

Animal is prepared: under the state of isoflurane-Nitrous Oxide anesthesia, respectively via femoral artery and vein, durable medical polyethylene (Tygon) conduit is implanted in aorta abdominalis and postcava.After implanting, by the independent stable breeding 7-10 of animal days until experiment day.

Experimental arrangement: before experiment, by training two cycles of all rats (each two hours), make them adapt to the restriction cage for experiment.In experiment day, animal is transferred in the restriction cage, and started to carry out the carrier soln that intravenous injection contains 150mM glucose.In whole experimentation, injection rate is 0.5ml/h.After the of short duration adaptation cycle, start injection lipopolysaccharides (LPS).Dosage with the 4mg/kg body weight gives LPS(e. coli serotype 0127B8, L3129, Sigma, St.Louis, USA), intravenous injection surpasses is carried in 1 hour.Within 60,90 and 120 minutes, get 0.3ml arterial blood sample after lps injection starts, and substitute with the blood from normal donor rat of heparinization immediately.

Experimental group:

All fast injection (bolus injection) processing for rat except lps injection:

Carrier (0.5mL isotonic saline solution);

α in one of following dosage-MSH:50 μ g/kg body weight; 200 μ g/kg body weight or 1000 μ g/kg body weight;

Experimental compound in one of following dosage: 50 μ g/kg body weight; 200 μ g/kg body weight or 1000 μ g/kg body weight.

The mensuration of Plasma TNF-α: there is 0.5mM EDTA, pH7.4, and 20x10 ⁶collect blood sample in the precooling experiment tube of IU/ml Trypsin inhibitor,Trasylol.After centrifugal through 4 ℃, by plasma sample, transfer in the precooling experiment tube and the TNF-α mensuration for back-20 ℃ of storages.By ELISA(Biotrak, Amersham, Britain) measure the TNF-α of blood plasma.

Statistical study: result means with mean number ± SE.Use two factor variance analyses (two-way ANOVA) the check group difference for replicate measurement.In the situation that p<0.05, the not t-check in pairs of proofreading and correct by the Bang Feiluoni (Bonferroni) with confidence level, estimate the difference between the corresponding phase.

Experimental program 3

The infiltration of neutrophilic granulocyte and eosinophilic granulocyte after inhibition rat suction LPS

Use is from M& B A/S, DK-8680Ry, the male Sprague-Dawley rat of Denmark is for all experiments.Rat is closed in 3 type standard cages, make its live in that temperature (22-24 ℃) and humidity (40-70%) are controlled, have 12 hours the bright-dark circulation (carrying out illumination from 6:00A.M. to 6:00P.M.) room in.Feed is the Altromin1324 special formulation that autoclave was processed, and this is filled a prescription by Altromin Denmark(Chr.Pedersen A/S, 4100Ringsted, Denmark) produce.Unrestrictedly give feed and water.

After domestication, rat is randomly assigned to experimental group, LPS induce while starting intravenous injection dosage give experimental compound and LPS induce after 8 hours again intravenous injection dosage give experimental compound.

To organize 3 rat anesthesia with 0.1ml fentanyl/many Meikang/100g, and intravenous injection dosage gives experimental compound.Immediately they are placed in respiratory chamber after dosed administration, wherein make their spraying LPS solution.The concentration of LPS is 1mg/ml.The dosed administration time is 15 minutes.Within 24 hours, put to death rat after with the administration of experiment dosages of substance.Finally use CO ₂/ O ₂put to death rat.

By injecting and extracting 6 * 2.5ml PBS out and carry out bronchoalveolar lavage to right lung.Carry out lavation with the state that makes lung still remain in the heart after removing breastbone and rib.To arrive the pipe connecting knotting of left lung in this process.Under 4 ℃, 1000rpm, centrifugal bronchoalveolar lavage fluid (BALF) is 10 minutes.Be resuspended in 0.5ml PBS by cell and carry out total cell count after removing supernatant liquor.To from each rat, with Ji's nurse Sa (May-Gr ü wald Giemsa) BALF that dyeing is crossed, prepare two smears.BALF from each rat is carried out to total cell count and Arneth's count.

Experimental group:

Carrier (0.5mL isotonic saline solution);

α-MSH:200 μ g/kg body weight;

Alpha-MSH analogue: 200 μ g/kg body weight.

Finally by vehicle treated, do not suck the time control group of LPS.

Statistics:

Data mean with mean number ± SE.The single-factor variable analysis that is expense house least significant difference method by back contrasts between being organized.Think that in 0.05 level difference is significant.

Experimental program 4

Release and the pulmonary hypertension of the cytokine that the LPS in inhibition pig body induces

By female landrace (Landrace pig) (～30kg) overnight fasting but allow freely to approach water source.Then intramuscular injection of ketamine (10mg/kg) and imidazoles reach logical sequence (0.25mg/kg) pig are carried out to premedicate.Intravenous injection ketamine (5mg/kg) causes anesthesia.Pig is inserted to pipe from oral cavity, and with continuous intravenous injection fentanyl (60 μ g/kg/h) and imidazoles reach logical sequence (6mg/kg/h) keep anesthesia.With 5cm H ₂vent-pipe (the Servo900 ventilator (ventilator) that the end-expiratory positive pressure of O is controlled with volume; Siemens (Siemens) Elema, Solna, Sweden) animal is ventilated.Tidal volume remains on 10-15ml/kg, and adjusts respiratory rate (20-25 time/min) to keep the constant (arterial carbon dioxide [PaCO of blood carbonic acid ₂] at 34-45mmHg).Ventilate and be intended to make arterial oxygen pressure (PaO with the oxygen of mixing air ₂) higher than 105mmHg.An arterial sheath and two venous sheath are arranged in carotid artery and corresponding vein, described vein for perfusion, conduit Measure blood pressure by being full of fluid, get blood and for introducing conduit.

Insert pulmonary artery floating catheter (Swan-Ganz) (Edward biotech firm (Edwards Lifescience Corp.), Irvine, California) in pulmonary artery via right superior vena cava.Because being right side by heart, it enters into pulmonary artery, can be by observing the Characteristic pressures curve on monitor and the position of determining balloon catheter by the X-ray.Insert another conduit (5French at left neck artery; St.Jude Medical Company, St.Paul, the Minnesota State) for continuous blood pressure monitoring and blood sampling.Insert catheter for collecting urine.When evaluate cardiac shows, interim pace-making conduit is inserted into to right atrium (guidance of X-ray) by venous sheath and is used for making the heart rate stdn.

Hemodynamic monitoring: carry out day-night observation arteriotony, heart rate (from electrocardiogram(ECG) and pulmonary artery pressure (PAP).

Lipopolysaccharides injects: before each experiment, 120min is by e. coli lipopolysaccharide intracellular toxin (E.coli026:_6, Bacto lipopolysaccharides; Enlightening husband laboratory but (Difco Laboratories), Detroit, the state of Michigan) be dissolved in salt solution to dissolve any throw out.After one period steady time, the speed that is 2.5 μ g/kg/h at baseline and progressively be increased to start injection lipopolysaccharides under the condition of 15 μ g/kg/h in 30 minutes.Afterwards, injection speed is remained on to 2.5 μ g/kg/h in 150 minutes, after this stop injection.

Intervention group: the carrier that gives immediately control group and intervention group equal volume before lps injection starts.Alpha-MSH analogue (dosage is 200 μ g/kg) is administered to the intervention group as single vein fast injection.

Cytokine: by according to the enzyme immunoassay of product description commodity in use, use the FFP sample that the blood stablized from EDTA obtains to measure TNF-α,

Statistics: data mean with mean number ± SE.The single-factor variable analysis that is expense house least significant difference method by back contrasts between being organized.Think that in 0.05 level difference is significant.

Experimental program 5

The myocardial infarction area that inhibition is induced by blocking rat left anterior descending coronary artery 60min

That isolation is fed and specified-pathogens free female Wistar rats (250g) is from Charles River, Hannover, Germany.Make animal live in that temperature (22-24 ℃) and humidity (40-70%) are controlled, have 12 hours the bright-dark circulation (carrying out illumination from 6:00A.M. to 6:00P.M.) room in.All animals allow contact free tap water and ball shape rat feed, and it approximately contains 140mmol/kg sodium, 275mmol/kg potassium and 23% albumen (Altromin catalogue no.1310, Altromin International, Lage, Germany).

Via femoral artery and vein, durable medical Tygon conduit is implanted in postcava and aorta abdominalis.Use the O that contains 4% isoflurane after one week in respiratory cavity ₂make rat anesthesia.After inserting tracheal catheter, use the O that contains 1.0% isoflurane for following Hugo Basile Rodent ventilator ₂animal is carried out to the artificial ventilation.Tidal volume is the 8-10ml/kg body weight, and respiratory rate is 75min ^-1, keep artery pH between 7.35-7.45.In surgical procedure, animal is placed on and can keeps rectal temperature on the operating table of the heating of 37-38 ℃.Use Hugo Sachs ECG instrument measurement standard ECG(the second wire), and collect standard ecg on the line of 4,000Hz on PowerLab.After parasternal cuts and opens pericardium, visual fixedly left anterior descending coronary artery (LAD).Arrange between the bottom righthand side of pulmonary trunk and left auricle around the LAD surrounding and there is the suture of the permission harmless 6-0 silk thread of the occluder of opening again.After 10 minutes, make left anterior descending coronary artery (LAD) interlock.The interlock success is confirmed in variation (the ST-fragment rises and the R-ripple amplifies increase) and the decline of MAP by ECG.After 60 minutes, by opening occluder, pour into again.Control rats is carried out sham-operation.

Rat is carried out to a kind of of following intravenous injection processing:

Carrier: 0.5ml150mM NaCl.

α-MSH: the 200 μ g or the 1000 μ g α-melanotropin/kg body weight that are dissolved in 0.5ml150mM NaCl.

Experimental compound: the 200 μ g or the 1000 μ g experimental compounds/kg body weight that are dissolved in 0.5ml150mM NaCl.

Within 5 minutes, giving above-mentioned processing before perfusion again.

The following size of determining the cardiac muscle of local asphyxia and necrosis

After the perfusion of local asphyxia/again, make rat remain on narcosis, carry out the heavily interlock of LAD after within three hours, pouring into again.In this period, continuously measured ECG and MAP.Then intravenous injection Evans Blue dyestuff is used for determining the size of local asphyxia area.Heart being excised and is cut into horizontal stratification is used for determining the size of local asphyxia area and the cardiac muscle of ischemic is divided and leaves from downright bad cardiac muscle.Ischemic area is separated, by its under 37 ℃ in 0.5%2,3,5-triphenyl tetrazolium chloride solution incubation 10 minutes.Then use the size of the tissue of computer generated image program determination necrosis.In order to estimate the therapeutic treatment effect to congestive heart failure, the animal of another program is postoperative with the buprenorphine processing, then turn back in cage and measured left ventricular end diastolic presssure (LVEDP) after two weeks.The micro-top of the 2F conduit that use is inserted into left ventricle via right carotid is measured LVEDP.Regulating isoflurane concentration makes average artery pressure (MAP) be stabilized in 85-90mmHg.

Statistics:

Data mean with mean number ± SE.With contrasting in the paired t check analysis of student group.The single-factor variable analysis that is expense house least significant difference method by back contrasts between being organized.Think that in 0.05 level difference is significant.

Experimental program 6

The renal failure that inhibition is induced by two-way obstruction rat renal artery in 40 minutes

Isolation nursing and specified-pathogens free female Wistar rats (250g) are from Charles River, Hannover, Germany.Make animal live in that temperature (22-24 ℃) and humidity (40-70%) are controlled, have 12 hours the bright-dark circulation (carrying out illumination from 6:00A.M. to 6:00P.M.) room in.All animals allow contact free tap water and ball shape rat feed, and it approximately contains 140mmol/kg sodium, 275mmol/kg potassium and 23% albumen (Altromin catalogue no.1310, Altromin International, Lage, Germany).

To implant and have the rat of long-term nephridioduct to be positioned in metabolic cage in advance, through after two day adaptive phase of metabolic cage, by stopping up arteriorenal two ends 60min, induce experimental ARF.In surgical procedure, with isoflurane-nitrous oxide anesthesia rat, place it on the operating table of heating to keep rectal temperature at 37 ℃.By the rib abdominal incision, two kidneys are exposed, by making it loosening from the perinephric fat excision it, then from vein, cut lightly the sub-fraction Renal artery.With ganoid blood vessel clip (60g pressure; World Precision Instruments, Britain) obstruction Renal artery 40min.Confirm complete local asphyxia by the whiting of observing whole kidney surface.During local asphyxia, temporarily sew up a wound to keep body temperature.After removing clip, then observe kidney 2-5min to guarantee the colour-change of indicating blood to reflux.Then use 3-0 silk suture wound.Rat is put back in metabolic cage, measure urinary volume and the water intake amount of 24h every day, totally 5 days.As a control group, rat carries out not stopping up the Renal artery with the sham-operation identical for ARF.The rat of parallel monitoring sham-operation and the rat with ARF.

Make rat carry out a kind of of following intravenous injection processing:

Vehicle group: 0.5ml150mM NaCl.

α-MSH group: be dissolved in 200 μ g α-melanotropin of 0.5ml150mM NaCl/kg body weight.

Experimental compound group: be dissolved in the 200 μ g experimental compounds of 0.5ml150mM NaCl/kg body weight.Kidney again the perfusion before 5 minutes, then after 6 hours and 24 hours, give above-mentioned processing.

Statistics:

Experimental program 7

The renal failure that suppresses cisplatin induction

To implant and have the rat of long-term nephridioduct to be positioned in metabolic cage in advance, through after the adaptive phase to metabolic cage, the cis-platinum 5.0mg/kg body weight or the carrier (0.5ml150mM NaCI) that are dissolved in 0.5ml150mM NaCI with abdominal injection be processed rat.Then, after 5 days, rat is put back to metabolic cage, measure and collect next 24h urinary volume and water intake amount in 5 days every day.Then at fluothane/N ₂by all rat anesthesias, collect the arterial blood sample in coated bottle at the EDTA of precooling in O.Blood sample is collected in and contains 0.5mM EDTA, pH7.4, and 20x10 ⁶in the precooling experiment tube of IU/ml Trypsin inhibitor,Trasylol.After centrifugal through 4 ℃, plasma sample is transferred to the precooling experiment tube, and is kept at-20 ℃ for measure creatinine and magnesium (Mg) later.In addition, also be determined at the creatinine in the urine of collecting during nearest 24 hours before blood collecting.Creatinine clearance (C as renal glomerulus infiltration rate index _cr) can be calculated as follows: C _cr=V _ux U _cr/ P _cr, V here _utwenty-four-hour urine output; U _crcreatinine concentration in urine, P _crcreatinine concentration in blood plasma.Use clinical chemistry system VITEOS950(to proofread and correct clinical diagnosis company (Ortho-Clinical Diagnostics Inc.), Johnson& Johnson, New Jersey) and Roche Hitachi module (Roche Hitachi Modular) (Roche diagnosis (Roche Diagnostics), mannheim (Mannheim), Germany) urinate with blood plasma in the mensuration of creatinine.

Make rat carry out a kind of of following intravenous injection processing:

Vehicle group: 0.5ml150mM NaCl.

Experimental compound group: be dissolved in the 200 μ g experimental compounds of 0.5ml150mM NaCl/kg body weight.Kidney again the perfusion before 5 minutes, then after 6h and 24h, give above-mentioned processing.

Statistics:

Result

Embodiment 1

Experimental compound is alpha-MSH analogue #1:

Ac-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Met-Glu-His-Phe-A rg-Trp-Gly-Lys-Pro-Val-NH ₂(sequence table sequence 1* is acetylation and is amidated at the C-end at the N-end)

This compound is tested at experimental program 1-7.

α-MSH and alpha-MSH analogue #1(sequence table sequence 1*) both dosage rely on ground and reduce the TNF-α accumulation that in the human leukocyte suspension, LPS induces.Surprisingly, inhibition alpha-MSH analogue #1(sequence table sequence 1*) is more remarkable than the antiphlogistic effects of native peptides α-MSH.α-MSH suppresses TNF-α and is accumulated to 73 ± 9% of maximum reaction (LPS-carrier).In contrast, alpha-MSH analogue #1(sequence table sequence 1*) can reduce by 47 ± 2%(that TNF-α is accumulated to carrier α-MSH is organized to p<0.01) (see figure 1)

Suppress the TNF-α production that the LPS in the rat body induces

In the process of intravenous injection LPS, α-MSH and alpha-MSH analogue #1(sequence table sequence 1*) both reduced TNF-α accumulation in rat.Obtained α-MSH and alpha-MSH analogue #1(sequence table sequence 1* under the dosage of 200 μ g/kg body weight) maximum suppression effect, and within 120 minutes after lps injection starts, demonstrate the maximum suppression effect that TNF-α is produced.Surprisingly, inhibition alpha-MSH analogue #1(sequence table sequence 1*) is more remarkable than the antiphlogistic effects of native peptides α-MSH.Although α-MSH suppresses TNF-α concentration in rat plasma to 17 ± 3% of maximum reaction (LPS-carrier), alpha-MSH analogue #1(sequence table sequence 1*) can reduce by 9 ± 1%(that TNF-α is accumulated to carrier α-MSH is organized to p=0.05) (see figure 2).

In the BALF collected in 24 hours after LPS sucks as shown in the remarkable reduction of eosinophilic granulocyte like that, α-MSH and alpha-MSH analogue #1(sequence table sequence 1*) both be reduced in the inflammatory reaction to the LPS suction in alveolar space.With the quantity of eosinophilic granulocyte in BALF be 164.6 ± 42.2 * 10 ⁵rat individual cell, vehicle treated is compared, and α-MSH processes the quantity to 26.7 that reduces the eosinophilic granulocyte in BALF ± 4.3 * 10 ⁵individual cell (to vehicle group, p<0.05), alpha-MSH analogue #1(sequence table sequence 1*) reduce the quantity to 49.0 of the eosinophilic granulocyte in BALF ± 10.5 * 10 ⁵individual cell (to vehicle group, p<0.05) (see figure 3).With upper identical, for α-MSH and alpha-MSH analogue #1(sequence table sequence 1*), the effect of the quantity of the neutrophilic granulocyte in BALF is similar.

The release and the pulmonary hypertension that suppress the cytokine that in the pig body, LPS induces

As with alpha-MSH analogue #1(sequence table sequence 1*) in the pig of processing as shown in the plasma concentration of significantly reduced TNF-α after lps injection, alpha-MSH analogue #1(sequence table sequence 1*) there is significant antiphlogistic effects.Except this antiphlogistic effects, surprisingly, alpha-MSH analogue #1(sequence table sequence 1*) also there is the ability that the pulmonary hypertension development is avoided in protection, this can be by with alpha-MSH analogue #1(sequence table sequence 1*) find in the rat of processing, significantly weaken the PAP amplification that LPS induces and confirm (maximum PAP amplification: vehicle group: 22 ± 4mmHg, alpha-MSH analogue #1(sequence table sequence 1*) group: 8 ± 2mmHg; P=0.05) (see figure 4).

, surprisingly, alpha-MSH analogue #1(sequence table sequence 1* contrary with α-MSH) reduced myocardial infarction area, myocardial infarction area is with necrosis area/infarct critical area(s) product representation, and measures in 3 hours after LAD pours into again.Obtained alpha-MSH analogue #1(sequence table sequence 1* under the dosage of 200 μ g/kg body weight) maximum suppression effect, wherein with the rat of vehicle treated, compare, the reduction of infarct size is～the 30%(vehicle group: 50.6 ± 2.6% of dangerous area, alpha-MSH analogue #1(sequence table sequence 1*) group: 35.7 ± 5.6% of dangerous area; P=0.01).Under the dosage of 1000 μ g/kg body weight, with the rat of vehicle treated, compare, the reduction of infarct size still～30%(alpha-MSH analogue #1(sequence table sequence 1*) group: 35.0 ± 4.4% of dangerous area, to vehicle group p<0.01) (see figure 5).Within 14 days after stopping up LAD60 minute, measure the left ventricular end diastolic presssure (LVEDP) of another program animal, result shows alpha-MSH analogue #1(sequence table sequence 1*) beneficial effect of infarct size is attended by the remarkable reduction (LVEDP: alpha-MSH analogue #1(sequence table sequence 1*) group: 10.4 ± 2.9mmHg that congested type cardiac failure develops after LVEDP and infarct; To vehicle group: 20.2 ± 2.2mmHg; P<0.01; Time is contrasted: 7.5 ± 2.3mmHg; Without significance) (see figure 6).

60 minutes two-way kidney local asphyxia (RIR) induce polyuria after obvious ischemic.The RIR rat has identical polyuria, with the control rats of sham-operation, compares, and the 5th day diuresis amount after the local asphyxia infringement increases 101%(RIR-vehicle group: 34.8 ± 3.3ml/24 hour, time contrast: 17.3 ± 2.1ml/24 hour, p<0.01).α-MSH processes can not alleviate polyuria (RIR-α-MSH group: 29.0 ± 2.9ml/24 hour; To the RIR-carrier without significance).Surprisingly, alpha-MSH analogue #1(sequence table sequence 1*) can induce complete normalizing (RIR-alpha-MSH analogue #1(sequence table sequence 1*) group: the 18.8 ± 3.6ml/24 hour of urine flow; To time contrast without significance; To RIR-vehicle group p<0.01) (see figure 7).

The renal failure that suppresses cisplatin induction

As GFR, decline is confirmed, cisplatin treated is induced obvious hypomagnesemia and nephrotoxicity.According to this point, with cis-platinum and vehicle treated rat, induce hypomagnesemia (blood plasma Mg:0.61 ± 0.04mM, control rats: 0.77 ± 0.05mM, p<0.05) and significantly GFR decline.In the rat of processing with cis-platinum and α-MSH, blood plasma Mg reduces (0.37 ± 0.04mM, to control rats p<0.05) equally.Surprisingly, with alpha-MSH analogue #1(sequence table sequence 1*) process the hypomagnesemia that prevented cisplatin induction (0.84 ± 0.04mM, with respect to control rats without significance) and prevented that the GFR of cisplatin induction from descending.

Embodiment 2

Experimental compound is alpha-MSH analogue #2:

Ac-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Nle-Glu-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-Val-NH ₂(sequence table sequence 5* is acetylation and is amidated at the C-end at the N-end)

By in site, 10 use Nle replace Met and, by 13 use (D-Phe) the stereochemistry replacement Phe in site, make alpha-MSH analogue #2(sequence table sequence 5) be different from alpha-MSH analogue #1(sequence table sequence 1*).

This compound of test in experimental program 1-3 and 5-7.

The TNF-α that the LPS that inhibition vitro human white corpuscle produces induces produces

α-MSH and alpha-MSH analogue #2(sequence table sequence 5*) both dosage rely on ground and reduce the TNF-α accumulation that in the human leukocyte suspension, LPS induces.Surprisingly, inhibition alpha-MSH analogue #2(sequence table sequence 5*) is more remarkable than the antiphlogistic effects of native peptides α-MSH.α-MSH suppresses TNF-α and is accumulated to 73 ± 9% of maximum reaction (LPS-carrier).In contrast, alpha-MSH analogue #2(sequence table sequence 5*) can reduce by 42 ± 11%(that TNF-α is accumulated to carrier α-MSH is organized to p<0.01) (see figure 8).

Suppressing the TNF-α that in the rat body, LPS induces produces

In the process of intravenous injection LPS, α-MSH and alpha-MSH analogue #2(sequence table sequence 5*) both reduced the TNF-α accumulation of rat.Obtained α-MSH and alpha-MSH analogue #2(sequence table sequence 5* under the dosage of 200 μ g/kg body weight) maximum suppression effect.Surprisingly, inhibition alpha-MSH analogue #2(sequence table sequence 5*) is more remarkable than the antiphlogistic effects of native peptides α-MSH.Although α-MSH suppresses TNF-α concentration in rat plasma to 17 ± 3% of maximum reaction (LPS-carrier), alpha-MSH analogue #2(sequence table sequence 5*) can reduce by 9 ± 1%(that TNF-α is accumulated to carrier α-MSH is organized to p<0.05) (see figure 9).

In the BALF collected in 24 hours after LPS sucks as shown in the remarkable reduction of eosinophilic granulocyte like that, α-MSH and alpha-MSH analogue #2(sequence table sequence 5*) both reduce the inflammatory reaction to the LPS suction in alveolar space.With the quantity of eosinophilic granulocyte in BALF be 164.6 ± 42.2 * 10 ⁵rat individual cell, vehicle treated is compared, and α-MSH processes the quantity to 26.7 that reduces the eosinophilic granulocyte in BALF ± 4.3 * 10 ⁵individual cell (to vehicle group, p<0.05), alpha-MSH analogue #2(sequence table sequence 5*) reduce the quantity to 34.0 of the eosinophilic granulocyte in BALF ± 8.6 * 10 ⁵individual cell (to vehicle group, p<0.05) (see figure 10).Surprisingly, alpha-MSH analogue #2(sequence table sequence 5*) have than α-MSH more obviously to inhibition (the alpha-MSH analogue #2(sequence table sequence 5*) group of the neutrophilic granulocyte in BALF: 9.1 ± 2.4 * 10 ⁵individual cell, α-MSH group: 20.1 ± 2.5 * 10 ⁵individual cell; P<0.05) (see Figure 11).

The myocardial infarction area that inhibition is induced by blocking the rat left anterior descending coronary artery in 60 minutes

, surprisingly, alpha-MSH analogue #2(sequence table sequence 5* contrary with α-MSH) reduced myocardial infarction area, myocardial infarction area is with necrosis area/infarct critical area(s) product representation, and measures in 3 hours after LAD pours into again.Obtained alpha-MSH analogue #2(sequence table sequence 5* under the dosage of 200 μ g/kg body weight) maximum suppression effect, wherein with the rat of vehicle treated, compare, the reduction of infarct size is～the 27%(vehicle group: 51.4 ± 2.1% of dangerous area, alpha-MSH analogue #2(sequence table sequence 5*) group: 37.4 ± 5.1% of dangerous area; P=0.01) (see Figure 12).Within 14 days after stopping up LAD60 minute, measure the left ventricular end diastolic presssure (LVEDP) of another program animal, result shows alpha-MSH analogue #2(sequence table sequence 5*) beneficial effect of infarct size is attended by the remarkable reduction that congested type cardiac failure develops after LVEDP and infarct.

60 minutes two-way kidney local asphyxia (RIR) induce polyuria after obvious ischemic.The RIR rat has identical polyuria, with the control rats of sham-operation, compares, and the 5th day diuresis amount after the local asphyxia infringement increases 101%(RIR-vehicle group: 34.8 ± 3.3ml/24 hour, time contrast: 17.3 ± 2.1ml/24 hour, p<0.01).α-MSH processes can not alleviate polyuria (RIR-α-MSH group: 29.0 ± 2.9ml/24 hour; To the RIR-vehicle group without significance).In contrast, with alpha-MSH analogue #2(sequence table sequence 5*) process and significantly reduced the polyuria degree of finding after RIR.

The renal failure that suppresses cisplatin induction

As GFR, decline is confirmed, cisplatin treated is induced obvious hypomagnesemia and nephrotoxicity.According to this point, with cis-platinum and vehicle treated rat, induce hypomagnesemia (blood plasma Mg:0.61 ± 0.04mM, control rats: 0.77 ± 0.05mM, p<0.05) and significantly GFR decline.In the rat of processing with cis-platinum and α-MSH, blood plasma Mg reduces (0.37 ± 0.04mM, to control rats p<0.05) equally.In contrast, alpha-MSH analogue #2(sequence table sequence 5*) prevented that the hypomagnesemia of cisplatin induction and the GFR of cisplatin induction from descending.

Embodiment 3

Experimental compound is alpha-MSH analogue #3:

Ac-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Nle-Glu-His-D-Nal-Arg-Trp-Gly-Lys-Pro-Val-NH ₂(sequence table sequence 9* is acetylation and is amidated at the C-end at the N-end)

By in site, 10 use Nle replace Met and, by 13 use D-Nal replace Phe in site, make alpha-MSH analogue #3(sequence table sequence 9) be different from alpha-MSH analogue #1(sequence table sequence 1*).

This compound of test in

experimental program

1,2 and 5.

Suppress the TNF-α generation that external LPS induces, wherein TNF-α is produced by human leukocyte.

α-MSH and alpha-MSH analogue #3(sequence table sequence 9*) both dosage rely on ground and reduce the TNF-α accumulation that in the human leukocyte suspension, LPS induces.Surprisingly, inhibition alpha-MSH analogue #3(sequence table sequence 9*) is more remarkable than the antiphlogistic effects of native peptides α-MSH.α-MSH suppresses TNF-α and is accumulated to 73 ± 9% of maximum reaction (LPS-carrier).In contrast, alpha-MSH analogue #3(sequence table sequence 9*) can reduce by 53 ± 13%(that TNF-α is accumulated to carrier α-MSH is organized to p<0.05) (seeing Figure 13).

Suppressing the TNF-α that in the rat body, LPS induces produces

In the process of intravenous injection LPS, α-MSH and alpha-MSH analogue #3(sequence table sequence 9*) both reduced the TNF-α accumulation of rat.Obtained α-MSH and alpha-MSH analogue #3(sequence table sequence 9* under the dosage of 200 μ g/kg body weight) maximum suppression effect.Surprisingly, inhibition alpha-MSH analogue #3(sequence table sequence 9*) is more remarkable than the antiphlogistic effects of native peptides α-MSH.Although α-MSH suppresses TNF-α concentration in rat plasma to 17 ± 3% of maximum reaction (LPS-carrier), alpha-MSH analogue #3(sequence table sequence 9*) can reduce by 11 ± 3%(that TNF-α is accumulated to carrier α-MSH is organized to p<0.05) (seeing Figure 14).

, surprisingly, alpha-MSH analogue #3(sequence table sequence 9* contrary with α-MSH) reduced myocardial infarction area, myocardial infarction area is with necrosis area/infarct critical area(s) product representation, and measures in 3 hours after LAD pours into again.Obtained alpha-MSH analogue #2(sequence table sequence 5* under the dosage of 200 μ g/kg body weight) maximum suppression effect, wherein with the rat of vehicle treated, compare, the reduction of infarct size is～the 24%(vehicle group: 51.3 ± 2.1% of dangerous area, alpha-MSH analogue #3(sequence table sequence 9*) group: 39.0 ± 3.4% of dangerous area; P=0.05) (see Figure 15).Within 14 days after stopping up LAD60 minute, measure the left ventricular end diastolic presssure (LVEDP) of another program animal, result shows alpha-MSH analogue #3(sequence table sequence 9*) beneficial effect of infarct size is attended by the remarkable reduction that congested type cardiac failure develops after LVEDP and infarct.

Embodiment 4

Experimental compound is alpha-MSH analogue #4:

Ac-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Ser-Ile-Ile-Ser-His-Phe-A rg-Trp-Gly-Lys-Pro-Val-NH ₂(sequence table sequence 13* is acetylation and is amidated at the C-end at the N-end)

By in site, 8 use Ser replace Tyr, in site, 9 use Ile replace Ser, in site, 10 use Ile replace Met and, by 11 use Ser replace Glu in site, make alpha-MSH analogue #4(sequence table sequence 13) be different from alpha-MSH analogue #1(sequence table sequence 1*).

This compound of test in

experimental program

1 and 2.

α-MSH and alpha-MSH analogue #4(sequence table sequence 13*) both dosage rely on ground and reduce the TNF-α accumulation that in the human leukocyte suspension, LPS induces.Surprisingly, inhibition alpha-MSH analogue #4(sequence table sequence 13*) is more remarkable than the antiphlogistic effects of native peptides α-MSH.

Suppressing the TNF-α that in the rat body, LPS induces produces

In the process of intravenous injection LPS, α-MSH and alpha-MSH analogue #4(sequence table sequence 13*) both reduced the TNF-α accumulation of rat.Obtained α-MSH and alpha-MSH analogue #4(sequence table sequence 13* under the dosage of 200 μ g/kg body weight) maximum suppression effect.Surprisingly, inhibition alpha-MSH analogue #4(sequence table sequence 13*) is more remarkable than the antiphlogistic effects of native peptides α-MSH.Although α-MSH suppresses TNF-α concentration in rat plasma to 17 ± 3% of maximum reaction (LPS-carrier), alpha-MSH analogue #4(sequence table sequence 13*) can reduce by 12 ± 2%(that TNF-α is accumulated to carrier α-MSH is organized to p<0.05) (seeing Figure 16).

Embodiment 5

Experimental compound is alpha-MSH analogue #5:

Ac-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Ser-Ile-Ile-Ser-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-Val-NH ₂(sequence table sequence 17* is acetylation and is amidated at the C-end at the N-end)

By in site, 8 use Ser replace Tyr, in site, 9 use Ile replace Ser, in site, 10 use Ile replace Met, by in site, 11 use Ser replace Glu and, by 13 use (D-Phe) the stereochemistry replacement Phe in site, make alpha-MSH analogue #5(sequence table sequence 17) be different from alpha-MSH analogue #1(sequence table sequence 1*).

This compound of test in

experimental program

1 and 2.

α-MSH and alpha-MSH analogue #5(sequence table sequence 17*) both dosage rely on ground and reduce the TNF-α accumulation that in the human leukocyte suspension, LPS induces.Surprisingly, inhibition alpha-MSH analogue #5(sequence table sequence 17*) is more remarkable than the antiphlogistic effects of native peptides α-MSH.

Suppressing the TNF-α that in the rat body, LPS induces produces

In the process of intravenous injection LPS, α-MSH and alpha-MSH analogue #5(sequence table sequence 17*) both reduced the TNF-α accumulation of rat.Obtained α-MSH and alpha-MSH analogue #5(sequence table sequence 17* under the dosage of 200 μ g/kg body weight) maximum suppression effect.Surprisingly, inhibition alpha-MSH analogue #5(sequence table sequence 17*) is more remarkable than the antiphlogistic effects of native peptides α-MSH.

Embodiment 6

Experimental compound is alpha-MSH analogue #6:

Ac-Glu-Glu-Glu-Glu-Glu-Glu-Ser-Tyr-Ser-Met-Glu-His-Phe-A rg-Trp-Gly-Ly s-Pro-Val-NH ₂(sequence table sequence 2* is acetylation and is amidated at the C-end at the N-end)

By at site 1-6, using (Glu) ₆replace (Lys) ₆, make alpha-MSH analogue #6(sequence table sequence 2) and be different from alpha-MSH analogue #1(sequence table sequence 1*).

This compound of experiment in

experimental program

1 and 2.

α-MSH and alpha-MSH analogue #6(sequence table sequence 2*) both dosage rely on ground and reduce the TNF-α accumulation that in the human leukocyte suspension, LPS induces.Surprisingly, inhibition alpha-MSH analogue #6(sequence table sequence 2*) is more remarkable than the antiphlogistic effects of native peptides α-MSH.

Suppressing the TNF-α that in the rat body, LPS induces produces

In the process of intravenous injection LPS, α-MSH and alpha-MSH analogue #6(sequence table sequence 2*) both reduced the TNF-α accumulation of rat.Obtained α-MSH and alpha-MSH analogue #6(sequence table sequence 2* under the dosage of 200 μ g/kg body weight) maximum suppression effect.Surprisingly, inhibition alpha-MSH analogue #6(sequence table sequence 2*) is more remarkable than the antiphlogistic effects of native peptides α-MSH.

Embodiment 7

Experimental compound is alpha-MSH analogue #7:

Ac-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Met-Glu-His-Phe-A rg-Trp-Gly-Lys-Pro-(D-Val)-NH ₂(sequence table sequence 3* is acetylation and is amidated at the C-end at the N-end)

By the three-dimensional Val that replaces of 19 use (D-Val) chemistry in site, make alpha-MSH analogue #7(sequence table sequence 3) be different from alpha-MSH analogue #1(sequence table sequence 1*).

This compound of experiment in

experimental program

1 and 2.

α-MSH and alpha-MSH analogue #7(sequence table sequence 3*) both dosage rely on ground and reduce the TNF-α accumulation that in the human leukocyte suspension, LPS induces.Surprisingly, inhibition alpha-MSH analogue #7(sequence table sequence 3*) is more remarkable than the antiphlogistic effects of native peptides α-MSH.

Suppressing the TNF-α that in the rat body, LPS induces produces

In the process of intravenous injection LPS, α-MSH and alpha-MSH analogue #7(sequence table sequence 3*) both reduced the TNF-α accumulation of rat.Obtained α-MSH and alpha-MSH analogue #7(sequence table sequence 3* under the dosage of 200 μ g/kg body weight) maximum suppression effect.Surprisingly, inhibition alpha-MSH analogue #7(sequence table sequence 3*) is more remarkable than the antiphlogistic effects of native peptides α-MSH.

The accompanying drawing explanation

The TNF-α accumulation that in Fig. 1 human leukocyte suspension, LPS induces

The figure illustrates alpha-MSH analogue #1(sequence table sequence 1* in experimental program 1) maximum antiphlogistic effects (MCA#1).For α-MSH and MCA#1 10 ^-7obtain the maximum suppression effect of the TNF-α production that LPS is induced in the M situation.N=6-9 in every group of mean value ± SE().*: to vehicle group p<0.05, #: α-MSH is organized to p<0.05.

The TNF-α accumulation that Fig. 2 plasma levels of LPS is induced

The figure illustrates alpha-MSH analogue #1(sequence table sequence 1* in experimental program 2) maximum antiphlogistic effects (MCA#1).Obtain for α-MSH and MCA#1 the maximum suppression effect that TNF-α that rat LPS is induced produces in intravenous injection 200 μ g/kg body weight situations.N=4-6 in every group of mean number ± SE().*: to vehicle group p<0.05, #: α-MSH is organized to p<0.05.

Fig. 3 eosinophilic granulocyte

The figure illustrates α-MSH and alpha-MSH analogue #1(sequence table sequence 1* in experimental program 3) (MCA#1) to alveolar in the effect of eosinophilic granulocyte accumulation.Two compound intravenous injection administrations under the dosage of 200 μ g/kg body weight.N=6-9 in every group of mean number ± SE().*: different from carrier.

Fig. 4 Ppa pulmonary artery pressure

The figure illustrates alpha-MSH analogue #1(sequence table sequence 1*) porcine pulmonary artery of (MCA#1) LPS being induced presses the effect changed.N=6-9 in every group of mean number ± SE().*: different from carrier.

The infarct size of 3 hours after Fig. 5 pours into again

The figure illustrates alpha-MSH analogue #1(sequence table sequence 1* in experimental program 4) (MCA#1) to the provide protection of myocardial infarction area.Give by intravenous injection the maximum efficiency that 200 μ g/kg body weight obtain MCA#1.N=5-10 in every group of mean number ± SE().*: different from carrier.

Fig. 6 LVEDP of two weeks after 60min LAD stops up

The figure illustrates alpha-MSH analogue #1(sequence table sequence 1* in experimental program 4) (MCA#1) to infarct after the provide protection of congestive heart failure.Obtain the effect of MCA#1 by 200 μ g/kg body weight.N=6-9 in every group of mean number ± SE().*: to sham operated rats p<0.05, #: to vehicle group p<0.05.

Fig. 7 diuresis amount

The figure illustrates alpha-MSH analogue #1(sequence table sequence 1* in experimental program 5) (MCA#1) to ischemic after the provide protection of polyuria development.Give by intravenous injection the effect that 200 μ g/kg body weight obtain MCA#1.N=5-7 in every group of mean number ± SE().*: different from carrier.

The TNF-α accumulation that in Fig. 8 human leukocyte suspension, LPS induces

The figure illustrates alpha-MSH analogue #2(sequence table sequence 5* in experimental program 1) maximum antiphlogistic effects (MCA#2).For α-MSH and MCA#2 10 ^-7obtain the maximum suppression effect of the TNF-α production that LPS is induced in the M situation.N=6-9 in every group of mean number ± SE().*: to vehicle group p<0.05, #: α-MSH is organized to p<0.05.

The TNF-α accumulation that Fig. 9 plasma levels of LPS is induced

The figure illustrates alpha-MSH analogue #2(sequence table sequence 5* in experimental program 2) maximum antiphlogistic effects (MCA#2).Obtain for α-MSH and MCA#2 the maximum suppression effect that TNF-α that rat LPS is induced produces in intravenous injection 200 μ g/kg body weight situations.N=4-6 in every group of mean number ± SE().*: to vehicle group p<0.05, #: α-MSH is organized to p<0.05.

Figure 10 eosinophilic granulocyte

The figure illustrates α-MSH and alpha-MSH analogue #2(sequence table sequence 5* in experimental program 3) (MCA#2) to alveolar in the effect of eosinophilic granulocyte accumulation.Two compound intravenous injection administrations under the dosage of 200 μ g/kg body weight.N=6-9 in every group of mean number ± SE().*: different from carrier.

Figure 11 neutrophilic granulocyte

The figure illustrates α-MSH and alpha-MSH analogue #2(sequence table sequence 5* in experimental program 3) (MCA#2) to alveolar in the effect of neutrophilic granulocyte accumulation.Two compound intravenous injection administrations under the dosage of 200 μ g/kg body weight.N=6-9 in every group of mean number ± SE().*: different from carrier.

The infarct size of 3 hours after Figure 12 pours into again

The figure illustrates alpha-MSH analogue #2(sequence table sequence 5* in experimental program 4) (MCA#2) to the provide protection of myocardial infarction area.Give by intravenous injection the maximum efficiency that 200 μ g/kg body weight obtain MCA#1.N=5-10 in every group of mean number ± SE().*: different from carrier.

The TNF-α accumulation that in Figure 13 human leukocyte suspension, LPS induces

The figure illustrates alpha-MSH analogue #3(sequence table sequence 9* in experimental program 1) maximum antiphlogistic effects (MCA#3).For α-MSH and MCA#3 10 ^-7obtain the maximum suppression effect of the TNF-α production that LPS is induced in the M situation.N=6-9 in every group of mean number ± SE().*: to vehicle group p<0.05, #: α-MSH is organized to p<0.05.

The TNF-α accumulation that Figure 14 plasma levels of LPS is induced

The figure illustrates alpha-MSH analogue #3(sequence table sequence 9* in experimental program 2) maximum antiphlogistic effects (MCA#3).Obtain for α-MSH and MCA#3 the maximum suppression effect that TNF-α that rat LPS is induced produces in intravenous injection 200 μ g/kg body weight situations.N=4-6 in every group of mean number ± SE().*: to vehicle group p<0.05, #: α-MSH is organized to p<0.05.

The infarct size of 3 hours after Figure 15 pours into again

The figure illustrates alpha-MSH analogue #3(sequence table sequence 9* in experimental program 4) (MCA#3) to the provide protection of myocardial infarction area.Give by intravenous injection the maximum efficiency that 200 μ g/kg body weight obtain MCA#3.N=5-10 in every group of mean number ± SE().*: different from carrier.

The TNF-α accumulation that Figure 16 plasma levels of LPS is induced

The figure illustrates alpha-MSH analogue #4(sequence table sequence 13* in experimental program 2) maximum antiphlogistic effects (MCA#4).Obtain for α-MSH and MCA#4 the maximum suppression effect that TNF-α that rat LPS is induced produces in intravenous injection 200 μ g/kg body weight situations.N=4-6 in every group of mean number ± SE().*: to vehicle group p<0.05, #: α-MSH is organized to p<0.05.

Reference

United States Patent (USP) the 4th, 288, No. 627

WO91/17243

WO99/46283

Beaucage,S.L.Caruthers,M.H.Tetrahedron?Letters22,1981,pp.1859-1869

Bodanszky, M. and Bodanszky, A., " synthetic put into practice (the The Practice of Peptide Synthesis) of peptide ", second edition, Springer-Verlag, 1994.

Catania, A., Rajora, F., Capsoni, F., Minonzio, R.A., Star, and Upton, J.M. peptide (Peptides) 17:675-679,1996.

Ehrlich,1978,Proc.Natl.Acad.Sci.USA75:1433).

Guo and Sherman, 1995, molecular cytobiology (Molecular Cellular Biology) 15:5983-5990.

Hartmeyer, M., Scholzen T., Becher E., Bhardwaj R.S., Schwarz T. and Luger T.A., J.Immunol., 159:1930-1937,1997.

Hiltz?M.E.et?al.(1991),Peptides,12,767-771.

Hruby?VJ.et?al.(1995),J.Med.Chem.,38,3454-3461.

Jones, J. " chemistry of peptides synthesizes (The Chemical Synthesis of Peptides) ", Clarendon Press, 1991.

Kullmann, W.1987, the enzymatic polypeptide synthesizes (Enzymatic Peptide Synthesis), CRC Press, Boca Raton, Florida, pp.41-59.

Lipton, J.M and Catania, A. immunology today (Immunol.Today) 18:140-145.1997

Liu et al., 1996, J.Am.Chem.Soc.118:307-312 and Dawson et al., 1996,226:776.

Luger, T.A., Scholzen T. and Grabbe S., J.Investig.Dermatol.Symp.Proc, 2:87-93,1997.

Matthes?et?al.,EMBO?Journal3,1984,pp.801-805.

MANIATIS, T., E.F.FRITSCH and J.SAMBROOK, 1982 molecular clonings: laboratory manual (Molecular Cloning:A Laboratory Manual) .Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Remington: " the pharmacy science with put into practice (The science and practice of pharmacy) " the 20th edition .Mack Publishing; Easton PA; 2000ISBN0-912734-04-3 and " pharmaceutical technology encyclopedia (Encyclopedia of Pharmaceutical Technology) "; Swarbrick edits, J.& J.C.Boylan, Marcel Dekker, Inc., New York, 1988ISBN0-8247-2800-9.

Rajora, N., Boccoli, G., Catania and Lipton J.M., Peptides, 18:381-385,1997.

Remington: the pharmacy science with put into practice (The science and practice of pharmacy) " the 20th edition .Mack Publishing, Easton PA, 2000ISBN0-912734-04-3.

Rizzi A.et al. (2002), pharmacology Britain magazine (British Journal of Pharmacology), 137,369-374.

Romanos et al., 1992, yeast (Yeast) 8:423-488.

Sawyer?T.K.(1980),Proc.Nat.Acad.Sci.,10,5754-5758.

?H.B.et?al.(1998),Eur.J.Pharm.,349,359-366.

Star, R.A., Rajora N., Huang J., Stock R.C., Catania A. and Upton J.M.; Proc.Natl.Acad.Sci.U.S.A, 92:8016-8020,1995.

Wong, K.Y., Rojora, G., Boccoli, A., Catania, A., and Upton J.M., neuroimmunomodulation (Neuroimmunomodulation), 4:37-41,1997.

Useful proteins matter (Useful proteins from recombinant bacteria) " the science U.S. (in Scientific American), 1980,242:74-94 from recombinant bacteria

Claims

1. the preparation method of peptide, the length of described peptide is 19 amino-acid residues and comprises following aminoacid sequence:

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 1),

Wherein, the aminoterminal of described peptide is CH ₃-C (=O)-, the carboxyl terminal of described peptide is-C (=O)-NH ₂,

By solution synthetic method, Merrifield type solid-phase synthesis or recombinant DNA technology, prepared by described peptide.

2. the preparation method of peptide, the length of described peptide is 19 amino-acid residues and comprises following aminoacid sequence:

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Nle-Glu-His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-Val(sequence table sequence 5), and

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Nle-Glu-His-D-Nal-Ar g-Trp-Gly-Lys-Pro-Val(sequence table sequence 9),

The aminoterminal of wherein said peptide is CH ₃-C (=O)-; And

The carboxyl terminal of wherein said peptide is-C (=O)-NH ₂;

Wherein said peptide has the ability that activates one or more melanocortin receptors that are selected from 1,3,4 and 5 type melanocortin receptors,

3. method according to claim 2, wherein, described peptide comprises following sequence:

4. method according to claim 2, wherein, described peptide comprises following sequence:

5. according to any one described method in claim 1-4, wherein, prepared by Merrifield type solid-phase synthesis by described peptide.

6. according to any one described method in claim 1-5, wherein, described method is used the resin of functionalization, the group that the resin of described functionalization selects polystyrene, latex and the Dai Nuo immunomagnetic beads of free polystyrene, polyacrylamide, polydimethylacrylamiin, polyoxyethylene glycol, Mierocrystalline cellulose, polyethylene, polyoxyethylene glycol grafting to form.

7. according to the described method of claim 5 or 6, wherein, by acid, peptide of the present invention is excised from the solid support material.

8. according to the described method of claim 5 or 6, wherein, by alkali, peptide of the present invention is excised from the solid support material.

9. according to any one described method in claim 1-4, wherein, prepared by solution synthetic method, Merrifield type solid-phase synthesis or recombinant DNA technology by described peptide.

10. according to any one described method in claim 1-4, wherein, prepared by recombinant DNA technology by described peptide, and it comprises the steps:

(a) under the condition that allows described peptide to produce, cultivate the recombinant host cell of the nucleotide sequence that contains coding for said peptides, and

(b) separate described peptide from described culture.

11., according to any one described method in claim 1-4, wherein, prepared by recombinant DNA technology by described peptide, it comprises the steps:

(a) nucleotide sequence of coding for said peptides is transferred in host cell,

(b) cultivate described host cell, and

(c) separate described peptide from described culture.