CN103497237B

CN103497237B - The alpha-MSH analogue of therapeutic activity

Info

Publication number: CN103497237B
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: 2015-10-28
Anticipated expiration: 2025-08-26
Also published as: CN103497237A

Abstract

The present invention relates to the alpha-MSH analogue of therapeutic activity.The invention describes the peptide analogs of alpha-Melanocyte stimulating hormone (α-MSH), described peptide analogs has effect of raising compared with natural α-MSH peptide.Compared with α-MSH, alpha-MSH analogue shows the antiphlogistic effects of raising and the ability preventing Ischemia Cases of raising.The present invention further discloses the purposes of this peptide for the preparation of pharmaceutical composition, and pharmaceutical composition, described pharmaceutical composition is used for the treatment of or prevents the patient's condition of tissue of mammiferous one or more organ.

Description

The alpha-MSH analogue of therapeutic activity

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

Technical field

The present invention relates to the peptide analogs of alpha-Melanocyte stimulating hormone (α-MSH), described peptide analogs has effect of raising compared with natural α-MSH peptide.Compared with α-MSH, alpha-MSH analogue show the antiphlogistic effects of raising and the treatment of raising or prevention and local asphyxia or be that the whole body that the local asphyxia of blood vessel Reperfu-sion is relevant damages below, the ability of organ damage or cell injury.

Background technology

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

Large quantifier elimination has been carried out about MC acceptor in many tissues.Know, 1 receptor (MC1) that α-MSH is bonded thereto with great avidity is expressed in some tissues and cell such as brain, comprises stellate cell, testis, ovary, scavenger cell and neutrophilic granulocyte.But MC1 may express in the tissue of more wide region, although this also needs to set up.The selectivity of MC receptors bind different MS H peptide is change.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 only in conjunction with ACTH, and not in conjunction with any one MSH peptide.γ-MSH(MC3-acceptor is comprised to the most high-affinity of the aglucon of other acceptors) and β-MSH(MC4-acceptor).On the contrary, MC5 in the mode identical with MC1 with low-down avidity in conjunction with MSH peptide (namely the highest to α-MSH avidity).

The MSH-peptide worked by activating MC-acceptor has various function, comprises the synthesis/release of immunity, anti-inflammatory, thermoregulation, pain, aldosterone synthesis, blood pressure regulation, heart rate, vascular tone, cerebral blood flow (CBF), nerve growth, placenta development, many hormones (such as aldosterone, thyroxine, prolactin, FSH).ACTH generates on (steroidoneogenesis) at activation ketosteroid has Main Function.α-MSH is induced skin chromogenesis also.

It is important to note that MSH peptide, particularly also not set up completely by the many effects related to about which acceptor of α-MSH.The anti-inflammatory action of α-MSH relates to various process and comprises interference NO production, endothelin-1 effect, IL-10 formation by inference, and this is associated with again the MC1 acceptor of expressing in scavenger cell and monocyte.

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

Hypoxia (local asphyxia) and reperfusion injury are all important factors in people's physiopathology.The example (anticipating in refilling process as damage) of tissue hypoxia comprises recycle system shock, myocardial ischaemia, apoplexy, transient kidney local asphyxia, large surgical operation and organ transplantation.Because owing to the M & M reason that ischemic disease is extremely common, and because organ transplantation is more and more frequent, in order to improve the health level of the public, be starved of the therapeutic strategy with restriction reperfusion injury ability.The underlying pathophysiology of ischemia reperfusion injury is complicated, not only relate to the typical inflammation Reperfu-sion reaction to neutrophilic granulocyte-infiltration, and relating to the genetic expression of Reperfu-sion tissue/organ based intracellular cvtokine, 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 directly suppressing shrinkability and cell death inducing, the TNF-α that local produces facilitates as caused post-ischemic organ's dysfunction in heart after infarct.Due to the character of local asphyxia and/or Reperfu-sion complexity; verified simple anti-inflammatory treatment concept is invalid: therefore; most of experimental study points to such fact, namely avoids reperfusion injury to protect, and needs the interaction with companion's row of more than one activation pathway.Verified, α-MSH has ability that is anti-inflammatory, oxidation resistant and anti-apoptotic simultaneously, and this gives good explanation for this compound to protect the effect being avoided reperfusion injury.

The receptor-specific of the receptor affinity (to such as 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 that extends or more in conjunction with profile (receptor-specific binding profile) ( deng, 1997; Hruby etc., 1995; Sawyer etc., 1980; Hiltz etc. 1991; Scarden ì ngs etc., 2000).But when being intended to produce 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, there is transformation period within the scope of several minutes usually.Proteolytic enzyme and other proteolytic ferments are immanent, particularly all the more so in the gastrointestinal tract, and therefore when oral, peptide is usually responsive to degraded in multiple site, and also responsive to degraded to a certain extent in blood, liver, kidney and blood vessel endothelium.In addition, responsive to degraded on more than one key of particular peptide usually in main chain; Each hydrolysis position is mediated by specific proteases.Even if overcome these obstacles, particularly for neuropeptide, encounter the difficulty in they leap blood brain barrier transports.

In order to improve the metabolic stability of peptide, the 1999(WO99/46283 such as Larsen) have developed one be called as SIP(structure induction probe) technology.Sip technique structure based induces the use of probe, and this structure induction probe take short peptide sequence as representative, is namely attached to C-end or the N-end of parent's peptide or is attached to (Lys) that C-holds and N-holds simultaneously ₆.Based on intramolecular hydrogen bond, the conformation that structure induction probe forces parent's peptide to become more orderly, whereby, contrary with the peptide being in random-coil conformation, peptide mosaic (being connected to the peptide of probe) is low to protease-sensitive degree.As structurized result, peptide mosaic is more difficult to be easily degraded by proteases.Additional SIP usually cause to biologically active peptides the raising of the enzyme stability of peptide and while biological activity remain unchanged (Rizzi etc. 2002).

Summary of the invention

Surprisingly, the present invention is verified, and compared with natural α-MSH peptide, the SIP-that α-MSH and alpha-MSH analogue are held at the N-of peptide modifies the maximum effect that improve peptide.Compared with natural α-MSH peptide, peptide of the present invention shows the ability of the antiphlogistic effects of raising and the prevention ischemic conditions of raising.

Therefore, the present invention relates to specific peptide, described peptide be included in the N-end portion of peptide SIP modify and at the α-MSH of the C-end portion of peptide or the aminoacid sequence of α-MSH variant.

First aspect, the invention provides the peptide adding 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 being selected from Lys-Pro-Val and Lys-Pro-(D-Val), and

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

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

The aminoterminal of wherein said peptide is (B4) HN-, (B4) (B5) N-or (B6) HN-, and wherein B4 and B5 is independently selected from H, the C that can be substituted _1-6alkyl, the C that can be substituted _2-6alkenyl, the C that can be substituted _6-10aryl, the C that can be substituted _7-16aralkyl and the C that can be substituted _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 of tissue being used for the treatment of or preventing mammiferous one or more organ.In addition, the present invention relates to composition such as pharmaceutical composition, according to peptide of the present invention purposes medically and the method for the patient's condition of tissue being used for the treatment of mammiferous one or more organ, wherein said pharmaceutical composition comprises one or more 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 being used for the treatment of the patient's condition caused by local asphyxia, inflammation and/or toxic effect that is poisoning or pharmacological agent.

Summary of the invention

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

As herein defined, if it can be used in treatment, alleviates or weaken the instruction of morbid state, the physiology patient's condition, symptom or etiology or evaluate or its diagnosis, so peptide sequence is " therapeutic activity ".If it can be used in preventing morbid state, the physiology patient's condition, symptom or etiology from indicating, so peptide sequence is " prophylactic activity ".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 similar physiologic function peptide, in vitro, the body of standard or analyzed in vitro method analyze.

Peptide of the present invention

The present invention relates to a kind of peptide, described peptide comprises the α-MSH of the C-end portion being included in peptide or the aminoacid sequence of α-MSH variant and induces probe (SIP) in the structure of the N-end portion of peptide.Peptide of the present invention is called as alpha-MSH analogue.In the present description and claims, 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, superposition in the sequence or modify.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 being selected from His-Phe-Arg, His-(D-Phe)-Arg, His-Nal-Arg and His-(D-Nal)-Arg, wherein Z comprises and is selected from Lys-Pro-Val and Lys-Pro-(D-Val), and wherein Aa ₁, Aa ₂, Aa ₃, Aa ₄, Aa ₅, Aa ₆and Aa ₇can be the amino-acid residue that exists of the naturally occurring or non-natural of any one or disappearance independently.

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

Natural amino-acid residue is defined as the amino-acid residue occurred at nature, such as 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 preferably natural amino-acid residue is Ser, Tyr, Met, Glu, Ile, Trp and Gly.

Non-natural amino-acid residue be defined as not nature occur but experiment create amino-acid residue.Non-natural amino-acid residue comprises the α of synthesis, the amino acid of β or gamma-amino acid residue (regardless of L-configuration or D-form) and side chain modification, the tyrosine such as modified, wherein aromatic ring also replaces such as one or more halogen, 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 wherein known according to the those of ordinary skill in chemistry of peptides field is protected amino acid side chain, such as be recorded in the method for such as Bodanszky etc. 1994 and J.Jones and Jones1991.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).

In the widest, the present invention relates to the peptide adding up to 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 being 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 " group be used to described in finger and can be selected from C by one or more _1-8alkyl, C _1-8alkoxyl group, oxo (enol form of isomery can be expressed as), carboxyl, amino, hydroxyl (keto-acid of isomery can be expressed as when being present in enol system), nitro, cyano group, dihalo-C _1-8alkyl, three halo-C _1-8the group of alkyl, halogen replaces once or several times, such as 1 ~ 5 time, preferably 1 ~ 3 time, more preferably 1 ~ 2 time.Usually, above-mentioned replacement can carry out other optional replacement.

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

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

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

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

Preferably " 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 2H-thiapyran, 3H-thiapyran, 4H-thiapyran, tetrahydric thiapyran, 2H-pyrans, tetrahydropyrans, piperidines, 1, 2-dithia tetrahydrobenzene, 1, 2-dithian, 1, 3-dithia tetrahydrobenzene, 1, 3-dithian, 1, 4-dithia tetrahydrobenzene, 1, 4-dithian, 1, 2-dioxine, 1, 2-dioxane, 1, 3-dioxine, 1, 3-dioxane, 1, 4-dioxine, 1, 4-dioxane, piperazine, 1, 2-oxathiene, 1, 2-oxathiane, 4H-1, 3-oxathiene, 1, 3-oxathiane, 1, 4-oxathiene, 1, 4-oxathiane, 2H-1, 2-thiazine, tetrahydrochysene-1, 2-thiazine, 2H-1, 3-thiazine, 4H-1, 3-thiazine, 5, 6-dihydro-4H-thiazine, 4H-1, 4-thiazine, tetrahydrochysene-1, 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, 3-trithian, 1, 3, 5-trithian, six hydrogen-1, 3, 5-triazine, tetramethylene sulfide, tetrahydrofuran (THF), pyrroline, tetramethyleneimine, pyrrolidone, pyrrolidine-diones, pyrazoline, pyrazolidine, tetrahydroglyoxaline, imidazolidine, 1, 2-dioxole, 1, 2-dioxolane, 1, 3-dioxole, 1, 3-dioxolane, 3H-1, 2-dithiole, 1, 2-dithiolane, 1, 3-dithiole, 1, 3-dithiolane, different azoles quinoline, different azoles alkane, 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-tri-thiacyclopentane, 1,2,4-tri-thiacyclopentane, 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.Can be at heteroatomic position or the carbon atom by heterocycle to the combination of heterocycle.

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

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

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

In one embodiment, the present invention relates to the peptide adding up to 12 ~ 19 amino-acid residues, described peptide comprises the aminoacid sequence in the group being selected from and being made up of following material:

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。

Wherein Aa ₁, Aa ₂, Aa ₃, Aa ₄, Aa ₅, Aa ₆and Aa ₇can be the natural or non-natural amino-acid residue of any one 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。

Wherein Aa ₁, Aa ₂, Aa ₃, Aa ₄, Aa ₅, Aa ₆and Aa ₇can be the natural or alpha-non-natural amino acid of any one independently.Thus, 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.

Have several method can be used for stabilized peptide to make its anti-degraded and reduce the ability that peptide and other compounds, reagent and/or peptide/protein (such as in blood plasma) carries out reacting.The invention still further relates to the peptide 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, wherein the aminoterminal of peptide is acetylation modification.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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Stabilization can be carried out, the N-end of such as acetylize peptide of the present invention and/or the C-end of amidation peptide of the present invention by the N-end and/or C-end modifying above-mentioned peptide.

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

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

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

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

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

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

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

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

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

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

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

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

The biology effect of contriver is verified peptides of the present invention:

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

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

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

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

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

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

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

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

In human leukocyte suspension (experimental program 1), all seven peptide dose-dependant ground suppresses the TNF-α of LPS induction to accumulate (embodiment 1-7).Surprisingly, we find compared with natural melanotropin α-MSH, all seven peptides are more effective and more available energy, the former defined by the maximum suppression effect produced TNF-α, and the latter is defined (embodiment 1-7) by providing maximum TNF-α to accumulate the compound concentration needed for suppressing.

The scheme (experimental program 2) of LPS inducing systemic inflammation is in rats being injected by vein; contriver is investigated the effect (sequence 1*, sequence 2*, sequence 3*, sequence 5*, sequence 9*, sequence 13*, sequence 17*, all * are acetylation at N-end and are amidated at C-end) of above listed seven peptides.We confirm, described peptide greatly inhibits the TNF-α of LPS induction in circulating to accumulate.Surprisingly; all seven peptides (sequence 1*, sequence 2*, sequence 3*, sequence 5*, sequence 9*, sequence 13*, sequence 17*, all * are acetylation at N-end and are amidated at C-end) can suppress the TNF-α of LPS induction in circulating to be accumulated to the degree (embodiment 1-7) higher than natural melanotropin α-MSH.

In the scheme being sucked LPS incite inflammation by rat (experimental program 3), contriver is investigated the effect of following peptide:

These two peptides (sequence 1* and 5*) verified greatly suppress eosinophil accumulation (embodiment 1 and embodiment 2) of LPS induction in alveolar.Surprisingly, peptide (sequence 5*) to except the effect of eosinophil, also suppresses neutrophil infiltrates to a lot of degree higher than the degree found in the rat processed with natural melanotropin α-MSH except this significantly.

The transient local asphyxia (temporal ischemia) of kidney is usually 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.This acute renal failure causing local asphyxia to be induced, for most of patient, deterioration is chronic renal failure by it.Also do not occur that effective methods for the treatment of is to prevent the development of renal failure at present.In the development that the common discovery of local asphyxia latter stage is the concentrated defect of urine, described urine concentrates the formation without solute urine (solute free urine) output that defect has raising.

People know, the EARF (ARF) of being induced by local asphyxia and the local asphyxia of the rat that Reperfu-sion is induced causes the characteristic structural of the renal cells relevant to the infringement of urinary concentrating mechanism to change.The ARF model of this local asphyxia induction provides suitable model to evaluate the effect of MSH analogue in the damage of local asphyxia induction.In the Serious acute renal failure of being induced by the temporary transient two-way obstruction Renal artery exhausts, The inventors have studied peptide Ac-(Lys) ₆-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH ₂the effect of (sequence 1* is acetylation at N-end and is amidated at C-end), and the effect of itself and native peptides α-MSH is compared (experimental program 6).Within 5 days after the temporary block Renal artery, when evaluating, develop into polyuria (defined by the diuresis amount of higher than control rats 101%) with the rat of vehicle treated, described control rats carries out false renal artery obstruction.Surprisingly; the described compound (sequence 1* holds to be acetylation and to hold at C-at N-and is amidated) taking the molar weight identical with native peptides α-MSH makes diuresis amount normalizing completely; this illustrates that described peptide has the ability protecting the ARF avoiding local asphyxia to induce, and can not make urine production normalizing with native peptides process in the 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 be had 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 obtain early stage Reperfu-sion (spontaneous Reperfu-sion rate is less than 30%) in the patient of 50-70%.The object of Reperfu-sion is the size reducing infraction, thus reduces the development of impaired myocardial function.Whole effects of fibrinolysis/PTCA are that short-term and long-term mortality decline 20%.But AMI is attended by inflammatory reaction, this is prerequisite for healing and cicatrization.Coronary occlusion seriously reduces the volume of blood flow of muscle portion, and this compromises energy metabolism significantly.Local asphyxia continue considerable time (>20min) cause infraction and cause inflammatory reaction, both can accelerate and expand when Reperfu-sion ischemic myocardial.

Ischemic/reperfusion (MIR) not only activates the typical inflammation Reperfu-sion reaction to neutrophilic granulocyte-infiltration, and activating the genetic expression of myocardial cell's 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 process LAN of cytokine not only plays an important role in the adjustment of infarct size but also in the development of myocardial dysfunction (comprising vessel wall recast, heart failure and cardiac hypertrophy).In addition, it is believed that, by directly suppressing shrinkability and cell death inducing, the TNF-α that local generates facilitates post-ischemic organ's dysfunction.

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

In the Ischemic/reperfusion model of rat, in described model, left anterior coronary artery gets clogged 60 minutes, before removing coronary occlusion, just give the process with peptide according to the present invention, and then follows the tracks of rat 3 hours.Then evaluate described peptide and reduce the ability of infarct size, and the effect of itself and native peptides α-MSH is compared (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 5* is acetylation at N-end and is amidated at C-end), and

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

According to the functional performance of the peptide above and described by embodiment, the present invention relates to the peptide with the following characteristic of at least one:

A) suppress the generation of the TNF-α of LPS induction, wherein TNF-α is produced by human leukocyte

B) Eosinophil Infiltration of inflammation-induced in lung is suppressed

C) neutrophil infiltrates of inflammation-induced in lung is suppressed

D) the TNF-α of inflammation-induced in circulating is suppressed to accumulate

E) acute renal failure of local asphyxia induction is alleviated

F) myocardial infarction area is reduced

G) degree of heart failure after myocardial infarction is alleviated

H) Pulmonary Vascular hypertension is alleviated

I) renal failure of cisplatin induction is alleviated

Peptide can have more than one these characteristics, and such as 2,3,4,5,6,7,8 or above-mentioned all characteristics.These characteristics can be tested with executing strictly according to the facts the method summarized in example.

As mentioned above, the feature of alpha-MSH analogue of the present invention is have the effect improved than natural α-MSH.

In the present description and claims, term " effect " is defined as the obtainable peak response of compound.In the various tests that embodiment is recorded, alpha-MSH analogue of the present invention can produce the peak response higher than natural α-MSH.

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

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

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

Compared with α-MSH, alpha-MSH analogue of the present invention can also suppress the TNF-α of inflammation-induced in circulating to produce minimum 10%, more preferably 25% and most preferably 40%.

In addition, alpha-MSH analogue of the present invention can alleviate the acute renal failure of local asphyxia induction, and this is measured by the ability alleviating polyuria degree after local asphyxia.When comparing with α-MSH, find that 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, is proved by the ability of the size reducing necrosis area in ischemic myocardial.When comparing with α-MSH, find that 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, is proved by the performance of directly measuring left ventricle end diastolic pressure or similar quantivative approach assess cardiac.When comparing with α-MSH, find that 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 Pulmonary Vascular vascular hypertension.When comparing with α-MSH, find that minimum expected effect is that Pulmonary Vascular 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 comparing with α-MSH, find that 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 native agonist of 1 type, 3 types, 4 types and 5 type melanocortin (MC) acceptors for people know, and ACTH is the natural ligand of 2 receptors (MC2).Because described peptide comprises the aminoacid sequence of α-MSH or its analogue, described peptide has the ability activating 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.Thus, α-MSH, α-MSH variant, alpha-MSH analogue and X primitive (motif) can be prepared by the peptide technology of preparing (such as solution synthetic method or Merrifield type solid-phase synthesis) of standard.

In a possible synthesis strategy; peptide of the present invention can be prepared by solid-phase synthesis: first use the protection of known standard, coupling and deprotection steps to build the peptide sequence (α-MSH, α-MSH variant, alpha-MSH analogue) of pharmacologically active; after this on bioactive peptide with the aminoacid sequence building mode like bioactive peptide and continue coupling primitive X, finally excise whole peptide from carrier.This strategy obtains a kind of peptide, and wherein peptide sequence X holds on nitrogen-atoms at the N-of peptide and is covalently bound on the peptide of this pharmacologically active.

Another possible strategy is the sequence being prepared α-MSH peptide/analogue and X-primitive (or its part) by solution synthetic method, solid-phase synthesis, recombinant technology or Enzyme optrode respectively, then in the solution or use solid phase technique or its be combined through known fragment condensation step coupling two sequences.In one embodiment, α-MSH peptide/analogue can be prepared by recombinant DNA method, and X primitive can be prepared by solid-phase synthesis.The joint of α-MSH peptide/analogue and X primitive can be undertaken by using chemical bond method.This technology allows under high specific mode, assemble complete unshielded peptide fragment (Liu etc., 1996).Can also be formed by the peptide bond of proteases catalyze and carry out described joint, the technology which providing a kind of high special is with by the complete unshielded peptide fragment of peptide keyed engagement (Kullmann, 1987).

The example of suitable solid support material (SSM) be such as functionalization polystyrene, the latex of resin as 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 solid support material by conventional connexon, conventional connexon is such as 2, 4-dimethoxy-4 ' '-hydroxy benzophenone, 4-(4-hydroxy-methyl-3-methoxyphenoxy)-butyric acid, 4-hydroxy-methyl phenylformic acid, 4-methylol-phenylium, 3-(4-hydroxymethylphenoxy) propionic acid, with p-[(R, S)-a [l-(9H-fluorenes-9-base) methoxymethylamide base]-2, 4-Dimethoxyphenyl]-phenoxy group-acetic acid.

The method that " scavenging agent " (such as dithioglycol, tri isopropyl silane, phenol, the thioanisole etc.) being optionally suitable for this object with one or more by acid (such as trifluoroacetic acid, trifluoromethanesulfonic acid, hydrogen bromide, hydrogenchloride, hydrogen fluoride etc.) combine, peptide of the present invention can be excised from solid support material, or, by method such as ammonia, hydrazine, alkoxide such as sodium ethylate, the oxyhydroxide such as sodium hydroxide etc. of alkali, peptide joiner of the present invention is excised from 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 such as phosphoamidite method (phosphoamidite method), the nucleotide sequence of peptide of the present invention of encoding can be prepared synthetically.According to phosphoamidite method, such as, in automatic dna synthesizer, synthesis on suitable carrier, purifying, annealing, connection and clone's oligonucleotide.

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

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

(it is used to guide transcribing of the nucleotide sequence of peptide of the present invention of encoding to suitable promotor, especially in bacterial host cell) example be the promotor obtained from following source: E coli lac operon, streptomyces coelicolor agarase gene (dagA), subtilis levansucrase 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 recombinantbacteria " Scientific American, 1980,242:74-94; The same with Sambrook etc., 1989().

The nucleotide sequence of the peptide invented for instructing code book in filamentous fungal host cell is transcribed, the example of suitable promotor is the promotor obtained from the gene of the following enzyme of coding: oryzae TAKA amylase, rhizomucor miehei (Rhizomucor miehei) aspartate protease, Aspergillus ni ger neutral α-amylase, aspergillus niger acid-resistant alpha-amylase, aspergillus niger or Aspergillus awamori amylase (glaA), rhizomucor miehei lipase, line protease, Aspergillus oryzae triose phosphate isomerase, 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.That TAKA amylase, NA2-tpi(are from the hybrid of the promotor of the gene of coding Aspergillus niger neutral α-amylase and Aspergillus oryzae triose phosphate isomerase for filamentous fungal host cell, particularly preferred promotor) 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.Other useful promotors for yeast host cell are recorded in Romanos etc. 1992, yeast (Yeast) 8:423-488.

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

For yeast host cell preferred terminator from coding following enzyme gene obtain: yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) Hydratase, phosphoenolpyruvate, S. cerevisiae cytochrome C(CYC1), Ethanol in Saccharomyces cerevisiae desaturase/glyceraldehyde-3-phosphate dehydrogenase.It is the same that other useful terminators for yeast host cell are recorded in the 1992(such as Romanos).

Described carrier can also comprise element such as polyadenylation signal (such as, from SV40 or adenovirus 5Elb district), transcription enhancer sequences (such as, SV40 enhanser) and translational enhancer (such as, 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 coding: oryzae TAKA amylase, aspergillus niger glucoamylase, Aspergillus nidulans aminobenzoate synthetic enzyme.Useful polyadenylation signal for yeast host cell is recorded in Guo and Sherman, 1995, molecular cytobiology (Molecular CellularBiology) 15:5983-5990.

Recombinant expression vector can also comprise the DNA sequence dna that described carrier can be made to 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 pBR322 plasmid, pUC19, pACYC177, pACYC184, pUBHO, pE194, pTA1060 and pAM β l.Example for the replication orgin of yeast host cell is 2 micron origin of replication (2micron origin of replication), the combination of CEN6 and ARS4, the combination of CEN3 and ARS1.Replication orgin can be the replication orgin with sudden change is temperature sensitive (see Ehrlich, 1978, Proc.Natl.Acad.Sci.USA75:1433) to make its function in host cell.

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

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

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

For transfection mammalian cell and express the method being transferred to the DNA sequence dna of cell can be any one method well known in the art (such as, MANIATIS, T., E.F.FRITSCH and J.SAMBROOK, 1982Molecular Cloning:A Laboratory Manual. CSH Press, cold spring port, New York).

Host cell can also be unicellular pathogenic agent (such as prokaryotic cell prokaryocyte) or non-unicellular pathogenic agent (eukaryotic cell).Useful unicellular cells is that bacterial cell such as gram-positive microorganism (includes but not limited to bacillus cell, such as Alkaliphilic bacillus (Bacillus alkalophilus), 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)), or streptomyces cell (such as, shallow Streptomyces glaucoviolaceus (Streptomyces lividans) or mouse ash streptomycete (Streptomyces murinus)), or Gram-negative bacteria (such as intestinal bacteria (E.Coli) and pseudomonas (Pseudomonas sp)).The conversion of bacterial host cell can such as by protoplast transformation, by using competent cell, carrying out by electroporation or by fusion method.

Host cell can be fungal cell." fungi " used herein comprises Ascomycota (phylaAscomycota), Basidiomycota (Basidiomycota), chytrid door (Chytridiomycota) and in conjunction with bacterium door (Zygomycota) and oomycetes door (Oomycota) and all mitosporic fungi (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 listed below.Fungal host cells also can be yeast cell.Here " yeast " used comprises ascosporogenous yeast (ascosporogenous yeast) (Endomycetaceae (Endomycetales)), basidiospore forms yeast (basidiosporogenous yeast) and belong to the yeast of imperfect fungi door (Fungi Imperfecti) (gemma guiding principle (Blastomycetes)).Substratum for culturing cell can be the conventional medium (such as containing suitable additive containing blood serum medium or serum free medium) of any suitable growth mammalian cell, or for growing the suitable culture medium of insect cell, yeast cell or fungal cell.Suitable substratum can obtain from commercial suppliers or prepare (such as, the catalogue at American Type Culture preservation center) according to published formula.

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

Therefore, the present invention relates to the method for the preparation of peptide according to the present invention, described method recombinant DNA technology, it comprises the steps: that the nucleotide sequence of coding for said peptides is transferred in host cell by (a), (b) cultivate described host cell, from described culture, be separated described peptide with (c); Or (a), under the condition allowing described peptide to produce, cultivates the recombinant host cell of the nucleotide sequence containing coding for said peptides, is separated 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, the purposes that the patient's condition particularly mentioned with one or more, discomfort or disease are relevant above or below.

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 tissue of mammiferous one or more organ.Described organ is selected from but is not limited to by 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 comprising small intestine, colon, rectum and anal tube and prostate gland.

As mentioned above, compared with α-MSH, peptide of the present invention demonstrates the antiphlogistic effects of raising and the ability preventing 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 tissue of mammiferous one or more organ, and the wherein said patient's condition is local asphyxia or inflammatory condition.The described patient's condition can also be the exhaustion owing to toxin or drug-induced cell, tissue or organ.

In the present description and claims, if context does not get rid of this explanation especially, term " treatment " generally includes the treatment of the existing patient's condition and the prevention (prophylactic treatment) of this patient's condition.

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

Local asphyxia

In the present description and claims, local asphyxia is defined as the volume of blood flow reduced one or more organ, the Oxygen deliver causing tissue to reduce and/or utilization.Local asphyxia can occur in one or more organ, and described organ comprises (non-fully 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 arterial blood supply completely, 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.Qualification can suppress or prevent the compound of (completely or partially) many cell/tissue/organ damages of occurring as ischemic result or destruction to be highly profitable.

Carry out the patient's condition for the treatment of owing to the local asphyxia of tissue or to be produced by the local asphyxia organized, such as 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 owing to blood pressure drops in operation or other interventional therapy processes.Therefore, the patient's condition of carrying out treating can be the reduction such as ypotension of any system volume of blood flow, and it is by the system volume of blood flow of impact 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 being used for the treatment of ischemic pharmaceutical composition, the wherein said patient's condition is 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-fully list) has 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 ypotension of systemic disease (comprising transmissible disease or anaphylaxis), the ypotension owing to one or more toxic chemical or poison or medicine.

In this second embodiment, the present invention relates to peptide according to the present invention for the preparation of the purposes being used for the treatment of ischemic pharmaceutical composition, the wherein said patient's condition is caused by secondary ischaemia.

The local asphyxia being secondary to disease or the patient's condition can be selected from following disease and the patient's condition at one or more to be observed: diabetes, hyperlipidaemia, Buerger's disease (Buerger's disease), Takayasu's syndrome, transient arteritis, mucocutaneous lymphnode syndrome (mucocutaneous lymphnode syndrome), cardiovascular syphilis, the connective tissue disease as Raynaud disease, blue phlebitis, vascular trauma (comprising Iatrogenic injury such as cannulation or operation or organ transplantation).In addition, this list comprises the local asphyxia caused by one or more organ surgery, one or more organ transplantation, operation insertion transplanting, device, graft, artificial limb or other biological medical cpds or equipment.

In the third embodiment, the present invention relates to the purposes according to peptide of the present invention, the wherein said patient's condition causes by owing to the local asphyxia of septic shock or the patient's condition relevant to systemic hypotension.

Inflammatory symptom

Term " inflammatory symptom " in this article refers to a kind of patient's condition, and in the described patient's condition, the such as mechanism of reaction of T lymphocyte specific reaction or antibody and antigen causes raising of inflammatory cell and Nei Sheng medium chemical substance.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 inflammatory 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 tissue of mammiferous one or more organ, and the wherein said patient's condition is inflammatory condition.

Inflammatory condition can be caused by inflammatory disease, and it comprises (non-fully list): sacroiliitis (comprising the disease relevant with sacroiliitis), osteoarthritis, rheumatoid arthritis; SpA (such as ankylosing spondylitis), reactive arthritis (sacroiliitis after comprising rheumatic fever), anaphylactoid purpura and Reiter are sick.In addition, inflammatory diseases comprises the vasculitis of connective tissue disease such as systemic lupus erythematous, polymyositis/dermatomyositis, systemic sclerosis, MCTD, sarcoidosis and primary Sjogren syndrome (comprising keratoconjunctivitis sicca), polymyalgia rheumatica and other types, crystallization deposition disease (comprising gout), tetra-sodium joint disease, acute Calcified periarthritis.In addition, inflammatory diseases comprises juvenile arthritis (still's disease), psoriasis, osteoarthritis, be secondary to the osteoarthritis of excessive mobile disease, congenital dysplasia, femoral head epiphysis gets loose, Perthes disease (Perthes ' disease), intra-articular fracture, meniscectomy, obesity, recurrent is dislocated, repetitive, crystallization deposition disease and cartilage metabolism abnormality disease (comprising tetra-sodium joint disease), ochronosis, hemochromatosis, ischemic necrosis (comprising sickle cell disease), by the therapeutics of adrenocortical hormone or other drug, decompression sickness, septic or infective arthritis (comprise tuberculous arthritis, meningococcal arthritis, gonococcal 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 infection and has rubella, mumps, hepatitis B, HIV or parvovirus), or recidivity hemarthrosis.In addition, inflammatory disease comprises vasculitis such as owing to the germy infectious vasculitis of infection, comprises spirochetal diseases as Lyme disease, syphilis, Rickettsiae and mycobacterial infections, fungi, virus or protozoan infection.In addition, inflammatory disease comprises non-infectious vasculitis, comprises polyarteritis under Takayasu's disease pulseless disease, giant cell arteritis (transient arteritis and polymyalgia rheumatica), Buerger's disease, polyarteritis nodosa, microscope, wegener granulomatosis, allergic granulomatous vasculitis (Churg-Strauss syndrome), the vasculitis that is secondary to connective tissue disease comprise systemic lupus erythematous, polymyositis/dermatomyositis, systemic sclerosis, MCTD, sarcoidosis and primary Sjogren syndrome.In addition, inflammatory disease comprises the vasculitis being secondary to rheumatic arthritis.

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

In addition, inflammatory disease comprises all types of childhood arthritis, and such as juvenile arthritis comprises still's disease, Juvenile rheumatoid joint, Juvenile ankylosing spondylitis.

In addition, inflammatory disease comprises the sick and downtake case of epithelium healing 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.

In addition inflammatory disease comprises all types of storage disorders, as gout, tetra-sodium joint disease, acute Calcified periarthritis.

In addition the inflammatory condition of inflammatory disease all types of generation backache comprise infection, septic diskitis, tuberculosis, malignant tumour (such as metastatic hepatic neoplasm, myelomatosis and other), tumor of spine, ankylosing spondylitis, disease/osteoarthritis, osteoporosis, richets between outstanding, chronic dish between acute dish.It also comprises scleromalacia, hyperparathyroidism, renal osteodystrophy, spondylolisthesis, Taper Pipe stenosis birth defect and fibromyalgia.

In addition inflammatory disease comprises all types of soft tissue rheumatism and comprises bursitis, tenosynovitis or aponeurositis, tendinous end inflammation, nerve compression, periarthritis or capsulitis, muscular tone and muscular dysfunction.

In addition the inflammatory disease that inflammatory disease comprises gastrointestinal system (comprises all types of gastritis, pemphigus, large blister quasi-Pemphigus, benign mucosal pemphigoid), disease of salivary gland (such as sarcoidosis, saliva blockage and Sjogren syndrome), (such as gastro oesophageal reflux disease (GORD) or infection have Candida species to esophageal 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, gluten enteropathy, dermatitis herpetiformis, tropical is suffered from diarrhoea, Whipple disease, radiation enteritis, systemic amyloidosis is sick, 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 tissue of mammiferous one or more organ, and the wherein said patient's condition is the inflammatory condition being 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 the present description and claims, " drug-induced cell, tissue or organ failure " is defined as the cell or tissue function and/or modal change of being induced by pharmacological compounds.Described pharmacological compounds includes but not limited to that cis-platinum drawn together by cancer chemotherapy cartridge bag, 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 (sub-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, microbiotic as 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: the toxic reaction (comprising drug-induced toxicity) of local asphyxia, acute and/or chronic inflammatory diseases, anaphylaxis, rheumatosis, infection (comprising virus, fungi, bacteriological infection), prion and other microorganisms and other infectious preparations well known in the art, form of ownership and acute and chronic injury.Chronic injury comprises the situation of the repeated injury of the time variations restored wholly or in part with organ or tissue's function.The patient's condition that wherein normal function of cell, tissue or organ has changed comprises damage, and it is relevant to one or more organ or the implantation for other devices transplanted, and it is expected to peptide of the present invention also by useful in the treatment or prevention of the described patient's condition.Described organ can derive from individuality self, animal body self or derive from other individual or animals.This comprises: organ transplantation, bone collection, soft tissue implant (silicone implantations), metal and plastics implantation, or other medically transplantable devices.Described individual representative and other Mammalss.

The patient's condition of carrying out treating also can be (such as comprise lung, bronchiole, epithelium healing to respiratory system by cancer or by having to organ, and/or on heart and/or on kidney and/or on gastro-intestinal system) precancerous lesion that affects causes, and comprises the transitivity malignant tumour of acute leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, lymphosarcoma, myelomatosis, any cause of disease.It is expected to peptide of the present invention also by useful in the treatment or prevention of the described patient's condition.

In addition, the patient's condition of carrying out treating also can be selected from following disease by any one to cause: diabetes, there is the patient's condition of the 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, there is the patient's condition of the Serum Triglyceride level of raising, there is the patient's condition of the empty stomach HDL-cholesterol levels of raising, retroperitoneal fibrosis, lupus erythematosus, many knots arteritis, scleroderma, polymyositis, dermatomyositis, rheumatic arthritis, allergy, serum sickness, hemolytic anemia and supersensitivity granulopenia.It is expected to peptide of the present invention also by useful in the treatment or prevention of the described patient's condition.

Many infection causing performance to reduce may have the influence organized and disturb normal function, and it can be improved by the peptide of the present invention giving effective dose.These infection comprise protozoon, virus, bacterium and fungi infestation, and comprise the patient's condition such as AIDS, bacterial septicemia, systemic fungal infection, rickettsiosis, toxic shock syndrome, infectious mononucleosis, chlamydia trachomatis, chlamydia psittaci, cytomegalovirus infection, arc bacillus, Salmonellas, influenza, poliomyelitis, toxoplasmosis, lassa fever is sick, 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 carrying out treating can be relevant with the chemical wound relating to one or more toxicants and/or medicine.These medicines comprise tricyclic antidepressant, lithium salts, prenylamine and phenothiazines, cis-platinum drawn together by cancer chemotherapy cartridge bag, 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 ((sub-Baudrillard enzyme element), etoposide, idarubicin, irinotecan, mitomycin, taxol, Plicamycin, topotecan, vincaleucoblastine, vincristine(VCR), Vinorelbine, amasacrine, asparaginase, hydroxyurea, mititane, mitoxantrone, microbiotic as aminoglycoside comprises Streptomycin sulphate, Liu Suanyan NEOMYCIN SULPHATE, kantlex, Amikacin Sulphate, gentamicin, tobramycin, sisomicin, netilmicin, immunosuppressive compounds is as ciclosporin.Physical trauma comprises electromagnetic radiation also can cause damage, it can be made to alleviate by the alpha-MSH analogue according to the present invention using effective dose.

Connective tissue disease such as scleroderma, systemic lupus erythematous can also be comprised according to the patient's condition that the present invention carries out treating, such as, or neuromyopathy, Du Xing (Duchenne) type progressive muscular dystrophy, Freed rely uncommon (Friedreich) ataxia, myotonia atrophica.The described patient's condition such as can relate to the tissue of mammal intestine.

The present invention relates to the purposes according to peptide of the present invention, the wherein said patient's condition is selected from by following formed group: 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 the method for the patient's condition of the tissue of one or more organs of the mammalian subject being used for the treatment of or preventing at its needs, described method comprise give effective dose one or more according to peptide of the present invention.The described patient's condition can be the result of ischemic conditions or inflammatory condition and/or the toxic action from poisoning or pharmacological agent.

To that produced by any one organ or vessel graft or the associated patient's condition, comprise the reaction preventing graft to host, methods for the treatment of of the present invention is effectively special.In these patient's condition, whole organ is to all changes extreme sensitivity about nutrition, metabolism, perfusion etc., and we believe, can stablize the described patient's condition and make the situation of described tissue to the function of any compressing organ more have resistance according to treatment of the present invention.Also be included in the process being transplanted to acceptor the peptide of the present invention giving organ graft effective dose according to method of the present invention, comprise the peptide of the present invention of interpolation effective dose in grafting matrix.

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

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

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

The patient's condition of carrying out treating 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 comprises acute infection (particularly those affect the acute infection of lung), pulmonary infarction, ypotension, apoplexy, hypoxemia or can accelerate myocardial ischemia and thus cause ARR anemia.Irregular pulse can worsen cycle penalty and thus set up mistake, self-perpetuating circulation.

We believe, increase is used for developing ARR threshold value by treatment according to the present invention, thus prevents ARR development.By directly described effect can be realized to delivery system effect or indirectly by carrying out effect to the patient's condition of guiding or ARR reason.

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 relating to abnormal cardiac muscle connection between atrium and ventricle.

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

It is expected to carry out treating by the ARR danger of reduction development with alpha-MSH analogue according to the present invention, irregular pulse produces due to the association treatment with other antiarrhythmic drugs.

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

Other patient's condition that can be alleviated by the peptide according to the present invention giving effective dose are that organ (such as heart) ionogen is chaotic and self is chaotic, comprise the exception of the relative concentration 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 being 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 according to the dosage of alpha-MSH analogue of the present invention for preventing the development of any sign of the patient's condition or the patient's condition.

Prevention or preventative treatment can be ongoing treatment or the heart attack of patient for preventing from suffering from coronary stricture in such as surgical procedure.Prophylactic treatment can also be used for short duration.Those skilled in the art can based on the concrete treatment procedure of emergency situation evaluation.Preferred embodiment, described treatment or prevention can reduce local asphyxia infarct size coronarius.Compared with untreated individuality, this infarct size can reduce 20%, and such as at least 30%, preferably at least 50%.

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

According to the dosage of alpha-MSH analogue of the present invention can as single dose, rule or continuous print administration, or as order administration and give.

The application that administration can be systemic applications, topical comprises medicine target system, conduit and implantation, oral administration, administered parenterally, such as subcutaneous administration, intramuscularly, intravenous injection, abdominal injection, intrathecal injection, pulmonary administration are such as by suction, external application, mucosa delivery, percutaneous dosing.

Therefore, administration comprises systemic applications; 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 comprise 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 the route of administration that any one is relevant or is prevented any one 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 the examples below, give containing one or more suitable compositions according to peptide of the present invention.For giving individual (animal or human) administration, material preferably makes pharmaceutical composition, and it contains described material and the optional acceptable vehicle of one or more pharmacy.

Composition can with such as solid-state, the form of semi-solid state or fluid composition exists such as, such as but not limited to, can 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 such as two containers separated, 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, such as implant or implant coating or a kind of form being suitable for implanting and use under transplanting condition.

Composition can be prepared by conventional pharmaceutical methods, see such as " 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 ofPharmaceutical Technology) "; Swarbrick; J. & J.C edits .Boylan; Marcel Dekker; Inc.; New York, 1988ISBN0-8247-2800-9.

Pharmaceutical composition containing active substance is used as delivery system.Term " delivery system " represents a kind of pharmaceutical composition (pharmaceutical preparation or formulation) in this article, and it is at the active substance of administration interval scale to human body or animal body.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, the pharmaceutical composition for purposes according to the present invention can comprise pharmacy or the acceptable vehicle of cosmetology.

In the composition for purposes according to the present invention, selection and the optimum concn thereof of the acceptable vehicle of pharmacy generally can not be predicted, and need to determine based on its measuring.Whether the acceptable vehicle of a kind of pharmacy is suitable for the type generally also depending on selected formulation in pharmaceutical composition.But the those of ordinary skill of pharmacy field can 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 the individuality that will give composition.This vehicle will meet the requirement that Drug Administration is promulgated usually.Official pharmacopoeias such as British Pharmacopoeia, American Pharmacopeia and European Pharmacopoeia all define the standard of the acceptable vehicle of known pharmacy.

Following present the summary of the relevant pharmaceutical composition for purposes according to the present invention.This summary is based on specific route of administration.It is to be understood, however, that, the acceptable vehicle of those pharmacy can be different formulation or when combinationally using, the application of the acceptable vehicle of specific pharmacy is not limited to the specific function of particular dosage form or this vehicle.

Parenteral composition

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

Composition used according to the invention comprises solid-state, semi-solid state and the fluid composition of form of ownership.Specific relevant composition is, such as solution, suspend night, emulsion, gel, implant and implant.

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.Composition for purposes according to the present invention can contain usually nontoxic pharmaceutically acceptable carrier and vehicle, comprises microballoon and liposome.

Composition for purposes according to the present invention comprises solid-state, semi-solid state and the fluid composition of form of ownership.Specific relevant composition is, such as, 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 (such as collagen sponge), pad, dressing (e.g., such 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

Composition for purposes according to the present invention comprises all types of solid-state, semi-solid state and fluid composition.Specific relevant composition be such as solution, suspension, emulsion, nude film, controlled release tablet, enteric coated tablet, mouth dispersible tablet, effervescent tablet, chewable tablet, soft capsule, hard capsule, controlled release capsule, enteric coated capsule, naked particle, effervescent granule, particle for the preparation of the liquid orally used, coated granule, enteric coated particles, controlled release granule, for the pulvis of oral administration and the pulvis for the preparation 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, (such as, 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), mineral oil, fatty oil, whiteruss, polyoxyethylene glycol, propylene glycol, glycerine, liquid gathers alkylsiloxane, 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.

Example for the sanitas of composition is but is not limited to parabens (such as methyl p-hydroxybenzoate, 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 (MDMhydantoin), IPBC, ethylenediamine tetraacetic acid (EDTA) (EDTA), benzalkonium chloride and phenylcarbinol, or 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, such as Sudan Gum-arabic or tragacanth, and natural exist phosphatide such as soybean lecithin; Sorbitan monooleate derivative; Lanolin; Wool wax alcohol; Sorb sugar ester; Direactive glyceride; Fatty alcohol; Fatty acid ester (tri-glyceride of such as lipid acid); And composition thereof.

The example of suspension agent is but is not limited to Mierocrystalline cellulose and derivatived cellulose, such as 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, such as starch or derivatived cellulose is carboxymethyl cellulose, Natvosol such as, and other derivatived celluloses, water-swellable hydrocolloid, carrageenin, hyaluronate (hyaluronic acid derivatives such as optionally containing sodium-chlor), and alginates (comprising propylene glycol alginate).

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, such as polyoxyethylene sorbitan monoleate (tween Tween).

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

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 fluid/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, glyceryl palmitostearate (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 dose weight, is preferably 0.01-75%, is more preferably 0.1-20%, especially 1-15% such as 1-10%.

Dose-dependant is in the patient's condition of carrying out treating.Various medicine can use under dosage well known in the art.Can be expected that, one or more are according to the scope of the dosage of peptide of the present invention in 1ng-100mg/kg body weight, are preferably 1 μ g-10mg/kg body weight, are more preferably 10 μ g-1mg/kg body weight, such as 50-500 μ g/kg body weight.

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

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

To record herein and claimed invention is not limited to the scope of embodiment disclosed herein, because these embodiments are used to illustrate several aspect of the present invention.Any equivalent embodiments all within the scope of the invention.In fact, except those amendments shown herein and describe, will become apparent to those skilled in the art from the various amendment of the present invention of above-mentioned specification sheets.These amendments also fall into the scope of accompanying claim.

Refer to various reference herein, content disclosed in it is incorporated to herein as a reference in the mode of their entirety.

Run through this specification sheets, word " comprises ", or its variation refers to containing the element pointed out, integer or step as " comprising " or " comprising " should be understood to, or the group of element, integer or step, but do not get rid of any other element, integer or step, or the group of element, integer or step.

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

Below the present invention is described the drawings and Examples by following unrestricted effect.

Embodiment

Below generally depict the method for testing 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 the ability of antiphlogistic effects and suppression or the cell/tissue/organ damage preventing the result as the toxic action of local asphyxia, inflammation or medicine and occur or destruction.

The feature of the deterioration of inflammatory reaction or chronic inflammatory diseases is the production of cell source medium (such as 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 leukocyte accumulation (comprising neutrophilic granulocyte and eosinophilic granulocyte) in alveolar space in lung.Comprised the inflammatory mediator of TNF-α by induction, the lipopolysaccharides (LPS) discharged from infectious agent is playing an important role in the inflammatory reaction infected.Therefore, the methods for the treatment of with suppression TNF-α throughput believes to have significant antiphlogistic effects.Contriver uses LPS to stimulate and produces inflammatory reaction (see experimental program 1-3) in many schemes, and the main mark for the antiphlogistic effects of peptide according to the present invention is the ability suppressing TNF-α to produce.

By reduce/arterial blood supply completely stops the local asphyxia of induction to induce many tissue reactions, comprises neutrophilic granulocyte accumulation, other inflammatory reactions and necrocytosis.The compound of most cell/tissue/organ damage that qualification can suppress or prevent (completely or partially) to occur as ischemic result or destruction is highly profitable.Contriver uses two transient local asphyxia models: 1) the myocardial ischemia-reperfusion model of rat, it is the development of the Acute Myocardial Infarction that blood supply recovers after simulating, because it obtains (experimental program 4) by fibrinolytic therapy or coronary angioplasty; 2) two-way renal artery obstruction, its inducing acute renal failure (ARF), its be similar to carry out seen in the patient of large surgical interventional therapy to, the ARF(that induced by the instantaneous reduction of kidney blood supply can carry out operation interventional therapy due to abdominal aortic aneurysm embodiment) (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, in therapeutic process, observe the obvious reduction of renal glomerulus infiltration rate.The renal toxicity of cis-platinum can regard the direct cytotoxin damage of the nephron of the thick section of ascending branch to medulla externa layer (especially the S3 fragment of proximal tubule) and Henle ring as.Therefore, plus cisplatin in treatment usually causes the heavy native defect of uriniferous tubules, comprises the ability of the dilute urine of damage.In the patient crossed with plus cisplatin in treatment of about 50%, observe hypomagnesemia, it is likely magnesium (Mg) the re-absorbed defect due to kidney.Nearest research shows that supplementary Mg is the important factor that protection avoids the Nephrotoxicity of cyclosporin A, and has indicated the possible relation between Mg loss and the nephrotoxicity of cisplatin induction recently.Therefore, be intended to prevent the treatment of hypomagnesemia to have useful effect, not only in order to reduce the needs of supplementary Mg but also the renal toxicity in order to reduce 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 TNF-α of the external LPS induction produced by human leukocyte

20mL human blood is collected in the vacuum blood collecting tube (vacutainer tube) containing EDTA.Ficoll-Paque+(the Ficoll-Paque Plus) of peace agate West Asia specification sheets (Amersham ' s Instruction) 71-7167-00AD, 2002-06 is used to carry out separating periphery blood monocytic cell (PBMC).Blue solution (Sigma (Sigma)) is to PBMC counting to use platform to expect, and with 5 × 10 ⁵the concentration of cell/mL by PBMC incubation at RPMI1640(Ai Puli chemical products (Applichem)) in, RPMI1640 is 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, at 37 DEG C, the PBMC incubation separated is had substratum, 10ng LPS/mL(Sigma) and experimental compound 24 hole flat undersides (Corning Incorporated (CorningIncorporated)) in.After 18 hours, sample is centrifugal, use the tumor necrosis factor alpha (Tumour Necrosis Factor Alpha) [(h) TNF-α] hanging down gram (HumanBiotrak) enzyme-linked immunosorbent assay (ELISA) system (Amersham) from the mankind hundred to detect the TNF-α of supernatant liquor.

Each donor adopts following mode by sample incubation:

PBMC is in RPMI (time contrast)

PBMC is at 10ng LPS/mL (carrier)

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

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

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

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

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

PBMC at 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.

Being processed to protect in the bottle crossed at BSA bag by all solution avoids compound to be bonded on the surface of bottle.

Data represent with mean number ± SE.The impact that the TNF-α that experimental compound is induced LPS discharges is expressed as the percentage ratio that in LPS-vehicle group, TNF-α accumulates.With student not pair t-test analyze all contrasts.Under the confidence level (p) of 0.05, think that difference is significant.

Experimental program 2

The TNF-α of the induction of the LPS in rat body is suppressed to produce

Laboratory animal: female Wistar rats (220-240g) is from Charlie river (Charles River), Sulzfeld, Germany, and make its live in temperature (22-24 DEG C) and humidity (40-70%) controls, in the room with 12 h light-dark cycle (carrying out illumination from 6:00A.M. to 6:00P.M.).The Rodent Diet (there is 140mmol/kg sodium, 275mmol/kg potassium and 23% protein, A Ertuomin international corporation (Altromin International), Lage, Germany) that rat is maintained the standard, and there is the freedom close to water source.

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

Experimental arrangement: before the experiments, by training all rats two cycles (each two hours), makes the restriction cage of they adaptations for testing.In experiment day, animal is transferred in restriction cage, and start 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 to inject lipopolysaccharides (LPS).LPS(e. coli serotype 0127B8 is given, L3129, Sigma, St.Louis, USA with the dosage of 4mg/kg body weight), intravenous injection was carried more than 1 hour.Within 60,90 and 120 minutes after lps injection starts, get 0.3ml arterial blood sample, and substitute with the blood from Normal donor rat of heparinization immediately.

Experimental group:

Except lps injection, all rats fast injection (bolus injection) processes:

Carrier (0.5mL isotonic saline solution);

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

Be in the experimental compound of 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 ⁶blood sample is collected in the precooling experiment tube of IU/ml Trypsin inhibitor,Trasylol.After 4 DEG C centrifugal, plasma sample transferred in precooling experiment tube and to measure at-20 DEG C of storage TNF-α later.By ELISA(Biotrak, Amersham, Britain) measure the TNF-α of blood plasma.

Statistical study: result represents with mean number ± SE.Two analysis of variance (two-way ANOVA) being used for replicate measurement are used to check group difference.When p < 0.05, the not pair t-test corrected by the Bang Feiluoni (Bonferroni) with confidence level, evaluates the difference between the corresponding phase.

Experimental program 3

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

Use from M & B A/S, DK-8680Ry, the male Sprague-Dawley rat of Denmark is used for all experiments.Rat is closed in 3 type standard cages, make its live in temperature (22-24 DEG C) and humidity (40-70%) controls, in the room with 12 h light-dark cycle (carrying out illumination from 6:00A.M. to 6:00P.M.).Feed is the Altromin1324 special formulation of autoclave process, this formula by Altromin Denmark(Chr.Pedersen A/S, 4100Ringsted, Denmark) produce.Unrestrictedly give feed and water.

After domestication, rat is randomly assigned experimental group, when LPS induction starts intravenous injection dosage give experimental compound and LPS induction after 8 hours again intravenous injection dosage give experimental compound.

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

Bronchoalveolar lavage is carried out to right lung by injecting and extracting 6 × 2.5ml PBS out.Lavation is carried out with the state making lung still remain in the heart after removing breastbone and rib.In this process the pipe connecting to left lung is tied a knot.At 4 DEG C, centrifugal limb bronchoalveolar lavage fluid (BALF) 10 minutes under 1000rpm.Cell resuspension is carried out total cell count in 0.5ml PBS after removing supernatant liquor.To from each rat, the BALF that crosses with Ji's nurse Sa (May-Gr ü wald Giemsa) dyeing, prepare two smears.Total cell count and Arneth's count are carried out to the BALF from each rat.

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.

The time control group of LPS is not finally sucked by vehicle treated.

Statistics:

Data represent with mean number ± SE.By be below expense house least significant difference method single-factor variable analysis group between contrast.Think that difference is significant in 0.05 level.

Experimental program 4

Suppress release and the pulmonary hypertension of the cytokine of the LPS induction in pig body

Female landrace (Landrace pig) (~ 30kg) overnight fasting is allowed freely close to water source.Then intramuscular injection of ketamine (10mg/kg) and imidazoles reach logical sequence (0.25mg/kg) and carry out premedicate to pig.Intravenous ketamine (5mg/kg) causes anesthesia.Pig is inserted pipe from oral cavity, and with continuous print 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) of the end-expiratory positive pressure fixing fabric structure of O; Siemens (Siemens) Elema, Solna, Sweden) to animal ventilation.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).Carry out ventilation with the oxygen of mixing air to be intended to make arterial oxygen tension (PaO ₂) higher than 105mmHg.An arterial sheath and two venous sheath are arranged in carotid artery and corresponding vein, described vein for pouring into, by be full of fluid conduit Measure blood pressure, get blood and for introducing conduit.

In pulmonary artery, pulmonary artery floating catheter (Swan-Ganz) (Edward biotech firm (Edwards Lifescience Corp.), Irvine, California) is inserted via right superior vena cava.Because it enters into pulmonary artery by the right side of heart, can by the Characteristic pressures curve on observation monitor and the position being determined balloon catheter by X-ray.Another conduit (5French is inserted at left neck artery; St.JudeMedical 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 right atrium (guidance of X-ray) by venous sheath and is used for making heart rate criteria.

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 can laboratory (Difco Laboratories), Detroit, the state of Michigan) be dissolved in salt solution to dissolve any throw out.After one period of steady time, be the speed of 2.5 μ g/kg/h at baseline and start to inject lipopolysaccharides be progressively increased to the condition of 15 μ g/kg/h in 30 minutes under.Afterwards, in 150 minutes, injection speed is remained on 2.5 μ g/kg/h, after this stop injection.

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

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

Statistics: data represent with mean number ± SE.By be below expense house least significant difference method single-factor variable analysis group between contrast.Think that difference is significant in 0.05 level.

Experimental program 5

Suppress the myocardial infarction area by blocking rat left anterior descending coronary artery 60min induction

Isolation feed and specified-pathogens free female Wistar rats (250g) from Charles River, Hannover, Germany.Make animal live in temperature (22-24 DEG C) and humidity (40-70%) controls, in the room with 12 h light-dark cycle (carrying out illumination from 6:00A.M. to 6:00P.M.).All animals allow contact free tap water and pellet rat feed, and it is approximately containing 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.In respiratory cavity, the O containing 4% isoflurane is used after one week ₂make rat anesthesia.After insertion tracheal catheter, use the O of following Hugo Basile Rodent ventilator containing 1.0% isoflurane ₂artificial ventilation is carried out to animal.Tidal volume is 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 DEG C.Use Hugo Sachs ECG instrument measurement standard ECG(second wire), and on the line of 4,000Hz, standard ecg is collected on PowerLab.After parasternal cuts and opens pericardium, visual fixing left anterior descending coronary artery (LAD).The harmless 6-0 silk thread having and allow the suture occluder of opening is again set between pulmonary trunk and the bottom righthand side of left auricle around LAD surrounding.After 10 minutes, left anterior descending coronary artery (LAD) is engaged.By the change of ECG (ST-fragment rise and R-ripple amplify increase) and the decline of MAP confirm to be engaged successfully.Reperfu-sion is carried out by leveling arch device after 60 minutes.Control rats carries out sham-operation.

Rat is carried out the one of following intravenous injection process:

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 compound/kg body weight that are dissolved in 0.5ml150mM NaCl.

Within 5 minutes before Reperfu-sion, give above-mentioned process.

Determine the size of the cardiac muscle of local asphyxia and necrosis as follows

After local asphyxia/Reperfu-sion, make rat remain on narcosis, after three hours of reperfusion, carry out the heavily occlusion of LAD.In this period, continuously measured ECG and MAP.Then intravenous injection Evans Blue dyestuff is used for determining the size of local asphyxia area.Cardiac ablation is cut into horizontal stratification and is used for determining the size of local asphyxia area and point is left from the cardiac muscle of necrosis by the cardiac muscle of ischemic.By ischemic Line Integral from, by its at 37 DEG C in 0.5%2,3,5-benzyltriphenylphosphonium chloride tetrazole solution incubation 10 minutes.Then the size of the tissue of computer generated image program determination necrosis is used.In order to evaluate the therapeutic treatment effect to congestive heart failure, by postoperative for the animal of another program buprenorphine process, then turning back in cage and measuring left ventricular end diastolic presssure (LVEDP) after two weeks.Use the 2F micro-top conduit measurement LVEDP being inserted into left ventricle via right carotid.Isoflurane concentration is regulated to make average artery pressure (MAP) be stabilized in 85-90mmHg.

Statistics:

Data represent with mean number ± SE.Contrast with in student's paired t check analysis group.By be below expense house least significant difference method single-factor variable analysis group between contrast.Think that difference is significant in 0.05 level.

Experimental program 6

Suppress the renal failure by the induction in 40 minutes of two-way blocking rat renal artery

Isolation nursing and specified-pathogens free female Wistar rats (250g) are from Charles River, Hannover, German.Make animal live in temperature (22-24 DEG C) and humidity (40-70%) controls, in the room with 12 h light-dark cycle (carrying out illumination from 6:00A.M. to 6:00P.M.).All animals allow contact free tap water and pellet rat feed, and it is approximately containing 140mmol/kg sodium, 275mmol/kg potassium and 23% albumen (Altromin catalogue no.1310, Altromin International, Lage, Germany).

The rat of long-term nephridioduct is had to be positioned in metabolic cage, after two day adaptive phase of metabolic cage, by blocking arteriorenal two ends 60min inducing experimental ARF by implanting in advance.In surgical procedure, with isoflurane-nitrous oxide anesthesia rat, to keep rectal temperature at 37 DEG C on the operating table placing it in heating.By rib abdominal incision, two kidneys being exposed, by making it loosen from perinephric fat excision it, then from vein, cutting the sub-fraction Renal artery lightly.With ganoid blood vessel clip (60g pressure; World Precision Instruments, Britain) block Renal artery 40min.Complete local asphyxia is confirmed by the whiting 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 to indicate the colour-change of blood backflow.Then 3-0 silk suture wound is used.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 blocking the Renal artery with for the identical sham-operation of ARF.Rat and the rat with ARF of parallel monitoring sham-operation.

Rat is made to carry out the one of following intravenous injection process:

Vehicle group: 0.5ml150mM NaCl.

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

Experimental compound group: the 200 μ g experimental compound/kg body weight being dissolved in 0.5ml150mM NaCl.5 minutes before the Reperfu-sion of kidney, then after 6 hours and 24 hours, give above-mentioned process.

Statistics:

Experimental program 7

Suppress the renal failure of cisplatin induction

There is the rat of long-term nephridioduct to be positioned in metabolic cage by implanting in advance, after adaptive phase to metabolic cage, being dissolved in the cis-platinum 5.0mg/kg body weight of 0.5ml150mM NaCI with abdominal injection or carrier (0.5ml150mM NaCI) processes rat.Then, after 5 days, rat is put back to metabolic cage, measure every day and collect 24h urinary volume and water intake amount in following 5 days.Then at fluothane/N ₂by all rat anesthesias in O, in the bottle of the EDTA bag quilt of precooling, collect arterial blood sample.By Blood Sample Collection containing 0.5mM EDTA, pH7.4, and 20x10 ⁶in the precooling experiment tube of IU/ml Trypsin inhibitor,Trasylol.After 4 DEG C centrifugal, plasma sample is transferred to precooling experiment tube, and be kept at-20 DEG C for measuring creatinine and magnesium (Mg) later.In addition, be also determined at blood collecting before nearest 24 hours period collect urine in creatinine.As the creatinine clearance (C of 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 _crconcentrations in urine, P _crit is Concentrations in blood plasma.Clinical chemistry systems VITEOS950(is used to correct clinical diagnosis company (Ortho-Clinical Diagnostics Inc.), Johnson & Johnson, New Jersey) and Roche Hitachi module (Roche Hitachi Modular) (Roche Diagnistics (Roche Diagnostics), mannheim (Mannheim), Germany) carry out urinating and the mensuration of creatinine in blood plasma.

Rat is made to carry out the one of following intravenous injection process:

Vehicle group: 0.5ml150mM NaCl.

Experimental compound group: the 200 μ g experimental compound/kg body weight being dissolved in 0.5ml150mM NaCl.5 minutes before the Reperfu-sion of kidney, then after 6h and 24h, give above-mentioned process.

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 1* is acetylation at N-end and is amidated at C-end)

This compound is tested at experimental program 1-7.

α-MSH and alpha-MSH analogue #1(sequence 1*) TNF-α that both dose-dependant ground reduces LPS induction in human leukocyte suspension accumulates.Surprisingly, alpha-MSH analogue #1(sequence 1*) inhibition more remarkable than the antiphlogistic effects of native peptides α-MSH.α-MSH suppresses TNF-α to be accumulated to 73 ± 9% of maximum reaction (LPS-carrier).In contrast, alpha-MSH analogue #1(sequence 1*) 47 ± 2%(that TNF-α is accumulated to carrier can be reduced p<0.01 is organized to α-MSH) (see figure 1)

The TNF-α of the induction of the LPS in rat body is suppressed to produce

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

As LPS suck after 24 hr collections BALF in eosinophilic granulocyte remarkable reduction as shown in, α-MSH and alpha-MSH analogue #1(sequence 1*) be both reduced in alveolar space to LPS suck inflammatory reaction.164.6 ± 42.2 × 10 with the quantity of the eosinophilic granulocyte in BALF ⁵individual cell, the rat of vehicle treated compares, α-MSH processes the quantity of the eosinophilic granulocyte reduced in BALF to 26.7 ± 4.3 × 10 ⁵individual cell (to vehicle group, p<0.05), alpha-MSH analogue #1(sequence 1*) reduce the quantity of the eosinophilic granulocyte in BALF to 49.0 ± 10.5 × 10 ⁵individual cell (to vehicle group, p<0.05) (see figure 3).Identical with upper, for α-MSH and alpha-MSH analogue #1(sequence 1*), the effect of the quantity of the neutrophilic granulocyte in BALF is similar.

Suppress release and the pulmonary hypertension of the cytokine of LPS induction in pig body

As with alpha-MSH analogue #1(sequence 1*) in the pig that processes after lps injection significantly reduced TNF-α plasma concentration as shown in, alpha-MSH analogue #1(sequence 1*) there is significant antiphlogistic effects.Except this antiphlogistic effects, surprisingly, alpha-MSH analogue #1(sequence 1*) also there is the ability protected and avoid pulmonary hypertension to develop, this can by with alpha-MSH analogue #1(sequence 1*) find in the rat that processes, the PAP amplification that significantly weakens LPS induction confirms (maximum PAP amplification: vehicle group: 22 ± 4mmHg, alpha-MSH analogue #1(sequence 1*) group: 8 ± 2mmHg; P=0.05) (see figure 4).

Contrary with α-MSH, surprisingly, alpha-MSH analogue #1(sequence 1*) reduce myocardial infarction area, myocardial infarction area is with necrosis area/infarct critical area(s) product representation, and 3 hours after LAD Reperfu-sion measure.Under the dosage of 200 μ g/kg body weight, obtain alpha-MSH analogue #1(sequence 1*) maximum suppression effect, wherein compared with the rat of vehicle treated, the reduction of infarct size is ~ 30%(vehicle group: 50.6 ± 2.6% of dangerous area, alpha-MSH analogue #1(sequence 1*) group: 35.7 ± 5.6% of dangerous area; P=0.01).Under the dosage of 1000 μ g/kg body weight, compared with the rat of vehicle treated, the reduction of infarct size still ~ 30%(alpha-MSH analogue #1(sequence 1*) group: 35.0 ± 4.4% of dangerous area, to vehicle group p<0.01) (see figure 5).After blocking LAD60 minute, within 14 days, measure the left ventricular end diastolic presssure (LVEDP) of another program animal, result display alpha-MSH analogue #1(sequence 1*) LVEDP and infarct are attended by the beneficial effect of infarct size after remarkable reduction (LVEDP: alpha-MSH analogue #1(sequence 1*) group: 10.4 ± 2.9mmHg of developing of congestion type cardiac failure; 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 asphyxias (RIR) induce polyuria after obvious ischemic.RIR rat has identical polyuria, compared with the control rats of sham-operation, the 5th day diuresis amount after Ischemic lesions increases 101%(RIR-vehicle group: 34.8 ± 3.3ml/24 hour, and the time contrasts: 17.3 ± 2.1ml/24 hour, p<0.01).α-MSH process can not alleviate polyuria (RIR-α-MSH group: 29.0 ± 2.9ml/24 hour; To RIR-carrier without significance).Surprisingly, alpha-MSH analogue #1(sequence 1*) complete normalizing (RIR-alpha-MSH analogue #1(sequence 1*) group: the 18.8 ± 3.6ml/24 hour urinating flow can be induced; To time contrast without significance; To RIR-vehicle group p<0.01) (see figure 7).

Suppress the renal failure of cisplatin induction

As GFR decline confirm, cisplatin treated induces 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 processed 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 1*) process prevent the hypomagnesemia of cisplatin induction (0.84 ± 0.04mM, relative to control rats without significance) and prevent the GFR of cisplatin induction to decline.

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 5* is acetylation at N-end and is amidated at C-end)

By replacing Met in site 10 with Nle and by replacing Phe in site 13 by (D-Phe) stereochemistry, making alpha-MSH analogue #2(sequence 5) be different from alpha-MSH analogue #1(sequence 1*).

This compound is tested in experimental program 1-3 and 5-7.

The TNF-α of the LPS induction suppressing vitro human white corpuscle to produce produces

α-MSH and alpha-MSH analogue #2(sequence 5*) TNF-α that both dose-dependant ground reduces LPS induction in human leukocyte suspension accumulates.Surprisingly, alpha-MSH analogue #2(sequence 5*) inhibition more remarkable than the antiphlogistic effects of native peptides α-MSH.α-MSH suppresses TNF-α to be accumulated to 73 ± 9% of maximum reaction (LPS-carrier).In contrast, alpha-MSH analogue #2(sequence 5*) 42 ± 11%(that TNF-α is accumulated to carrier can be reduced p<0.01 is organized to α-MSH) (see figure 8).

The TNF-α of LPS induction in rat body is suppressed to produce

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

As shown in the remarkable reduction of eosinophilic granulocyte in the BALF of 24 hr collections after LPS sucks, α-MSH and alpha-MSH analogue #2(sequence 5*) both reduce the inflammatory reaction that LPS is sucked in alveolar space.164.6 ± 42.2 × 10 with the quantity of the eosinophilic granulocyte in BALF ⁵individual cell, the rat of vehicle treated compares, α-MSH processes the quantity of the eosinophilic granulocyte reduced in BALF to 26.7 ± 4.3 × 10 ⁵individual cell (to vehicle group, p<0.05), alpha-MSH analogue #2(sequence 5*) reduce the quantity of the eosinophilic granulocyte in BALF to 34.0 ± 8.6 × 10 ⁵individual cell (to vehicle group, p<0.05) (see figure 10).Surprisingly, alpha-MSH analogue #2(sequence 5*) to have than α-MSH more obviously to inhibition (the alpha-MSH analogue #2(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).

Suppress the myocardial infarction area by blocking the induction in 60 minutes of rat left anterior descending coronary artery

Contrary with α-MSH, surprisingly, alpha-MSH analogue #2(sequence 5*) reduce myocardial infarction area, myocardial infarction area is with necrosis area/infarct critical area(s) product representation, and 3 hours after LAD Reperfu-sion measure.Under the dosage of 200 μ g/kg body weight, obtain alpha-MSH analogue #2(sequence 5*) maximum suppression effect, wherein compared with the rat of vehicle treated, the reduction of infarct size is ~ 27%(vehicle group: 51.4 ± 2.1% of dangerous area, alpha-MSH analogue #2(sequence 5*) group: 37.4 ± 5.1% of dangerous area; P=0.01) (see Figure 12).After blocking LAD60 minute, within 14 days, measure the left ventricular end diastolic presssure (LVEDP) of another program animal, result display alpha-MSH analogue #2(sequence 5*) LVEDP and infarct are attended by the beneficial effect of infarct size after the remarkable reduction that develops of congestion type cardiac failure.

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

Suppress the renal failure of cisplatin induction

As GFR decline confirm, cisplatin treated induces 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 processed 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 5*) prevent the hypomagnesemia of cisplatin induction and the GFR of cisplatin induction to decline.

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 9* is acetylation at N-end and is amidated at C-end)

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

This compound is tested in experimental program 1,2 and 5.

The TNF-α suppressing external LPS to induce produces, and wherein TNF-α is produced by human leukocyte.

α-MSH and alpha-MSH analogue #3(sequence 9*) TNF-α that both dose-dependant ground reduces LPS induction in human leukocyte suspension accumulates.Surprisingly, alpha-MSH analogue #3(sequence 9*) inhibition more remarkable than the antiphlogistic effects of native peptides α-MSH.α-MSH suppresses TNF-α to be accumulated to 73 ± 9% of maximum reaction (LPS-carrier).In contrast, alpha-MSH analogue #3(sequence 9*) 53 ± 13%(that TNF-α is accumulated to carrier can be reduced p<0.05 is organized to α-MSH) (see Figure 13).

The TNF-α of LPS induction in rat body is suppressed to produce

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

Contrary with α-MSH, surprisingly, alpha-MSH analogue #3(sequence 9*) reduce myocardial infarction area, myocardial infarction area is with necrosis area/infarct critical area(s) product representation, and 3 hours after LAD Reperfu-sion measure.Under the dosage of 200 μ g/kg body weight, obtain alpha-MSH analogue #2(sequence 5*) maximum suppression effect, wherein compared with the rat of vehicle treated, the reduction of infarct size is ~ 24%(vehicle group: 51.3 ± 2.1% of dangerous area, alpha-MSH analogue #3(sequence 9*) group: 39.0 ± 3.4% of dangerous area; P=0.05) (see Figure 15).After blocking LAD60 minute, within 14 days, measure the left ventricular end diastolic presssure (LVEDP) of another program animal, result display alpha-MSH analogue #3(sequence 9*) LVEDP and infarct are attended by the beneficial effect of infarct size after the remarkable reduction that develops of congestion type cardiac failure.

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 13* is acetylation at N-end and is amidated at C-end)

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

This compound is tested in experimental program 1 and 2.

α-MSH and alpha-MSH analogue #4(sequence 13*) TNF-α that both dose-dependant ground reduces LPS induction in human leukocyte suspension accumulates.Surprisingly, alpha-MSH analogue #4(sequence 13*) inhibition more remarkable than the antiphlogistic effects of native peptides α-MSH.

The TNF-α of LPS induction in rat body is suppressed to produce

In the process of intravenous injection LPS, α-MSH and alpha-MSH analogue #4(sequence 13*) the TNF-α that both reduces rat accumulates.Under the dosage of 200 μ g/kg body weight, obtain α-MSH and alpha-MSH analogue #4(sequence 13*) maximum suppression effect.Surprisingly, alpha-MSH analogue #4(sequence 13*) inhibition more remarkable than the antiphlogistic effects of native peptides α-MSH.Although α-MSH suppresses the TNF-α concentration in rat plasma to 17 ± 3%, alpha-MSH analogue #4(sequence 13* of maximum reaction (LPS-carrier)) TNF-α can be reduced be accumulated to 12 ± 2%(of carrier to α-MSH group p<0.05) (see 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 17* is acetylation at N-end and is amidated at C-end)

By replacing Tyr in site 8 with Ser, Ser is replaced with Ile in site 9, Met is replaced with Ile in site 10, by replacing Glu in site 11 with Ser and by replacing Phe in site 13 by (D-Phe) stereochemistry, making alpha-MSH analogue #5(sequence 17) be different from alpha-MSH analogue #1(sequence 1*).

This compound is tested in experimental program 1 and 2.

α-MSH and alpha-MSH analogue #5(sequence 17*) TNF-α that both dose-dependant ground reduces LPS induction in human leukocyte suspension accumulates.Surprisingly, alpha-MSH analogue #5(sequence 17*) inhibition more remarkable than the antiphlogistic effects of native peptides α-MSH.

The TNF-α of LPS induction in rat body is suppressed to produce

In the process of intravenous injection LPS, α-MSH and alpha-MSH analogue #5(sequence 17*) the TNF-α that both reduces rat accumulates.Under the dosage of 200 μ g/kg body weight, obtain α-MSH and alpha-MSH analogue #5(sequence 17*) maximum suppression effect.Surprisingly, alpha-MSH analogue #5(sequence 17*) inhibition 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-Lys-Pro-Val-NH ₂(sequence 2* is acetylation at N-end and is amidated at C-end)

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

This compound is tested in experimental program 1 and 2.

α-MSH and alpha-MSH analogue #6(sequence 2*) TNF-α that both dose-dependant ground reduces LPS induction in human leukocyte suspension accumulates.Surprisingly, alpha-MSH analogue #6(sequence 2*) inhibition more remarkable than the antiphlogistic effects of native peptides α-MSH.

The TNF-α of LPS induction in rat body is suppressed to produce

In the process of intravenous injection LPS, α-MSH and alpha-MSH analogue #6(sequence 2*) the TNF-α that both reduces rat accumulates.Under the dosage of 200 μ g/kg body weight, obtain α-MSH and alpha-MSH analogue #6(sequence 2*) maximum suppression effect.Surprisingly, alpha-MSH analogue #6(sequence 2*) inhibition 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 3* is acetylation at N-end and is amidated at C-end)

By replacing Val in site 19 with (D-Val) chemical stereo, make alpha-MSH analogue #7(sequence 3) be different from alpha-MSH analogue #1(sequence 1*).

This compound is tested in experimental program 1 and 2.

α-MSH and alpha-MSH analogue #7(sequence 3*) TNF-α that both dose-dependant ground reduces LPS induction in human leukocyte suspension accumulates.Surprisingly, alpha-MSH analogue #7(sequence 3*) inhibition more remarkable than the antiphlogistic effects of native peptides α-MSH.

The TNF-α of LPS induction in rat body is suppressed to produce

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

Accompanying drawing explanation

In Fig. 1 human leukocyte suspension, the TNF-α of LPS induction accumulates

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

The TNF-α of Fig. 2 plasma levels of LPS induction accumulates

The figure illustrates alpha-MSH analogue #1(sequence 1* in experimental program 2) the maximum antiphlogistic effects of (MCA#1).Maximum suppression effect to the TNF-α production that rat LPS induces is obtained under intravenous injection 200 μ g/kg body weights for α-MSH and MCA#1.Mean number ± SE(is N=4-6 in group often).*: to vehicle group p<0.05, #: p<0.05 is organized to α-MSH.

Fig. 3 eosinophilic granulocyte

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

Fig. 4 Ppa pulmonary artery pressure

The figure illustrates alpha-MSH analogue #1(sequence 1*) (MCA#1) effect of porcine pulmonary artery pressure change that LPS is induced.Mean number ± SE(is N=6-9 in group often).*: different from carrier.

The infarct size of 3 hours after Fig. 5 Reperfu-sion

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

Fig. 6 is the LVEDP of two weeks after 60min LAD blocks

The figure illustrates alpha-MSH analogue #1(sequence 1* in experimental program 4) (MCA#1) to infarct after the provide protection of congestive heart failure.The effect of MCA#1 is obtained by 200 μ g/kg body weight.Mean number ± SE(is N=6-9 in group often).*: 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 1* in experimental program 5) (MCA#1) to ischemic after polyuria development provide protection.The effect of 200 μ g/kg body weight acquisition MCA#1 is given by intravenous injection.Mean number ± SE(is N=5-7 in group often).*: different from carrier.

In Fig. 8 human leukocyte suspension, the TNF-α of LPS induction accumulates

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

The TNF-α of Fig. 9 plasma levels of LPS induction accumulates

The figure illustrates alpha-MSH analogue #2(sequence 5* in experimental program 2) the maximum antiphlogistic effects of (MCA#2).Maximum suppression effect to the TNF-α production that rat LPS induces is obtained under intravenous injection 200 μ g/kg body weights for α-MSH and MCA#2.Mean number ± SE(is N=4-6 in group often).*: to vehicle group p<0.05, #: p<0.05 is organized to α-MSH.

Figure 10 eosinophilic granulocyte

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

Figure 11 neutrophilic granulocyte

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

The infarct size of 3 hours after Figure 12 Reperfu-sion

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

In Figure 13 human leukocyte suspension, the TNF-α of LPS induction accumulates

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

The TNF-α of Figure 14 plasma levels of LPS induction accumulates

The figure illustrates alpha-MSH analogue #3(sequence 9* in experimental program 2) the maximum antiphlogistic effects of (MCA#3).Maximum suppression effect to the TNF-α production that rat LPS induces is obtained under intravenous injection 200 μ g/kg body weights for α-MSH and MCA#3.Mean number ± SE(is N=4-6 in group often).*: to vehicle group p<0.05, #: p<0.05 is organized to α-MSH.

The infarct size of 3 hours after Figure 15 Reperfu-sion

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

The TNF-α of Figure 16 plasma levels of LPS induction accumulates

The figure illustrates alpha-MSH analogue #4(sequence 13* in experimental program 2) the maximum antiphlogistic effects of (MCA#4).Maximum suppression effect to the TNF-α production that rat LPS induces is obtained under intravenous injection 200 μ g/kg body weights for α-MSH and MCA#4.Mean number ± SE(is N=4-6 in group often).*: to vehicle group p<0.05, #: p<0.05 is organized to α-MSH.

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Claims

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

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

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

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

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

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

Lys-Lys-Lys-Lys-Lys-Lys-Ser-Tyr-Ser-Nle-Glu-His-D-Nal-Ar g-Trp-Gly-Lys-Pro-Val (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 activating and be selected from one or more melanocortin receptors of 1,3,4 and 5 type melanocortin receptors,

3. method according to claim 2, wherein, described peptide is made up of following sequence:

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

4. method according to claim 2, wherein, described peptide is made up of following sequence:

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

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

6. method according to claim 5, wherein, the resin of described method using function, the resin of described functionalization is selected from the group be made up of the polystyrene of polystyrene, polyacrylamide, polydimethylacrylamiin, polyoxyethylene glycol, Mierocrystalline cellulose, polyethylene, polyoxyethylene glycol grafting, latex and Dai Nuo immunomagnetic beads.

7. method according to claim 6, wherein, is excised described peptide from solid support material by acid.

8. method according to claim 6, wherein, is excised described peptide from solid support material by alkali.

9. according to the method in claim 1-4 described in any one, wherein, described peptide is prepared by solution synthetic method or recombinant DNA technology.

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

A (), under the condition allowing described peptide to produce, cultivates the recombinant host cell of the nucleotide sequence containing coding for said peptides, and

B () is separated described peptide from described culture.

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

A the nucleotide sequence of coding for said peptides is transferred in host cell by (),

B () cultivates described host cell, and

C () is separated described peptide from described culture.