CN1264858C - Angiogenesis-inhibitory tripeptides, compositions and their methods of use - Google Patents

Abstract

This invention pertains to methods and compositions for inhibiting endothelial cell tube formation, the initial step of tumor angiogenesis. More specifically, the present invention relates to tripeptides that show inhibition of angiogenesis-mediated processes. The most preferred amino acid sequences are SNS and SQS.

Description

Angiogenesis-inhibitory tripeptides, compoistion and method of use

Invention field

The present invention relates to suppress animal and human's endotheliocyte pipe and form, the initial step of tumor-blood-vessel growth and tissue blood vessel generate the compoistion and method of use of dependence disease.More particularly, the present invention relates to show the process that suppresses the vasculogenesis mediation such as the tripeptides of cancer, eye neovascularization and inflammatory diseases, its analogue, stand-in and chemical derivative.Disclosed anti-angiogenic agent also can be united use with operation, chemotherapy, radiotherapy and laser therapy.

Background of invention

Vasculogenesis is that the blood vessel by preexist develops into neovascularity (Mousa, S.A., InAngiogenesis Inhibitors and Stimulators:PotentialTherapeutic Implications, Landes Bioscience, Georgetown, TX; Chapter 1, (2000)).From physiology, vasculogenesis is guaranteed ripe biological suitable growth, and the uterus of the preparation implantation of ovum also plays a crucial role in wound healing.On the other hand, vasculogenesis support and a lot of morbid states such as the relevant pathologic condition of cancer, inflammation and illness in eye.

In embryo's generation or normal and the pathological angiogenesis generative process, the growth of blood vessel network depends on interaction (Breier etc., the Trends inCell Biology 6:454-456 (1996) between somatomedin and cell and the extracellular matrix; Folkman, Nature Medicine1:27-31 (1995); Risau, Nature 386:671-674 (1997)).In embryo's generating process, blood vessel occurs through two processes: blood vessel takes place and vasculogenesis (Blood etc., Bioch.Biophys.Acta 1032:89-118 (1990)).Vascular endothelial growth factor (VEGF), bFGF, IL-8 is some somatomedins (Folkman etc., Science 235:442-447 (1987)) that work in the relevant pathological angiogenesis of noumenal tumour, diabetic retinopathy and rheumatoid arthritis generates with TNF-α.In adult or mature tissue, lack vasculogenesis usually, although there be (Moses etc., Science 248:1408-1410 (1990)) in it really in wound healing and embryo's generation.

Vasculogenesis or " neovascularization " are the preceding multistep process of controlling with balance anti-angiogenesis.The later stage of this process comprises that endotheliocyte (EC) hyperplasia and machine turn to the pipe spline structure.Think that somatomedin such as FGF2 and VEGF are the key substance that promotes endothelial cell growth and differentiation.Endotheliocyte is the key component in the angiogenesis and by its cell surface receptor and intracellular signal transduction mechanism a lot of cytokines is made a response.Endotheliocyte in the culture can form the pipe spline structure with chamber.It seems that therefore, endotheliocyte is not only the prerequisite of neovascularization, and also be the basic structure needs.

Angiogenesis-dependent disease comprises following: inflammatory is disorderly as immune and non-immune inflammation, rheumatoid arthritis, psoriasis; Eye is disorderly as diabetic retinopathy, neovascularization glaucoma, retinopathy of prematurity, relevant macular degeneration of age, corneal graft rejection; With cancer associated disorders such as entity onch-tumor metastasis kitchen range with come from the tumour of blood such as leukemia, hemangiofibroma, Kaposi sarcoma, innocent tumour, and need neovascularization to support other cancer of tumor growth.

The inhibition that has proposed vasculogenesis will be effective therapy of restriction tumor growth.The inhibition of vasculogenesis can be finished the reaction of vasculogenesis stimulator by suppressing endotheliocyte, as Folkman etc., (Cancer Biology 3:89-96 (1992)) advises, wherein described example such as angiostatic steroid, the product that derives from fungi such as fumidil, platelet factor 4, glycoprotein G, alpha-interferon, novel vitamin D analogues and the Beracilline of those endotheliocyte reaction suppressors.For the angiogenesis inhibitor of other proposition, see Blood etc., Bioch.Biophys.Acta 1032:89-118 (1990); Moses etc., Science248:1408-1410 (1990); With United States Patent (USP) 5,092,885,5,112,946,5,192,744 and 5,202,352.

1997, Kefalides and co-worker thereof have reported that at the joint tissue of the University of Pennsylvania research institute medical board peptide of the residue sequence 185-203 of noncollagenous 1 (NC1) structural domain that is equivalent to IV type basilar membrane collagen α 3 chains suppresses the polymorphonuclear leukocyte (activation (Han etc., J.Biol.Chem.272:20395-20401 (1997)) of PMN ' s).Peptide α 3 (IV) the residue 185-203 that discovery has a sequence C NYYSNSYSFWLASLNPER (SEQ ID NO:1) promotes the human melanoma cell to adhere to surpass contrast 50-60%, and the hyperplasia that suppresses them surpasses 40%.The L-Ala displacement that runs through this peptide sequence shows that observed activity depends on the existence of residue 189-191, is called the SNS sequence.Kefalides group had reported afterwards that the IV Collagen Type VI needed the cAMP level to increase (Shahan etc. to the inhibition of melanoma cells, Connective Tissue Res.40:221-232 (1999)), identification CD47/ integrin related protein (IAP) and α v β 3 are as two acceptor (Shahan etc. of melanoma and prostatic cell IV Collagen Type VI α 3 (IV) chain, Cancer Res., 59:4584-4590 (1999)).Recently, they have reported the Ca2+ dependency (Shahan etc. in the tumour cell chemotaxis, J.Biol.Chem.275:4796-4802 (2000)) and the NC1 structural domain of IV Collagen Type VI Expression of Matrix Metalloproteinases and activatory are suppressed (Pasco etc., CancerRes.60:467-473 (2000)).

Independently, Kalluri ' s group from Harvard University medical science school has also been reported the external and in vivo test of use, the characteristic of two kinds of dissimilar anti-tumor activities (anti-hyperplasia and angiogenesis inhibitor) of α 3 (IV) NC1 structural domain (Maeshima etc., J.Biol.Chem.275:21340-21348 (2000)).In a word, these reports have effectively been given prominence to difference and the unique antitumor performance and the potential purposes as small molecules antitumor drug design lead thereof of α 3 (IV) NC1 structural domain.

What these reports result lacked is the evaluation that keeps the active less identification epi-position of larger peptide.Therefore, problem to be solved provides for the evaluation of the little identification epi-position that becomes decisive step in the template is provided towards the medicinal design strategy based on structure of small molecules analogue or peptide mimics.This small molecules will comprise cyclic peptide of intervening angiogenesis-dependent disease such as cancer and the peptide isostere with better physiochemistry and pharmacokinetics performance.

Summary of the invention

The present invention relates to the angiogenesis-inhibitory tripeptides of general formula aa1-aa2-aa3, have first amino acid (aa1), second amino acid (aa2) and the 3rd amino acid (aa3), wherein,

(a) described first amino acid is selected from Ser, Thr, Ala, Phe, Tyr, Cys, Gly, Leu, Lys, Pro, Arg, Gln, Glu, Asp, Asn, His, Met, Ile, Trp, Val, the group that diaminopropionic acid (diaminoproprionic acid) and anti--4-hydroxyl-proline(Pro) are formed;

(b) described second amino acid is selected from Asn, Ala, Gly, Asp, Glu, Gln, the group that diaminopropionic acid and anti--4-hydroxyl-proline(Pro) are formed; With

(c) described the 3rd amino acid is selected from Ser, Thr, Ala, Phe, Tyr, Cys, Gly, Leu, Lys, Pro, Arg, Gin, Glu, Asp, Asn, his, met, Ile, Trp, Val, the group that diaminopropionic acid and anti--4-hydroxyl-proline(Pro) are formed.

The method that tripeptides suppresses vasculogenesis that gives also is provided.

The accompanying drawing summary

Following accompanying drawing is the illustration of embodiment of the present invention, does not limit the scope of the present invention that claim comprises.

Fig. 1 has illustrated SNS and has been suppressed to the effect in forming of fibroblast growth factor basis (FGF2) inductive human endothelial cell pipe.

Fig. 2 has illustrated in CAM (CAM) model, the effect of SNS in suppressing FGF2 inductive vasculogenesis.

Fig. 3 has illustrated in the presence of SNS, the inhibition per-cent of TSU-Pr (human prostate) tumor growth.

Fig. 4 has illustrated the blood vessel formation against function of SNS tripeptide analog thing in formation of human endothelial cell pipe and CAM model.

Fig. 5 has illustrated the blood vessel formation against function of SNS tripeptide analog thing in the CAM model.

Detailed Description Of The Invention

The applicant suppresses the little identification epi-position that the endothelial cell pipe formed and showed inhibition angiogenesis-mediated process by providing, three amino acid residue peptides, and namely tripeptides has solved the problem of statement. The using method of tripeptides also is provided.

Term used herein " tripeptides " means to have the peptide of three amino acid residues, and comprises any analog, peptide mimics and chemical derivative discussed here.

Term used herein " peptide " means covalent bond two or more amino acid residues together. Peptide of the present invention comprises having hundreds of or the polypeptide of amino acids residue more. Usually, the covalent bond between two or more amino acid residues is amido link. Yet amino acid can link together by known to the skilled various other methods of peptide and chemical field. Therefore, term " peptide " is intended to comprise all or part of molecule that contains non-amido link between amino acid, amino acid analogue and the analogies. Similarly, this term also comprises cyclic peptide and other conformation restraining structure.

SNS and SQS tripeptides

Tripeptides of the present invention has at tissue, and three amino acid residues of angiogenesis inhibiting activity are provided in animal and the individuality.

The feature of a preferred tripeptides of the present invention is sequence Ser-Asn-Serine, and structure 1 is described, and also represents with Ser-Asn-Ser, also is abbreviated as the single-letter amino acid code, is called " SNS " here.Preferred amino acids be levorotatory form and with acetyl blocked N-terminal and carboxamide end-blocking C-terminal.

(structure 1)

Another preferred embodiment of the present invention is Ser-Gln-Ser, and also being abbreviated as the single-letter amino acid code is SQS, wherein amino acid be levorotatory form and with acetyl blocked N-terminal and carboxamide end-blocking C-terminal.

The other embodiment of tripeptides of the present invention is analogue, peptide mimics and the chemical derivative that tripeptides is contained be not the Ser-Asn-Ser of natural other chemical part that has a normal part of albumen or modified amino acid, as will further discussing here.

Term " end-blocking " is meant on tripeptides amino or C-terminal and adds group.The end of preferred tripeptides of the present invention by with the amino bonded ethanoyl of the N-terminal (CH of tripeptides of the present invention ₃CO-) and with C-terminal carboxyl bonded amido (NH ₂), it also is abbreviated as " carboxamide ", sealing or " end-blocking ".This tripeptides can be by any other group end capping.

In this disclosure, use abbreviation in a large number.Following definition is provided.

" endotheliocyte " is abbreviated as EC.

" fibroblast growth factor basis " is abbreviated as FGF2.

" CAM " is abbreviated as CAM.

" vascular endothelial growth factor " is abbreviated as VEGF.

" anti--the 4-oxyproline " is abbreviated as t4Hyp.

" I-hydroxybenzotriazole " is abbreviated as HOBt.

" 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl-uronium phosphofluoric acid " is abbreviated as HBTU.

" diisopropyl ethyl amine " is abbreviated as DIEA.

" dimethyl formamide " is abbreviated as DMF.

" trifluoracetic acid " is abbreviated as TFA.

" tri isopropyl silane " is abbreviated as TIS.

" Human umbilical vein endothelial cells " is abbreviated as HUVEC.

" endothelial cell growth substratum " is abbreviated as EGM.

" endothelial basal medium " is abbreviated as EBM.

" bovine serum albumin " is abbreviated as BS.

" 9-fluorenyl methyl oxygen carbonyl " is abbreviated as FMOC.

The conventional chemical synthetic method

Provide their short length, preferably use solid phase synthesis to prepare tripeptides of the present invention, as Chan etc., (In FMOC Solid Phase Peptide Synthesis:A PracticalApproach, Oxford University Press; Chapter 3 (2000)) describe usually, although known in the art other is equal to chemosynthesis also effective (Merrifield, J.Amer.Chem.Soc.85:2149-54 (1963)).Solid-phase peptide is synthetic can be from the C-terminal of peptide, by a-amino acid and the appropriate resin coupling with protection.Can be connected with chloromethyl resin or with hydroxymethyl resin by the amino acid of ester bond, or be connected with bha resin or mbha resin, prepare this parent material by amido linkage with a-amino acid-protection.BHA and the mbha resin carrier is commercial obtains only uses when C-terminal has non-displacement acid amides when synthetic expectation polypeptide usually.

Can use technology well known in the art that the peptide chain of amino acid and growth is connected coupling formation peptide bond.For example, method comprises and changes amino acid into derivative that the easier amino reaction of free N-terminal with growthing peptide chain of amino acid whose carboxyl will be provided.Specifically be, by with the reaction of ethyl chloroformate, Phenyl Chloroformate 99, sec-butyl chloride manthanoate, isobutyl chloroformate or trimethyl-acetyl chloride or other similar chlorination acid, the amino acid whose C-terminal of protection can change mixed acid anhydride into.Alternatively, amino acid whose C-terminal can change active ester into, as 2,4, and 5-trichlorine phenyl ester, pentachlorobenzene phenolic ester, penta fluoro benzene phenolic ester, p-nitrophenyl ester, N-hydroxy-succinamide ester or the ester that forms by I-hydroxybenzotriazole.Another coupling method comprises the use suitable coupling agents, as N, and N '-dicyclohexylcarbodiimide or N, N '-DIC.

Each the amino acid whose alpha-amino group that uses during peptide is synthetic should be protected in linked reaction, prevents to relate to the side reaction of their active alpha-amino-functional.Some amino acid contain the reactive side chain functional group (as; sulfydryl, amino, carboxyl and hydroxyl); this functional group also should get up with suitable protectiveness radical protection, prevents the chemical reaction in reaction side chain site in (1) alpha-amino group site or the follow-up coupling step of (2) initial sum.

In the selection of the specific protectiveness group that is ready to use in synthetic peptide, the typical case follows following general rule.Specifically be; be inertia under the condition that alpha-amino group protectiveness group (1) should make the alpha-amino group official to use in linked reaction; (2) after linked reaction; do not removing the side chain protected group and do not changing under the condition of peptide fragment structure; should remove easily and (3) time should reduce the possibility of racemization greatly in activation before coupling.

On the other hand; be inertia under the condition that side chain protected group (1) should make the side chain functional group use in linked reaction; (2) under the condition of removing alpha-amino group protectiveness group; should stablize; (3) under the condition that does not change peptide chain structure, should from the complete secretory piece of expectation, remove easily.

To those skilled in the art, known reactivity for the synthetic effective protectiveness group of peptide and the reagent that is used to remove them changes clearly.For example, some protectiveness group, as trityl and 2-(p-biphenyl) sec.-propyl oxygen carbonyl, very unstable and under solutions of weak acidity, can be ruptured.Other protectiveness group, unstable slightly and remove them and need suitable intensity acid as t-butyl oxygen carbonyl (BOC), t-amyl group oxygen carbonyl, adamantyl-oxygen carbonyl and p-methoxyphenyl oxygen carbonyl, as trifluoracetic acid, hydrochloric acid, three fluoroboric acid in acetic acid.Still have other protectiveness group; as benzyloxycarbonyl (CBZ), halogen benzyloxycarbonyl, p-nitro carbobenzoxy-(Cbz) and sec.-propyl oxygen carbonyl even unstable slightly and remove them even need more strong acid, as hydrogen fluoride, hydrogen bromide or trifluoracetic acid boron in trifluoracetic acid.

In the classification of the amino acid protectiveness group of effectively protecting alpha-amino group or protection side-chain radical, comprise following:

(1) for alpha-amino group, the protectiveness group of three quasi-representatives is:

(a) fragrant urethane type protectiveness group, as fluorenyl methoxy carbonyl (FMOC), the CBZ of CBZ and replacement is as p-chlorine benzyloxycarbonyl, p-nitro carbobenzoxy-(Cbz), p-bromo-benzyloxycarbonyl and p-methoxy carbobenzoxy-(Cbz), o-chlorine benzyloxycarbonyl, 2,4-dichloro-benzyloxy carbonyl, 2,6-dichloro-benzyloxy carbonyl, or the like;

(b) fatty urethane type protectiveness group, as BOC, t-amyl group oxygen carbonyl, sec.-propyl oxygen carbonyl, 2-(p-biphenyl) sec.-propyl oxygen carbonyl, allyloxy carbonyl or the like; With

(c) cycloalkyl urethane type protectiveness group, as cyclopentyl oxygen carbonyl, adamantyl oxygen carbonyl and cyclohexyl oxygen carbonyl.

Preferred alpha-amino group protectiveness group is BOC and FMOC.

(2) for the side chain amino that exists among the Lys, can be with any radical protection of mentioning in top (1), as BOC, 2-chlorine benzyloxycarbonyl or the like.

(3) for the guanidine radicals of Arg, nitro, tosyl group, CBZ, adamantyl oxygen carbonyl, 2,2,5,7,8-pentamethyl-benzo dihydropyrane-6-alkylsulfonyl, 2,3,6-trimethylammonium-4-methoxyphenyl alkylsulfonyl or BOC can provide protection.

(4) for the hydroxyl of Ser or Thr, for example t-butyl, benzyl (BZL); Or the BZL that replaces, as p-methoxybenzyl, p-nitrobenzyl, p-benzyl chloride base, o-benzyl chloride base and 2, the 6-dichloro benzyl can be protected.

(5) for the carboxyl of Asp or Glu, for example use and to protect as group esterifications such as BZL, t-butyl, cyclohexyl, cyclopentyl.

(6) for the imidazoles nitrogen of His, benzyloxymethyl (BOM) or tolylsulfonyl base section are suitable as the protectiveness group.

(7), be fit to use protectiveness group such as THP trtrahydropyranyl, the tertiary butyl, trityl, BZL, toluene(mono)chloride, 4-bromobenzyl and 2, the 6-toluene dichloride for the phenolic hydroxyl group of Tyr.Preferred protectiveness group is a bromobenzyl oxygen carbonyl.

(8), preferably use xanthyl (Xan) for the side chain amino of Asn or Gln.

(9) for Met, the preferred amino acid left side is not protected.

(10) for the sulfenyl of Cys, the typical case uses the p-methoxybenzyl.

Suitably select protectiveness group such as BOC typically at first amino acid of C-terminal of alpha-amino group guarded by location growthing peptide chain, as Glu.Can use the sec.-propyl carbodiimide, 25 ℃ at first with BOC-Glu-(γ-cyclohexyl)-OH and the coupling of benzyl hydrogen polyimide resin two hours, stir simultaneously, or with the chloromethyl resin coupling.Behind the amino acid and resin carrier coupling of BOC protection, remove alpha-amino group protectiveness group usually, typically use the solution of trifluoracetic acid (TFA) in methylene dichloride or independent TFA.The alpha-amino group protective reaction can take place under the wide region temperature, but usually implements under the temperature between about 0 ℃ and the room temperature.

The alpha-amino group of other standard removes to protect reagent, as HCl in dioxane and the condition of removing special alpha-amino group protectiveness group belong to the state of the art scope.After removing alpha-amino group protectiveness group, unprotected alpha-amino group, side chain is still protected usually, coupling in the plan sequence in a step-wise fashion.

The amino acid of each protection or aminoacid sequence import solid phase reactor to surpass stoichiometrical amount usually, are coupled in organic solvent such as dimethyl formamide (DMF), methylene dichloride or its mixture and suitably carry out.If incomplete coupling takes place, preparing and next amino acid coupling, before the removal N-amido protecting group, repeat coupling step usually.After alpha-amino group protectiveness group is removed, the coupling in the plan sequence in a step-wise fashion of the amino acid of last alpha-amino group and side chain protected.Can monitor the whether success of linked reaction in synthetic each stage.Monitoring synthetic preferred method is to pass through ninhydrin reaction.Also can use business method and the device known, for example Beckman 990 peptide synthesizers automatically perform linked reaction.

After the expectation peptide sequence is finished, should should remove all protectiveness groups from the peptide of resin carrier fracture protection.The removal of cleavage reaction and protectiveness group is suitably worked in coordination with or is finished continuously with protective reaction.When peptide and resin fixed key were ester bond, it can be interrupted ester bond and be penetrated any reagent fracture of resin medium.A special effective means is to handle with anhydrous liquid hydrogen fluoride.This reagent not only can also can be removed all acid labile protectiveness groups from the resin peptide that ruptures usually, therefore, will directly provide and remove to protect peptide fully.When existing when not being the other protectiveness group of acid labile, should implement other going and protect step.According to concrete needs and environment, can before or after above-mentioned hydrogen fluoride trealment, carry out these steps.

When using chloromethyl resin, hydrogen fluoride fracture/protective reaction causes free peptide acid to form usually.When using the benzhydrylamine resin, hydrogen fluoride trealment produces the free peptide acid amides usually.In the presence of methyl-phenoxide and imethylsulfide, 0 ℃ with hydrogen fluoride reaction one hour, typically can remove the side chain protected group, peptide discharges from resin.

Do not remove the peptide that ruptures under the situation of protectiveness group when expectation, the peptide-resin of protection can be accepted methanolysis, therefore produces the peptide of the methylated protection of C-terminal carboxyl.Can obtain free C-terminal carboxyl with this methyl ester of posthydrolysis under weak basic condition.Follow available strong acid treatment,, remove the protectiveness group on the peptide chain as liquid hydrogen fluoride.The special effective technology of methanolysis is wherein in the presence of crown ether, with the peptide-resin of methyl alcohol and potassium cyanide processing protection.

When using chloromethyl resin, comprise that from other method of the peptide of resin fracture protection (1) ammonia is separated and (2) hydrazinolysis.If expectation, gained C-terminal acid amides or hydrazides hydrolyzable are free C-terminal carboxy moiety, and the protectiveness group can be removed as usual.The protectiveness group that exists on the N-terminal alpha-amino group can be removed before or after the carrier fracture at protective peptide.

The purifying of peptide of the present invention typically uses chromatographic technique to finish, as preparation HPLC (comprising reversed-phase HPLC), gel infiltration, ion-exchange, partition chromatography, affinity chromatography (comprising the monoclonal anti scapus) or the like, or other routine techniques such as adverse current distribution or the like.

Also can use recombinant DNA technology to prepare SNS tripeptides of the present invention.

Amino acid analogue

Term used herein " amino acid " means the amino acid of natural existence and non-natural existence, and amino acid analogue and stand-in.Naturally occurring amino acid comprises 20 (L)-amino acid utilizing in the protein biology building-up process and other, as, 4-oxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and orthinine.The amino acid that non-natural exists comprises, for example, and (D)-amino acid, nor-leucine, norvaline, p-fluorophenylalanine, ethionine or the like.

Amino acid used herein " analogue " or " peptide analogs " comprise that there is amino acid in the natural and non-natural of modified forms.This modification can comprise, for example, and displacement or the replacement or amino acid whose the deriving of chemical group and part on the amino acid.

What comprise among the present invention is tripeptides, and wherein at least one amino-acid residue is different with general formula (1) tripeptides (Ser-Asn-Ser).Detailed description for albumen chemistry and structure is seen Schulz, G.E. etc., In Principles of Protein Structure, Springer-Verlag, New York, 1979, and Creighton, T.E., InProteins:Structure and Molecular Principles, W.H.Freeman﹠amp; Co., San Francisco, 1984.The displacement type of carrying out in general formula of the present invention (1) tripeptides (Ser-Asn-Ser) can be a preservative replacement, is defined as follows the exchange in one of group here.Yet, the invention is not restricted to these displacements.

(1) D-amino-acid substitution L-amino acid

(2) little aliphatics, nonpolar or low-pole residue: as, Ala, Ser, Thr, Gly;

(3) polarity, negative charge residue and acid amides thereof: as, Asp, Asn, Glu, Gln;

(4) polarity, positive charge residue: as, His, Arg, Lys;

Even its accurate effect of precognition is very difficult before displacement, and those skilled in the art will figure out available conventional shaker test and estimate this effect, preferred following biological test.The modification of the method detection of peptides performance of knowing with those of ordinary skills comprises the susceptibility of redoxomorphism or thermostability, hydrophobicity, proteolytic degradation or with the gathering of carrier or assemble and be polymeric tendency.

Those skilled in the art will know that and to determine that maybe what structure composition function is equal to amino acid analogue.

Chemical derivative

It is not the other chemical part of a tripeptides part that " chemical derivative " of tripeptides of the present invention used herein contains normally.The covalent modification of tripeptides comprises within the scope of the invention.This modification can be by peptide the target amino acid residue with can introduce in the molecule with organic derivation agent reaction of selected side chain or terminal residue reaction.

Preferably use ethanoyl (CH ₃CO-) sealing or " end-blocking " amino and C-terminal are with N-terminal N and amido (NH ₂) combine with the C-terminal carboxyl; Also be abbreviated as " carboxamide ", but can be easily by other any group end capping.The correct selection of capping group allows other promoting agent to add to peptide.For example, the existence of the sulfydryl that is connected with N or C-terminal cap will allow combining of derived peptide and other molecule.Some N-terminal capping groups are the compounds that are selected from the group of ethanoyl, benzoyl, alkyl sulphonyl, aryl sulfonyl, alkylamino acyl group, arylamino acyl group, formyl radical, peptide and polymkeric substance composition.The C-terminal examples of groups is to be selected from NH ₂, the group formed of OH and NHR compound, wherein R is selected from the group that alkyl, aromatic base, peptide and polymkeric substance are formed.

End capped peptide is natural (L amino acid configuration) not example of the preferred chemical derivative of end-blocking peptide.The combination of analogue or chemical derivative above can be with any capping group end-blocking disclosed herein any.

Amino acid and peptide mimics

" amino acid analog thing " used herein for example comprises, the Presentation Function similar performance is as the organic structure with reference to amino acid whose electric charge and electric charge spaced features.Stand-in also comprise feasible optimal spacing and the coulombic interaction of keeping amino acid or amino-acid functional group of restraining structure.Those skilled in the art will know that and to determine that maybe what structure composition function is equal to the amino acid analog thing.

" peptide mimics " used herein or peptide mimics be keep with corresponding peptides in the organic molecule of the similar peptide chain pharmacophoric group that exists.Peptide mimics also can be the function equivalent of particular peptide.SNS tripeptides of the present invention can divide into groups based on the pharmacophore performance.Term used herein " pharmacophore " definition is that compound produces specific reaction or has the particular arrangement of the required functional group of expectation activity.

Preferred peptide stand-in compound of the present invention is the compound of end-blocking or not end capped simulation SNS biological action.Peptide mimics reagent can be that non-natural exists peptide (D amino acid configuration) or end-blocking or not end capped non-peptide reagent with stereochemistry performance of tripeptides of the present invention, make it have end-blocking or not end capped tripeptides of the present invention in conjunction with activity or biological activity.

The peptide mimics compound of large number of biological bioactive peptide has been described, agonist, substrate or inhibitor are as opioid peptides, VIP, zymoplasm, hiv protease etc.Design and the method for preparing the peptide mimics analogue are (Kempf, D.J.Methods Enzymol.241:334-354 (1994) known in the art; Hruby, V.J., Biopolymers 33:1073-82 (1993); Wiley etc., Med.Res.Rev.13:327-384 (1993); Claeson, G., Blood Coagul.Fibrinolysis 5:411-436 (1994)).These methods can be used to prepare end-blocking or not end capped peptide mimics, and it has the binding ability of tripeptides and specificity and preferred also biologically active at least.Available chemistry of peptides of those skilled in the art and general vitochemical knowledge design and detect this compound.

For example, the three-dimensional structure of check crystallography deutero-peptide of the present invention can be identified this peptide mimics.Alternatively, can obtain structure with the tripeptides of the present invention of its receptors bind with the NMR (Nuclear Magnetic Resonance) spectroscopy technology.For example, the stereochemical better knowledge of end-blocking or not end capped SNS and its acceptor interaction will allow this peptide mimics reagent of appropriate design.

Vasculogenesis and angiogenesis-dependent disease

Term used herein " vasculogenesis inhibition ", " vasculogenesis inhibition " or " angiogenesis inhibitor " comprise that blood vessel takes place, and means the reduction of the degree, amount or the speed that cause neovascularization.Cause that it is the specific examples that suppresses vasculogenesis that endotheli ocytosis or the degree of dividing a word with a hyphen at the end of a line, amount or speed reduce in tissue.

Term " vasculogenesis inhibition composition " is meant and suppresses the vasculogenesis mediated process such as endotheliocyte is divided a word with a hyphen at the end of a line, hyperplasia, pipe form and suppress neovascularity subsequently and produced by existing blood vessel, so the composition of inhibition angiogenesis-dependent disease.

Term used herein " angiogenesis-dependent disease " means vasculogenesis or the blood vessel generating process is supported or the disease of increase pathologic condition.Vasculogenesis is by preceding existing kapillary or high endothelials venules to form neovascularity.Blood vessel takes place to be produced and cause from endotheliocyte precursor-angioblast by neovascularity.Two processes cause neovascularization and are included in the meaning of term angiogenesis-dependent disease.Similarly, term used herein " vasculogenesis " is intended to comprise that vascular forms again as take place producing from blood vessel and from existing vascular, kapillary and venular branch and the generation of sprouting.

Vasculogenesis comprises that blood vessel takes place, and is the important physical process, does not have it, and fetal development and wound healing will can not take place.Yet, vasculogenesis also as a kind of give influence in-house cell and enough blood and nutrition supply be provided and cause a large amount of pathologic conditions inadequately.A lot of these pathologic conditions comprise abnormal cells hyperplasia or adjustment.It is believed that vasculogenesis is known as angiogenesis-dependent disease here to its important this illness.Yet method of the present invention also can usefully be used to suppress the relevant normal physiological processes of vasculogenesis.For example, the inhibition of the menstrual cycle that vasculogenesis is relevant can preventative effective ways as birth control.Therefore, the following description about the angiogenesis-dependent disease treatment is applicable to that also inhibition existence prevention or treatment need or the normal blood vessels formation reaction of benefit.

Angiogenesis-dependent disease comprises that for example, inflammatory is disorderly as immunity and non-immune inflammation, rheumatoid arthritis, chronic rheumatic arthritis and psoriasis; Inappropriate or kapillary hyperplasia and osteoporosis in vascular infringement associated disorders such as diabetic retinopathy, neovascularization glaucoma, retinopathy of prematurity, macular degeneration, corneal graft rejection, retrolental fibroplasia, flush, the atherosclerotic plaque unfortunately; With the cancer associated disorders, comprise for example solid tumor, metastases kitchen range and come from the tumour of blood vessel such as leukemia, hemangiofibroma, Kaposi sarcoma, innocent tumour such as vascular tumor, acoustic tumor, neurofibroma, trachoma, with the gathering rheumatic granulomas, and need neovascularization to support other cancer of tumor growth.The other example of angiogenesis-dependent disease for example comprises, Osler-Webber syndrome; Angiogenesis of cardiac muscle; Plaque neovessels forms; Telangiectasis; Hemophilia joint and wound granulation take place.Other disease that vasculogenesis works in keeping the pathological state progress is well known by persons skilled in the art and is included in similarly in the meaning of term used herein.

The external biological test of angiogenesis inhibiting activity

In external several pilot systems, detect the angiogenesis inhibiting activity of compound of the present invention.Can prepare or the commercial endotheliocyte that obtains, for example, Human umbilical vein endothelial cells (HUVEC) or human microvascular endothelial cell (mvec) (HMVEC) are with 2 * 10 ⁵1: 1 (v/v) ratio of the concentration of individual cell/ml and Fibrinogen (5mg/mL in phosphate-buffered saline (PBS)) mixes.(final concentration 5 units/mL), mixture is transferred to 24 orifice plates (0.5mL/ hole) immediately to add zymoplasm.The permission fibrin gel forms, and then vascular endothelial growth factor (VEGF) and fibroblast growth factor basis (FGF2) add (every hole is with the final concentration of 5ng/mL) in the hand-hole with test compounds.Cell is at 37 ℃, 5%CO ₂In cultivated 4 days, count the cell in each hole and be categorized as circular, that no branch extends, branch extends or 2 or more multiple-limb extension.The result is expressed as average 5 the different holes of compound of each concentration.Typically, in the presence of angiogenesis inhibitor, cell keeps circular or forms differentiation pipe (as, 0 or 1 branch).This area thinks that this test is prediction (Grant etc., the In Vitro CellDev.Biol.27A:327-336 (1991) of angiogenic effect (or angiogenesis inhibiting activity) in the body; Min etc., Cancer Res.56:2428-2433 (1996)).

In alternative test, when endotheliocyte from Becton Dickinson ofBedford, the commercial obtainable Matrigel of PA ^_When cultivating on the matrix coated panel, observe the endotheliocyte pipe and form (Schnaper etc., J.Cell.Physiol.165:107-118 (1995)).Endotheliocyte (1 * 10 ⁴Individual cells/well) transfers to Matrigel ^_Matrix is coated with on 24 orifice plates, and quantity tube forms after 48 hours.When adding endotheliocyte or at each time point thereafter, add inhibitor, it is tested.

This test is by giving the basilar membrane of endotheliocyte particular type, promptly divides a word with a hyphen at the end of a line and hypothallus that the expectation of the endotheliocyte that breaks up may at first be run into imitates vasculogenesis.Except binding growth factor, Matrigel ^_The matrix components that (with in the basilar membrane original position) found in the matrix or its protein hydrolysate also can form by the stimulating endothelial cell pipe, make this model replenish the fibrin gel angiogenesis model.

In addition, estimate the angiogenic activity (Oikawa etc., Cancer Lett.59:57-66 (1991)) of The compounds of this invention with chicken CAM (CAM) test.

Medication

The invention provides the method that suppresses to organize the medium vessels generation by SNS tripeptides, analogue, stand-in or its chemical derivative that gives the vasculogenesis amount of suppression.

The present invention further provides the method that generates dependence disease by SNS tripeptides, analogue or its function equivalent treatment animal blood vessels that gives the vasculogenesis amount of suppression.

The present invention also provides the method that suppresses people's angiogenesis-dependent disease by the SNS tripeptides that gives the vasculogenesis amount of suppression, analogue or its function equivalent.

SNS tripeptides, its analogue, or chemical derivative can have the animal of tumour are determined and the contrast of non-anti-angiogenic peptides is compared, and this peptide is to the inhibition activity or the effect of tumor growth.The reduction of tumor growth rate or degree, or the disappearance of tumour is relevant with the effect of anti-angiogenesis activity and angiogenesis inhibitor dependence disease progress.For the model description of the animal that has tumour, see for example United States Patent (USP) 5,639,725, be incorporated herein by reference here.

Dosage

Any in the above method is determined in the activity and/or effect of SNS tripeptides, analogue or chemical derivative, can give can indicate in special test the peptide that suppresses in the active concentration range known in the art.For example, the concentration of the indicative result's of generation peptide inhibitor is typically about 100-1000ng/ml in the test of ox hair capillary endothelial cells.Similarly, the peptide inhibitor of positive angiogenesis inhibitor produces the peptide inhibitor concentration of positive findings in CAM, is typically about for example 0.5-20 μ g/ml, 10-20 μ g/ plate, and in concentration is the scope of 0.1-100 μ g/ plate, or 25 μ g/ plates.Be desirably in the representative concentration that produces the peptide inhibitor of positive findings in the rabbit corneal test and be typically about 40 μ g/ Hydron grains.At last, the concentration that produces indicative result's peptide inhibitor in metastases kitchen range and above-mentioned carrying animal model for tumour is respectively approximately weekly twice 250 μ g or 10mg/kg/ days, 10 days; With every days 12.5 μ g or 1mg biweekly.Can carry out further selected, for example, by changing the concentration of the SNS tripeptides in the active concentration scope, to determine to suppress the optimum concn or the amount of vasculogenesis.

In addition, above-mentioned model, and other method well known by persons skilled in the art can be used for determining about suppressing or the administration time of SNS tripeptides, analogue, stand-in or the chemical derivative of treatment angiogenesis-dependent disease is selected, the suitable dosage system of administration quantity and each dosage similarly.Similarly, aforesaid method also can conventional be used to prepare and identify new, modification or improved SNS tripeptides, analogue, stand-in or chemical derivative.After having provided instruction described here and having instructed, one skilled in the art will know that the significant quantity that maybe can determine to suppress angiogenesis-dependent or treat SNS tripeptides, analogue, stand-in or the chemical derivative of angiogenesis-dependent disease.

Term used herein " vasculogenesis amount of suppression " mean when organize, animal or when individual, cause that degree, amount or the speed of neovascularization reduce the amount of required SNS tripeptides of the present invention, analogue, stand-in or chemical derivative.The dosage of SNS tripeptides, analogue, stand-in or chemical derivative that treatment is effectively required will depend on angiogenesis-dependent disease for example to be treated, route of administration and form, the usefulness of the molecule that gives and big active transformation period, tissue, animal or individual body weight and situation and former or concurrent treatment.Here the guidance that provides is provided, and those skilled in the art can determine the suitable amount of present method application.For example, can infer this amount from vasculogenesis test in the above-mentioned external or body.Those skilled in the art will recognize that the amount of the patient's that whole therapeutic process need be monitored situation and the composition that is given can correspondingly be adjusted.

For suppressing vasculogenesis or treatment angiogenesis-dependent disease, the peptide of vasculogenesis amount of suppression of the present invention for example can be, between about 10 μ g/kg to 500mg/kg body weight, for example, approximately between the 0.1mg/kg to 100mg/kg, or between the preferably approximately 1mg/kg to 50mg/kg, this depends on the treatment system.For example, if gave one in one day to peptide several times, so with weekly or every month or more do not give peptide continually and compare, then need more low dosage.Similarly, allowing regularly, the preparation of release peptide will provide than the continuous release that gives single bolus dosage peptide more in a small amount.For example, can give peptide with 4mg/kg/ week.

Carry system

SNS tripeptides of the present invention, analogue, stand-in or chemical derivative can be carried by whole body, as intravenously or intra-arterial.SNS tripeptides, analogue, stand-in or chemical derivative also can be at vasculogenesis position topical administrations.The suitable position of the known SNS of the giving tripeptides of those skilled in the art, analogue, stand-in or chemical derivative or can determine according to the clinical indication of the individuality for the treatment of.For example, use the known compound method of those of ordinary skills, have the active SNS tripeptides of above-mentioned inhibition, analogue, stand-in or chemical derivative and can be used as medicine and can accept the albumen of separation in the preparation and basic purifying and protein fragments and insoluble polymer and provide.Can give these preparations with standard way, for example comprise, local, in skin, nose, in the intraperitoneal, encephalic, the cerebrovascular, in the brain, intravaginal, intrauterine, oral, rectum, parenteral (as, intravenously, backbone is interior, subcutaneous or intramuscular) approach.In addition, SNS tripeptides, analogue, stand-in or chemical derivative can mix and allow to continue to discharge in the biodegradable polymer of compound, the implanted expectation of this polymkeric substance is carried near the position of medicine, for example, tumour or implant site make the whole body release in time of SNS tripeptides, analogue, stand-in or chemical derivative.Permeate little pump peptide, analogue, stand-in or chemical derivative and also can be used to provide the controlled delivery of high density SNS tripeptides, analogue, stand-in or chemical derivative, arrive interested position by sleeve pipe, as the growth or the tumor vessel supply of directly arriving metastasis.Biodegradable polymer and uses thereof is at for example Brem etc.:, J.Neurosurg 74:441-446 has a detailed description in (19911, be incorporated herein by reference here).

SNS tripeptides, analogue, stand-in or chemical derivative also can be united with the other treatment compound that is selected from chemotherapy, microbiotic, antiviral, anti-inflammatory, target-seeking compound, cytokine, immunotoxin, anti-tumour antibody, angiogenesis inhibitor, Ivy extract agent, radiosensitizer and composition thereof and given.

The invention provides SNS tripeptides, analogue, stand-in or chemical derivative and medicine and can accept medium and preparation composition together.This composition can suppress vasculogenesis or treatment angiogenesis-dependent disease with in the methods of the invention.For example, SNS tripeptides, analogue, stand-in or chemical derivative can be used as solution or suspension and can accept medium with medicine and give.It for example can be that this medicine can be accepted medium, and water, sodium phosphate buffer, phosphate buffered saline (PBS), common salt solution or Ringer solution or other physiological buffer salt solution or other solvent or carrier such as ethylene glycol, glycerine, oil are as sweet oil or injectable organic ester.

SNS tripeptides, analogue, stand-in or chemical derivative preparation comprise the preparation that those are applicable to parenteral admin, as in subcutaneous, intraperitoneal, intramuscular, intravenously, intradermal, encephalic, the tracheae and epidural administration.And the preparation that is applicable to oral, rectum, eye (comprising in the vitreum or intracameral), nose, part (comprising cheek or hypogloeeis) preparation, intrauterine or vagina administration.SNS tripeptides, analogue, stand-in or chemical derivative can exist and available pharmaceutical technology preparation well known to those skilled in the art with unit dosage form.This technology comprises the step in conjunction with activeconstituents and pharmaceutical carrier or vehicle.SNS tripeptides of the present invention, analogue, stand-in or chemical derivative also can flow to individuality by the coding nucleic acid that gives this peptide and be used to suppress vasculogenesis or treatment angiogenesis-dependent disease.Therefore, the nucleic acid of encode SNS tripeptides of the present invention, analogue, stand-in or chemical derivative can be used to transmit the peptide or the varient of vasculogenesis amount of suppression effectively in conjunction with various gene therapy methods widely known in the art.Here technology and the guidance provide are provided, and the expression that the nucleic acid of encode one or more SNS tripeptides, analogue, stand-in or chemical derivative or its combination can be introduced carrier known in the art or delivery system and be used to transmit encoding sequence is to reach the vasculogenesis amount of suppression.Suitable carrier known in the art and delivery system comprise, for example, retroviral vector, adenovirus carrier, the related virus of adenovirus, part are in conjunction with particle and target-seeking nucleic acid, separated DNA and RNA, liposome, polylysine and cell therapy, comprise the acid anhydride cell therapy, utilize and modified the Transplanted cells of expressing the SNS tripeptides, and various other gene carrying method and improvement well known by persons skilled in the art, as Shea etc., Nature Biotechnol.17:551-559 (1999) is described, is incorporated herein by reference here.

The specific examples of method well known in the art is at for example United States Patent (USP) 5,399,346; United States Patent (USP) 5,580,859; 5,589,466; 5,460,959; 5,656,965; 5,643,578; 5,620,896; 5,460,959; 5,506,125; European patent application EP 0 779 365A2; PCT WO 97/10343; PCT WO 97/09441; Among the PCT WO 97/10343 description is arranged, all introduce here as a reference.Also there are other method well known by persons skilled in the art and also similar being applicable to by expressing SNS tripeptides, analogue, stand-in or the chemical derivative of nucleic acid sequence encoding delivery of vascular generation amount of suppression.

The present invention also relates in the gene therapy method effectively and the nucleic acid and the carrier of the coding that methods known in the art can prepare.Also provide and contained the composition that this nucleic acid, carrier and medicine can be accepted medium.Medicine can be accepted the element that medium should not contain degradable expectation nucleic acid.Use the method for SNS tripeptides, analogue, stand-in or chemical derivative can utilize any various types of SNS tripeptides listed earlier, analogue, stand-in and chemical derivative.

Non-SNS sequence can give peptide structure of the present invention or functional performance.The position that SNS tripeptides, analogue, stand-in or chemical derivative are aimed at the abnormal vascular generation provides the other treatment advantage that peptide is fixed on the pathologic condition position.Therefore, this result supports the peptide of high effective concentration to be diffused into the vasculogenesis zone in time and to allow basically and gives SNS tripeptides, analogue, stand-in or chemical derivative to vasculogenesis position continuous and local.

In addition, two or more SNS tripeptides of the present invention, analogue, stand-in or chemical derivative be can give in the methods of the invention and vasculogenesis or treatment angiogenesis-dependent disease suppressed.Similarly, one or more SNS tripeptides, analogue, stand-in or chemical derivative can be united with one or more SNS tripeptides, analogue, stand-in or chemical derivative and given, and suppress vasculogenesis or angiogenesis-dependent disease.Therefore, in the inventive method, the various combination and permutation of SNS tripeptides, SNS analogue, SNS stand-in and SNS chemical derivative and combination thereof be can give, vasculogenesis and treatment angiogenesis-dependent disease effectively suppressed.

SNS tripeptides, analogue, stand-in and chemical derivative and composition thereof and can alternately give make and make up their angiogenesis suppression action at any time.For example, the SNS analogue can give by single bolus dosage, repeatedly give subsequently one or more independent SNS tripeptides or with SNS tripeptides, analogue, stand-in or chemical derivative combination.Whether simultaneously or Alternating Transportation SNS tripeptides, analogue, stand-in or chemical derivative or its combination, mode of administration can be any aforementioned administration type, and depends on that particular treatment needs and for the effect of the selected SNS tripeptides of purpose, analogue, stand-in or chemical derivative.Determine in the mixture combination or in temporary transient administration system which kind of SNS tripeptides of combination, analogue, stand-in or chemical derivative will depend on angiogenesis-dependent disease and the concrete physical characteristics of the individuality that catches.Those skilled in the art the instruction that provides here are provided and are instructed together with particular blood vessel and generate known diagnosis and clinical criteria in the dependence disease field, can know and maybe can determine effective actual mixt of application-specific or administration system.

Treatment is used

Give in addition method that SNS tripeptides, analogue, stand-in or chemical derivative suppress vasculogenesis or treatment angiogenesis-dependent disease can with the other therapies Joint Implementation.The possible embodiment of the present invention comprises the application with other therapies such as the direct anti entity tumour and this administration of conventional chemical therapy bonded of control metastasis foundation.By promoting vasculogenesis to recover, provide blood supply and nutrition to tumor tissues, tumor tissues should be attacked aitiogenic the time toxicity, typically during chemotherapy or give angiogenesis inhibitor afterwards.In addition, preferably after the noumenal tumour that carries out in order preventing to shift to be removed operation, to carry out this vasculogenesis and suppress step.

The present invention also can unite use with other angiogenesis inhibitor.Angiogenesis inhibitor is currently known methods preparation known in the art and available.For example, angiogenesis inhibitor comprises that integrin inhibition compound such as α υ integrin inhibiting antibody, cell adhesion protein or its contain the function fragment of cell adhesion binding sequence.Other angiogenesis inhibitor for example comprises, other mechanism of prolactin N-terminal fragment, Thalidomide and the inhibition vasculogenesis of the function fragment of angiostatin, angiostatin, endostatin, fibroblast growth factor (FGF) inhibitor, FGF acceptor inhibitor, VEGF inhibitor, vegf receptor inhibitor, blood vessel permeability factor (VPF) inhibitor, VPF acceptor inhibitor, glycoprotein G, platelet factor 4, interferon-alpha, IFN-, interferon-inducible protein 10, interleukin 12, gro-beta and 16kDa.

Angiostatin is the theme of above-mentioned United States Patent (USP) 5,639,725.Endostatin is the theme of above-mentioned PCT publication WO 97/15666.For the residue angiogenesis inhibitor of listing above and the description of target, respectively referring to for example, Chen etc., Cancer Res.55:4230-423 3 (1995); Good etc., Proc.Natl.Acad.Sci.USA 87:6629-6628 (1990); O ' Reilly etc., Cel 179:315-328 (19943; Parangi etc., Proc.Natl.Acad.Sci.USA 93:2002-2007 (1996); Rastinejad etc., Cell56:345-355 (1989); Gupta etc., Proc.Natl.Acad.Sci.USA 92:7799-7803 (1995); Maione etc., Science 247:77-79 (1990): Angiolillo etc., J.Exp.Med.182:155-162 (1995); Strieter etc., Biochem.Biophys.Res.Comm.210:51-57 (1995); Voest etc., J.Natl.Cancer Inst 87:581-586 (1995); Cao etc., J.Exp.Med.182:2069-2077 (1995); Clapp etc., Endocrinology 133:1292-1299 (1993). for the description of other angiogenesis inhibitor, referring to for example Blood etc., Bioch.Biophys Acta., 1032:89-118 (1990); Moses etc., Science, 248:1408-1410 (1990); Ingber etc., Laf Invest., 59:44-51 (1988) and United States Patent (USP) 5,092,885; 5,112,946.

Preparation

The preparation that is fit to administered parenterally comprises that moisture and anhydrous aseptic parenteral solution such as above-mentioned medicine can accept medium.This solution can for example contain in addition, antioxidant, damping fluid, bacteriostatic agent and provide and plan the solute of the isoosmotic preparation of acceptor blood.Other preparation for example comprises, can comprise the moisture and anhydrous sterile suspensions of suspension agent and thickening material.Preparation can be present in single dose or the multi-dose container, and for example, sealing peace is cutd open and bottle, and can be kept at and add immediately under the lyophilize condition of sterile liquid carrier before need for example using.Can be by the interim injection liquid of sterilized powder, particle and tablet preparation and the suspension of aforementioned type.

Medicine can be accepted medium and for example can contain in addition as stable or increase the physiology that SNS tripeptides, analogue, stand-in or chemical derivative absorb and can accept compound.This physiology can be accepted compound and comprise for example carbohydrate such as glucose, sucrose or dextran; Antioxidant such as xitix or Triptide; Sequestrant such as EDTA, its destroy microorganisms film; Divalent-metal ion such as calcium or magnesium; Low molecular weight protein (LMWP); Lipid or liposome; Or other stablizer or vehicle.SNS tripeptides, analogue, stand-in or chemical derivative also can be accepted medium such as biodegradable polymer is prepared with medicine.

Polymeric preparations

What the present invention also comprised is the use that the polymeric preparations medium vessels generates inhibition tripeptides, analogue, stand-in or chemical derivative.Tripeptides can mix medium or make capsule as biomedical material by surface grafting, copolymerization, non-covalent combination.The adjusting of neovascularization and control are the essential parts that obtains keep equilibrated tissue engineering material between preceding angiogenesis inhibitor and the anti-angiogenesis.Tripeptides can be united use with artificial organs material in the support of known vasculogenesis promotor, functional living being medical material such as the implantation of regulating neovascularization in the tissue or repair materials, organizational project, wound healing material, the non-body.Polymeric preparations also can be used for continuing to discharge the angiogenesis inhibitor inhibitory peptide that suppresses neovascularization.This is an example of medicament delivery method, comprises that the angiogenesis inhibitor inhibitory peptide combines with the solid support material that can be used for the blood vessel formation against function of this preparation of localized delivery in angiogenesis-dependent disease.

Embodiment

Generally now described the present invention, will be more readily understood the present invention by reference the following example, embodiment provides in the elaboration mode, is not intended to limit the present invention, unless stated otherwise.

General method

Chemical reagent:

All reagent be chemical grade and available from Sigma Chemical Co. (St.Louis, MO) or by VWR Scientific (Bridgeport NJ) buys.Cortisone acetate, bovine serum albumin (BSA) and gelatin solution (from the 2%B type of ox-hide skin) contain Earl ' s salt, basic FGF, Regular Insulin-Transferrins,iron complexes-selenium G additive (I-T-Se) 100X, contain or do not contain Ca available from SigmaChemical Co.M199 growth medium ^{+ 2}With the Dulbecco ' s phosphate buffered saline(PBS) (PBS) of Mg+2 and 0.5M EDTA available from Gibco BRL (Grand Island, NY).Human umbilical vein endothelial cells (HUVEC), endothelial basal medium (serum-free, EBM), EGM (replenishing somatomedin, foetal calf serum) and 0.025% trypsinase/0.01%EDTA solution derive from Clonetics Inc. (San Diego, CA).Human prostate (TSU-Pr) tumour cell derive from American type culture collection (Rockyille, MD).Matrigel ^_Matrix and human III type collagen available from Becton Dickinson (Bedford, MA).HEMA-3 fixing agent and dyeing solution be available from Biochemical Sciences, and Inc. (Swedesboro, NJ).Chicken oosperm is available from Charles River Laboratories, SPAFAS Avian Products ﹠amp; Services (North Franklin, CT).

Peptide is synthetic:

Use the FMOC chemical process, prepare described all peptides (Chan etc., In FMOC Solid Phase Pepetide Synthesis:A PracticalApproach, Oxford University Press by the standard solid-phase synthetic schemes; Chapter 3 (2000)).The amino acid of solid phase, high capacity PAL-PEG-PS resin and all FMOC protection derive from PerseptiveBiosystems (Framingham, MA).Peptide synthesizes and comprised for three steps:

(1) amino acid coupling: I-hydroxybenzotriazole (HOBt)/2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/diisopropyl ethyl amine (DIEA), the amino acid (4.0 equivalent) of (the 4.0/4.0/8.0 equivalent is based on resin-carried) and FMOC protection is in dimethyl formamide (DMF), and room temperature was carried out 4 hours;

(2) FMOC goes protection: 20%v piperidines in DMF, 1.5 hours;

(3) rupture from resin: 95: 5 trifluoracetic acid (TFA)/tri isopropyl silanes (TIS), carried out 6 hours in room temperature.Grind crude product and from the aqueous solution lyophilize with ethyl ether.With electrospray mass spectrometry prove conclusively all peptides molecular weight (MW=347.32, Obs.M+1=348.23).The structure of N-acetyl-Ser-Asn-Serine-(Ac-Ser-Asn-Ser-carboxamide (1)) and purity are estimated to pass through ¹H NMR spectral test and HPLC carry out, and its moderate purity estimates about 80%.Principal pollutant be from the PAL-PEG-PS resin the polyoxyethylene glycol residue ( ¹H NMR spectrum peak is at δ 3.6ppm).Use the identical bigger peptide of peptide synthetic method preparation, Ac-Asn-Tyr-Tyr-Ser-Asn-Ser (SEQ ID NO:2), Ac-Ser-Asn-Ser-Tyr-Ser-Phe-Trp-Leu (SEQ ID NO:3) and Ac-Cys-Asn-Tyr-Tyr-Ser-Asn-Ser-Tyr-Ser-Phe-Trp Leu (SEQ ID NO:4).

Synthesizing of SNS analogue library:

Conventional-

In Quest 210 synthesizers, use 5mL Teflon ^_(registered trade mark E.I.de Nemours and Company of Wilmington DE) carries out the library and synthesizes reaction tubes.Using the FMOC method to carry out solid-phase peptide on Argogel Rink resin synthesizes.Go to protect with coupling and circulate repeatedly up to the peptide that forms desired length.Then rupture with this peptide acidylate and from resin.

Step-

Go protection:

Weighing, (200mg 0.064mmol) washs in the 5mL reaction vessel and with 3mL DMF Argogel Rink-FMOC.Added 20% piperidines/DMF (4mL) solution and agitating resin 2 minutes.Filter reaction mixture also repeats this step with 20% piperidine solution of the fresh equivalent of 4mL and carried out other 20 minutes.Then, discharge resin also with 5mL DMF washing four times.Use in each washing step and stirred in 5 minutes.

Coupling:

Add 0.6mL and do DMF in reaction vessel, add each 3 a normal N-FMOC-amino acid and I-hydroxybenzotriazole (HOBt) subsequently, it is for doing the 0.5M solution among the DMF.Agitating resin 1 minute is added 3 normal HBTU subsequently, and it is for doing the 0.5M solution among the DMF.Agitating resin 4 hours is discharged then and with 5 * 4mL DMF washing, was stirred 5 minutes when again washing at every turn.

The second and the 3rd FMOC amino acid is repeated to protect step and coupling step.After the 3rd the amino acid coupling, go to protect step once more.

Acidylate:

Use the 3mL acetic anhydride: diisopropyl ethyl amine: the resin that 1: 1: 2 mixture process above-mentioned steps of DMF obtains 1 hour.Discharge resin and use the DMF repeated washing.

Fracture:

In resin, add 3mL 50%TFA/ dichloromethane solution.Agitating resin 1 hour.Filter resin and collect filtrate.Filtrate is slowly added on the ice-cold ether, makes required product precipitation.Filter collecting precipitation and be dissolved in 50% acetonitrile solution.Freeze-drying solution obtains the required product of solid.

The meaning of abbreviation is as follows: " sec " meaning is second, " min " meaning is a branch, " h " meaning is hour, " d " meaning is the sky, " μ L " meaning is a microlitre, and " mL " meaning is a milliliter, and " L " meaning is to rise, " mM " meaning be mmole/liter, " M " meaning is that mol and " mmol " meaning is a mmole.

Embodiment 1

The inhibition that the endotheliocyte pipe forms

The method of (In Vitro Cell Dev.Biol.27A:327-336 (1991)) exploitations such as use Grant detects the differentiation of endotheliocyte.With Matrige l ^_Matrix, no phenol red (from Becton Dickinson, Bedford, MA commerce obtains) thaws in 4 ℃ and spends the night.Use the enfleurage tip, with 3.0mg/ hole Matrigel ^_Matrix is positioned over cold 24 porous plates (Falcon).37 ℃ of incubations 30 minutes, make Matrigel ^_The matrix polymerization.

With Human umbilical vein endothelial cells (HUVEC) remain under 37 ℃ at 5%CO ₂In the endothelial cell growth substratum that contains 2% foetal calf serum (EGM) in 95% humidity.(I-T-Se manages test in endothelial basal medium 100X) (EBM) at the Regular Insulin-Transferrins,iron complexes-selenium-G additive that replenishes 0.5% bovine serum albumin (BSA) and dilution in 1: 100.HUVEC is by trypsin acting and centrifugal, washed twice in phosphate buffered saline (PBS) (PBS) subsequently.Behind the counting, cell density is adjusted to 35,000 cell/mL.

With 100ng/mL recombinant human fibroblast growth factor basis (FGF2) with to handle final concentration with the peptide (as follows) that the concentration of 0.015 μ mol is dissolved in the EBM substratum be 35,000 cells/mL/ hole.The cell of handling under 37 ℃ at 5%CO ₂With overnight incubation under 95% humidity, make cell adhesion.

Subsequently, the sucking-off substratum, fixed cell is also used the dyeing of improvement HEMA-3 staining kit.With DKC50003-CCD colourvideo camera arrangement (Toshiba America, New York, NY) collect the digital picture of microtitration bore portion also with Image Pro Plus software (Media Cybernetics, Silver Spring, MD) analysis.Matrigel ^_Stromal surface (calculate from 5 images/hole for Becton Dickinson, Bedford by the area and the greatest axis length that have the staining cell of tubulose form on PA).

Detect following four peptides: ethanoyl-Ser-Asn-Ser-carboxamide (structure 1); ethanoyl-Asn-Tyr-Tyr-Ser-Asn-Ser (SEQ ID NO:2), ethanoyl-Ser-Asn-Ser-Tyr-Ser-Phe-Trp-Leu (SEQ ID NO:3) and ethanoyl-Cys-Asn-Tyr-Tyr-Ser-Asn-Ser-Tyr-Ser-Phe-Trp-Leu (SEQ ID NO:4).Clear ethanoyl-Ser-Asn-Ser-the carboxamide (structure 1) of having illustrated of table 1 and Fig. 1 is the extremely strong power inhibitor that external FGF2 stimulates the EC pipe to form.Area data is with 10 ⁴The unit representation of square micron, length data is with mm/mm ²The length/area of unit is represented.

Table 1

The microscopical analysis that the EC pipe forms

Sample	Concentration	The cell pipe forms (area)	The cell pipe forms (length)
				PBS	-	1.0554+/-0.11	41.3611+/-2.61
FGF2	100ng/mL	2.2501+/-0.28	79.8272+/-8.66
				Ethanoyl-SNS-carboxamide	0.015μmol	1.1011+/-0.12	47.5267+/-4.31

It is the inhibitor that the external FGF2 more powerful than bigger peptide stimulates the EC pipe to form that table 2 has been illustrated ethanoyl-Ser-Asn-Ser-carboxamide (structure 1).Suppress the per-cent data and deduct the quotient representation of negative control value (EBM substratum) divided by the difference between positive control value and the negative control value with experimental value.

Table 2

The average inhibition % that peptide forms the EC pipe

Sample	MW	0.015 μmol	The inhibition % (area) that the cell pipe forms	The inhibition % (length) that the cell pipe forms
					Ethanoyl-SNS-carboxamide	347.33	5.20μg	97％+/-8	84％+/-9
SEQ ID NO：3	787.79	11.80μg	75％+/-7	65％+/-3
					SEQ ID NO：4	1044.14	15.65μg	71％+/-6	54％+/-3
SEQ ID NO：5	1587.74	23.8μg	55％+/-2	49％+/-1

Embodiment 2

The microscopical analysis of last neovascularization of CAM and CAM section

With neovascularization in (J.Dev.Biol.41:391-394 (1974)) described method detection bodies such as Auerbach.The embryo in ten day age is available from Spafas, and Inc. (CT) and at 37 ℃, hatch under 55% relative humidity by Preston.In the dark, by means of candle lamp, on the shell that hides air bag, pierce through a duck eye with hypodermic needle.Directly cross in the horizontal side of ovum on the shell of embryo's film vasculature part and pierce through second hole, as observed in the candle light checking process.Apply negative pressure for first hole, below second hole, produce a false air bag, cause that CAM (CAM) separates with shell.On the shell on the sagging CAM, (Dremel, Division of Emerson Electric Company Racine WI) cuts about 1.0cm to emery wheel to use the unskilled labourer to plant ²Window, allow the CAM of directly arrival under it.(Sigma, St.Louis's filter disc of #1 filter paper (WhatmanInternational, United Kingdom) drench in 95% ethanol MO) and the aqueous solution, subsequently dry air under aseptic condition containing the 3mg/mL cortisone acetate.(Life Technologies, Gaithersburg MD) are used for the vascular of growing to FGF2 on the CAMs of 10 days instar chicken embryos.Be positioned on the CAMs of growth having absorbed the aseptic filter disc that is dissolved in the FGF2 of PBS with 1 μ g/ml.In the time of 24 hours, directly add detection compound or control vector to Local C AMs.

From organizing with the CAM under the filter disc that excision FGF2 is saturated compound or 48 hours the embryo of control treatment before.Organize three times with the PBS washing.Section be positioned over the 35mm Petri dish (Nalge Nunc, Rochester, NY) in and at the SV6 stereoscopic microscope (KarlZeiss, Thornwood check down with 50 * ratio of enlargement under NY).With 3-CCD colourvideo camera arrangement (Toshiba America, New York, NY) digital picture of the CAM section of the contiguous filter of collection is also with Image-Pro Plus software (Media Cybernetics, Silver Spring, MD) analyze, as shown in Figure 2.Table 3 contains the quantity of contained vessel branchings point in the annular section, and this annular section equals the filter membrane area of each section.Suppress the per-cent data and deduct of the quotient representation of negative control value divided by the difference between positive control value and the negative control value with experimental value.

Table 3

In the CAM model, the average inhibition % of vasculogenesis

Sample	The mean number # of tapping point
		The PBS contrast	94.714
The FGF2 contrast	263.88
		Difference	169.16
FGF-2+16 μ g ethanoyl-SNS-carboxamide	91.60
		The inhibition % that tapping point forms	100.23

Embodiment 3

The test of chicken CAM tumour

10,000,000 tumour cells are positioned over each content-addressable memory face and cultivate a week.Cutting gained tumour also is cut into the 50mg fragment.These fragments place on the additional C AMs, and intravenous injection next day detection reagent is carried out part or whole body and handled.After 48 hours, cutting CAM s and calculating enter the blood vessel quantity (as the vessel branchings point) of tumour from ovum.Data are with the mean vascular quantity of each treatment group (+/-bioassay standard error) expression.Ten tumours are introduced in the each test of each treatment group at least.Follow the tumor weight of also definite each tumour of tumor resection from ovum.Data are with the average tumor weight of each treatment group in the table 3 (+/-bioassay standard error) expression.Use Student ' s t-check carrying out statistical study.The result of this test is as described in Table 3, shows that 16 μ g ethanoyl-Ser-Asn-Ser-carboxamides (structure 1) control FGF2 and stimulate in the neovascularization effectively (when 16 μ g, 100% on average suppresses) in CAM.

Embodiment 4

The inhibition of TSU-Pr (prostate gland) tumor growth

10,000,000 tumour cells are positioned over each content-addressable memory face (7 days instar embryos) and cultivate a week.Cutting gained tumour also is cut into the 50mg fragment.These fragments place on the additional C AMs, inferior daily ethanoyl-Ser-Asn-Ser-carboxamide (structure 1) or carrier Local treatment.After seven days, cutting CAM s and calculating enter the blood vessel quantity (as the vessel branchings point) of tumour from ovum.Data are with the mean vascular quantity of each treatment group (+/-bioassay standard error) expression.Ten tumours are introduced in the each test of each treatment group at least.Typical case's tumour is taken a picture with 10 * ratio of enlargement.Follow the tumor weight of also definite each tumour of tumor resection from ovum.Data are with the average tumor weight of each treatment group (+/-standard error of mean) expression.Use Student ' s t-check carrying out statistical study.

Demonstration ethanoyl-Ser-Asn-Ser-carboxamide (structure 1) is given tumour by direct injection, suppresses human prostate tumor growth (Fig. 3) in the body.After seven days, there are six to show tangible weight savings (on average suppressing (at 16 μ g time 100% on average suppress) 45 o'clock 111%) in ten tumours with 45 μ g ethanoyl-Ser-Asn-Ser-carboxamides (structure 1) injections.

Embodiment 5

Prepare the library based on ethanoyl-Ser-Asn-Ser-carboxamide

Based on the anti-angiogenesis activity of ethanoyl-Ser-Asn-Ser-carboxamide (structure 1) of observing, use following 15 peptide: the SAS of standard FMOC solid-phase peptide chemical method (seeing general method) preparation, SQS; sNS, snS, SGS; SES, sNs, sns; SDS; SnS, SNs, Sns; t4Hyp-NS, t4Hyp-N-t4Hyp and SN-t4Hyp.Capitalization is represented L amino acid (natural), and lowercase represents that D amino acid (non-natural) and t4Hyp represent instead-the 4-oxyproline.

SAS C ₁₁H ₂₀N ₄O ₄SQSC ₁₃H ₂₂N ₆O ₇SNS C ₁₂H ₂₁N ₆O ₇SnS C ₁₂H ₂₁N ₆O ₇Accurate mass: 304.14 accurate masses, 361.18 accurate masses: 347.14 accurate masses: 347.14 Mol.WL:304.30 Mol.WL:361.35 Mol.WL:347.32 Mol.WL:347.32

C.43.42；H.6.82； N.18.41；O.31.55

C.43.21；H.8.42： N.19.38；O.30.90

C.41.50;H.6.08 N.20.16；O.32.25

C.41.50;H.6.09 N.20.16;O.32.25

SGSC ₁₀H ₁₈N ₄O ₄SESC ₁₃H ₂₂N ₄O ₈SNs C ₁₂H ₂₁N ₆O ₇Sns C ₁₂H ₂₁N ₅O ₇Accurate mass: 290.12 accurate masses: 362.14 accurate masses: 347.14 accurate masses: 347.14 Mol.WL:200.27 Mol.WL:362.34 Mol.WL:347.32 Mol.WL:347.32 C.41.36; H.6.25; N.19.30; O.33.07 C.43.09; H.5.12; N.15.46; O.35.33 C.41.50; H.6.09; N.20.16; O.32.25 C.41.50; H.6.09; N.20.16; O.32.26

SDSC ₁₂H ₂₀N ₄O ₈SnSC ₁₂H ₂₁N ₅O ₇SNsC ₁₂H ₂₁N ₆O ₇SnsC ₁₂H ₂₁N ₄O ₇Accurate mass: 348.13 accurate masses: 347.14 accurate masses: 347.14 accurate masses: 347.14 Mol.WL:348.31 Mol.WL:347.32 Mol.WL:347.32 Mol.WL:347.32 C.41.38; H.5.70; N.16.09; O.36.75 C.41.50; H.6.09; N.20.16; O.32.25 C.41.50; H.6.06; N.20.16; O.32.25 C.41.50; H.6.00; N.20.16; O.32.25

14Hyp-NS C ₁₄H ₂₃N ₆O ₇T4Hyp-N-t4Hyp C16H ₂₅N ₆O ₇SN-t4Hyp C ₁₄H ₂₃N ₅O ₇Accurate mass: 373.15 accurate masses: 309.18 accurate matter are heavy: 373.16 Mol.WL.:373.36 Mol.WL:399.40 Mol.WL:373.36 C.45.04; H.6.21; N.18.76; O.30.00 C.48.12; H.6.31:N.17.53; O.28.04 C.45.04; H.6.21; N.18.76; O.30.00

Detect above-mentioned 15 peptides and the blood vessel formation against function of ethanoyl Ser-Asn-Ser-carboxamide (structure 1) in the human endothelial cell pipe forms, as (Fig. 4) as described in the embodiment 1.In the CAM test, also detecting three compound S QS, SNS and SN-t4Hyp the most powerful, as (also at Fig. 4) as described in the embodiment 2. Fig. 5 has illustrated the angiogenesis suppression action of SNS tripeptide analog thing in the CAM model.The dose response data of SQS, SNs and SN-t4Hyp in the table 4 demonstration CAM test.

Table 4The inhibition % of tapping point in the FGF2 inductive CAM test

Concentration μ g/CAM	SQS	SNs	SN-t4Hyp
				1	101％+/-7	62％+/-7	39％+/-11
5	121％+/-6	78％+/-6	60％+/-7
				15	82％+/-7	69％+/-8	71％+/-8

Claims (18)

1. the angiogenesis-inhibitory tripeptides that general formula is aa1-aa2-aa3 has first amino acid (aa1), second amino acid (aa2) and the 3rd amino acid (aa3), and described tripeptides is selected from:

Ser-Asn-Serine (SNS),

Serine-L-Ala-Serine (SAS),

Serine-glutamine-Serine (SQS),

(D-Serine)-l-asparagine-Serine (sNS),

(D-Serine)-(D-l-asparagine)-Serine (snS),

Serine-glycine-Serine (SGS),

Serine-L-glutamic acid-Serine (SES),

(D-Serine)-l-asparagine-(D-Serine) (sNs),

(D-Serine)-(D-l-asparagine)-(D-Serine) (sns),

Serine-aspartic acid-Serine (SDS),

Serine-(D-l-asparagine)-Serine (SnS),

Ser-Asn-(D-Serine) (SNs),

Serine-(D-l-asparagine)-(D-Serine) (Sns),

(anti--the 4-oxyproline)-l-asparagine-Serine (t4Hyp-NS),

(anti--the 4-oxyproline)-l-asparagine-(anti--the 4-oxyproline) (t4Hyp-N-t4Hyp)

And Ser-Asn-(anti--the 4-oxyproline) (SN-t4Hyp).

2. the angiogenesis-inhibitory tripeptides of claim 1, it is selected from

Ser-Asn-Serine (SNS),

Serine-L-Ala-Serine (SAS),

Serine-glutamine-Serine (SQS),

(D-Serine)-l-asparagine-Serine (sNS),

Serine-glycine-Serine (SGS),

(D-Serine)-l-asparagine-(D-Serine) (sNs),

Serine-aspartic acid-Serine (SDS),

Serine-(D-l-asparagine)-Serine (SnS),

Ser-Asn-(D-Serine) (SNs),

Serine-(D-l-asparagine)-(D-Serine) (Sns),

(anti--the 4-oxyproline)-l-asparagine-Serine (t4Hyp-NS),

And Ser-Asn-(anti--the 4-oxyproline) (SN-t4Hyp).

3. the angiogenesis-inhibitory tripeptides of claim 1, it is selected from

Ser-Asn-Serine (SNS),

Serine-glutamine-Serine (SQS),

Ser-Asn-(D-Serine) (SNs) and

Ser-Asn-(anti--the 4-oxyproline) (SN-t4Hyp).

4. the angiogenesis-inhibitory tripeptides of claim 1, wherein N-terminal by acetyl blocked and C-terminal by the amide group end-blocking.

5. vasculogenesis composite inhibiting comprises the angiogenesis-inhibitory tripeptides of claim 1.

6. one kind effectively as the pharmaceutical composition of angiogenesis inhibitor, and said composition comprises the angiogenesis-inhibitory tripeptides of the claim 1 of vasculogenesis amount of suppression.

7. claim 1,2,3 or 4 any one tripeptides are used to make the purposes that is used to suppress to organize the medicine that medium vessels generates.

8. claim 1,2,3 or 4 any one tripeptides are used to make the purposes that is used to suppress the medicine that the animal medium vessels generates.

9. claim 1,2,3 or 4 any one tripeptides are used to make the purposes of the medicine that is used to suppress the philtrum vasculogenesis.

10. the purposes of claim 7, wherein said tissue has inflammation.

11. the purposes of claim 7, wherein said tissue are selected from the group that noumenal tumour, noumenal tumour metastasis, retinal tissue and tela chorioidea form.

12. the purposes of claim 7, wherein vasculogenesis be selected from a neovascularization disease, choroid neovascularization disease, retina neovascularization disease, the neovascularization of angle, bartonellosis, chronic inflammatory diseases, osteoarthritis, rheumatoid arthritis, atherosclerosis phemphigoid, the situation of the group that trachoma or hereditary hemorrhagic telangiectasia are formed is relevant.

13. the purposes of claim 7, wherein said medicine comprise that medicine can accept medium.

14. the purposes of claim 7, wherein said medicine comprises biodegradable polymer.

15. the purposes of claim 13, wherein said medicine comprises carrier, and described carrier is selected from the group that retrovirus, adenovirus, part bind nucleic acid, separated DNA, isolating RNA, liposome and polylysine are formed.

16. having, the purposes of claim 13, wherein said medicine be selected from following formulation: oral, and the part, nose, through skin, intraperitoneal, encephalic, in the brain, vagina, intrauterine, rectum, parenteral and ocular administration formulation.

17. the purposes of claim 13, wherein said medicine comprises the treatment compound, and this treatment compound is selected from the group that chemotherapy, microbiotic, antiviral, anti-inflammatory, target-seeking compound, cytokine, immunotoxin, anti-tumour antibody, angiogenesis inhibitor, Ivy extract agent, radiosensitizer and combination thereof are formed.

18. the composition of claim 5 or 6 is used to make the purposes that is used to suppress to organize the medicine that medium vessels generates.

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