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

Abstract

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

Description

Angiogenesis inhibitory tripeptides, compositions and methods of use

field of invention

The present invention relates to compositions and methods of use thereof for inhibiting animal and human endothelial cell tube formation, the first steps in tumor angiogenesis and tissue angiogenesis-dependent diseases. More particularly, the present invention relates to tripeptides, analogs, mimetics and chemical derivatives thereof shown to inhibit angiogenesis-mediated processes such as cancer, ocular neovascularization and inflammatory diseases. The disclosed anti-angiogenic agents may also be used in conjunction with surgery, chemotherapy, radiation therapy, and laser therapy.

Background of the invention

Angiogenesis is the development of new blood vessels from pre-existing vessels (Mousa, S.A., In Angiogenesis Inhibitors and Stimulators: Potential Therapeutic Implications, Landes Bioscience, Georgetown, TX; Chapter 1, (2000)). Physiologically, angiogenesis ensures the proper development of mature organisms, prepares the uterus for egg implantation and plays a key role in wound healing. On the other hand, angiogenesis supports pathological conditions associated with many disease states such as cancer, inflammation and eye diseases.

During embryogenesis or normal and pathological angiogenesis, the development of the vascular network is dependent on growth factors and the interaction between cells and the extracellular matrix (Breier et al., Trends in Cell Biology 6:454-456 (1996); Folkman, Nature Medicine 1 : 27-31 (1995); Risau, Nature 386: 671-674 (1997)). During embryogenesis, blood vessels emerge through two processes: angiogenesis and angiogenesis (Blood et al., Bioch. Biophys. Acta 1032:89-118 (1990)). Vascular endothelial growth factor (VEGF), bFGF, IL-8, and TNF-α are some of the growth factors that play a role in pathological angiogenesis associated with solid tumors, diabetic retinopathy, and rheumatoid arthritis (Folkman et al., Science 235: 442-447 (1987)). In adult or mature tissues, angiogenesis is usually absent, although it does occur in wound healing and embryogenesis (Moses et al., Science 248:1408-1410 (1990)).

Angiogenesis or "neovascularization" is a multistep process controlled by the balance of pro- and anti-angiogenic factors. Later stages of the process include endothelial cell (EC) proliferation and organization into tube-like structures. Growth factors such as FGF2 and VEGF are considered to be key substances that promote the growth and differentiation of endothelial cells. Endothelial cells are key components of the angiogenesis process and respond to many cytokines through their cell surface receptors and intracellular signaling mechanisms. Endothelial cells in culture are capable of forming tube-like structures with lumens. Thus, endothelial cells are not only a prerequisite for neovascularization, but also appear to be a fundamental structural requirement.

Angiogenesis-dependent diseases include the following: inflammatory disorders such as immune and nonimmune inflammation, rheumatoid arthritis, psoriasis; ocular disorders such as diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, age-related macular degeneration, corneal transplantation Rejection; and cancer-related disorders such as solid tumors, tumor metastases, and tumors of hematological origin such as leukemias, angiofibromas, Kaposi's sarcoma, benign tumors, and other cancers that require neovascularization to support tumor growth.

It has been suggested that inhibition of angiogenesis would be an effective therapy to limit tumor growth. Inhibition of angiogenesis can be accomplished by inhibiting endothelial cell responses to angiogenic stimuli, as suggested by Folkman et al., (Cancer Biology 3:89-96 (1992)), which describe examples of those inhibitors of endothelial cell responses such as angiostatic steroids , products from fungi such as fumagillin, platelet factor 4, glycoprotein G, alpha-interferon, vitamin D analogues and D-penicillamine. For additional proposed angiogenesis inhibitors, see Blood et al., Bioch. Biophys. Acta 1032:89-118 (1990); Moses et al., Science 248:1408-1410 (1990);

In 1997, Kefalides and co-workers at the Joint Tissue Research School of Medicine at the University of Pennsylvania reported that a peptide corresponding to the residue sequence 185-203 of the noncollagenous 1 (NC1) domain of the α3 chain of type IV basement membrane collagen inhibited polymorphonuclear leukocytes Activation of (PMN's) (Han et al., J. Biol. Chem. 272:20395-20401 (1997)). Peptide α3(IV) residues 185-203 with the sequence CNYYSNSYSFWLASLNPER (SEQ ID NO: 1) were found to promote human melanoma cell adhesion by 50-60% over controls and also inhibit their proliferation by more than 40%. Alanine substitutions throughout the peptide sequence indicated that the observed activity was dependent on the presence of residues 189-191, referred to as the SNS sequence. The Kefalides group later reported that inhibition of melanoma cells by type IV collagen required increased levels of cAMP (Shahan et al., Connective Tissue Res. 40:221-232 (1999)), recognizing CD47/integrin-associated protein (IAP) and αvβ3 As two receptors for the α3(IV) chain of type IV collagen in melanoma and prostate cells (Shahan et al., Cancer Res., 59:4584-4590 (1999)). Recently, they reported the Ca2+ dependence in tumor cell chemotaxis (Shahan et al., J. Biol. Chem. 275:4796-4802 (2000)) and the role of the NC1 domain of type IV collagen on matrix metalloproteinase expression and activation. Inhibition (Pasco et al., Cancer Res. 60:467-473 (2000)).

Independently, Kalluri's group from Harvard Medical School also reported the properties of two different types of antitumor activity (antiproliferative and antiangiogenic) of the α3(IV)NC1 domain using in vitro and in vivo assays (Maeshima et al. , J. Biol. Chem. 275:21340-21348 (2000)). Taken together, these reports effectively highlight the distinct and unique antitumor properties of the α3(IV)NC1 domain and its potential use as a lead for small molecule antitumor drug design.

Lacking these reported results is the identification of smaller recognition epitopes that retain the activity of the larger peptide. Thus, the problem to be solved was to provide the identification of small recognition epitopes that would be a decisive step in providing templates for structure-based drug design strategies towards small molecule analogs or peptidomimetics. Such small molecules will include cyclic peptides that intervene in angiogenesis-dependent diseases such as cancer and peptide isosteres with better physiochemical and pharmacokinetic properties.

Summary of the invention

The present invention relates to an angiogenesis inhibitory tripeptide of general formula aa1-aa2-aa3, having a first amino acid (aa1), a second amino acid (aa2) and a third amino acid (aa3), wherein,

(a) the 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 consisting of diaminoproprionic acid (diaminoproprionic acid) and trans-4-hydroxyl-proline;

(b) said second amino acid is selected from the group consisting of Asn, Ala, Gly, Asp, Glu, Gln, diaminopropionic acid and trans-4-hydroxy-proline; and

(c) the third 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 , a group consisting of diaminopropionic acid and trans-4-hydroxy-proline.

Also provided are methods of administering a tripeptide to inhibit angiogenesis.

Brief description of the drawings

The following drawings are illustrations of embodiments of the invention and do not limit the scope of the invention encompassed by the claims.

Figure 1 illustrates the role of SNS in inhibiting fibroblast growth factor basal (FGF2)-induced tube formation in human endothelial cells.

Figure 2 illustrates the role of SNS in inhibiting FGF2-induced angiogenesis in a chorioallantoic membrane (CAM) model.

Figure 3 illustrates the percent inhibition of TSU-Pr (human prostate) tumor growth in the presence of SNS.

Figure 4 illustrates the anti-angiogenic effects of SNS tripeptide analogues in human endothelial cell tube formation and CAM models.

Figure 5 illustrates the anti-angiogenic effect of SNS tripeptide analogues in a CAM model.

Detailed description of the invention

Applicants have solved the stated problem by providing a small recognition epitope, a three amino acid residue peptide, ie a tripeptide, that inhibits tube formation in endothelial cells and has been shown to inhibit angiogenesis-mediated processes. Methods of use of the tripeptides are also provided.

The term "tripeptide" as used herein means a peptide having three amino acid residues and includes any analogs, peptidomimetics and chemical derivatives discussed herein.

The term "peptide" as used herein means two or more amino acid residues covalently bound together. Peptides of the present invention include polypeptides having several hundred or more amino acid residues. Typically, a covalent bond between two or more amino acid residues is an amide bond. However, amino acids can be linked together by various other methods known to those skilled in the art of peptide and chemistry. Thus, the term "peptide" is intended to include molecules containing non-amide bonds in whole or in part between amino acids, amino acid analogs and mimetics. Similarly, the term also includes cyclic peptides and other conformationally constrained structures.

SNS and SQS tripeptides

The tripeptides of the present invention have three amino acid residues that provide angiogenesis inhibitory activity in tissues, animals and individuals.

A preferred tripeptide of the invention is characterized by the sequence Serine-Asparagine-Serine, described in Structure 1, also represented by Ser-Asn-Ser, also abbreviated by the single letter amino acid code, referred to herein as "SNS". Preferred amino acids are in the left-handed form and are amino-terminally capped with an acetyl group and carboxy-terminally capped with a carboxyl group.

(Structure 1)

Another preferred embodiment of the invention is Ser-Gln-Ser, also abbreviated by the one-letter amino acid code SQS, wherein the amino acid is in the left-handed form and is amino-terminally capped with an acetyl group and carboxy-terminally capped with a carbamoyl group.

Additional embodiments of the tripeptides of the present invention are analogs, peptidomimetics and chemical derivatives of Ser-Asn-Ser that contain additional chemical moieties that are not the normal part of naturally occurring proteins or modified amino acids, as will be discussed further herein. .

The term "capped" refers to the addition of a group at the amino or carboxyl terminus of a tripeptide. Preferably, the end of the tripeptide of the present invention is acetyl group (CH ₃ CO-) bonded to the N-terminal amino group of the tripeptide of the present invention and an amido group (-NH ₂ ) bonded to the C-terminal carboxyl group of the tripeptide of the present invention, which is also abbreviated as "carbamoyl ", closed or "capped". The tripeptide can be capped by any other group.

In this disclosure, a number of abbreviations are used. Provide the following definitions.

"Endothelial cells" are abbreviated as EC.

"Fibroblast Growth Factor Basis" is abbreviated as FGF2.

"Chorrioallantoic membrane" is abbreviated as CAM.

"Vascular endothelial growth factor" is abbreviated as VEGF.

"Trans-4-hydroxyproline" is abbreviated as t4Hyp.

"1-Hydroxybenzotriazole" is abbreviated as HOBt.

"2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate" is abbreviated as HBTU.

"Diisopropylethylamine" is abbreviated as DIEA.

"Dimethylformamide" is abbreviated as DMF.

"Trifluoroacetic acid" is abbreviated as TFA.

"Triisopropylsilane" is abbreviated as TIS.

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

"Endothelial cell growth medium" is abbreviated as EGM.

"Endothelial Cell Basal Medium" is abbreviated as EBM.

"Bovine serum albumin" is abbreviated as BS.

"9-Fluorenylmethyloxycarbonyl" is abbreviated as FMOC.

Conventional chemical synthesis method

Given their short length, it is preferred to prepare the tripeptides of the invention using solid phase synthesis, as generally described by Chan et al. (In FMOC Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press; Chapter 3 (2000)), although the art Other equivalent chemical syntheses are known to work as well (Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963)). Solid-phase peptide synthesis can start from the C-terminus of the peptide by coupling the protected α-amino acid to a suitable resin. This starting material can be prepared by linking α-amino acid-protected amino acids to chloromethyl resins or to methylol resins via ester linkages, or to BHA resins or MBHA resins via amide linkages. BHA and MBHA resin supports are commercially available and are generally only used when the desired polypeptide is synthesized with a non-substituted amide at the C-terminus.

Amino acids can be coupled to growing peptide chains to form peptide bonds using techniques well known in the art. For example, one approach involves converting an amino acid into a derivative that will render the carboxyl group of the amino acid more reactive with the free N-terminal amino group of the growing peptide chain. In particular, protection of The C-terminus of the amino acid can be converted to a mixed anhydride. Alternatively, the C-terminus of the amino acid can be converted to an active ester such as 2,4,5-trichlorophenyl ester, pentachlorophenol ester, pentafluorophenol ester, p-nitrophenyl ester, N-hydroxysuccinyl Amino esters or esters formed from 1-hydroxybenzotriazoles. Another coupling method involves the use of a suitable coupling agent such as N,N'-dicyclohexylcarbodiimide or N,N'-diisopropylcarbodiimide.

The α-amino group of each amino acid used in peptide synthesis should be protected during the coupling reaction to prevent side reactions involving their reactive α-amino function. Certain amino acids contain reactive side-chain functional groups (e.g., sulfhydryl, amino, carboxyl, and hydroxyl), which should also be protected with suitable protective groups to prevent occurrence at (1) α-amino sites or (2) Chemical reaction of reactive side chain sites in initial and subsequent coupling steps.

In the selection of particular protecting groups to be used in the synthesis of peptides, the following general rules are typically followed. Specifically, the α-amino protecting group (1) should render the α-amino function inert under the conditions used in the coupling reaction, (2) after the coupling reaction, without removing the side chain protecting group and It should be easy to remove without changing the structure of the peptide fragments, and (3) the possibility of racemization should be greatly reduced upon activation prior to conjugation.

On the other hand, the side chain protecting group (1) should render the side chain functional group inert under the conditions used in the coupling reaction and (2) should be stable under the conditions of removal of the α-amino protecting group. , and (3) should be easily removed from the desired fully assembled peptide without changing the structure of the peptide chain.

Variations in the reactivity of protecting groups known to be effective for peptide synthesis with the reagents used to remove them will be apparent to those skilled in the art. For example, certain protective groups, such as trityl and 2-(p-biphenyl)isopropyloxycarbonyl, are very unstable and can be cleaved under mildly acidic conditions. Other protective groups such as t-butyloxycarbonyl (BOC), t-pentyloxycarbonyl, adamantyl-oxycarbonyl and p-methoxyphenyloxycarbonyl are slightly unstable and their removal requires acids of moderate strength, such as tris Fluoroacetic acid, hydrochloric acid, trifluoroboric acid in acetic acid. Still other protective groups like benzyloxycarbonyl (CBZ), halobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl and isopropyloxycarbonyl are even slightly less stable and their removal requires even stronger acids like hydrogen fluoride , hydrogen bromide or boron trifluoroacetate in trifluoroacetic acid.

Among the classes of amino acid protecting groups effective for protecting α-amino groups or protecting side chain groups include the following:

(1) For α-amino groups, three typical protective groups are:

(a) Aromatic urethane type protective groups such as fluorenylmethoxycarbonyl (FMOC), CBZ and substituted CBZ such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl Oxycarbonyl and p-methoxybenzyloxycarbonyl, o-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, etc.;

(b) Aliphatic urethane-type protective groups, such as BOC, t-pentyloxycarbonyl, isopropyloxycarbonyl, 2-(p-biphenyl)isopropyloxycarbonyl, allyloxycarbonyl, etc.; and

(c) Cycloalkylurethane type protecting groups such as cyclopentyloxycarbonyl, adamantyloxycarbonyl and cyclohexyloxycarbonyl.

Preferred alpha-amino protecting groups are BOC and FMOC.

(2) For the side chain amino group present in Lys, it can be protected with any of the groups mentioned in (1) above, such as BOC, 2-chlorobenzyloxycarbonyl and the like.

(3) For the guanidine group of Arg, nitro, tosyl, CBZ, adamantyloxycarbonyl, 2,2,5,7,8-pentamethylchroman-6-sulfonyl, 2,3 , 6-trimethyl-4-methoxyphenylsulfonyl or BOC can provide protection.

(4) For the hydroxyl group of Ser or Thr, such as t-butyl, benzyl (BZL); or substituted BZL, such as p-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o- Chlorobenzyl and 2,6-dichlorobenzyl can be protected.

(5) For the carboxyl group of Asp or Glu, for example, it can be protected by esterification with groups such as BZL, t-butyl, cyclohexyl, and cyclopentyl.

(6) For the imidazole nitrogen of His, a benzyloxymethyl (BOM) or tosyl moiety is suitable as a protecting group.

(7) For the phenolic hydroxyl group of Tyr, protective groups such as tetrahydropyranyl, tert-butyl, trityl, BZL, chlorotoluene, 4-bromobenzyl and 2,6-dichlorotoluene are suitably used. A preferred protecting group is bromobenzyloxycarbonyl.

(8) For the side-chain amino group of Asn or Gln, xanthyl (Xan) is preferably used.

(9) For Met, it is preferred that the left side of the amino acid is not protected.

(10) For the thio group of Cys, p-methoxybenzyl is typically used.

Appropriately chosen protecting groups such as BOC typically protect the C-terminal first amino acid of the growing peptide chain at the α-amino position, such as Glu. Isopropylcarbodiimide can be used to first couple BOC-Glu-(γ-cyclohexyl)-OH to benzylhydrogenamine resin at 25°C for two hours while stirring, or to chloromethyl resin. Following coupling of the BOC-protected amino acid to the resin support, the α-amino protecting group is usually removed, typically using a solution of trifluoroacetic acid (TFA) in dichloromethane or TFA alone. The α-amino deprotection reaction can take place over a wide range of temperatures, but is generally carried out at a temperature between about 0°C and room temperature.

Other standard α-amino deprotecting reagents, such as HCl in dioxane, and conditions for removal of a particular α-amino protecting group are within the skill of the art. After removal of the α-amino protecting group, the unprotected α-amino group, usually with side chains still protected, can be coupled in a planned sequence in a stepwise manner.

Each protected amino acid or amino acid sequence is usually introduced into a solid phase reactor in a superstoichiometric amount and the coupling is suitably carried out in an organic solvent such as dimethylformamide (DMF), dichloromethane or a mixture thereof. If incomplete coupling occurs, the coupling step is usually repeated before removing the N-amino protecting group in preparation for coupling to the next amino acid. After removal of the α-amino protecting group, the remaining α-amino and side chain protected amino acids can be coupled in a planned sequence in a stepwise manner. The success of the coupling reactions at each stage of the synthesis can be monitored. A preferred method of monitoring the synthesis is via the ninhydrin reaction. Coupling reactions can also be performed automatically using well-known commercial methods and apparatus, such as the Beckman 990 Peptide Synthesizer.

After completion of the desired peptide sequence, the protected peptide should be cleaved from the resin support and all protective groups should be removed. The cleavage reaction and removal of the protecting group is suitably carried out concurrently or sequentially with the deprotection reaction. When the bond between the peptide and the resin immobilization is an ester bond, it can be broken by any reagent capable of breaking the ester bond and penetrating the resin medium. A particularly effective method is treatment with liquid anhydrous hydrogen fluoride. This reagent usually not only cleaves the peptide from the resin, but also removes any acid-labile protecting groups, and thus, will directly provide the fully deprotected peptide. Additional deprotection steps should be performed when additional protecting groups are present that are not acid labile. These steps may be performed before or after the above-mentioned hydrogen fluoride treatment according to specific needs and circumstances.

When using chloromethylated resins, the hydrogen fluoride cleavage/deprotection reaction usually results in the formation of free peptide acids. When using benzhydrylamine resins, hydrogen fluoride treatment usually yields free peptide amides. Reaction with hydrogen fluoride at 0°C for one hour in the presence of anisole and imethylsulfide typically removes the side chain protecting groups and the peptide is released from the resin.

When it is desired to cleave the peptide without removing the protective group, the protected peptide-resin can be subjected to methanolysis, thus yielding a protected peptide with C-terminal carboxymethylation. This methyl ester can then be hydrolyzed under mildly basic conditions to give the free C-terminal carboxyl group. The protective group on the peptide chain can then be removed by treatment with a strong acid, such as liquid hydrogen fluoride. A particularly efficient technique for methanolysis is where the protected peptide-resin is treated with methanol and potassium cyanide in the presence of a crown ether.

When using chloromethyl resins, other methods of cleaving protected peptides from the resin include (1) aminolysis and (2) hydrazinolysis. If desired, the resulting C-terminal amide or hydrazide can be hydrolyzed to the free C-terminal carboxyl moiety and the protecting group can be routinely removed. The protective group present on the N-terminal [alpha]-amino group can be removed before or after the protective peptide is cleaved from the support.

Purification of the peptides of the invention is typically accomplished using chromatographic techniques such as preparative HPLC (including reverse phase HPLC), gel permeation, ion exchange, partition chromatography, affinity chromatography (including monoclonal antibody columns), and the like, or Other conventional techniques such as countercurrent distribution and the like.

The SNS tripeptides of the invention can also be prepared using recombinant DNA techniques.

Amino acid analogs

The term "amino acid" as used herein means naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimetics. Naturally occurring amino acids include the 20 (L)-amino acids utilized in protein biosynthesis and others, e.g., 4-hydroxyproline, hydroxylysine, desmosin, isodesmosin, homocysteine, citrulline amino acid and orthinine. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, and the like.

Amino acid "analogues" or "peptide analogs" as used herein include modified forms of naturally and non-naturally occurring amino acids. Such modifications may include, for example, substitution or substitution of chemical groups and moieties on amino acids or derivatization of amino acids.

Included in the present invention are tripeptides in which at least one amino acid residue differs from the tripeptide of general formula (1) (Ser-Asn-Ser). For a detailed description of protein chemistry and structure see Schulz, G.E. et al., In Principles of Protein Structure, Springer-Verlag, New York, 1979, and Creighton, T.E., In Proteins: Structure and Molecular Principles, W.H. Freeman & Co., San Francisco, 1984 . The type of substitution performed in the tripeptide of the general formula (1) (Ser-Asn-Ser) of the present invention may be a conservative substitution, and the exchange within one of the following groups is defined here. However, the present invention is not limited to these substitutions.

(1) D-amino acid replacement for L-amino acid

(2) Small aliphatic, non-polar or weakly polar residues: such as Ala, Ser, Thr, Gly;

(3) Polar, negatively charged residues and their amides: such as Asp, Asn, Glu, Gln;

(4) Polar, positively charged residues: eg, His, Arg, Lys;

It is difficult to predict its exact effect even prior to substitution, and those skilled in the art will appreciate that this effect can be estimated using routine screening assays, preferably the biological assays described below. Modifications in peptide properties, including redox or thermal stability, hydrophobicity, susceptibility to proteolytic degradation, or tendency to aggregate with the support or into multimers, are detected using methods well known to those of ordinary skill in the art.

Those skilled in the art know or can determine what structural components are functionally equivalent amino acid analogs.

chemical derivatives

As used herein, "chemical derivatives" of the tripeptides of the invention contain additional chemical moieties that are not normally part of the tripeptides. Covalent modification of tripeptides is within the scope of the present invention. Such modifications can be introduced into the molecule by reacting targeted amino acid residues of the peptide with organic derivatizing agents capable of reacting with selected side chains or terminal residues.

Capping or "capping" the amino and carboxyl termini with an acetyl group ( _CH3CO- ), bonded to the amino-terminal N and amido ( _-NH2 ) to the C-terminal carboxyl group; also abbreviated "carbamoyl", but can Easily blocked by any other group. Proper choice of capping group allows addition of other active agents to the peptide. For example, the presence of a sulfhydryl group attached to the N- or C-terminal cap will allow conjugation of the derivatized peptide to other molecules. Some amino terminal capping groups are compounds selected from the group consisting of acetyl, benzoyl, alkylsulfonyl, arylsulfonyl, alkylaminoacyl, arylaminoacyl, formyl, peptide, and polymer. Examples of carboxyl end groups are compounds selected from the group consisting of _NH2 , OH and NHR, where R is selected from the group consisting of alkyl, aryl, peptide and polymer.

Capped peptides are examples of preferred chemical derivatives of natural (L amino acid configuration) uncapped peptides. Combinations of any of the above analogs or chemical derivatives can be capped with any capping group disclosed herein.

Amino Acids and Peptidomimetics

As used herein, "amino acid mimetic" includes, for example, organic structures that exhibit functionally similar properties, such as the charge and charge spacing properties of a reference amino acid. Mimetics also include constraining structures such that optimal spacing and charge interactions of amino acids or amino acid functional groups are maintained. Those skilled in the art know or are able to determine what structural constituents are functionally equivalent amino acid mimetics.

A "peptidomimetic" or peptidomimetic, as used herein, is an organic molecule that retains a peptide chain pharmacophore similar to that present in the corresponding peptide. A peptidomimetic can also be a functional equivalent of a specific peptide. The SNS tripeptides of the invention can be grouped based on pharmacophore properties. The term "pharmacophore" as used herein is defined as a particular arrangement of functional groups of a compound required to produce a particular reaction or possess a desired activity.

Preferred peptidomimetic compounds of the invention are blocked or unblocked compounds that mimic the biological effects of the SNS. The peptidomimetic agent may be a non-naturally occurring peptide (D amino acid configuration) or a non-peptidic agent having the stereochemistry of the tripeptide of the invention, capped or uncapped, such that it has the stereochemical properties of the tripeptide of the invention, either capped or uncapped. Binding activity or biological activity.

A large number of peptidomimetic compounds, agonists, substrates or inhibitors of biologically active peptides have been described, such as opioid peptides, VIP, thrombin, HIV protease, etc. Methods for designing and preparing peptidomimetic analogs are known in the art (Kempf, D.J. Methods Enzymol. 241:334-354 (1994); Hruby, V.J., Biopolymers 33:1073-82 (1993); Wiley et al., Med. 13:327-384 (1993); Claeson, G., Blood Coagul. Fibrinolysis 5:411-436 (1994)). These methods can be used to prepare capped or uncapped peptidomimetics, which have at least the binding capacity and specificity of a tripeptide, and preferably also have biological activity. Knowledge of peptide chemistry and general organic chemistry is available to those skilled in the art to design and test such compounds.

For example, examination of crystallographically derived three-dimensional structures of peptides of the invention can identify such peptidomimetics. Alternatively, the structure of the tripeptide of the invention bound to its receptor can be obtained using nuclear magnetic resonance spectroscopy techniques. For example, better knowledge of the stereochemistry of the interaction of blocked or uncapped SNS with its receptors would allow the rational design of such peptidomimetic agents.

Angiogenesis and angiogenesis-dependent diseases

As used herein, the terms "angiogenesis inhibitory", "angiogenesis inhibitory" or "anti-angiogenic" include angiogenesis, meaning a reduction in the extent, amount or rate of new blood vessel formation that results. Causing a reduction in the extent, amount or speed of endothelial cell proliferation or migration in tissue are specific examples of inhibition of angiogenesis.

The term "angiogenesis-inhibiting composition" refers to a composition that inhibits angiogenesis-mediated processes such as endothelial cell migration, proliferation, tube formation, and subsequently inhibits the generation of new blood vessels from existing vessels, thereby inhibiting angiogenesis-dependent diseases.

The term "angiogenesis-dependent disease" as used herein means a disease in which angiogenesis or the process of angiogenesis supports or augments a pathological condition. Angiogenesis is the formation of new blood vessels by pre-existing capillary or post-capillary venules. Angiogenesis is caused by the generation of new blood vessels from endothelial cell precursors - hemangioblasts. Two processes cause neovascularization and are included within the meaning of the term angiogenesis-dependent disease. Similarly, the term "angiogenesis" as used herein is intended to include vessel re-formation as arising from angiogenesis as well as from branching and sprouting of existing vessels, capillaries and venules.

Angiogenesis, including angiogenesis, is an important physiological process without which embryonic development and wound healing would not occur. However, angiogenesis also inappropriately causes a number of pathological conditions as a means of providing adequate blood and nutrient supply to the cells within the affected tissue. Many of these pathological conditions involve abnormal cell proliferation or regulation. Such conditions for which angiogenesis is believed to be important are referred to herein as angiogenesis-dependent diseases. However, the methods of the invention may also be beneficially used to inhibit normal physiological processes associated with angiogenesis. For example, angiogenesis-associated inhibition of the menstrual cycle could be used prophylactically as an effective method of birth control. Accordingly, the following descriptions regarding the treatment of angiogenesis-dependent diseases also apply to the inhibition of normal angiogenic responses where there is a need or benefit for prophylaxis or treatment.

Angiogenesis-dependent diseases include, for example, inflammatory disorders such as immune and nonimmune inflammation, rheumatoid arthritis, chronic rheumatoid arthritis, and psoriasis; disorders associated with inappropriate or unfortunate vascular invasion such as diabetic retinopathy, neovascular Formation of glaucoma, retinopathy of prematurity, macular degeneration, corneal graft rejection, retrolentic fibrogenesis, flushing, capillary hyperplasia in atherosclerotic plaques, and osteoporosis; and cancer-related disorders including, for example, solid tumors, tumor metastasis Foci and tumors of vascular origin such as leukemia, angiofibroma, Kaposi's sarcoma, benign tumors such as hemangioma, acoustic neuroma, neurofibroma, trachoma, and pyogenic rheumatoid granuloma, and tumors requiring neovascularization to support Other cancers that grow. Additional examples of angiogenesis-dependent diseases include, eg, Osler-Webber syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasias; hemophilic joint and wound granulation. Other diseases in which angiogenesis plays a role in maintaining the progression of the pathological state are known to those skilled in the art and are similarly included within the meaning of the term as used herein.

In Vitro Biological Assay for Angiogenesis Inhibitory Activity

The angiogenesis inhibitory activity of the compounds of the present invention was tested in several test systems in vitro. Prepared or commercially available endothelial cells, for example, human umbilical vein endothelial cells ⁽ HUVEC) or human microvascular endothelial cells (HMVEC) were mixed with fibrinogen (in phosphate-buffered saline (PBS) 5mg/mL) in a 1:1 (v/v) ratio. Thrombin was added (final concentration 5 units/mL), and the mixture was immediately transferred to a 24-well plate (0.5 mL/well). Fibrin gels were allowed to form, then vascular endothelial growth factor (VEGF) and fibroblast growth factor basal (FGF2) were added to the wells along with test compounds (at a final concentration of 5 ng/mL per well). Cells were cultured at 37°C, 5% _CO2 for 4 days, and cells in each well were counted and classified as round, without branch extension, with one branch extension, or with 2 or more branch extensions. Results are expressed as an average of 5 different wells for each concentration of compound. Typically, cells remain round or form undifferentiated tubes (eg, 0 or 1 branch) in the presence of an angiogenesis inhibitor. This assay is considered in the art to be predictive of angiogenic effects (or angiogenesis inhibitory activity) in vivo (Grant et al., In Vitro Cell Dev. Biol. 27A: 327-336 (1991); Min et al., Cancer Res. 56: 2428-2433 ( 1996)).

In an alternative test, endothelial cell tube formation was observed when endothelial cells were cultured on Matrigel® matrix ^- coated plates commercially available from Becton Dickinson of Bedford, PA (Schnaper et al., J. Cell. Physiol. 165: 107-118 (1995)). Endothelial cells (1 x ¹⁰⁴ cells/well) were transferred to Matrigel® matrix ^- coated 24-well plates, and quantitative tubes formed after 48 hours. Inhibitors were tested simultaneously with the addition of endothelial cells or at various time points thereafter.

This assay mimics angiogenesis by giving endothelial cells a specific type of basement membrane, the stromal layer that migrating and differentiating endothelial cells are expected to encounter first. In addition to binding growth factors, matrix components found in the ^Matrigel® matrix (and in situ in the basement membrane) or their proteolytic products can also stimulate endothelial tube formation, making this model complementary to the fibrin gel angiogenesis model.

In addition, the angiogenic activity of the compounds of the present invention was estimated using the chick chorioallantoic membrane (CAM) assay (Oikawa et al., Cancer Lett. 59:57-66 (1991)).

Method of administration

The present invention provides methods of inhibiting angiogenesis in tissue by administering an angiogenesis inhibiting amount of an SNS tripeptide, analog, mimetic or chemical derivative thereof.

The present invention further provides a method for treating angiogenesis-dependent diseases in animals by administering an angiogenesis-inhibiting amount of SNS tripeptide, analogs or functional equivalents thereof.

The present invention also provides a method for inhibiting human angiogenesis-dependent diseases by administering an angiogenesis-inhibiting amount of SNS tripeptide, analogs or functional equivalents thereof.

SNS tripeptides, analogs thereof, or chemical derivatives can be administered to tumor-bearing animals to determine the inhibitory activity or efficacy of the peptides on tumor growth compared to non-anti-angiogenic peptide controls. A reduction in the rate or extent of tumor growth, or tumor disappearance, is associated with anti-angiogenic activity and efficacy against the progression of an angiogenesis-dependent disease. For a model description of tumor bearing animals, see, eg, US Patent No. 5,639,725, incorporated herein by reference.

Dosage

In determining the activity and/or efficacy of an SNS tripeptide, analog or chemical derivative in any of the above methods, the peptide may be administered within a concentration range known in the art that is indicative of inhibitory activity in a particular assay. For example, concentrations of polypeptide inhibitors that produce indicative results in bovine capillary endothelial cell assays typically range from about 100-1000 ng/ml. Similarly, polypeptide inhibitors that are active against angiogenesis in the CAM produce a positive result at a concentration of the polypeptide inhibitor, typically about e.g. , or 25 μg/plate. A representative concentration of a polypeptide inhibitor expected to produce a positive result in the rabbit cornea test is generally about 40 μg/Haichang grain. Finally, the concentrations of the polypeptide inhibitors that produced indicative results in tumor metastases and in the aforementioned tumor-bearing animal models were approximately 250 μg or 10 mg/kg/day twice weekly for 10 days; and 12.5 μg daily or 1 mg twice weekly, respectively . Further refinement can be performed, for example, by varying the concentration of the SNS tripeptide within the active concentration range, to determine the optimal concentration or amount for inhibiting angiogenesis.

In addition, the above models, as well as other methods known to those skilled in the art, can similarly be used to determine the timing, timing, and The amount administered and the appropriate dosage regime for each dose. Similarly, the methods described above can also be routinely used to prepare and identify new, modified or improved SNS tripeptides, analogs, mimetics or chemical derivatives. Given the teachings and guidance described herein, one skilled in the art will know or be able to determine the effectiveness of SNS tripeptides, analogs, mimetics, or chemical derivatives for inhibiting angiogenesis-dependent or treating angiogenesis-dependent diseases. quantity.

The term "angiogenesis-inhibiting amount" as used herein means an SNS tripeptide, analog, mimetic, or chemically derivative of the present invention required to cause a decrease in the degree, amount, or rate of neovascularization when administered to a tissue, animal, or individual. amount of things. The dosage of SNS tripeptides, analogs, mimetics or chemical derivatives required to be therapeutically effective will depend on, for example, the angiogenesis-dependent disease to be treated, the route and form of administration, the potency and maximum active half-life of the administered molecule, the tissue , animal or individual body weight and condition, and previous or concurrent treatments. Using the guidance provided herein, one skilled in the art can determine appropriate amounts for use in the present method. For example, the amount can be inferred from the in vitro or in vivo angiogenesis assays described above. Those skilled in the art will recognize that the condition of the patient to be monitored throughout the course of treatment and the amount of the composition administered can be adjusted accordingly.

For inhibiting angiogenesis or treating angiogenesis-dependent diseases, the angiogenesis-inhibiting amount of the peptide of the present invention can be, for example, between about 10 μg/kg and 500 mg/kg body weight, for example, between about 0.1 mg/kg and 100 mg/kg, Or preferably between about 1 mg/kg and 50 mg/kg, depending on the treatment regime. For example, if the peptide is administered one to several times a day, a lower dosage may be required than if the peptide is administered weekly or monthly, or less frequently. Similarly, formulations that allow timed release of peptides will provide continuous release of smaller amounts of peptide than administration of a single bolus dose. For example, the peptide can be administered at 4 mg/kg/week.

delivery system

The SNS tripeptides, analogs, mimetics or chemical derivatives of the invention can be delivered systemically, eg intravenously or intraarterially. SNS tripeptides, analogs, mimetics or chemical derivatives can also be administered locally at the site of angiogenesis. The appropriate site for administration of the SNS tripeptide, analog, mimetic or chemical derivative is known to those skilled in the art or may be determined according to the clinical indications of the individual being treated. For example, using the formulation methods known to those of ordinary skill in the art, the SNS tripeptides, analogs, mimetics or chemical derivatives with the above-mentioned inhibitory activity can be used as isolated and substantially purified proteins and protein fragments in pharmaceutically acceptable preparations and Insoluble polymer provided. These formulations can be administered by standard routes, including, for example, topical, transdermal, intranasal, intraperitoneal, intracranial, intravascular, intracerebral, intravaginal, intrauterine, oral, rectal, parenteral (e.g., intravenous, Intraspinal, subcutaneous or intramuscular) routes. In addition, SNS tripeptides, analogs, mimetics or chemical derivatives can be incorporated into biodegradable polymers allowing sustained release of the compound that are implanted near the site where drug delivery is desired, e.g., tumor or implant site , to release the SNS tripeptide, analogue, mimetic or chemical derivative systemically over time. Osmotic minipump peptides, analogs, mimetics or chemical derivatives can also be used to provide controlled delivery of high concentrations of SNS tripeptides, analogs, mimetics or chemical derivatives through the cannula to the site of interest, e.g. direct Growth or tumor vascular supply to metastases. Biodegradable polymers and their uses are described in detail, eg, in Brem et al., J. Neurosurg 74:441-446 (19911, incorporated herein by reference).

SNS tripeptides, analogs, mimetics or chemical derivatives can also be combined with a group selected from chemotherapy, antibiotics, antiviral, anti-inflammatory, targeting compounds, cytokines, immunotoxins, anti-tumor antibodies, angiogenesis inhibitors, anti-edema agents, radiosensitizers, and combinations thereof with additional therapeutic compounds.

The present invention provides a composition of SNS tripeptide, analogue, mimetic or chemical derivative together with pharmaceutically acceptable medium and formulation. Such compositions may be used in the methods of the invention to inhibit angiogenesis or to treat angiogenesis-dependent diseases. For example, the SNS tripeptide, analog, mimetic or chemical derivative can be administered as a solution or suspension with a pharmaceutically acceptable vehicle. Such pharmaceutically acceptable medium may be, for example, water, sodium phosphate buffer, phosphate buffered saline, normal saline or Ringer's solution or other physiologically buffered saline or other solvents or vehicles such as glycol, glycerol, oils such as olive oil or Inject organic esters.

SNS tripeptide, analog, mimetic, or chemical derivative formulations include those suitable for parenteral administration, such as subcutaneous, intraperitoneal, intramuscular, intravenous, intradermal, intracranial, intratracheal, and epidural medication. As well as formulations suitable for oral, rectal, ocular (including intravitreal or intracameral), nasal, topical (including buccal or sublingual), intrauterine or vaginal administration. SNS tripeptides, analogs, mimetics or chemical derivatives may be presented in unit dosage form and may be prepared using pharmaceutical techniques well known to those skilled in the art. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier or excipient. The SNS tripeptide, analogue, mimetic or chemical derivative of the present invention can also be delivered to an individual by administering the nucleic acid encoding the peptide for the purpose of inhibiting angiogenesis or treating angiogenesis-dependent diseases. Therefore, the nucleic acids encoding the SNS tripeptides, analogs, mimetics or chemical derivatives of the present invention can be effectively combined with a wide variety of gene therapy methods known in the art for the delivery of angiogenesis-inhibiting amounts of peptides or variants. Using the techniques and guidance provided herein, nucleic acids encoding one or more SNS tripeptides, analogs, mimetics, or chemical derivatives, or combinations thereof, can be introduced into vectors or delivery systems known in the art and used to deliver expression of the coding sequences to reach angiogenesis inhibitory dose. Suitable vectors and delivery systems known in the art include, for example, retroviral vectors, adenoviral vectors, adeno-associated viruses, ligand-binding particles and targeting nucleic acids, isolated DNA and RNA, liposomes, polylysine Acid and cell therapy, including anhydrous cell therapy, transplantation using cells modified to express the SNS tripeptide, and various other gene delivery methods and modifications known to those skilled in the art, such as Shea et al., Nature Biotechnol. 17:551-559 (1999), incorporated herein by reference.

Specific examples of methods well known in the art are found in, for example, US Patent 5,399,346; US Patent 5,580,859; 5,589,466; 5,460,959; 5,656,965; 5,643,578; 97/09441; PCT No. WO 97/10343, all of which are incorporated herein by reference. Other methods known to those skilled in the art also exist and are similarly suitable for delivering an angiogenesis-inhibiting amount of an SNS tripeptide, analogue, mimetic or chemical derivative by expression of a coding nucleic acid sequence.

The invention also relates to encoding nucleic acids and vectors which are useful in gene therapy methods and can be prepared by methods known in the art. Compositions comprising such nucleic acids, carriers and pharmaceutically acceptable medium are also provided. The pharmaceutically acceptable medium should contain no elements that could degrade the desired nucleic acid. Methods of using SNS tripeptides, analogs, mimetics or chemical derivatives may utilize any of the various types of SNS tripeptides, analogs, mimetics and chemical derivatives previously listed.

Non-SNS sequences may confer structural or functional properties on the peptides of the invention. Targeting the SNS tripeptide, analog, mimetic or chemical derivative to the site of abnormal angiogenesis offers the additional therapeutic advantage of immobilizing the peptide at the site of the pathological condition. Thus, this result supports that high effective concentrations of the peptide diffuse over time to the angiogenic region and essentially allow continuous local administration of SNS tripeptides, analogs, mimetics or chemical derivatives to the angiogenic site.

Additionally, two or more SNS tripeptides, analogs, mimetics or chemical derivatives of the invention may be administered in the methods of the invention to inhibit angiogenesis or treat angiogenesis-dependent diseases. Similarly, one or more SNS tripeptides, analogs, mimetics or chemical derivatives may be administered in combination with one or more SNS tripeptides, analogs, mimetics or chemical derivatives to inhibit angiogenesis or angiogenesis Dependent diseases. Therefore, in the method of the present invention, various combinations and permutations of SNS tripeptides, SNS analogs, SNS mimics and SNS chemical derivatives and combinations thereof can be administered to effectively inhibit angiogenesis and treat angiogenesis-dependent diseases.

SNS tripeptides, analogs, mimetics and chemical derivatives and combinations thereof may be administered alternately so that their angiogenesis inhibitory effects are combined at any time. For example, an SNS analog may be administered in a single bolus dose followed by multiple administrations of one or more SNS tripeptides alone or in combination with SNS tripeptides, analogs, mimetics or chemical derivatives. Whether the SNS tripeptides, analogs, mimetics or chemical derivatives or combinations thereof are delivered simultaneously or alternately, the mode of administration may be any of the foregoing types of administration, and depends on the particular therapeutic need and the SNS tripeptides, similar effects of substances, mimetics or chemical derivatives. Determining which SNS tripeptides, analogs, mimetics or chemical derivatives to combine in a mixture or in a transient dosing regime will depend on the specific physical characteristics of the angiogenesis-dependent disease and the individual infected with the disease. A particular mixture or administration regime effective for a particular application will be known or can be ascertained by one skilled in the art using the teaching and guidance provided herein, along with diagnostic and clinical criteria known in the art for a particular angiogenesis-dependent disease.

therapeutic application

Additional methods of administering SNS tripeptides, analogs, mimetics or chemical derivatives to inhibit angiogenesis or treat angiogenesis-dependent diseases can be practiced in conjunction with other therapies. Possible embodiments of the invention include the use of such administration in combination with other therapies such as conventional chemotherapy directed against solid tumors and to control the establishment of metastases. Angiogenesis inhibitors are typically administered during or after chemotherapy at a time when tumor tissue should respond to a toxic challenge by promoting resumption of angiogenesis, providing blood supply and nutrients. Furthermore, it is preferable to perform such an angiogenesis inhibition step after solid tumor removal surgery for the prevention of metastasis.

The present invention may also be used in combination with other angiogenesis inhibitors. Angiogenesis inhibitors are known in the art and can be prepared by known methods. For example, angiogenesis inhibitors include integrin inhibitory compounds such as αυ integrin inhibitory antibodies, cell adhesion proteins or functional fragments thereof that contain cell adhesion binding sequences. Additional angiogenesis inhibitors include, for example, angiostatin, functional fragments of angiostatin, endostatin, fibroblast growth factor (FGF) inhibitors, FGF receptor inhibitors, VEGF inhibitors, VEGF receptor inhibitors , vascular permeability factor (VPF) inhibitors, VPF receptor inhibitors, glycoprotein G, platelet factor 4, interferon alpha, interferon gamma, interferon-inducible protein 10, interleukin 12, gro-beta, and prolactin of 16 kDa N-terminal fragments, thalidomide, and other mechanisms of inhibition of angiogenesis.

Angiostatin is the subject of the aforementioned US Patent 5,639,725. Endostatin is the subject of the aforementioned PCT publication WO 97/15666. For a description of the remaining angiogenesis inhibitors and targets listed above, respectively, see, e.g., Chen et al., Cancer Res. 55: 4230-423 3 (1995); Good et al., Proc. Natl. Acad. Sci. USA 87: 6629 -6628 (1990); O'Reilly et al., Cel 179:315-328 (19943; Parangi et al., Proc.Natl.Acad.Sci.USA 93:2002-2007 (1996); Rastinejad et al., Cell 56:345-355( 1989); Gupta et al, Proc. 162 (1995); Strieter et al., Biochem. Biophys. Res. Comm. 210: 51-57 (1995); Voest et al., J. Natl. Cancer Inst 87: 581-586 (1995); Cao et al., J. Exp. Med. 182: 2069-2077 (1995); Clapp et al., Endocrinology 133: 1292-1299 (1993). For a description of additional angiogenesis inhibitors see, e.g., Blood et al., Bioch. Biophys Acta., 1032: 89-118 (1990); Moses et al., Science, 248:1408-1410 (1990); Ingber et al., Laf Invest., 59:44-51 (1988) and US Patents 5,092,885; 5,112,946.

preparation

Formulations suitable for parenteral administration include aqueous and anhydrous sterile injection solutions such as pharmaceutically acceptable media above. The solution may additionally contain, for example, antioxidants, buffers, bacteriostats, and solutes to render the formulation isotonic with the blood of the intended recipient. Other formulations include, for example, aqueous and dry sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in single-dose or multi-dose containers, eg, hermetically sealed ampoules and vials, and may be stored under lyophilized conditions requiring, eg, the addition of sterile liquid carriers immediately before use. Extemporaneous injections and suspensions can be prepared from sterile powders, granules and tablets of the type previously described.

The pharmaceutically acceptable medium may additionally contain physiologically acceptable compounds, eg for stabilizing or increasing the absorption of the SNS tripeptide, analogue, mimetic or chemical derivative. Such physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents such as EDTA, which disrupt microbial membranes; divalent metal ions such as calcium or magnesium; proteins; lipids or liposomes; or other stabilizers or excipients. SNS tripeptides, analogs, mimetics or chemical derivatives can also be formulated with a pharmaceutically acceptable vehicle such as a biodegradable polymer.

polymeric preparation

Also included in the invention is the use of angiogenesis-inhibiting tripeptides, analogs, mimetics or chemical derivatives in polymeric formulations. Tripeptides can be incorporated into the medium through surface grafting, copolymerization, non-covalent bonding, or encapsulated as biomedical materials. Regulation and control of neovascularization is an essential part to obtain tissue engineering materials that should maintain a balance between pro-anti-angiogenic and anti-angiogenic factors. Tripeptides can be used in combination with known angiogenic promoters that regulate neovascularization in tissues, functional biomedical materials such as implant or repair materials, scaffolds for tissue engineering, wound healing materials, non-vivo artificial organ materials. Polymeric formulations can also be used for the sustained release of anti-angiogenic inhibitory peptides that inhibit neovascularization. This is an example of a method of drug delivery comprising binding an anti-angiogenic inhibitory peptide to a carrier material that can be used to locally deliver the anti-angiogenic effect of such an agent in an angiogenesis dependent disease.

Example

Having now generally described this invention, it will be more readily understood by reference to the following Examples, which are provided by way of illustration and are not intended to be limiting unless specifically indicated.

general method

Chemical reagents:

All reagents were of chemical grade and purchased from Sigma Chemical Co. (St. Louis, MO) or through VWR Scientific (Bridgeport, NJ). Cortisone acetate, bovine serum albumin (BSA) and gelatin solution (2% type B from bovine skin) were purchased from Sigma Chemical Co. M199 growth medium containing Earl's salt, basal FGF, insulin-transferrin- Selenium G supplement (IT-Se) 100X, Dulbecco's phosphate buffered saline (PBS) with or without Ca ⁺² and Mg+2, and 0.5M EDTA were purchased from Gibco BRL (Grand Island, NY). Human umbilical vein endothelial cells (HUVEC), endothelial cell basal medium (serum-free, EBM), EGM (supplemented growth factors, fetal calf serum), and 0.025% trypsin/0.01% EDTA solution were obtained from Clonetics Inc. (San Diego , CA). Human prostate (TSU-Pr) tumor cells were obtained from the American Type Culture Collection (Rockyille, MD). ^Matrigel® matrix and human type III collagen were purchased from Becton Dickinson (Bedford, MA). HEMA-3 fixative and staining solutions were purchased from Biochemical Sciences, Inc. (Swedesboro, NJ). Fertilized chicken eggs were purchased from Charles River Laboratories, SPAFAS Avian Products & Services (North Franklin, CT).

Peptide Synthesis:

All peptides described were prepared by standard solid phase synthesis protocols using FMOC chemistry (Chan et al., In FMOC Solid Phase Pepetide Synthesis: A Practical Approach, Oxford University Press; Chapter 3 (2000)). Solid phase, highly loaded PAL-PEG-PS resin and all FMOC protected amino acids were obtained from PerseptiveBiosystems (Framingham, MA). Peptide synthesis consists of three steps:

(1) Amino acid coupling: 1-hydroxybenzotriazole (HOBt)/2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/ Diisopropylethylamine (DIEA), (4.0/4.0/8.0 equivalents based on resin loading) and FMOC-protected amino acid (4.0 equivalents) in dimethylformamide (DMF) for 4 hours at room temperature;

(2) FMOC deprotection: 20% v piperidine in DMF, 1.5 hours;

(3) Fracture from resin: 95:5 trifluoroacetic acid (TFA)/triisopropylsilane (TIS), at room temperature for 6 hours. The crude product was triturated with ethyl ether and lyophilized from aqueous solution. The molecular weights of all peptides were confirmed by electrospray mass spectrometry (MW = 347.32, Obs. M+1 = 348.23). The structure and purity of N-acetyl-serine-asparagine-serine-(Ac-Ser-Asn-Ser-carbamoyl(1)) were estimated by ¹ H NMR spectroscopy and HPLC, where the purity was estimated to be about 80%. The main contaminant was polyethylene glycol residues from PAL-PEG-PS resin ( ¹ H NMR spectral peak at δ 3.6 ppm). The same peptide synthesis method was used to prepare larger peptides, 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).

Synthesis of SNS analog library:

conventional-

Library synthesis was performed in a Quest 210 synthesizer using 5 mL ^Teflon® reaction tubes (registered trademark EIde Nemours and Company of Wilmington, DE). Solid-phase peptide synthesis was performed on Argogel Rink resin using the FMOC method. The cycle of deprotection and coupling is repeated until a peptide of the desired length is formed. The peptide is then acylated and cleaved from the resin.

step-

To protect:

Argogel Rink-FMOC (200 mg, 0.064 mmol) was weighed into a 5 mL reaction vessel and washed with 3 mL DMF. A 20% piperidine/DMF (4 mL) solution was added and the resin was stirred for 2 minutes. The reaction mixture was filtered and the procedure was repeated with 4 mL of a fresh aliquot of 20% piperidine solution for an additional 20 minutes. Next, the resin was drained and washed four times with 5 mL of DMF. 5 minutes of agitation was used in each wash step.

Coupling:

To the reaction vessel was added 0.6 mL of dry DMF followed by 3 equivalents each of the first N-FMOC-amino acid and 1-hydroxybenzotriazole (HOBt) as a 0.5M solution in dry DMF. The resin was stirred for 1 min before adding 3 equivalents of HBTU as a 0.5M solution in dry DMF. The resin was stirred for 4 hours, then drained and washed with 5 x 4 mL of DMF, again stirring for 5 minutes per wash.

The deprotection step and coupling step are repeated for the second and third FMOC amino acids. After the third amino acid has been coupled, the deprotection step is performed again.

Acylation:

The resin obtained in the above procedure was treated with 3 mL of a 1:1:2 mixture of acetic anhydride:diisopropylethylamine:DMF for 1 hour. The resin was drained and washed repeatedly with DMF.

fracture:

Add 3 mL of 50% TFA/dichloromethane solution to the resin. The resin was stirred for 1 hour. Filter the resin and collect the filtrate. The filtrate was slowly added onto ice-cold ether to precipitate the desired product. The precipitate was collected by filtration and dissolved in 50% acetonitrile solution. Freeze drying of the solution afforded the desired product as a solid.

The meanings of the abbreviations are as follows: "sec" means seconds, "min" means minutes, "h" means hours, "d" means days, "μL" means microliters, "mL" means milliliters, " L" means liter, "mM" means millimole/liter, "M" means mole/liter and "mmol" means millimole.

Example 1

Inhibition of endothelial tube formation

Differentiation of endothelial cells was detected using the method developed by Grant et al. (In Vitro Cell Dev. Biol. 27A:327-336 (1991)). ^Matrigel® matrix, without phenol red (commercially obtained from Becton Dickinson, Bedford, MA) was thawed overnight at 4°C. Using a cold pipette tip, 3.0 mg/well ^Matrigel® matrix was placed into a cold twenty-four multiwell plate (Falcon). Incubate at 37°C for 30 min to polymerize the ^Matrigel® matrix.

Human umbilical vein endothelial cells (HUVEC) were maintained at 37°C in endothelial cell growth medium containing 2% fetal calf serum (EGM) in 5% _CO2 and 95% humidity. Tube experiments were performed in endothelial cell basal medium (EBM) supplemented with 0.5% bovine serum albumin (BSA) and 1:100 dilution of insulin-transferrin-selenium-G supplement (IT-Se, 100X). HUVECs were trypsinized and centrifuged, followed by two washes in phosphate buffered saline (PBS). After counting, the cell density was adjusted to 35,000 cells/mL.

Treat with 100 ng/mL recombinant human fibroblast growth factor base (FGF2) and peptide (see below) dissolved in EBM medium at a concentration of 0.015 μmol to a final concentration of 35,000 cells/mL/well. Treated cells were incubated overnight at 37 °C in 5% _CO2 and 95% humidity to allow cell adhesion.

Subsequently, the medium was aspirated, and cells were fixed and stained with a modified HEMA-3 staining kit. Digital images of microtiter well sections were collected with a DKC50003-CCD color video camera system (Toshiba America, New York, NY) and analyzed with Image Pro Plus software (Media Cybernetics, Silver Spring, MD). The area and maximum axial length of stained cells with tubular morphology on the ^Matrigel® matrix surface (Becton Dickinson, Bedford, PA) were calculated from 5 images/well.

The following four peptides were detected: Acetyl-Ser-Asn-Ser-carbamoyl (structure 1), Acetyl-Asn-Tyr-Tyr-Ser-Asn-Ser (SEQ ID NO: 2), Acetyl-Ser- Asn-Ser-Tyr-Ser-Phe-Trp-Leu (SEQ ID NO: 3) and Acetyl-Cys-Asn-Tyr-Tyr-Ser-Asn-Ser-Tyr-Ser-Phe-Trp-Leu (SEQ ID NO: 4). Table 1 and Figure 1 clearly demonstrate that acetyl-Ser-Asn-Ser-carbamoyl (structure 1) is a very potent inhibitor of FGF2-stimulated EC tube formation in vitro. Area data are expressed in units of 10 ⁴ square microns and length data are expressed in length/area in mm/mm ² units.

Table 1

Microscopic analysis of EC tube formation sample concentration Cell tube formation (area) Tube formation (length) PBS - 1.0554+/-0.11 41.3611+/-2.61 FGF2 100ng/mL 2.2501+/-0.28 79.8272+/-8.66 Acetyl-SNS-carbamoyl 0.015μmol 1.1011+/-0.12 47.5267+/-4.31

Table 2 demonstrates that Acetyl-Ser-Asn-Ser-carbamoyl (structure 1) is a more potent inhibitor of FGF2-stimulated EC tube formation in vitro than the larger peptide. Inhibition percentage data are expressed as the quotient of the experimental value minus the negative control value (EBM medium) divided by the difference between the positive control value and the negative control value.

Table 2

Mean % inhibition of EC tube formation by peptides sample MW 0.015μmol % Inhibition of Cell Tube Formation (Area) % Inhibition of Tube Formation (Length) Acetyl-SNS-carbamoyl 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

Example 2

Neovascularization on the CAM and Microscopic Analysis of CAM Sections

Neovascularization in vivo was assayed by the method described by Auerbach et al. (J. Dev. Biol. 41:391-394 (1974)). Ten day old embryos were purchased from Spafas, Inc. (Preston, CT) and incubated at 37°C, 55% relative humidity. In the dark, with the aid of a candle light, a small hole is pierced with a hypodermic needle in the shell covering the air sac. Pierce a second hole in the shell on the lateral side of the egg directly across the vascular portion of the embryonic membrane, as observed during candling. Applying negative pressure to the first hole creates a pseudo-air sac beneath the second hole, causing the chorioallantoic membrane (CAM) to separate from the shell. On the shell over the sagging CAM, a small craft grinding wheel (Dremel, Division of Emerson Electric Company Racine, WI) was used to cut an approximately 1.0 ^cm2 window allowing direct access to the CAM below it. Discs of #1 filter paper (Whatman International, United Kingdom) were soaked in 95% ethanol and water containing 3 mg/mL cortisone acetate (Sigma, St. Louis, MO) and then air dried under sterile conditions. FGF2 (Life Technologies, Gaithersburg, MD) was used to grow vessels on CAMs of 10-day-old chicken embryos. Sterile filter discs imbibed with FGF2 dissolved in PBS at 1 μg/ml were placed on the growing CAMs. At 24 hours, test compounds or control vehicle were added directly to topical CAMs.

CAM tissue just below FGF2-saturated filter discs was excised from embryos previously treated with compound or control for 48 hr. Wash the tissue three times with PBS. Sections were placed in 35mm Petri dishes (Nalge Nunc, Rochester, NY) and examined under a SV6 stereomicroscope (Karl Zeiss, Thornwood, NY) at 50X magnification. Digital images of CAM slices adjacent to the filter were collected with a 3-CCD color video camera system (Toshiba America, New York, NY) and analyzed with Image-Pro Plus software (Media Cybernetics, Silver Spring, MD), as shown in Figure 2. Table 3 contains the number of vessel branch points contained within the annular area equal to the filter area per section. Percent inhibition data are expressed as the quotient of the experimental value minus the negative control value divided by the difference between the positive control value and the negative control value.

table 3

Mean % inhibition of angiogenesis in CAM model sample Average number of branch points# PBS control 94.714 FGF2 control 263.88 Difference 169.16 FGF-2 + 16 μg Acetyl-SNS-carbamoyl 91.60 % inhibition of branch point formation 100.23

Example 3

Chick Chorioallantoic Membrane Tumor Test

Ten million tumor cells were placed on the surface of each CAM and cultured for one week. The resulting tumors were dissected and cut into 50 mg pieces. These fragments were placed on additional CAMs and the assay reagent was injected intravenously the next day for local or systemic treatment. Forty-eight hours later, CAMs were excised from the eggs and the number of blood vessels entering the tumor (as vessel branch points) was counted. Data are presented as the mean number of vessels (+/- standard error of assay) per treatment group. At least ten tumors were introduced per trial per treatment group. Tumors were then excised from the eggs and the tumor weight of each tumor was determined. Data are presented in Table 3 as mean tumor weight (+/- standard error of assay) for each treatment group. Statistical analysis was performed using Student's t-test. The results of this experiment, described in Table 3, show that 16 μg of Acetyl-Ser-Asn-Ser-carbamoyl (structure 1) is effective in controlling FGF2-stimulated neovascularization in the CAM (100% mean inhibition at 16 μg).

Example 4

Inhibition of TSU-Pr (Prostate) Tumor Growth

Ten million tumor cells were placed on the surface of each CAM (7 day old embryo) and cultured for one week. The resulting tumors were dissected and cut into 50 mg pieces. These fragments were placed on additional CAMs and treated topically with acetyl-Ser-Asn-Ser-carbamoyl (construct 1) or vehicle the next day. Seven days later, CAMs were excised from the eggs and the number of blood vessels entering the tumor (as vessel branch points) was counted. Data are presented as the mean number of vessels (+/- standard error of assay) per treatment group. At least ten tumors were introduced per trial per treatment group. Typical tumors were photographed at 10X magnification. Tumors were then excised from the eggs and the tumor weight of each tumor was determined. Data are presented as mean tumor weight (+/- standard error of the mean) for each treatment group. Statistical analysis was performed using Student's t-test.

Acetyl-Ser-Asn-Ser-carbamoyl (structure 1) was shown to inhibit human prostate tumor growth in vivo by direct injection into the tumor (Figure 3). Seven days later, six of ten tumors injected with 45 μg of Acetyl-Ser-Asn-Ser-carbamoyl (construct 1) showed significant weight loss (111% mean inhibition at 45 (100% mean at 16 μg) inhibition)).

Example 5

Preparing Libraries Based on Acetyl-Ser-Asn-Ser-Carboxamide

Based on the observed anti-angiogenic activity of acetyl-Ser-Asn-Ser-carbamoyl (structure 1), the following fifteen peptides were prepared using standard FMOC solid-phase peptide chemistry (see General Methods): SAS, SQS, sNS , snS, SGS, SES, sNs, sns, SDS, SnS, SNs, Sns, t4Hyp-NS, t4Hyp-N-t4Hyp and SN-t4Hyp. Uppercase letters indicate L amino acids (natural), lowercase letters indicate D amino acids (unnatural) and t4Hyp indicates trans-4-hydroxyproline.

                  

        

                  

        

SAS C ₁₁ H ₂₀ N ₄ O ₄ SQSC ₁₃ H ₂₂ N ₆ O ₇ sNS C ₁₂ H ₂₁ N ₆ O ₇ snS C ₁₂ H ₂₁ N ₆ O ₇ Exact mass: 304.14 Exact mass: 361.18 Exact mass: 347.14 Exact mass: 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.6.08N.20.16;O.32.25

C.41.50;H.6.09N.20.16;5O.32.2

                           

                           

SGSC ₁₀ H ₁₈ N ₄ O ₄ SESC ₁₃ H ₂₂ N ₄ O ₈ sns C ₁₂ H ₂₁ N ₆ O ₇ sns C ₁₂ H ₂₁ N ₅ O ₇ Exact mass: 290.12 Exact mass: 362.14 Exact mass: 347.14 Exact mass: 347.14 Mol.WL: 200.27 Mol.WL: 362.34 Mol.WL: 347.32 Mol.WL: 347.32C.41.36; H.6.25; N.19.30; O.33.07C.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 ₁₂ H ₂₀ N ₄ O ₈ SnSC ₁₂ H ₂₁ N ₅ O ₇ SNsC ₁₂ H ₂₁ N ₆ O ₇ SnsC ₁₂ H ₂₁ N ₄ O ₇ Exact mass: 348.13 Exact mass: 347.14 Exact mass: 347.14 Exact mass: 347.14 Mol. WL: 348.31Mol.WL: 347.32Mol.WL: 347.32Mol.WL: 347.32C.41.38; H.5.70; N.16.09; O.36.75C.41.50; H.6.09; N.20.16; O.32.25C. 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 ₁₄ H ₂₃ N ₆ O ₇ t4Hyp-N-t4Hyp C16H ₂₅ N ₆ O ₇ SN-t4Hyp C ₁₄ H ₂₃ N ₅ O ₇ Exact mass: 373.15 Exact mass: 309.18 Exact mass: 373.16 Mol.WL. : 373.36 Mol.WL: 399.40 Mol.WL: 373.36C.45.04; H.6.21; N.18.76; O.30.00C.48.12; N.18.76; O.30.00

The anti-angiogenic effect of the above fifteen peptides and the acetyl Ser-Asn-Ser-carbamoyl group (Structure 1) on tube formation in human endothelial cells was tested as described in Example 1 (Figure 4). In the CAM assay, the three most potent compounds SQS, SNS and SN-t4Hyp were also tested, as described in Example 2 (also Figure 4). Figure 5 illustrates the angiogenesis inhibitory effect of SNS tripeptide analogues in a CAM model. Table 4 shows the dose-response data of SQS, SNs and SN-t4Hyp in the CAM trial.

Table 4 Inhibition % of branch point in FGF2-induced CAM assay Concentrationμg/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.

Images