EP1137659A1 - Inhibition de l'angiogenese par du kininogene de poids moleculaire eleve et analogues peptidiques dudit kininogene - Google Patents

Inhibition de l'angiogenese par du kininogene de poids moleculaire eleve et analogues peptidiques dudit kininogene

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Publication number
EP1137659A1
EP1137659A1 EP99962723A EP99962723A EP1137659A1 EP 1137659 A1 EP1137659 A1 EP 1137659A1 EP 99962723 A EP99962723 A EP 99962723A EP 99962723 A EP99962723 A EP 99962723A EP 1137659 A1 EP1137659 A1 EP 1137659A1
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EP
European Patent Office
Prior art keywords
gly
lys
asn
amino acid
asp
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EP99962723A
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German (de)
English (en)
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EP1137659A4 (fr
Inventor
R. Keith Mccrae
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Temple Univ School of Medicine
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Temple Univ School of Medicine
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Publication of EP1137659A1 publication Critical patent/EP1137659A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to therapeutic compounds and methods for inhibiting angiogenesis.
  • Angiogenesis is the process in which new blood vessels grow into an area which lacks a sufficient blood supply. Angiogenesis commences with the erosion of the basement membrane surrounding endothelial cells and pericytes forming capillary blood vessels. Erosion of the basement membrane is triggered by enzymes released by endothelial cells and leukocytes. The endothelial cells then migrate through the eroded basement membrane when induced by angiogenic stimulants. The migrating cells form a "sprout" off the parent blood vessel. The migrating endothelial cells proliferate, and the sprouts merge to form capillary loops, thus forming a new blood vessel.
  • angiostatin which contains kringles 1-4 plasminogen (O'Reilly et al., Cell 79:315-328, 1994), endostatin, a 20 kD C-terminal fragment of collagen XVIII (O'Reilly et al., Cell 88:277-285, 1997), PEX, the hemopexin domain of matrix metalloprotease 2 (Brooks et al., Cell 92:391-400, 1998), the C- terminal 16 kD fragment of prolactin (Clapp et al., Endoc ⁇ nol 133:1292- 1299, 1993) and a 29 kD fragment of fibronectin (Homandberg et al., Am JPathol 120:327-332, 1985).
  • High molecular weight kininogen is a 120 kD glycoprotein containing heavy and light chains, comprised of domains 1 through 3, and 5 and 6, respectively (Kaplan et al., Blood 70: 1-15, 1987). The heavy and light chains are linked by domain 4, which contains bradykinin, a nonapeptide which mediates several events including NO- dependentvasodilation (Weimerera/., JPharmExp Therapeutics 262:729- 733, 1992).
  • HK also referred to as “single chain high molecular weight kininogen" binds with high affinity to endothelial cells, where it is cleaved to two-chain high molecular weight kininogen (HK a ) by plasma kallikrein. Bradykinin is released from HK through cleavage mediated by plasma kallikrein (Kaplan et al., Blood 70:1-15, 1987). This event occurs on the surface of endothelial cells following the activation of prekallikrein to kallikrein by an endothelial cell protease (Motta et al., Blood 91 :515-528, 1998).
  • HK a contains a 62 kD heavy chain and a 56 kD light chain linked by a disulfide bond.
  • HK a Conversion of HK to HK a is accompanied by a dramatic structural rearrangement, which has been demonstrated using rotary shadowing electron microscopy (Weisel et al. J. Biol Chem 269:10100- 10106, 1994). HK a , but not HK, has been shown to inhibit the adhesion of endothelial and other cell types to vitronectin (Asakura, J. Cell Biol 116:465-476, 1992).
  • the compounds of the present invention are in the form of peptides which possess anti-angiogenic activity.
  • the peptide may optionally comprise an amino-terminal and/or carboxy-terminal protecting group.
  • a compound of the formula X His-Lys-X-Lys-X 2 (hereinafter "X ⁇ His-Lys-X-Lys-Xs peptide") is provided wherein
  • X is any amino acid
  • X 2 is from zero to twelve amino acids, more preferably from zero to six amino acids, most preferably from zero to three amino acids.
  • X is an amino acid having a nonpolar side chain, i.e., Ala, Leu, He, Val, Pro, Phe, Trp, or Met; or X is an amino acid having a polar side group which is uncharged at pH 6.0 to 7.0, the zone of physiological pH, i.e., Ser, Thr, Tyr, Asn, Gin, Cys, or Gly.
  • X is Asn, Phe or His.
  • Preferred compounds comprise fragments of HK. In one group of such preferred compounds,
  • Gln-Gly-Gly-His-Val SEQ ID NO:2
  • C-terminal truncation fragment thereof containing at least one amino acid.
  • the compound has a substantial amino acid homology to either the amino acid sequence His-Gly-His-Glu-Gln-Gln-His-Gly-Leu-Gly-His-Gly-His-Lys-
  • Exemplary and preferred compounds include:
  • the invention also encompasses a method of inhibiting endothelial cell proliferation comprising contacting endothelial cells with HK, HK a or a X r His-Lys-X-Lys-X 2 peptide.
  • the invention also encompasses a method of inducing apoptosis of endothelial cells comprising contacting endothelial cells with HK, HK a or a X r His-Lys-X-Lys-X 2 peptide.
  • the invention is also a composition comprising a pharmaceutically effective carrier and HK, HK a or a X r His-Lys-X-Lys-X 2 peptide.
  • the invention is also a method of inhibiting angiogenesis in a mammal in need of such treatment comprising administering to said mammal a therapeutically effective amount of a composition comprising a pharmaceutically effective carrier and HK, HK a or a X r His-Lys-X-Lys-X 2 peptide.
  • the mammal treated is preferably a human being.
  • bFGF is recombinant human basic fibroblast growth factor.
  • HK means the mature form of high molecular weight kininogen, and any allelic variations thereof.
  • mature is meant the post- translationally-modified form of HK which results from cleavage of an eighteen amino acid leader from the initially translated molecule. All numbering with respect to amino acid positions of HK is from the N- terminus of the mature form as position 1.
  • HK is synonymous with “single chain HK", the mature form of high molecular weight kininogen prior to cleavage by kallikrein and the formation of two-chain high molecular weight kininogen.
  • HK a means two-chain high molecular weight kininogen, the product of kallikrein cleavage of mature high molecular weight kininogen, and any allelic variations thereof.
  • HDMVEC human dermal microvascular endothelial cells
  • HGF hepatocyte growth factor
  • Apoptosis means a process of programmed cell death.
  • a "peptide” is a compound comprised of amino acid residues covalently linked by peptide bonds.
  • amino acid as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids.
  • Natural amino acid means any of the twenty primary, naturally occurring amino acids which typically form peptides, polypeptides, and proteins.
  • Synthetic amino acid means any other amino acid, regardless of whether it is prepared synthetically or derived from a natural source.
  • synthetic amino acid also encompasses chemically modified amino acids, including but not limited to salts, derivatives (such as amides), and substitutions.
  • Amino acids contained within the peptides of the present invention can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the peptide's circulating half life without adversely affecting their activity. Additionally, a disulfide linkage may be present or absent in the peptides of the invention, as long as anti- angiogenic activity is maintained.
  • Amino acids have the following general structure:
  • Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.
  • Peptides comprising a large number of amino acids are sometimes called "polypeptides".
  • the amino acids of the peptides described herein and in the appended claims are understood to be either D or L amino acids with L amino acids being preferred.
  • Homology means similarity of sequence reflecting a common evolutionary origin. Peptides or proteins are said to have homology, or similarity, if a substantial number of their amino acids are either (1) identical, or (2) have a chemically similar R side chain. Nucleic acids are said to have homology if a substantial number of their nucleotides are identical.
  • protecting groups refers to a terminal amino group of a peptide, which terminal amino group is coupled with any of various amino-terminal protecting groups traditionally employed in peptide synthesis.
  • protecting groups include, for example, acyl protecting groups such asformyl, acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic urethane protecting groups such as benzyloxycarbonyl; and aliphatic urethane protecting groups, for example, tert-butoxycarbonyl or adamantyloxycarbonyl. See Gross and Mienhofer, eds., The Peptides, vol. 3, pp. 3-88 (Academic Press, New York, 1981) for suitable protecting groups.
  • protected with respect to a terminal carboxyl group refers to a terminal carboxyl group of a peptide, which terminal carboxyl group is coupled with any of various carboxyl-terminal protecting groups.
  • protecting groups include, for example, tert-butyl, benzyl or other acceptable groups linked to the terminal carboxyl group through an ester or ether bond.
  • Substantial amino acid sequence homology means an amino acid sequence homology greater than about 30 %, preferably greater than about 60%, more preferably greaterthan about 80%, and most preferably greater than about 90 %.
  • N-terminal truncation fragment with respect to an amino acid sequence is meant a fragment obtained from a parent sequence by removing one or more amino acids from the N-terminus thereof.
  • C-terminal truncation fragment with respect to an amino acid sequence is meant a fragment obtained from a parent sequence by removing one or more amino acids from the C-terminus thereof.
  • Fig. 1 shows the inhibition of endothelial cell proliferation over time following contact with HK a .
  • Fig. 2 shows the concentration-dependent inhibition of endothelial cell proliferation by HK a (black bars), HK (white bars) and low molecular weight kininogen (stippled bars). Low molecular weight kininogen is non-inhibitory.
  • Fig. 3 shows the ability of 30 nM HK a to inhibit endothelial cell proliferation stimulated by a variety of growth factors.
  • Fig. 4 shows that HK a inhibits proliferation of two types of endothelial cells (HUVEC and HDMVEC), but not human aortic smooth muscle cells (HASMC).
  • Fig. 5 shows the inhibition of endothelial cell proliferation as a function of HK a concentration and cell density in the culture.
  • Fig. 6A shows endothelial cells after staining with 4', 6'- diamidino-2-phenylindole hydrochloride.
  • Fig. 6B shows endothelial cells upon staining with 4',6'-diamidino-2-phenylindole hydrochloride after four hours of exposure to HK a .
  • Figs. 7A and 7B show DNA fragmentation in endothelial cells exposed to HK a as a function of time (at 30 nM concentration) and concentration (at 12 hours), respectively.
  • Figs. 8A and 8B show athymic Ncr nude mice injected subcutaneously with chilled Matrigel ® containing bFGF which resulted in formation of a visible "plug". The plug was photographed four days post implantation.
  • Figs. 8C and 8D are similar to 8A and 8B except that the plug contained HK a . The arrows in the figures point to the plug periphery. Plug vascularization is visible in Figs. 8A and 8B, but absent in Figs. 8C and 8D.
  • the present invention is based upon the discovery that HK a and peptide analogs of certain sites in the HK domain 5 inhibit endothelial cell proliferation and/or induce endothelial cell apoptosis. These activities confer upon HK a and the X r His-Lys-X-Lys-X 2 peptides the ability to inhibit cytokine-d riven angiogenesis in vivo.
  • HK plasma concentration (670 nM) of the parent molecule, HK, is considered, it is apparent that the anti-angiogenic activity of HK a and the X ⁇ His-Lys-X-Lys-X;, peptides is physiologically significant.
  • HK a potently inhibits the proliferation of human umbilical vein and microvascular endothelial cells in vitro in response to various pro-angiogenic growth factors: bFGF, VEGF, hepatocyte growth factor, TGF- ⁇ and PDGF. Inhibition of endothelial cell proliferation is detected within 6 hours of exposure of the cells to HK a , and is accompanied by morphologic and biochemical evidence of cell apoptosis.
  • HK a is effective in an in vivo model of angiogenesis. HK a inhibits the ingrowth of new blood vessels into a reconstituted extracellular matrix (Matrigel) containing the pro-angiogenic growth factor bFGF implanted subcutaneously into mice.
  • Motrigel extracellular matrix
  • HK a but not single chain HK, inhibits endothelial cell proliferation suggests that the structural change which the molecule undergoes following kallikrein-mediated cleavage is important for expression of its anti- angiogenic activity.
  • HK a generated by plasma kallikrein cleavage of HK differs from HK in that it lacks the nine amino acid segment comprising HK amino acids 363-371. This segment is released from HK as the nonapeptide bradykinin.
  • the two chains of HK resulting from the elimination of bradykinin remain linked by a disulfide bond between cysteine residues at positions 10 and 656 of mature HK.
  • HK a The N-terminal and C-terminal chains of HK a generated by plasma kallikrein cleavage of HK and release of bradykinin are known as HK "heavy" and “light” chains, respectively.
  • HK a may be generated by treating HK with plasma kallikrein, according to well- known methods. HK a is also commercially available.
  • other enzymes such as plasmin, chymotrypsin or matrix metalloproteases, for example, may degrade HK to release peptides similar to those described herein.
  • HK domain 5 spans HK residues 384-502. Located within domain 5 are two separate segments characterized by the sequence His- Lys-X-Lys. The first such segment occurs at position 457-460. The second segment occurs at position 488-491. HK a -derived peptides containing the His-Lys-X-Lys sequence inhibit endothelial cell proliferation and are useful as anti-angiogenic agents.
  • the X r His-Lys-X-Lys-X 2 peptides of the present invention may be recombinant peptides, natural peptides, or synthetic peptides. They may also be chemically synthesized, using, for example, solid phase synthesis methods.
  • the peptide chain can be prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence.
  • various N-protecting groups e.g., the carbobenzyloxy group or the t-butyloxycarbonyl group
  • various coupling reagents e.g., dicyclohexylcarb ⁇ diimide or carbonyldimidazole, various active esters, e.g., esters of N-hydroxyphthalimide or N-hydroxy- succinimide
  • the various cleavage reagents e.g., trifluoroacetic acid (TFA), HCI in dioxane, boron tris-(trifluoracetate) and cyanogen bromide
  • reaction in solution with isolation and purification of intermediates is well-known classical peptide methodology.
  • Peptide synthesis includes both manual and automated techniques employing commercially available peptide synthesizers.
  • the X His-Lys-X-Lys-X 2 peptides may be prepared by chemical synthesis and biological activity can be tested using the methods disclosed herein.
  • the X r His-Lys-X-Lys-X 2 peptides may be prepared utilizing recombinant DNA technology, which comprises combining a nucleic acid encoding the peptide thereof in a suitable vector, inserting the resulting vector into a suitable host cell, recovering the peptide produced by the resulting host cell, and purifying the polypeptide recovered.
  • recombinant DNA technology comprises combining a nucleic acid encoding the peptide thereof in a suitable vector, inserting the resulting vector into a suitable host cell, recovering the peptide produced by the resulting host cell, and purifying the polypeptide recovered.
  • synthetic genes encoding HK a -derived peptides may be synthesized directly on a DNA synthesizer, or may be synthesized as complementary oligonucleotides which are ligated together to form the synthetic gene.
  • the nucleic acids encoding HK a -derived peptides may be operatively linked to one or more regulatory regions. Regulatory regions include promoters, polyadenylation signals, translation initiation signals (Kozak regions), termination codons, peptide cleavage sites, and enhancers.
  • the regulatory sequences used must be functional within the cells of the vertebrate to be immunized. Selection of the appropriate regulatory region or regions is a routine matter, within the level of ordinary skill in the art.
  • Promoters that may be used in the present invention include both constitutive promoters and regulated (inducible) promoters.
  • the promoters may be prokaryotic or eukaryotic depending on the host.
  • prokaryotic (including bacteriophage) promoters useful for practice of this invention are lacl, lacZ, T3, T7, lambda Pr' PI' and trp promoters.
  • eukaryotic (including viral) promoters useful for practice of this invention are ubiquitous promoters (e.g. HPRT, vimentin, actin, tubulin), intermediate filament promoters (e.g. desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters (e.g.
  • tissue-specific promoters e.g. actin promoter in smooth muscle cells
  • promoters which respond to a stimulus e.g. steroid hormone receptor, retinoic acid receptor
  • tetracycline-regulated transcriptional modulators cytomegalovirus immediate-early, retroviral LTR, metallothionein, SV-40, E1a, and MLP promoters.
  • Tetracycline-regulated transcriptional modulators and CMV promoters are described in WO 96/01313, US 5,168,062 and 5,385,839, the entire disclosures of which are incorporated herein by reference.
  • polyadenylation signals examples include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals.
  • the X His-Lys-X-Lys-X 2 peptides prepared by either chemical synthesis or recombinant DNA technology may then be assayed for biological activity according to the assay methods described herein.
  • the peptides of the present invention may be used in the form of a pharmaceutically acceptable salt.
  • Suitable acids which are capable of forming salts with the peptides include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like
  • organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, mal
  • Suitable bases capable of forming salts with the peptides include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanol- amines (e.g., ethanolamine, diethanolamine and the like).
  • inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like
  • organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanol- amines (e.g., ethanolamine, diethanolamine and the like).
  • the present invention provides methods for inhibiting angiogenesis.
  • a preferred embodiment is a method of inhibiting the proliferation of endothelial cells, or obtaining apoptosis of such cells.
  • HK, HK a and/or one or more X r His-Lys-X-Lys-X 2 peptides is administered to a patient in need of such treatment.
  • a therapeutically effective amount of the drug may be administered as a composition in combination with a pharmaceutically carrier.
  • HK is considerably less anti-angiogenic than HK a it is possible that upon administration HK will be converted to HK a , and may therefore serve as a prodrug for HK a .
  • HK may be rapidly converted to HK a in tumors, or further cleaved by tumor-derived enzymes to release peptides the same or similar to the peptides disclosed herein.
  • HK a to inhibit the proliferation of endothelial cells cultured on different extracellular matrix (ECM) proteins was determined.
  • ECM extracellular matrix
  • HK a caused apoptosis of endothelial cells cultured on gelatin, but not on collagen, and of cells cultured at low density, but not under confluent or near-confluent conditions. Without wishing to be bound by any theory, it appears that mature endothelial cells residing on an intact, collagen-rich basement membrane may be protected from HK a -induced apoptosis, and that HK a might selectively target angiogenic endothelial cells in a protease-rich tumor milieu in which ECM is partially degraded.
  • compositions include physiologically tolerable or acceptable diluents, excipients, solvents, adjuvants, or vehicles, for parenteral injection, for intranasal or sublingual delivery, for oral administration, for rectal or topical administration or the like.
  • the compositions are preferably sterile and nonpyrogenic.
  • suitable carriers include but are not limited to water, saline, dextrose, mannitol, lactose, or other sugars, lecithin, albumin, sodium glutamate cysteine hydrochloride, ethanol, polyols (propyleneglycol, ethylene, polyethyleneglycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, ethoxylated isosteraryl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methahydroxide, bentonite, agar-agarand tragacanth, or mixtures of these substances, and the like.
  • compositions may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, pH buffering agents, antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, and the like). If desired, absorption enhancing or delaying agents (such as liposomes, aluminum monostearate, or gelatin) may be used.
  • auxiliary substances such as wetting agents, emulsifying agents, pH buffering agents, antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, and the like).
  • absorption enhancing or delaying agents such as liposomes, aluminum monostearate, or gelatin
  • the compositions can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • compositions may be administered by any convenient route which will result in delivery to the site of undesired angiogenesis in an amount effective for inhibiting that angiogenesis from proceeding.
  • Modes of administration include, for example, orally, rectally, parenterally (intravenously, intramuscularly, intraarterially, or subcutaneously), intracisternally, intravaginally, intraperitoneally, locally (powders, ointments or drops), or as a buccal or nasal spray or aerosol.
  • the compositions can also be delivered through a catheter for local delivery at a target site, or via a biodegradable polymer.
  • the compositions may also be complexed to ligands, or antibodies, for targeted delivery of the compositions.
  • compositions are most effectively administered parenterally, preferably intravenously or subcutaneously.
  • intravenous administration they may be dissolved in any appropriate intravenous delivery vehicle containing physiologically compatible substances, such as sodium chloride, glycine, and the like, having a buffered pH compatible with physiologic conditions.
  • physiologically compatible substances such as sodium chloride, glycine, and the like
  • the vehicle is a sterile saline solution. If the peptides are sufficiently small (e.g., less than about 8-10 amino acids) other preferred routes of administration are intranasal, sublingual, and the like.
  • Intravenous or subcutaneous administration may comprise, for example, injection or infusion.
  • compositions according to the invention can be administered in any circumstance in which inhibition of angiogenesis is desired.
  • Disease states which may be treated include but are not limited to cancer, rheumatoid arthritis, and certain ocular disorders characterized by undesired vascularization of the retina. Because the peptides of the invention are anti-angiogenic, cancers characterized by the growth of solid tumors through angiogenesis of the tissue surrounding the tumor site may be treated according to the invention.
  • the amount of active agent administered depends upon the degree of the anti-angiogenic effect desired. Those skilled in the art will derive appropriate dosages and schedules of administration to suit the specific circumstances and needs of the patient. Typically, dosages are from about 0.1 to about 100, preferably from about 0.5 to about 50, most preferably from about 1 to about 20, mg/kg of body weight.
  • the active agent may be administered by injection daily, over a course of therapy lasting two to three weeks, for example. Alternatively, the agent may be administered by continuous infusion, such as via an implanted subcutaneous pump.
  • Peptides which inhibit endothelial ceil proliferation by at least 30%, more preferably by at least 50%, most preferably by at least 70%, when incubated with such cells at a concentration of 50 ⁇ M are preferred.
  • percent inhibition of proliferation is determined according to the procedure and formula set forth in Example 1 , part A, below.
  • Tissue culture medium and reagents were obtained from Mediatech (Herndon, VA). Fetal bovine serum was from Hyclone (Logan, Utah). Endothelial growth supplement was purified from bovine hypothalamii, as previously described (Maciag et al., Proc natl Acad Sci 76:5674-5678, 1979). Recombinant human basic fibroblast growth factor (bFGF), vascular endothelial cell growth factor (VEGF) and hepatocyte growth factor (HGF) were obtained from Collaborative Biomedical Products/Becton Dickinson (Bedford, MA).
  • bFGF basic fibroblast growth factor
  • VEGF vascular endothelial cell growth factor
  • HGF hepatocyte growth factor
  • PDGF Platelet-derived growth factor
  • TGF- ⁇ transforming growth factor ⁇
  • Gelatin was purchased from Sigma (St. Louis, MO).
  • Single and two-chain high molecular weight kininogen were obtained from Enzyme Research Labs (South Bend, IN).
  • HK a was >98% two-chain, as determined using 10% SDS-PAGE after reduction.
  • Low molecular weight kininogen was purchased from American Research Products, (Belmont, MA). Bradykinin was from Peninsula Laboratories (Belmont, CA), and rabbit anti-bradykinin antiserum from Sigma. All HK a preparations used in these studies contained less than ⁇ 0.01 EU/ml of endotoxin, as determined using the E-Toxate (Limulus Amoebocyte) assay (Sigma).
  • Synthetic Peptides Synthetic peptides were synthesized on a Rainin Symphony multiple peptide synthesizer, using Fmoc chemistry. All resins (AnaSpec) used for solid phase synthesis were Wang resins preloaded with the first amino acid. Fmoc amino acids were purchased from Perseptive Biosystems, with side chain protective groups as follows: trityl (Asn, Cys, Gin, and His), Boc (Lys and Trp), Ombu (Asp and Glu), T.U. (Ser, Thr and Tyr) and P.G. (Arg). Deprotection of the Fmoc group was performed in piperidine in dimethtylformamide (DMF).
  • DMF dimethtylformamide
  • Coupling was carried out in HBTU in N-methylmorpholine/DMF as the activator. Standard synthesis cycles were 3 x 30" washes with DMF, 3 x 15" deprotection with piperidine, 6 x 20" DMF washes, 45 minute coupling with amino acid and activator followed by 3 x 30" DMF washes.
  • Peptides were cleaved off the solid phase support with cleavage cocktail consisting of 88:5:5:2 (TFA:water:phenol: triisopropylsilane). Cleavage was done on the synthesizer. Peptides were precipitated with ether, pelleted by centrifugation, washed three times with ether and then allowed to dry. HPLC was carried out on a Beckman HPLC system using Rainin Dynamax Reversed Phase columns and an acetonitrile gradient in water. The desired peptide was detected during elution by off line MALDI-TOF mass spectrophotometry using a Perseptive Biosystems Voyager instrument. Purified peptides were lyophilized and then reanalyzed by MALDI-TOF mass spectrophotemetry.
  • cleavage cocktail consisting of 88:5:5:2 (TFA:water:phenol: triisopropylsilane). Cleavage was done on the synthesizer. P
  • % inhibition (1 - [(A 490 (+GF, HKa) - A 490 ( . GF) ) / (A 490 ( - GF) - A 490 ( . GF) )]) x 100,
  • (+GF) and (+GF) represent proliferation in the presence or absence of added growth factor
  • (+GF, HK a represents proliferation in the presence of both growth factor and HK a .
  • the significance of differences in relative endothelial cell proliferation cell numbers at the end of the proliferation assays was determined using the Student's two-tailed T-test for paired samples.
  • HK a inhibited endothelial cell proliferation to a much greater extent than single-chain HK.
  • Low molecular weight kininogen which is derived from alterative splicing of the kininogen gene and contains the entire HK light chain, domain 4 and a truncated domain 5 containing only 12 amino acids, had no effect on endothelial cell proliferation (Figure 2).
  • bradykinin did not inhibit proliferation, and anti-bradykinin antibodies did not inhibit the anti-proliferative effect of HK a (not shown). The latter results exclude the possibility that contamination of HK a with trace amounts of bradykinin was responsible for its ability to inhibit endothelial cell proliferation.
  • Single-chain HK modestly inhibited endothelial cell proliferation.
  • HK a is a potent and rapid inhibitor of endothelial cell proliferation in vitro, and that these activities occur within a concentration range similar to that of previously-described anti-angiogenic polypeptides.
  • HK a inhibited the proliferation of human endothelial cells in response to a number of mitogens, including bFGF, VEGF, HGF, TGF- ⁇ and PDGF, equally well (Figure 3).
  • the mitogenic activity of each of these factors is mediated through interactions with distinct receptors.
  • HK a In other experiments, the ability of HK a to inhibit the proliferation of endothelial cells cultured on different extracellular matrix (ECM) proteins. HK a (10nM) potently inhibited the proliferation of HUVEC cultured on gelatin, laminin and Matrigel, though slightly less potent inhibition, largely overcome by high concentrations of HK a (50 nM), occurred when cells were cultured on fibronectin or vitronectin.
  • ECM extracellular matrix
  • Cells plated on glass coverslips were cultured in the absence or presence of 30 nM HK a for periods of 2-24 hours. Cells were then fixed for 1 hour in phosphate-buffered saline (PBS) containing 1 % formaldehyde, and stained for 2 hours with a solution containing 1 ⁇ g/ml of 4',6'-diamidino- 2-phenylindole dihydrochloride (DAPI) and 10 ⁇ g/ml of sulforhodamine 101 (Molecular Probes, Eugene OR). Stained cells were visualized by UV illumination using a Nikon Microphot FXA microscope (objective 40X, Neofluor). Nuclear condensation, fragmentation and hyperchromaticity were considered to reflect apoptosis.
  • PBS phosphate-buffered saline
  • DAPI 4',6'-diamidino- 2-phenylindole dihydrochloride
  • sulforhodamine 101 Molecular Probes, Eugene OR
  • DNA fragmentation after exposure of cells to HK a was also directly assessed. Briefly, cells were cultured in the absence or presence of 30 nM HK a , for varying periods, and DNA isolated following cell lysis in a buffer containing 20 mm Ths-HC1 , pH 7.4, 5 mM ethylenediamine tetraacetic acid (EDTA) and 0.4% Triton X-100. After centrifugation to remove nuclei and insoluble material, the supernatant was extracted with an equal volume of phenol:chloroform:1-isopropanol (25:24:1), and DNA precipitated by the addition of 50 ⁇ l of 4 M LiCI and 500 ⁇ l 2-propanol.
  • EDTA ethylenediamine tetraacetic acid
  • Precipitated DNA was dried using a Speed-Vac (Savant, Holbrook, NY), resuspended in 20 mM Tris-HC1 , pH 7.4, containing 5 mM EDTA and incubated for 30 minutes in the presence of 0.1 mg/ml RNase A. Samples were then analyzed by 0.8% agarose gel electrophoresis and visualized under UV light after staining with ethidium bromide. Apoptosis was also confirmed by using the TUNEL reaction
  • HK a on cytokine-stimulated angiogenesis in vivo was determined using a previously-described assay in which the neovascularization of a Matrigel "plug" containing bFGF is assessed (Passaniti et al., Lab Invest 67:519-528, 1992).
  • mice 7-8 weeks old , females were injected subcutaneously on the left and right mid-back with 0.25 ml of chilled Matrigel containing 400 ng bFGF and 50 ⁇ g heparin, to which either 25 ⁇ l of PBS (left mid-back injection) or an equal volume of PBS containing 0.4 mg/ml HK a (right mid-back injection) had been added.
  • the Matrigel solidified and remained as a solid, subcutaneous "plug" through the 4 day duration of the experiment.
  • mice were sacrificed, and the skin incised along the mid back and peeled back over the flanks to expose the Matrigel plugs. Plugs were then photographed prior to their excision, fixation and processing, as previously described (Passaniti et al., Lab Invest 67:519- 528, 1992).
  • HK a The effect of HK a on cytokine-stimulated angiogenesis in vivo was also determined using a rat corneal micropocket angiogenesis assay as previously described (Polverini et al., Meth. Enzymol. 198:440-450, 1991 ; Fournier et al., Inv. Opthat. Vis. Sci. 21 :354, 1981). Pellets were prepared using 12% hydron. Control pellets contained bFGF (50 ng/pellet), while test pellets contained bFGF and HK a (final concentration 10 ⁇ M). A single pellet was implanted in a 2 mm pocket prepared in each cornea, 1 mm from the limbus.
  • the left eye of each animal received the control pellet, while the right eye received the HK a -containing pellet.
  • Corneal neovascularization was measured after 7 days, at which time a digital image of each eye was obtained using a Nikon NS-1 slit lamp.
  • digital images were analyzed using a Leica-Qwin (Northvale, NJ) image analysis system (Conrad et al., Lab. Invest. 70:434, 1994).
  • the cells which had migrated into these plugs appeared rounded and apoptotic, in contrast to the elongated, migratory phenotype of the cells invading the control plugs.
  • bFGF- containing hydron pellets implanted into control corneas induced a robust angiogenic response.
  • the length and density of neovessels were significantly reduced in corneas in which the implanted pellets contained bFGF and HK a .
  • the endothelial cell proliferation assay of Example 1 was performed, utilizing bFGF as the growth factor to stimulate angiogenesis and 50 ⁇ M of the above peptides. The same mathematical formula was employed but proliferation in the presence of GF plus peptide substituted for proliferation in the presence of GF plus HK a
  • the percent inhibition of endothelial cell proliferation attributable to the peptides is given in Table 1.
  • the value for HK a is also reported.
  • the greater than 100% inhibition level achieved in this assay with HK a (100% being no endothelial cell proliferation, that is, the level of proliferation in medium containing 2% serum alone, without added growth factor) reflects the fact that HK a induces endothelial cell apoptosis.

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Abstract

Le kininogène de poids moléculaire élevé à deux chaînes et des analogues peptidiques dudit kininogène ayant des homologies avec des sites situés dans le domaine 5 du kininogène sont de puissants inhibiteurs de l'angiogenèse. Lesdits peptides sont de formule X1-His-Lys-X-Lys-X2, dans laquelle X représente un acide aminé quelconque, X1 représente de zéro à douze acides aminés, de préférence de zéro à six acides aminés, et idéalement de zéro à trois acides aminés et X2 représente de zéro à douze acides aminés, de préférence de zéro à six acides aminés et idéalement de zéro à trois acides aminés. X est de préférence un acide aminé ayant une chaîne latérale non polaire, ou une chaîne latérale polaire qui est non chargée à un pH de 6,0 à 7,0. X est idéalement Asn, Phe ou His. Des méthodes d'inhibition de la prolifération de cellules endothéliales et de l'angiogenèse sont également décrites.
EP99962723A 1998-11-10 1999-11-05 Inhibition de l'angiogenese par du kininogene de poids moleculaire eleve et analogues peptidiques dudit kininogene Withdrawn EP1137659A4 (fr)

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WO2001034195A1 (fr) * 1999-11-12 2001-05-17 Temple University Of The Commonwealth System Of Higher Education Inhibition de l'angiogenese par des anticorps avec du kininogene de haut poids moleculaire du domaine 5
US6994852B1 (en) 1999-11-12 2006-02-07 Temple University-Of The Commonwealth System Of Higher Education Inhibition of angiogenesis by antibodies against high molecular weight kininogen domain 5
JP2004515222A (ja) * 2000-07-24 2004-05-27 アテニュオン， エルエルシー ヒトキニノゲンｄ５ドメインポリペプチドおよびこれらの使用
US7098187B2 (en) 2002-09-13 2006-08-29 Attenuon, Llc Human kininogen D3 domain polypeptide as an anti-angiogenic and anti-tumor agent
SE0301431D0 (sv) 2003-05-19 2003-05-19 Dermagen Novel antimicrobial peptides
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CN104211807B (zh) * 2014-08-22 2017-10-20 北京蛋白质组研究中心 一种抗两种多肽的单克隆抗体及其制备方法和应用
CN104155457B (zh) * 2014-08-22 2016-11-16 北京蛋白质组研究中心 一种结直肠癌相关“多肽-蛋白组合式标志物”检测试剂盒

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US5596079A (en) * 1991-12-16 1997-01-21 Smith; James R. Mimetics of senescent cell derived inhibitors of DNA synthesis
US5756291A (en) * 1992-08-21 1998-05-26 Gilead Sciences, Inc. Aptamers specific for biomolecules and methods of making
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JPH08208692A (ja) * 1994-09-28 1996-08-13 Sumitomo Pharmaceut Co Ltd 新規な細胞接着抑制ペプチド誘導体
EP0808627A2 (fr) * 1996-05-22 1997-11-26 Hoechst Aktiengesellschaft Utilisation des antagonistes de la bradykinine non-peptidiques pour le traitement et la prévention des maladies de foie fibrotiques chroniques, des maladies de foie aigues et complications
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