AU773862B2 - Inhibition of angiogenesis by high molecular weight kininogen and peptide analogs thereof - Google Patents

Description

WO 00/27866 PCT/US99/26419 -1- INHIBITION OF ANGIOGENESIS BY HIGH MOLECULAR WEIGHT KININOGEN AND PEPTIDE ANALOGS

THEREOF

Field of the Invention The invention relates to therapeutic compounds and methods for inhibiting angiogenesis.

Background of the Invention 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 15 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.

Angiogenesis can occur under certain normal conditions in mammals such as in wound healing, in fetal and embryonic development, and in the formation of the corpus luteum, endometrium and placenta.

Angiogenesis also occurs in certain disease states such as in tumor i WO 00/27866 PCT/US99/26419 formation and expansion, or in the retina of patients with certain ocular disorders. Angiogenesis can also occur in a rheumatoid joint, hastening joint destruction by allowing an influx of leukocytes with subsequent release of inflammatory mediators.

The evidence for the role of angiogenesis in tumor growth was extensively reviewed by O'Reilly and Folkman in U.S. Pat. 5,639,725, the entire disclosure of which is incorporated herein by reference. It is now generally accepted that the growth of tumors is critically dependent upon this process. Primary or metastatic tumor foci are unable to achieve a size of more than approximately 2 mm in the absence of neovascularization.

Serial evaluation of transgenic mice predisposed to develop neoplasms has demonstrated that neovascularization of premalignant lesions precedes their evolution into tumors (Folkman et al., Nature 339:58-61, 1989), and that inhibition of angiogenesis delays the growth of such lesions, as well as their assumption of a malignant phenotype (Hanahan et al., Cell 86:353- 364, 1996). In humans, several studies have demonstrated that increased density of microvessels within a tumor is associated with a poor clinical outcome (Weidner et al., J Natl Cancer Inst 84:1875-1887, 1992).

An emerging paradigm is that proteolytic fragments of plasma or extracellular matrix proteins regulate angiogenesis. To date, several polypeptides with such activities have been identified. These include 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 etal., Cell 88:277-285, 1997), PEX, the hemopexin domain of matrix metalloprotease 2 (Brooks et al., Cell 92:391-400, 1998), the Cterminal 16 kD fragment of prolactin (Clapp et al., Endocrinol 133:1292- 1299, 1993) and a 29 kD fragment of fibronectin (Homandberg et al., Am J Pathol 120:327-332, 1985). In addition, both intact thrombospondin 1 as well as peptides derived from its procollagen domain and properdin-like type-1 repeats express potent anti-angiogenic activity (Good et al., Proc Nat Acad Sci USA 87:6624-6628, 1990); Tolsma et al., J Cell Biol 122:497- 511, 1993. In preclinical models, several of these fragments inhibited tumor growth, and some induced tumor regression and dormancy (Boehm et al., Nature 390:404-407, 1997).

WO 00/27866 PCT[US99/26419 -3- High Molecular Weight Kininogen High molecular weight kininogen (HK) 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 NOdependent vasodilation (Weimer etal., 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 (HKa) 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). Cleavage of HK to form HKa and release bradykinin occurs between Lys(362) and Arg(363). HKa contains a 62 kD heavy chain and a 56 kD light chain linked by a disulfide bond.

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). HKa, 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).

Although the binding of HK to endothelial cells has been well characterized, comparatively little attention has been devoted to endothelial cell binding of HKa Furthermore, although binding of bradykinin to endothelial cells induces well-defined responses, functional consequences of the direct binding of HKa have not been reported.

Summary of the Invention The compounds of the present invention are in the form of peptides which possess anti-angiogenic activity.

In all embodiments; the peptide may optionally comprise an amino-terminal and/or carboxy-terminal protecting group.

WO 00/27866 PCT[S99/26419 -4- A compound of the formula X,-His-Lys-X-Lys-X 2 (hereinafter "X,-His-Lys-X-Lys-X 2 peptide") is provided wherein X is any amino acid, X, is from zero to twelve amino acids, more preferably from zero to six amino acids, most preferably from zero to three amino acids, and

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.

Preferably, X is an amino acid having a nonpolar side chain, Ala, Leu, lie, 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, Ser, Thr, Tyr, Asn, Gin, Cys, or Gly. Most preferably, X is Asn, Phe or His.

Preferred compounds comprise fragments of HK. In one group of such preferred compounds, X, is zero amino acids, or (ii) the segment His-Gly-His-Glu-GIn- GIn-His-Gly-Leu-Gly-His-Gly (SEQ ID NO:1) or N-terminal truncation fragment thereof containing at least one amino acid, and

X

2 is zero amino acids, or (ii) the segment Leu-Asp-Asp-Asp-Leu-Glu-His- GIn-Gly-Gly-His-Val (SEQ ID NO:2) or C-terminal truncation fragment thereof containing at least one amino acid.

In another group of such preferred compounds, X, is zero amino acids, or (ii) the segment Gly-His-Lys-His-Lys-His-Gly- His-Gly-His-Gly-Lys (SEQ ID NO:3) or N-terminal WO 00/27866 PCTIUS99/26419 truncation fragment thereof containing at least one amino acid, and

X

2 is zero amino acids, or (ii) the segment Gly-Lys-Lys-Asn-Gly-Lys-H is- Asn-Gly-Trp-Lys-Thr (SEQ ID NO:4) or C-terminal truncation fragment thereof containing at least one amino acid.

Accord ing to a further preferred embodiment of the invention, the compound has a substantial amino acid homology to either the amino acid sequence His-Gly-His-Glu-Gln-GlIn-His-Gly-Leu-Gly-H is-Gly-His-Lys- Phe-Lys-Leu-Asp-Asp-Asp-Leu-Glu.H is-GIn-Gly-Gly-His-Val (SEQ ID or the amino acid sequence Gly-His-Lys-His-Lys-His-Gly-His-Gly.

H is-G ly-Lys-H is-Lys-Asn-Lys-G ly- Lys- Lys-Asn-G ly..Lys-.H isAsn..GlyTrp- Lys-Thr (SEQ ID NO:6).

Exemplary and preferred compounds include: His-Gly-His-Glu-Gln-Gln-H is-Gly-Leu-Gly-His-Gly- His-Lys-Phe-Lys-Leu-Asp-Asp-Asp.Leu.Glu-His-Gin-Gly-Gly- His-Val (SEQ ID Gly-His-Lys-Phe-Lys-Leu-Asp-Asp-AspLeu-Glu- His-Gin-Gly-Gly-His (SEQ ID NO:7); Gly-His-Lys-His-Lys-His-Gly-HisGlyHisGly.Lys- H is-Lys-Asn-Lys-Gly-Lys-Lys-Asn-Gly.Lys.H is-Asn-Gly-Trp- Lys-Thr (SEQ ID NO:6); Lys-His-Gly-His-Gly-His-Gly-Lys-His-Lys.Asn-Lys.

Gly-Lys-Lys-Asn (SEQ ID NO:8); and His-Lys-Asn-Lys-Gly-Lys-Lys..Asn.GlyLysHis-Asn- Gly-Trp-Lys-Thr (SEQ ID NO:9).

The invention also encompasses a method of inhibiting endothelial cell proliferation comprising contacting endothelial cells with HK, HK, or a X 1 -His-Lys-X-Lys-X 2 peptide.

WO 00/27866 PCTIUS99/26419 -6- The invention also encompasses a method of inducing apoptosis of endothelial cells comprising contacting endothelial cells with HK, HKa or a X,-His-Lys-X-Lys-X 2 peptide.

The invention is also a composition comprising a pharmaceutically effective carrier and HK, HKa or a X,-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, HKa or a X,-His-Lys-X-Lys-X 2 peptide. The mammal treated is preferably a human being.

Other aspects and advantages of the present invention are described in the drawings and in the following detailed description of the preferred embodiments thereof.

Abbreviations and Short Forms The following abbreviations and short forms are used in this specification.

"bFGF" is recombinant human basic fibroblast growth factor.

"HK" means the mature form of high molecular weight kininogen, and any allelic variations thereof. By "mature" is meant the posttranslationally-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 Nterminus 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.

"HKa" means two-chain high molecularweight kininogen, the product of kallikrein cleavage of mature high molecular weight kininogen, and any allelic variations thereof.

"HDMVEC" means human dermal microvascular endothelial cells.

"HGF" means hepatocyte growth factor.

"HUVEC" means human umbilical vein endothelial cell WO 00/27866 PCT/US99/26419 -7- "PDGF" is platelet-derived growth factor.

"TGF-p" is transforming growth factor-p.

"VEGF" means vascular endothelial cell growth factor.

"X,-His-Lys-X-Lys-X 2 peptide" means a compound of the indicated formula wherein X, X, and X 2 are defined as above.

Amino Acid Abbreviations The nomenclature used to describe polypeptide compounds of the present invention follows the conventional practice wherein the amino group is presented to the lest and the carboxy group to the right of each amino acid residue. In the formulae representing selected specific embodiments of the present invention, the amino-and carboxy-terminal groups, although not specifically shown, will be understood to be in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by a one-letter or three-letter designation, corresponding to the trivial name of the amino acid, in accordance with the following schedule: A Alanine Ala C Cysteine Cys D Aspartic Acid Asp E Glutamic Acid Glu F Phenylalanine Phe G Glycine Gly H Histidine His I Isoleucine lie K Lysine Lys L Leucine Leu M Methionine Met N Asparagine Asn P Proline Pro Q Glutamine Gin R Arginine Arg S Serine Ser T Threonine Thr V Valine Val W Tryptophan Trp Y Tyrosine Tyr Definitions The following definitions, of terms used throughout the specification, are intended as an aid to understanding the scope and practice of the present invention.

WO 00/27866 PCT/US99/26419 -8- "Angiogenesis" means the generation of new blood vessels into a tissue or organ.

"Apoptosis" means a process of programmed cell death.

A"peptide" is a compound comprised of amino acid residues covalently linked by peptide bonds.

The expression "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. As used herein, "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, and particularly at the carboxy- or amino-terminus, 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 antiangiogenic activity is maintained.

Amino acids have the following general structure: H I

R-C-COOH

NH

2 Amino acids are classified into seven groups on the basis of the side chain R: aliphatic side chains, side chains containing a hydroxylic (OH) group, side chains containing sulfur atoms, side chains containing an acidic or amide group, side chains containing a basic group, side chains containing an aromatic ring, and 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 WO 00/27866 PCTIUS99/26419 -9understood 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 identical, or have a chemically similar R side chain. Nucleic acids are said to have homology if a substantial number of their nucleotides are identical.

As used herein, "protected" with respect to a terminal amino group 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. Such protecting groups include, for example, acyl protecting groups such as formyl, 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.

As used herein, "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. Such 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 greaterthan about 60%, more preferably greaterthan about 80%, and most preferably greater than about 90 By "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.

By "C-terminal truncation fragment" with respect to an amino acid sequence is meant a fragment obtained from a parent WO 00/27866 PCTIUS99/26419 sequence by removing one or more amino acids from the C-terminus thereof.

Description of the Figures Fig. 1 shows the inhibition of endothelial cell proliferation over time following contact with HKa.

Fig. 2 shows the concentration-dependent inhibition of endothelial cell proliferation by HKa (fine hatched bars), HK (white bars) and low molecular weight kininogen (coarse hatched). Low molecular weight kininogen is non-inhibitory.

Fig. 3 shows the ability of 30 nM HKa to inhibit endothelial cell proliferation stimulated by a variety of growth factors.

Fig. 4 shows that HKa 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 HKa concentration and cell density in the culture. White bars 1,500 cells/well; coarse hatched bars 3,000 cells/well; fine hatched bars 60,000 cells/well; very fine hatched bars 12,000 cells/well; vertically hatched bars 24,000 cells/well.

Fig. 6A shows endothelial cells after staining with diamidino-2-phenylindole hydrochloride. Fig. 6B shows endothelial cells upon staining with 4',6'-diamidino-2-phenylindole hydrochloride after four hours of exposure to HKa.

Figs. 7A and 7B show DNA fragmentation in endothelial cells 25 exposed to HK, 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,. 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.

s r r Detailed Description of the Invention WO 00/27866 PCT[US99/26419 -11 The present invention is based upon the discovery that HKa 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, and the X,-His-Lys-X-Lys-X 2 peptides the ability to inhibit cytokine-driven angiogenesis in vivo.

Antiproliferative effects are observed at concentrations below nM. The use among different laboratories of endothelial cells of different origin, and/or varying concentrations of endothelial cell growth factors, makes a direct comparison of the relative potency of HKa and previously-reported anti-angiogenic polypeptides difficult. However, the observations made herein suggest that the in vitro potency of HKa in this regard is similar to that of angiostatin (O'Reilly et al., Cell 79:315-328, 1994), endostatin (O'Reilly etal., Cell 88:277-285, 1997) and TSP-1 (Good et al., Proc Nat Acad Sci USA 87:6624-6628,1990). Furthermore, when the 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 2 peptides is physiologically significant.

The effects of HKa, and thus the effects of the X,-His-Lys-X- Lys-X 2 peptides also, are cell specific. No inhibition of the proliferation of either human aortic smooth muscle cells or HEK 293 cells by HK, is observed. According to the assays utilized herein, 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-P and PDGF. Inhibition of endothelial cell proliferation is detected within 6 hours of exposure of the cells to HKa, and is accompanied by morphologic and biochemical evidence of cell apoptosis.

Furthermore, as shown herein, HKa is effective in an in vivo model of angiogenesis. HK, inhibits the ingrowth of new blood vessels into a reconstituted extracellular matrix (Matrigel) containing the pro-angiogenic growth factor bFGF implanted subcutaneously into mice.

Without wishing to be bound by any theory, the observation that HKa, but not single chain HK, inhibits endothelial cell proliferation suggests that the structural change which the molecule undergoes WO 00/27866 PCT/US99/26419 -12following kallikrein-mediated cleavage is important for expression of its antiangiogenic activity.

The mature human HK amino acid sequence is set forth in the recent review by Colman and Schmaier, Blood, 90:3819-3843 (1997), for example. HKa 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. The N-terminal and C-terminal chains of HKa generated by plasma kallikrein cleavage of HK and release of bradykinin are known as HK "heavy" and "light" chains, respectively. HKa may be generated by treating HK with plasma kallikrein, according to wellknown methods. HK, is also commercially available. Furthermore, 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. HKa-derived peptides containing the His-Lys-X-Lys sequence inhibit endothelial cell proliferation and are useful as anti-angiogenic agents.

The X,-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.

In conventional solution phase peptide synthesis, 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. The use of various N-protecting groups, the carbobenzyloxy group or the t-butyloxycarbonyl group, various coupling reagents dicyclohexylcarbodiimide or carbonyldimidazole, various active esters, esters of N-hydroxyphthalimide or N-hydroxysuccinimide, and the various cleavage reagents, trifluoroacetic acid WO 00/27866 PCT/US99/26419 13- (TFA), HCI in dioxane, boron tris-(trifluoracetate) and cyanogen bromide, and reaction in solution with isolation and purification of intermediates is well-known classical peptide methodology. The preferred peptide synthesis method follows conventional Merrifield solid-phase procedures. See Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963) and Science 50:178-85 (1965). Additional information about the solid phase synthesis procedure can be had by reference to the treatise by Steward and Young (Solid Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, 1969, and the review chapter by Merrifield in Advances in Enzymology 32:221-296, F.F. Nold, Ed., Interscience Publishers, New York, 1969; and Erickson and Merrifield, The Proteins 2:255 et seq. (ed. Neurath and Hill), Academic Press, New York, 1976. The synthesis of peptides by solution methods is described in Neurath et al., eds. (The Proteins, Vol. II, 3d Ed., Academic Press, NY (1976)).

Crude peptides may be purified using preparative high performance liquid chromatography. The amino terminus may be blocked according, for example, to the methods described by Yang et al. (FEBS Lett. 272:61-64 (1990)).

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.

Alternatively, the X,-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. The techniques of recombinant DNA technology are known to those of ordinary skill in the art. General methods for the cloning and expression of recombinant molecules are described in Maniatis (Molecular Cloning, Cold Spring Harbor Laboratories, 1982), and in Sambrook (Molecular Cloning, Cold Spring Harbor Laboratories, Second Ed., 1989), and in Ausubel (Current Protocols in Molecular Biology, Wiley and Sons, 1987), which are incorporated by reference. The complete cDNA of human HK is reported, for example, by Takagi etal., J. Biol. Chem. 260:8601-8609 WO 00/27866 PCTIUS9926419 -14- (1985), the entire disclosure of which is incorporated herein by reference.

From this nucleic acid sequence, synthetic genes encoding HKa-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 HKa-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. Among the prokaryotic (including bacteriophage) promoters useful for practice of this invention are lad, lacZ, T3, T7, lambda Pr' PI' and trp promoters.

Among the eukaryotic (including viral) promoters useful for practice of this invention are ubiquitous promoters HPRT, vimentin, actin, tubulin), intermediate filament promoters desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters MDR type, CFTR, factor VIII), tissue-specific promoters actin promoter in smooth muscle cells), promoters which respond to a stimulus steroid hormone receptor, retinoic acid receptor), tetracycline-regulated transcriptional modulators, cytomegalovirus immediate-early, retroviral LTR, metallothionein, Ela, 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.

Examples of polyadenylation signals that can be used in the present invention include but are not limited to SV40 polyadenylation signals and LTR polyadenylation'signals.

WO 00/27866 PCT/US99/26419 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.

In some embodiments, 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.

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-, diand tri-alkyl and aryl amines triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanolamines 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.

Accordingly, HK, HKa and/or one or more X,-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 25 combination with a pharmaceutically carrier. Although HK is considerably less anti-angiogenic than HKa it is possible that upon administration HKwill be converted to HKa, and may therefore serve as a prodrug for HK.. In particular, it is believed that HK may be rapidly converted to HKa in tumors, or further cleaved by tumor-derived enzymes to release peptides the same or similar to the peptides disclosed herein.

The ability of HKa to inhibit the proliferation of endothelial cells S. cultured on different extracellular matrix (ECM) proteins was determined.

HK (10nM) potently inhibited the proliferation of HUVEC cultured on gelatin, laminin and Matrigel, though slightly less potent inhibition, largely 35 overcome by high concentrations of HK, (50 nM), occurred when cells were WO 00/27866 PCT[US99/26419 -16cultured on fibronectin or vitronectin. Intermediate effects were observed when cells were cultured on fibrinogen, though cells cultured on collagen types I or IV were resistant to the antiproliferative activity of HKa. In keeping with the results of proliferation assays, HK, 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 HKa-induced apoptosis, and that HK, might selectively target angiogenic endothelial cells in a protease-rich tumor milieu in which ECM is partially degraded.

Pharmaceutically acceptable carriers 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. Examples of 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-agar and tragacanth, or mixtures of these substances, and the like.

The pharmaceutical 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. 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.

The compositions may be administered by any convenient route which will result in delivery to the site of undesired angiogenesis in an WO 00/27866 PCT/US99/26419 -17amount 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.

The compositions are most effectively administered parenterally, preferably intravenously or subcutaneously. For 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. Such intravenous delivery vehicles are known to those skilled in the art. In a preferred embodiment, the vehicle is a sterile saline solution. If the peptides are sufficiently small 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.

The 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 WO 00/27866 PCT/US99/26419 -18administered by continuous infusion, such as via an implanted subcutaneous pump.

Peptides which inhibit endothelial cell proliferation by at least more preferably by at least 50%, most preferably by at least when incubated with such cells at a concentration of 50pM are preferred.

For purposes of this preference, percent inhibition of proliferation is determined according to the procedure and formula set forth in Example 1, part A, below.

Examples The present invention is illustrated by the following nonlimiting examples.

Materials The materials utilized in the Examples were sourced as follows. 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 hypothalami, 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 20 growth factor (HGF) were obtained from Collaborative Biomedical Products/Becton Dickinson (Bedford, MA). Platelet-derived growth factor (PDGF) and transforming growth factor P (TGF-) were from R&D Systems (Minneapolis, MN). 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 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. preparations used in these studies contained less than <0.01 EU/ml ofendotoxin, as determined using the E-Toxate (Limulus Amoebocyte) assay (Sigma).

Synthetic Peptides WO 00/27866 PCT[US99/26419 19- 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). Coupling was carried out done 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.

Cell Culture Methods The basic cell culture methods of the Examples are described as follows. Human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells were isolated and cultured as previously described (Graham et al., Blood 91:3300-7 1998). Human dermal microvascular endothelial cells (HDMVEC) were obtained from Clonetics (San Diego, CA) and cultured under identical conditions. All cells in these studies were of passage 3 or lower.

WO 00/27866 PCTIUS9926419 Example 1 Effect of Single-Chain and Two-Chain High Molecular Weight Kininogen on Endothelial Cell Proliferation A. Experimental To assess the effect of HK, on endothelial cell proliferation, cells were suspended at a concentration of 30,000 cells/ml in Medium 199 (M199) containing 2% FCS. One hundred microliters of this suspension was then plated in individual wells of a 96 well microplate precoated with 1% gelatin. After incubation for 2 hours, at 37"C, to allow cells to adhere and spread, medium was removed and replaced with fresh M199 containing 2% FCS, (ii) 10 pM ZnCI 2 (iii) 10 ng/ml bFGF, VEGF, HGF, TGF-p or PDGF and (iv) 50 pM HK or HKa. Cells were then incubated for 48 hours at 37 0 C, at which time the relative numbers of cells in each well were determined using the Cell Titer e Aqueous cell proliferation assay (Promega, Madison, WI). Briefly, 20 pl of a 19:1 (VN) mixture of dimethylthiazol-2-yl)-5-(3-carboxymethylphenyl)-2-(4-sulfophenyl)-2Htetrazolium (MTS) and phenazine methosulfate (PMS) was added to each well, and after an additional hour of incubation, A 49 o was measured using a BioRad model EL311 microplate reader. The percent inhibition of cell proliferation by HKa was determined using the formula: inhibition (1 [(A 490 HKa) A 49 0

(A

490

A

49 0 X 100 where and represent proliferation in the presence or absence of added growth factor, and HK. represents proliferation in the !presence of both growth factor and HK,. The significance of differences in relative endotheial cell proliferation cell numbers at the end of the lIU III; I Q III l Ill I I L1 proliferation assays was determined using the Student's two-tailed T-test for paired samples.

B. Results Inhibition of endothelial cell proliferation by HKa was apparent within 6 hours after its addition to cells, at which time cell spreading appeared to be diminished, and the cells began to display a more rounded morphology. However, the extent to which proliferation was inhibited o *ee WO 00/27866 PCTIUS9926419 -21increased progressively with longer exposure of cells to HK, (Figure 1).

Inhibition of endothelial cell proliferation by HKa occurred in a concentration-dependent manner; modest inhibition of bFGF ng/ml) stimulated proliferation occurred at an HKa concentration of nM, while HK, inhibited proliferation by 50% at a concentration of approximately 8 nM (Figure 2).

Single-chain HK, low molecular weight kininogen and bradykinin were tested in the same manner. HKa 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 Finally, bradykinin did not inhibit proliferation, and anti-bradykinin antibodies did not inhibit the anti-proliferative effect of HKa (not shown). The latter results exclude the possibility that contamination of HK, with trace amounts of bradykinin was responsible for its ability to inhibit endothelial cell proliferation. Single-chain HK modestly inhibited endothelial cell proliferation.

These results demonstrate that HK, 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.

HKa inhibited the proliferation of human endothelial cells in response to a number of mitogens, including bFGF, VEGF, HGF, TGF-3 and PDGF, equally well (Figure The mitogenic activity of each of these factors is mediated through interactions with distinct receptors. Thus, without wishing to be bound by any theory, these results imply that the mechanism(s) by which HKa and the HK, peptides inhibits endothelial cell proliferation is unlikely to depend upon inhibition of growth factor binding.

HKa inhibited the proliferation of HUVEC and HDMVEC with similar potency but did not affect the proliferation of human aortic smooth muscle cells (Fig. 4) or HEK 293 cells (not shown), demonstrating that HKa's anti-proliferative effects were endothelial cell-specific. Furthermore, the ability of HKa to inhibit endothelial cell proliferation was inversely proportional to the density of the cell culture (Figure suggesting that its WO 00/27866 PCT/US99/26419 -22effects may be at least partially dependent upon the rate of cell proliferation and DNA synthesis.

In other experiments, the ability of HKa to inhibit the proliferation of endothelial cells cultured on different extracellular matrix (ECM) proteins. HKa (10nM) potently inhibited the proliferation of HUVEC cultured on gelatin, laminin and Matrigel, though slightly less potent inhibition, largely overcome by high concentrations of HKa (50 nM), occurred when cells were cultured on fibronectin or vitronectin.

Intermediate effects were observed when cells were cultured on fibrinogen, though cells cultured on collagen types I or IV were resistant to the antiproliferative activity of HKa.

Example 2 Apoptosis of Endothelial Cells Induced by Two-Chain High Molecular Weight Kininogen A. Experimental Induction of endothelial cell apoptosis after exposure of cells to HKa was determined using three assays.

Cells plated on glass coverslips were cultured in the absence or presence of 30 nM HKa 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 pg/ml of4',6'-diamidino- 2-phenylindole dihydrochloride (DAPI) and 10 pg/ml of sulforhodamine 101 (Molecular Probes, Eugene OR). Stained cells were visualized by UV illumination using a Nikon Microphot FXA microscope (objective Neofluor). Nuclear condensation, fragmentation and hyperchromaticity were considered to reflect apoptosis.

DNA fragmentation after exposure of cells to HKa was also directly assessed. Briefly, cells were cultured in the absence or presence of 30 nM HKa, for varying periods, and DNA isolated following cell lysis in a buffer containing 20 mm Tris-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 pi of 4 M LiCI and 500 pl 2-propanol.

Precipitated DNA was dried using a Speed-Vac (Savant, Holbrook, NY), WO 00/27866 PCT/US99/26419 23 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 (In Situ Cell Death Detection Kit, Boehringer Mannheim, Indianapolis, IN) to label control cells and those exposed to HKa with fluorescein-conjugated dUTP, per the manufacturer's protocol. Labeled cells were then analyzed by flow cytometry.

B. Results Staining of cells with DAPI revealed nuclear condensation and fragmentation only in cells that had been exposed to HKa; these changes were observed in 30-50% of the cells within 6 hours after HKa addition (Figure 6A). Parallel studies in which cells were incubated with Trypan blue after incubation with HKa revealed no evidence of dye uptake, demonstrating that HKa did not induce cell lysis, and that its effects were due to the induction of apoptosis rather than cytotoxicity. Consistent with this observation, analysis of DNA isolated from cells incubated with HK, revealed striking fragmentation, which was first apparent approximately 6 hours after addition of the HKa (Figure 7A). Consistent with these results, a specific "laddering" pattern of DNA fragmentation, characteristic of apoptosis, was apparent upon electrophoretic analysis of DNA from cells exposed to HKa (data not shown). Finally, flow cytometric analysis of cells incubated in the absence or presence of HKa, and labeled with fluorescein dUTP by the TUNEL reaction, and revealed a rightward shift of the major peak only in cell populations exposed to HKa (not shown). As with DAPI staining and DNA fragmentation, these changes were apparent approximately 6 hours after exposure of cells to HKa. Taken together, these studies demonstrate that the antiproliferative activity of HKa reflects its ability to induce endothelial cell apoptosis.

Example 3 Inhibition of Cytokine-Stimulated Angioenesis by Two-Chain High Molecular Weight Kininogen In Vivo WO 00/27866 PCTIUS99/26419 -24- A. Experimental The effect of HKa 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). Briefly, athymic Ncr nude 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 pg heparin, to which either 25 pl of PBS (left mid-back injection) or an equal volume of PBS containing 0.4 mg/ml HK, (right mid-back injection) had been added. Immediately after injection, the Matrigel solidified and remained as a solid, subcutaneous "plug" through the 4 day duration of the experiment. At this point, 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).

The effect of HKa 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. Opthal. Vis. Sci. 21:354, 1981). Pellets were prepared using 12% hydron. Control pellets contained bFGF (50 ng/pellet), while test pellets contained bFGF and HKa (final concentration 10 pM). 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 HKa-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. To determine the total area of neovessels in each eye, digital images were analyzed using a Leica-Qwin (Northvale, NJ) image analysis system (Conrad et al., Lab. Invest. 70:434, 1994).

B. Results As depicted in Figures 8A and 8B, Matrigel plugs containing bFGF induced exuberant vessel ingrowth within 4 days after implantation.

WO 00/27866 PCTIUS99/26419 In contrast, no neovascularization of Matrigel plugs which contained bFGF and HKa was observed (Figures 8C and 8D). In addition, these plugs remained transparent, as opposed to the opaque appearance acquired by the plugs, suggesting that HK, blocked the intravasation of migratory cells into the Matrigel. The latter hypothesis was confirmed by histological analysis, which demonstrated markedly fewer cells within the HKacontaining Matrigel plugs. Furthermore, 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.

In the corneal micropocket angiogenesis assay, bFGFcontaining hydron pellets implanted into control corneas induced a robust angiogenic response. In comparison, the length and density of neovessels were significantly reduced in corneas in which the implanted pellets contained bFGF and HK,. Computer analysis of digital images revealed that the total vessel area within corneas that received HKa-containing pellets (293,807 pm 2 was reduced by 82% in comparison to those in which pellets contained bFGF only (53,931 pm 2 (P<0.000000005).

Example 4 Effect of Peptide Analogs of Two-Chain High Molecular Weight Kininogen on Endothelial Cell Proliferation The following HKa-derived peptides were synthesized: Gly-His-Lys-Phe-Lys-Leu-Asp-Asp-Asp-Leu-Glu-His-Gln-Gly- Gly-His (SEQ ID NO:7); Gly-His-Gly-His-Gly-His-Gly-Lys-His-Lys-Asn-Lys-Gly-Lys- Lys-Asn (SEQ ID NO:8); and His-Lys-Asn-Lys-Gly-Lys-Lys-Asn-Gly-Lys-His-Asn-Gly-Trp- Lys-Thr (SEQ ID NO:9).

The endothelial cell proliferation assay of Example 1 was performed, utilizing bFGF as the growth factor to stimulate angiogenesis and 50 pM 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 HKa The percent inhibition of WO 00/27866 PCTIUS99/26419 -26endothelial cell proliferation attributable to the peptides is given in Table 1.

The value for HKa is also reported. The greater than 100% inhibition level achieved in this assay with HKa (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, induces endothelial cell apoptosis.

Table 1 Inhibition of Endothelial Cell Proliferation by HKa and HKa-Derived Peptides Inhibitor (50 pM) Inhibition of Endothelial IC Cell Proliferation SEQ ID NO:7 59% n.d.

SEQ ID NO:8 81% 8 pM SEQ ID NO:9 92% 14 pM HK. 135% 10 nM r r r r r All references discussed herein are incorporated by reference. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

EDITORIAL NOTE APPLICATION NUMBER 19106/00 The following Sequence Listing pages 1 to 4 are part of the description. The claims pages follow on pages 27 to 34.

WO 00/27866 SEQUENCE LISTING <110> Temple University Of The Commonwealth System of <120> Inhibition of Angiogenesis By High Molecular Weight Kininogen and Peptide Analogs Thereof <130> 6056-257 PC <140> <141> <150> 60/107,833 <151> 1998-11-10 <160> 9 PCT/US99/26419 <170> PatentIn Ver. <210> <211> <212> <213> <220> <223> 1 12

PRT

Artificial Sequence Description of Artificial Sequence: Human high molecular weight kininogen (HK) domain 5 fragment <400> 1 His Gly His Glu Gln Gln His Gly Leu Gly His Gly 1 5 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Human HK domain 5 fragment <400> 2 Leu Asp Asp Asp Leu Glu His Gin Gly Gly His Val <210> 3

F

WO 00/27866 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Human HK domain 5 fragment <400> 3 Gly His Lys His Lys His Gly His Gly His Gly Lys 1 5 PCT/US99/26419 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial domain 5 fragment Sequence: Human HK Gly Trp Lys Thr <400> 4 Gly Lys Lys Asn Gly Lys His Asn <210> <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial domain 5 fragment Sequence: Human HK <400> His Gly His Glu Gin Gin His 1 5 Leu Asp Asp Asp Leu Glu His <210> 6 <211> 28 <212> PRT <213> Artificial Sequence Gly Leu Gly His Gly 10 Gin Gly Gly His Val His Lys Phe Lys WO 00/27866 PCT/US99/26419 <220> <223> Description of Artificial domain 5 fragment Sequence: Human HK <400> 6 Gly His Lys His Lys His Gly 1 5 Gly Lys Lys Asn Gly Lys His <210> 7 <211> 16 <212> PRT <213> Artificial Sequence His Gly His 10 Gly Lys His Lys Asn Lys Asn Gly Trp Lys Thr <220> <223> Description of Artificial domain 5 fragment <400> 7 Gly His Lys Phe Lys Leu Asp Asp Sequence: Human HK Asp Leu Glu His Gin Gly Gly His <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial domain 5 fragment <400> 8 Lys His Gly His Gly His Gly Lys 1 5 Sequence: Human HK His Lys Asn Lys Gly Lys Lys Asn 10 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Human HK WO 00/27866 domain 5 fragment <400> 9 His Lys Asn Lys Gly Lys Lys Asn Gly Lys His Asn Gly Trp Lys Thr 1 5 10 PCTIUS99/2641 9

Claims (39)

1. Use of a substance selected from the group of two- chain kininogen, single-chain kininogen, and a compound of the formula X1-His-Lys-X-Lys-X 2 wherein X is any amino acid, X 1 is from zero to twelve amino acids, and X 2 is from zero to twelve amino acids, and wherein said compound optionally comprises an amino-terminal and/or carboxy-terminal protecting group, for the manufacture of a medicament for inhibiting angiogenesis.

2. Use of a substance according to claim 1 wherein the substance is a compound of the formula X 1 -His-Lys-X-Lys-X 2

3. Xi is Use of a compound according to claim 2 wherein 20 oO09 OOO0 O000 *0 20 zero amino acids, or (ii) the segment His-Gly-His-Glu-GIn-Gln-His- Gly-Leu-Gly-His-Gly (SEQ ID NO:1), or N-terminal truncation fragment thereof containing at least one amino acid, and X 2 is zero amino acids, or (ii) the segment Leu-Asp-Asp-Asp-Leu-Glu-His-GIn- Gly-Gly-His-Val (SEQ ID NO:2), or C-terminal truncation fragment thereof containing at least one amino acid. :~25 4. X 1 is Use of a compound according to claim 2 wherein zero amino acids, or (ii) the segment Gly-His-Lys-His-Lys-His-Gly-His- PHIP\340948\1 Gly-His-Gly-Lys (SEQ ID NO:3) or N-terminal truncation fragment thereof containing at least one amino acid, and X 2 is zero amino acids, or (ii) the segment Gly-Lys-Lys-Asn-Gly-Lys-His-Asn- Gly-Trp-Lys-Thr (SEQ ID NO:4), or C-terminal truncation fragment thereof containing at least one amino acid. Use of a compound according to any of claims 2-4 wherein 20 25 o o o o o* *o *o o X 1 is from zero to six amino acids, and X 2 is from zero to six amino acids.

6. Use of compound according to any of claims wherein X is selected from the group consisting of Ala, Leu, Ile, Val, Pro, Phe, Trp, Met, Ser, Thr, Tyr, Asn, Gin, Cys, and Gly.

7. Use of a compound according to any of claims 2-6 wherein X is Asn, Phe or His.

8. Use of a compound according to claim 2 wherein the compound has substantial amino acid sequence homology to the amino acid sequence His-Gly-His-Glu-Gln-Gln-His-Gly-Leu-Gly-His-Gly- His-Lys-Phe-Lys-Leu-Asp-Asp-Asp-Leu-Glu-His-Gn-Gly-Gly-His-Val (SEQ ID

9. Use of a compound according to claim 3 wherein the compound has the amino acid sequence His-Gly-His-Glu-Gln-Gln- His-Gly-Leu-Gly-His-Gly-His-Lys-Phe-Lys-Leu-Asp-Asp-Asp-Leu-Glu- His-GIn-Gly-Gly-His-Val (SEQ ID

10. Use of a compound according to claim 3 wherein the compound has the amino acid sequence Gly-His-Lys-Phe-Lys-Leu- PHIP\340948\1 Asp-Asp-Asp-Leu-Glu-His-Gn-Gly-Gly-His (SEQ ID NO:7).

11. Use of a compound according to claim 2 wherein the compound has substantial amino acid sequence homology to the amino acid sequence Gly-His-Lys-His-Lys-His-Gly-His-Gly-His-Gly-Lys- His-Lys-Asn-Lys-Gly-Lys-Lys-Asn-Gly-Lys-His-Asn-Gly-Trp-Lys-Thr (SEQ ID NO:6).

12. Use of a compound according to claim 4 wherein the compound has the amino acid sequence Gly-His-Lys-His-Lys-His- Gly-His-Gly-His-Gly-Lys-His-Lys-Asn-Lys-Gly-Lys-Lys-Asn-Gly-Lys-His- Asn-Gly-Trp-Lys-Thr (SEQ ID NO:6).

13. Use of a compound according to claim 4 wherein the compound has the amino acid sequence Lys-His-Gly-His-Gly-His- Gly-Lys-His-Lys-Asn-Lys-Gly-Lys-Lys-Asn (SEQ ID NO:8).

14. Use of a compound according to claim 4 wherein the compound has the amino acid sequence His-Lys-Asn-Lys-Gly-Lys- Lys-Asn-Gly-Lys-His-Asn-Gly-Trp-Lys-Thr (SEQ ID NO:9). Use of a substance according to claim 1 wherein the substance is a two-chain kininogen.

16. Use of a substance according to claim 1 wherein the substance is a single-chain kininogen.

17. Use of a substance according to any preceding claim wherein inhibition of angiogenesis includes inhibition of endothelial S cell proliferation.

18. Use of a substance according to claim 17 wherein 25 inhibition of endothelial cell proliferation includes induction of endothelial cell apoptosis. PHIP\340948\1

19. A compound of the formula X1-His-Lys-X-Lys-X 2 wherein X is any amino acid, X1 is the segment His-Gly-His-Glu-Gln-Gln-His-Gly-Leu- Gly-His-Gly (SEQ ID NO:1) or N-terminal truncation fragment thereof containing at least one amino acid, and X 2 is zero amino acids, or (ii) the segment Leu-Asp-Asp-Asp-Leu-Glu-His-Gln- Gly-Gly-His-Val (SEQ ID NO:2), or C-terminal truncation fragment thereof containing at least one amino acid, and wherein said compound optionally comprises an amino-terminal and/or carboxy-terminal protecting group. A compound according to claim 19 wherein X is selected from the group consisting of Ala, Leu, lie, Val, Pro, Phe, Trp, Met, Ser, Thr, Tyr, Asn, Gin, Cys, and Gly. oooo oeoo oooo eoooe 20 eeeee

21. is Asn, Phe or His. A compound according to claim 19 or 20 wherein X

22. A compound according to claim 19 having the amino acid sequence His-Gly-His-Glu-Gln-Gln-His-Gly-Leu-Gly-His-Gly- His-Lys-Phe-Lys-Leu-Asp-A-AsAp-Leu-Glu-His-Gln-Gly-Gly-His-Val (SEQ ID NO:5), and wherein said compound optionally comprises an amino-terminal and/or carboxy-terminal protecting group.

23. A compound according to claim 19 having the amino acid sequence Gly-His-Lys-Phe-Lys-Leu-Asp-Asp-Asp-Leu-Glu- His-GIn-Gly-Gly-His (SEQ ID NO:7), and wherein said compound optionally comprises an amino-terminal and/or carboxy-terminal PHIP\340948\1 protecting group.

24. A compound having the amino acid sequence Lys- His-Gly-His-Gly-His-Gly-Lys-His-Lys-Asn-Lys-Gly-Lys-Lys-Asn (SEQ ID NO:8), said compound optionally comprising an amino-terminal and/or carboxy-terminal protecting group. A compound having the amino acid sequence His- Lys-Asn-Lys-Gly-Lys-Lys-Asn-Gly-Lys-His-Asn-Gly-Trp-Lys-Thr (SEQ ID NO:9), said compound optionally comprising an amino-terminal and/or carboxy-terminal protecting group.

26. A compound according to any of claims 19 to 25, for use in medicine.

27. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound according to any of claims 19 to 26.

28. A method of inhibiting angiogenesis comprising administering to a mammal an effective amount of two-chain high molecular weight kininogen.

29. A method of inhibiting endothelial cell proliferation comprising administering to a mammal an effective amount of two-chain high molecular weight kininogen. A method of inducing endothelial cell apoptosis comprising administering to a mammal an effective amount of two-chain high molecular weight kininogen. S.31. A method of inhibiting angiogenesis comprising 25 administering to a mammal an effective amount of single-chain high PHIP\340948\1 molecular weight kininogen.

32. A method of inhibiting endothelial cell proliferation comprising contacting endothelial cells with a compound of the formula X 1 -His-Lys-X-Lys-X 2 wherein X is any amino acid, Xi is from zero to twelve amino acids, and X 2 is from zero to twelve amino acids, and wherein said compound optionally comprises an amino-terminal and/or carboxy-terminal protecting group.

33. The method according to claim 32 wherein X 1 is from zero to six amino acids, and X 2 is from zero to six amino acids.

34. The method according to claim 32 or 33 wherein X is selected from the group consisting of Ala, Leu, lie, Val, Pro, Phe, Trp, Met, Ser, Thr, Tyr, Asn, Gin, Cys, and Gly.

35. The method according to any of claims 32 to 34 wherein X is Asn, Phe or His.

36. The method according to any of claims 32 to wherein X1 is zero amino acids, or (ii) the segment His-Gly-His-Glu-Gln-Gln-His-Gly- Leu-Gly-His-Gly (SEQ ID NO:1), or N-terminal truncation fragment thereof containing at least one amino acid, and 25 X2 is zero amino acids, or PHIP\340948\1 (ii) the segment Leu-Asp-Asp-Asp-Leu-Glu-His-GIn- Gly-Gly-His-Val (SEQ ID NO:2), or C-terminal truncation fragment thereof containing at least one amino acid.

37. The method according to any of claims 32, 34, or wherein Xi is zero amino acids, or (ii) the segment Gly-His-Lys-His-Lys-His-Gly-His- Gly-His-Gly-Lys (SEQ ID NO:3) or N-terminal truncation fragment thereof containing at least one amino acid, and X 2 is zero amino acids, or (ii) the segment Gly-Lys-Lys-Asn-Gly-Lys-His-Asn- Gly-Trp-Lys-Thr (SEQ ID NO:4) or C-terminal truncation fragment thereof containing at least one amino acid.

38. The method according to claim 32 wherein the compound has substantial amino acid sequence homology to the amino acid sequence His-Gly-His-Glu-Gln-GIn-His-Gly-Leu-Gly-His-Gly-His- Lys-Phe-Lys-Leu-Asp-Asp-Asp-Leu-Glu-His-GIn-Gly-Gly-His-Val (SEQ 20 ID

39. The method according to claim 38 wherein the compound has the amino acid sequence His-Gly-His-Glu-Gln-Gln-His- Gly-Leu-Gly-His-Gly-His-Lys-Phe-Lys-Leu-Asp-Asp-Asp-Leu-Glu-His- GIn-Gly-Gly-His-Val (SEQ ID

40. The method according to claim 36 wherein the cl compound has the amino acid sequence Gly-His-Lys-Phe-Lys-Leu-Asp- Asp-Asp-Leu-Glu-His-Gln-Gly-Gly-His (SEQ ID NO:7).

41. The method according to claim 32 wherein the PHIP\340948\1 compound has substantial amino acid sequence homology to the amino acid sequence Gly-His-Lys-His-Lys-His-Gly-His-Gly-His-Gly-Lys-His- Lys-Asn-Lys-Gly-Lys-Lys-Asn-Gly-Lys-His-Asn-Gly-Trp-Lys-Thr (SEQ ID NO:6).

42. The method according to claim 41 wherein the compound has the amino acid sequence Gly-His-Lys-His-Lys-His-Gly- His-Gly-His-Gly-Lys-His-Lys-Asn-Lys-Gly-Lys-Lys-Asn-Gly-Lys-His-Asn- Gly-Trp-Lys-Thr (SEQ ID NO:6).

43. The method according to claim 37 wherein the compound has the amino acid sequence Lys-His-Gly-His-Gly-His-Gly- Lys-His-Lys-Asn-Lys-Gly-Lys-Lys-Asn (SEQ ID NO:8).

44. The method according to claim 37 wherein the compound has the amino acid sequence His-Lys-Asn-Lys-Gly-Lys-Lys- Asn-Gly-Lys-His-Asn-Gly-Trp-Lys-Thr (SEQ ID NO:9).

45. The method according to any of claims 32 to 44 wherein inhibition of proliferation includes apoptosis of the endothelial cells.

46. Use according to any one of Claims 1 to 18 or a compound 20 according to any one of Claims 19 to 26 or a pharmaceutical composition according to Claim 27 or a method according to any one of Claims 28 to substantially as hereinbefore defined with reference to the Figures and/or Examples. PHIP\340948\1