AU774596B2 - Pharmaceutical compositions comprising troponin subunits, fragments and homologs thereof and methods of their use to inhibit angiogenesis - Google Patents

Description

WO 00/54770 PCT/US00/06667 PHARMACEUTICAL COMPOSITIONS COMPRISING TROPONIN SUBUNITS, FRAGMENTS AND HOMOLOGS THEREOF AND METHODS OF THEIR USE TO INHIBIT ANGIOGENESIS This is a continuation-in-part of copending U.S.

patent application Serial No. 09/268,274, filed March 15, 1999 which .is a continuation-in-part of copending U.S. application Serial No. 08/961,264, filed October 30, 1997 which is a continuation of U.S. application Serial No. 08/602,941, filed February 16, 1996, now U.S. Patent No. 5,837,680.

1. INTRODUCTION The present invention provides for novel pharmaceutical compositions, and methods of use thereof for the treatment of diseases or disorders involving abnormal angiogenesis.

More particularly, the present invention is based, in part, on the discovery that troponin subunits C, I and T and fragments thereof inhibit stimulated endothelial cell proliferation. Pharmaceutical compositions containing therapeutically effective amounts of troponin C, I, or T, subunits, fragments, or homologs and methods of therapeutic use thereof are provided.

2. BACKGROUND Angiogenesis, the process of new blood vessel development and formation, plays an important role in numerous physiological events, both normal and pathological.

Angiogenesis occurs in response to specific signals and involves a complex process characterized by infiltration of the basal lamina by vascular endothelial cells in response to angiogenic growth signal(s), migration of the endothelial cells toward the source of the signal(s), and subsequent proliferation and formation of the capillary tube. Blood flow -1- WO 00/54770 PCT/US00/06667 through the newly formed capillary is initiated after the endothelial cells come into contact and connect with a preexisting capillary.

The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate. Rastinejad et al., 1989, Cell 56:345-355. In those rare instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail.

Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, reviews by Moses et al., 1991, Biotech.

9:630-634; Folkman et al., 1995, N. Engl. J. Med., 333:1757- 1763; Auerbach et al., 1985, J. Microvasc. Res. 29:401-411; Folkman, 1985, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203; Patz, 1982, Am. J. Opthalmol. 94:715-743; and Folkman et al., 1983, Science 221:719-725. In a number of pathological conditions, the process of angiogenesis contributes to the disease state.

For example, significant data have accumulated which suggest that the growth of solid tumors is dependent on angiogenesis.

Folkman and Klagsbrun, 1987, Science 235:442-447.

The maintenance of the avascularity of the cornea, lens, and trabecular meshwork is crucial for vision as well as to ocular physiology. There are several eye diseases, many of which lead to blindness, in which ocular neovascularization occurs in response to the diseased state. These ocular -2- WO 00/54770 PCT/US00/06667 disorders include diabetic retinopathy, neovascular glaucoma, inflammatory diseases and ocular tumors retinoblastoma). There are also a number of other eye diseases which are also associated with neovascularization, including retrolental fibroplasia, uveitis, retinopathy of prematurity, macular degeneration, and approximately twenty eye diseases which are associated with choroidal neovascularization and approximately forty eye diseases associated with iris neovascularization. See, reviews by Waltman et al., 1978, Am. J. Ophthal. 85:704-710 and Gartner et al., 1978, Surv. Ophthal. 22:291-312. Currently, the treatment of these diseases, especially once neovascularization has occurred, is inadequate and blindness often results. Studies have suggested that vaso-inhibitory factors which are present in normal ocular tissue (cornea and vitreous) are lost in the diseased state.

An inhibitor of angiogenesis could have an important therapeutic role in limiting the contributions of this process to pathological progression of the underlying disease states as well as providing a valuable means of studying their etiology. For example, agents that inhibit tumor neovascularization could play an important role in inhibiting metastatic tumor growth.

The components of angiogenesis relating to vascular endothelial cell proliferation, migration and invasion, have been found to be regulated in part by polypeptide growth factors. Experiments in culture, indicate that endothelial cells exposed to a medium containing suitable growth factors can be induced to evoke some or all of the angiogenic responses. Several polypeptides with in vitro endothelial growth promoting activity have been identified. Examples include acidic and basic fibroblast growth factors, transforming growth factors a and P, platelet-derived endothelial cell growth factor, granulocyte colony-stimulating factor, interleukin-8, hepatocyte growth factor, proliferin, -3- WO 00/54770 PCT/US00/06667 vascular endothelial growth factor and placental growth factor. See, review by Folkman et al., 1995, N. Engl.

J. Med., 333:1757-1763.

Although extracts from several different tissue sources have been shown to contain anti-angiogenic activity, several molecules such as platelet factor-4, thrombospondin, protamine, and transforming growth factor B, have been found to negatively regulate different aspects of angiogenesis, such as cell proliferation or cell migration. No single tissuederived macromolecule capable of inhibiting angiogenesis has been identified in the prior art. See, reviews by Folkman, 1995, N. Engl. J. Med. 333:1757-1763 and D'Amore, 1985, Prog. Clin. Biol. Res. 221:269-283. There is therefore a great need for the further identification and characterization of chemical agents which can prevent the continued deregulated spread of vascularization and which would potentially have broad applicability as a therapy for those diseases in which neovascularization plays a prominent role.

Capillary endothelial cells proliferate in response to an angiogenic stimulus during neovascularization.

Ausprunk and Folkman, 1977, J. Microvasc. Res. 14:153-65. An in vitro assay assessing endothelial cell proliferation in response to known angiogenesis simulating factors, such as acidic or basic fibroblast growth factor (aFGF and bFGF, respectively), has been developed to mimic the process of neovascularization in vitro. This type of assay is the assay of choice to demonstrate the stimulation of capillary EC proliferation by various angiogenic factors. Shing et al., 1984, Science 223:1296-1298.

The process of capillary EC migration through the extracellular matrix towards an angiogenic stimulus is also a critical event required for angiogenesis. See, review by Ausprunk et al., 1977, J. Microvasc. Res. 14:53-65. This -4- WO 00/54770 PCT/US00/06667 process provides an additional assay by which to mimic the process of neovascularization in vitro. A modification of the Boyden chamber technique has been developed to monitor EC migration. Boyden et al., 1962, J. Exptl. Med. 115:453-456, Example 4. To date, only a few tissue-derived EC cell migration inhibitors are known. See, review by Langer et 1976, Science 193:70-72.

In the early 1970's, a number of in vivo angiogenesis model bioassays were widely used. These model systems included rabbit corneal pocket, chick chorioallantoic membrane rat dorsal air sac and rabbit air chamber bioassays. For review, see, Blood et al., 1990, Biochem. et Biophys. Acta 1032:89-118. The development of controlled release polymers capable of releasing large molecules such as angiogenesis stimulators and inhibitors was critical to the use of these assays. Langer et al., 1976, Nature 263:797-800.

In the CAM bioassay, fertilized chick embryos are cultured in Petri dishes. On day 6 of development, a disc of a release polymer, such as methyl cellulose, impregnated with the test sample or an appropriate control substance is placed onto the vascular membrane at its advancing edge. On day 8 of development, the area around the implant is observed and evaluated. Avascular zones surrounding the test implant indicate the presence of an inhibitor of embryonic neovascularization. Moses et al., 1990, Science, 248:1408- 1410 and Taylor et al., 1982, Nature, 297:307-312. The reported doses for previously described angiogenesis inhibitors tested alone in the CAM assay are 50 pg of protamine (Taylor et al. (1982)), 200 pg of bovine vitreous extract (Lutty et al., 1983, Invest. Opthalmol. Vis. Sci.

24:53-56), and 10 pg of platelet factor IV (Taylor et al.

(1982)). The lowest reported doses of angiogenesis inhibitors effective as combinations include heparin (50 pg) and hydrocortisone (60 pg), and B-cyclodextrin tetradecasulfate WO 00/54770 PCT/US00/06667 (14 pg) and hydrocortisone (60 pg), reported by Folkman et al., 1989, Science 243:1490.

According to the rabbit corneal pocket assay, polymer pellets of ethylene vinyl acetate copolymer ("EVAC") are impregnated with test substance and surgically implanted in a pocket in the rabbit cornea approximately 1 mm from the limbus. Langer et al., 1976, Science 193:707-72. To test for an angiogenesis inhibitor, either a piece of carcinoma or some other angiogenic stimulant is implanted distal to the polymer 2 mm from the limbus. In the opposite eye of each rabbit, control polymer pellets that are empty are implanted next to an angiogenic stimulant in the same way. In these control corneas, capillary blood vessels start growing towards the tumor implant in 5-6 days, eventually sweeping over the blank polymer. In test corneas, the directional growth of new capillaries from the limbal blood vessel towards the tumor occurs at a reduced rate and is often inhibited such that an avascular region around the polymer is observed. This assay is quantitated by measurement of the maximum vessel lengths with a stereospecific microscope.

Troponin, a complex of three polypeptides is an accessory protein that is closely associated with actin filaments in vertebrate muscle. The troponin complex, acts in conjunction with the muscle form of tropomyosin to mediate the Ca 2 dependency of myosin ATPase activity and thereby regulate muscle contraction. The troponin polypeptides T, I, and C, are named for their tropomyosin binding, inhibitory, and calcium binding activities, respectively. Troponin T binds to tropomyosin and is believed to be responsible for positioning the troponin complex on the muscle thin filament. Troponin I binds to actin, and the complex formed by troponins I and T, and tropomyosin, inhibits the interaction of actin and myosin.

Troponin C is capable of binding up to four calcium molecules.

Studies suggest that when the level of calcium in the muscle is raised, troponin C causes troponin I to loose its hold on -6- WO 00/54770 PCT/US00/06667 the actin molecule, causing the tropomyosin molecule shift, thereby exposing the myosin binding sites on actin and stimulating myosin ATPase activity.

The citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.

3. SUMMARY OF THE INVENTION The present invention relates to pharmaceutical compositions containing troponin subunits C, I, or T, or fragments thereof, in therapeutically effective amounts that are capable of inhibiting angiogenesis, for example, by inhibiting endothelial cell proliferation. The invention also relates to pharmaceutical compositions containing homologs of troponin subunits C, I, or T and homologs of their fragments, in therapeutically effective amounts that are capable of inhibiting angiogenesis, for example, by inhibiting endothelial cell proliferation. The invention further relates to treatment of neovascular disorders by administration of a therapeutic compound of the invention. Such therapeutic compounds (termed herein "Therapeutics"), include: troponin subunits C, I, and T, and fragments and homologs thereof, in particular, fragments of troponin subunit I comprising the inhibitory and carboxy terminal regions. In one embodiment, a Therapeutic of the invention is administered to treat a cancerous condition, for example, to inhibit the growth or reduce the volume of a solid tumor, or to prevent progression from the pre-neoplastic or pre-malignant state into a neoplastic or a malignant state or to inhibit metastasis. In other specific embodiments, a Therapeutic of the invention is administered to treat ocular disorders -7associated with neovascularization. As used herein, the term "troponin subunit", when not preceding the terms C, I or T, means generically any of troponin subunits C, I, or T. The amino-terminal, inhibitory and carboxy-terminal regions of troponin I are designated and respectively.

The present invention provides method of inhibiting: i) angiogenesis associated with a disease or disorder; ii) the growth or reducing the volume of a solid tumor in a subject; or iii) metastasis in a subject, which comprise administering an effective amount of a peptide in which the peptide is: less than 100 amino acids in length; and 15 greater than 80% homology with the inhibitory and carboxy terminal region of tropinin subunit I (SEQ ID NO:14).

*e* The present invention provides a pharmaceutical composition comprising a peptide in which the peptide is: less than 100 amino acids in length; and greater than 80% homology with the inhibitory and carboxy terminal region of troponin subunit I (SEQ ID NO:14); and a pharmaceutically acceptable carrier.

25 The present invention also provides a pharmaceutical composition comprising a recombinant cell containing a nucleotide sequence encoding a peptide in which the peptide is: less than 100 amino acids in length; and greater than 80% homology with the inhibitory and carboxy terminal region of troponin subunit I (SEQ ID NO:14), such that said peptide is expressed in a subject.

The present invention further provides a pharmaceutical composition comprising a peptide in which the peptide is: an inhibitor of bFGF-stimulated bovine endothelial cell proliferation having an ICso of at least 10 jM; X:\Volet\Nge\651921\65121 Nodelete.doc at least 100 continuous amino acids in length; and greater than 80% homology with a subunit selected from the group consisting of fast-twitch troponin subunit C (SEQ ID NO:1), troponin subunit I (SEQ ID NO:2), fast-twitch troponin subunit T (SEQ ID NO:3); and a pharmaceutically acceptable carrier.

The present invention yet further provides a pharmaceutical composition comprising a peptide in which the peptide is: an inhibitor of bFGF-stimulated bovine endothelial cell proliferation having an ICs 0 of at least selected from the group consisting of fast-twitch troponin e.

subunit C (SEQ ID NO:1), troponin subunit I (SEQ ID NO:2), 15 fast-twitch troponin subunit T (SEQ ID NO:3); and a pharmaceutically acceptable carrier.

The present invention provides a method for inhibiting the growth or reducing the volume of a solid tumor in a subject 20 comprising administering a recombinant cell containing a nucleotide sequence encoding a peptide in which the peptide is: less than 100 amino acids in length; and greater than 80% homology with the inhibitory and carboxy terminal region of troponin subunit I (SEQ ID NO:14), 25 such that said peptide is expressed in the subject.

The present invention also provides a method of inhibiting metastases in a subject comprising administering to a subject a recombinant cell containing a nucleotide sequence encoding a peptide in which the peptide is: less than 100 amino acids in length; and greater than 80% homology with the inhibitory and carboxy terminal region of troponin subunit I (SEQ ID NO:14), such that said peptide is expressed in the subject.

W-.XV-vehNNeR5192tMI 921 Ndelete~doc The present invention provides a pharmaceutical composition comprising a recombinant cell containing a nucleotide sequence encoding a peptide in which the peptide is: an inhibitor of bFGF-stimulated bovine endothelial cell proliferation having an IC 0 of at least 10 AM; at least 100 continuous amino acids in length; and greater than 80% homology with a subunit selected from the group consisting of fast-twitch troponin subunit C (SEQ ID NO:1), troponin subunit I (SEQ ID NO:2), and fast-twitch troponin subunit T (SEQ ID NO:3), such that said peptide is expressed in a subject.

The present invention also provides a pharmaceutical ego composition comprising a recombinant cell containing a 15 nucleotide sequence encoding a peptide in which the peptide is: an inhibitor of bFGF-stimulated bovine endothelial cell S" proliferation having an IC, 0 of at least selected from the group consisting of fast-twitch troponin subunit C (SEQ ID NO:1), troponin subunit I (SEQ ID NO:2), 20 and fast-twitch troponin subunit T (SEQ ID NO:3), such that said peptide is expressed in a subject.

The present invention provides a peptide comprising a region of troponin subunit I having amino acid residues 118-137 25 (huTnI 118 1 37 of SEQ ID NO:2.

The present invention provides a method of inhibiting angiogenesis associated with a disease or disorder which comprises administering an effective amount of a peptide in which the peptide is greater than 80% homology with amino acid residues 118-137 (huTnIn 8 13 of the inhibitory region of troponin subunit I (SEQ ID NO:2).

W:\VioletNigel1651921\651921 Nodelete.doc The present invention also provides a method of inhibiting angiogenesis associated with a disease or disorder which comprises administering an effective amount of a peptide having at least 10 continuous amino acid residues of the amino acid sequence of residues 118-137 (huTnIne,,,-) of SEQ ID NO:2 so that angiogenesis is inhibited.

The present invention further provides a method of inhibiting metastasis in a mammal comprising administering an effective amount of a peptide in which the peptide is greater than 80% homology with a region of troponin subunit I having amino acid residues 118--137 (huTnI, 11 8 1 3 of SEQ ID NO:2.

*The present invention provides a pharmaceutical 15 composition for inhibiting angiogenesis associated with a disease or disorder comprising a peptide: in which the peptide is greater than 80% homology with *amino acid residues 118-137 (huTnI 118 n 1 of SEG ID NO:2 and a pharmaceutically acceptable carrier; or S 20 (ii) having at least 10 continuous amino acid residues of the amino acid sequence of residues 118-137 (huTnIs- 137 of SEQ ID NO:2.

The present invention provides a pharmaceutical S 25 composition comprising a recombinant cell containing a nucleotide sequence encoding a peptide in which the peptide: is greater than 80% homology with region of troponin subunit I having amino acid residues 118-137 (huTnI,, 1 13 7 of SEQ ID NO:2, such that said peptide is expressed in a host; or (ii) is a region of troponin subunit I having at least continuous amino acid residues of the amino acid sequence of residues 118-137 (huTnI,,- 1 8 of SEQ ID NO:2 such that peptide is expressed in a host.

W-.IV-beeh~ige6 192 1\661921 Nodeleteddo The present invention also provides a method for inhibiting the growth or reducing the volume of a solid tumor in a subject comprising administering a recombinant cell containing a nucleotide sequence encoding a peptide in which the peptide is greater than 80% homology with a region of troponin subunit I having amino acid residues 118-137 (huTnIn- 8 1 3 of SEQ ID NO:2, such that said peptide is expressed in the host.

The present invention further provides a method of inhibiting metastases in a subject comprising administering to a subject a recombinant cell containing a nucleotide sequence encoding a peptide in which the peptide is greater than homology with a region of troponin subunit I having amino acid residues 118-137 (huTn1 1 ,-i 37 of SEQ ID NO:2, such that said 15 peptide is expressed in the host.

The present invention provides a peptide comprising a region of troponin subunit I having amino acid residues selected from the group consisting of 116-123 (huTnIns 123 120-127 (huTnI 120 27 122-129 (huTnI1 2212 9 124-131 (huTnI 24 -i 3 126-133 (huTnI 126 33 128-135 (huTnI 28 1 3 130-137 (huTnI 13 0 o- 37 132-139 o"o (huTnI 32 13 9 and 134-141 (huTnI 341 1 of SEQ ID NO:2.

The present invention a method of inhibiting angiogenesis 25 associated with a disease or disorder which comprises administering an effective amount of a peptide: in which the peptide is greater than 80% homology with amino acid residues selected from the group consisting of 116-123 (huTn 116 -1 23 120-127 (huTnI1 2012 7 122-129 (huTnI 22 129), 124-131 (huTnI1 241 31 126-133 (huTn1 1 26 133 128-135 (huTn1- 28 135 130-137 (huTnI, 3 o- 0 37 132-139 (huTnI 3 2- 139 and 134-141 (huTn1 134 1 1 of the inhibitory region of troponin subunit I (SEQ ID NO:2); or W:\VioletlNIgel\651928\651921Nodelete.doc (ii) having at least 8 continuous amino acid residues of the amino acid sequence selected from the group consisting of 116-123 (huTn1 11612 1 3 120-127 (huTn1 1 122-129 (huTn1 122 129) 1 124-131 (huTn1 12 41 31 126-133 (huTn1 1 5 133 128-135 (huTn 1 2 8 135 130-137 (huTn1 130 13 0) 132-139 (huTnI 132 139 and 134-141 (huTn1 1 3 4 1 1 of residues 118-137 (huTn1 118 137 of SEQ ID NO:2 so that angiogenesis is inhibited.

The present invention also provides a method of inhibiting metastasis in a mammal comprising administering an effective amount of a peptide in which the peptide is greater than homology with a region of troponin subunit I select ed from the group consisting of 116-123 (huTnI 116 123 120-127 (huTn1 120 127 122-129 (huTn1 122 1 1 9 124-131 (huTn1 1 2 4 131 126-133 (huTn1 1 26 1 33 128--135 (huTnI,,, 8 13 5 130-137 (huTn1 13 0 13 132-139 (huTn 1 32 139 and 134-141 (huTn1 134 141 having amino acid residues of SEQ ID NO :2.

The present invention provides a pharmaceutical composition for inhibiting angiogenesis associated with a disease or disorder comprising a peptide: in which the peptide is greater than 80% homology with amino acid residues selected from the group consisting of 116-123 (huTn1 11 1 3 120-127 (huTn1 12 0 12 7 122-129 (huTn1 122 129) 124-131. (huTnI 124 131 126-133 (huTn1 126 13 1) 128-135 (huTn1 128 13 0) 130-137 (huTn 13013 1 7 132-139 (huTn1 132 139 and 134-141 (huTn1 134 141 of SEQ ID NO:2 and a pharmaceutically acceptable carrier; or (ii) having at least 8 continuous amino acid residues of the amino acid sequence of residues selected from the group consisting of 116-123 (huTn1 1 6 123 120-127 (huTn 120 127 122-129 (huTn1 12 2 1 2 9 124-131 (huTn 124 131 126-133 (huTn 1 26 133) 1 128-135 (huTn 1281 3 130-137 (huTn1 13 0 137 ,132-139 (huTnI,-), and 134-141 (huTnI,3 4 1 of SEQ ID NO:2.

W.Wiolet\NigeASS1921\651921NOdelete.doc The present invention provides a pharmaceutical composition comprising a recombinant cell containing a nucleotide sequence encoding a peptide in which the peptide: is greater than 80% homology with region of troponin subunit I having amino acid residues selected from the group consisting of 116-123 (huTnIn 1 6 1 3 120-127 (huTnI 120 122-129 (huTn1 1 2 2 1 2 9 124-131 (huTnI, 2 41 3 126-133 (huTn1 26 1 33 128-135 (huTnI 28 13 5 130-137 (huTnI 130 13 132-139 (huTnI 1 32 139 and 134-141 (huTnI 134 41 of SEQ ID NO:2, such that said peptide is expressed in a host; or (ii) is a region of troponin subunit I having at least 8 continuous amino acid residues of the amino acid sequence of residues selected from the group consisting of 116-123 (huTn 1 1 6 -1 23 120-127 (huTnI 20 127 122-129 (huTnI 22 129 o 15 124-131 (huTnIl 24 131 126-133 (huTnIl 2 6 133 128-135 (huTnI 2 8 13,) 130-137 (huTnI 30 13 7 132-139 (huTnI1 32 1 3 9 and 134-141 (huTnI 131 4 1 of SEQ ID NO:2 such that said peptide is expressed in a host.

20 The present invention also provides a method for inhibiting the growth or reducing the volume of a solid tumor in a subject comprising administering a recombinant cell containing a nucleotide sequence encoding a peptide in which the peptide is greater than 80% homology with a region of troponin subunit I having amino acid residues selected from the group consisting of 116-123 (huTnI 1 1 6- 123 120-127 (huTnI 20 127 122-129 (huTnI 22 129 124-131 (huTnI 24 133 126-133 (huTnI 12 6 133 128-135 (huTnI1 28 135 130-137 (huTnI 13 0 1 37 132-139 (huTn1 132 139 and 134-141 (huTnIl 3 11) of SEQ ID NO:2, such that said peptide is expressed in the host.

The present invention further provides a method of inhibiting metastases in a subject comprising administering to a subject a recombinant cell containing a nucleotide sequence w.-vjoIetNe65921\65121 Nodelete.dc encoding a peptide in which the peptide is greater than homology with a region of troponin subunit I having amino acid residues selected from the group consisting of 116-123 (huTnI,, 1 123), 120-127 (huTnI 120 127 122-129 (huTnI1 22 129 124-131 (huTnI1 24 131) 126-133 (huTn1 26 133 128-135 (huTnI 1 2 1 s) 130-137 (huTnI 30 o 137), 132-139 (huTnI1 32 1 39 and 134-141 (huTnI 134 4 1 of SEQ ID NO:2, such that said peptide is expressed in the host.

4. BRIEF DESCRIPTION OF THE FIGURES Figure 1. Inhibition of bovine capillary Endothelial Cell (BCE) proliferation by troponin C. Percent inhibition of bFGFstimulated BCE proliferation is shown as a function of troponin C concentration (pg/well) Percent inhibition was determined by comparing results obtained for cells treated with stimulus alone 15 with those obtained for samples exposed to both stimulus and S* inhibitor. Well volume was 200 il.

Figure 2. Inhibition of capillary BCE proliferation by troponin I. Percent inhibition of bFGF-stimulated

BCE

proliferation is shown as a function of troponin I concentration 20 (g/well). Percent inhibition was determined as described in Figure 1. Well volume was 200 Al.

Figure 3. Inhibition of capillary BCE proliferation by troponin T. Percent inhibition of bFGF-stimulated BCE proliferation is shown as a function of troponin T concentration 25 (jg/well) Percent inhibition was determined as described in Figure 1. Well volume was 200 Al.

Figure 4. Inhibition of BCE proliferation by troponins C and I. Percent inhibition of bFGF-stimulated BCE proliferation is shown as a function of troponin I and C concentration (jg/well). Percent inhibition was determined as described in Figure 1. Well volume was 200 Al.

Figure 5. Inhibition of capillary BCE proliferation by troponin C, I and T. Percent inhibition of bFGF-stimulated

BCE

proliferation is shown as a function of troponin C, I, and w:\VolWNe ige 1921651 921 No dlee.doc WO 00/54770 PCT/US00/06667 T concentration (pg/well). Percent inhibition was determined as described in Figure. Well volume was 200 pl.

Figure 6. Schematic representation of amino acid sequences of tryptic peptides purified from cartilage as described in Methods. Sequence similarity to human TnI is indicated by alignment with the amino acid sequence of the human isoform.

Figure 7. RT-PCR products amplified from total RNA purified from two separate human intercostal cartilage specimens. Gene-specific primers were designed based on the cDNA sequence of human fast-twitch skeletal muscle TnI.

Nucleotide sequence of these PCR products showing identity to the cDNA sequence of human fast-twitch skeletal muscle TnI (nt 189 nt 384) (SEQ ID NO:16). RT-PCR amplification, from total RNA (20 ng each lane) purified from rat skeletal muscle (lane xyphoid (lane chondrosarcoma (lane 3) and liver (lane Gene-specific primers were designed based on the cDNA sequence of rat fast-twitch skeletal muscle TnI as described in Methods.

Figure 8. SDS-PAGE of recombinant human TnI before (lane A) and after (lane B) purification. In both cases, approximately 1 pg of total protein was electrophoresed, followed by silver staining as described in Methods.

Recombinant TnI migrates at a molecular weight of approximately 21,000 Da.

Figure 9. Inhibition of capillary EC proliferation by rTnI. Percent inhibition was determined by comparing wells exposed to the angiogenic stimulus bFGF and VEGF with those exposed to stimulus and inhibitor. Each point represents the mean of duplicate control and inhibitor wells.

This is a representative experiment of four different

EC

proliferation assays, each testing different TnI preparations.

Figure 10. Inhibition of embryonic angiogenesis in vivo by rTnI. After a 48 h exposure to rTnI as described in -9- WO 00/54770 PCT/US00/06667 Methods, avascular zones, free of capillaries and small vessels were observed using a binocular dissecting microscope at x7-10 magnification. This zone was produced by approximately 380 pmoles of TnI A normal chorioallantoic membrane(CAM) implanted with a methylcellulose disk containing buffer alone is shown in Figure 11. Inhibition of FGF-induced angiogenesis by systemic administration of TnI. TnI (50 mg/kg) was administered systemically every 12 hours to mice whose corneas had been implanted with pellets containing bFGF (40ng/ml) on Day 1. After-six days of treatment, significant inhibition of FGF-induced neovascularization was observed in TnI-treated corneas as compared to control corneas Figure 12. Derived amino acid sequence of recombinant human TnI (Hu)(SEQ ID NO:17) and its sequence comparison with recombinant rabbit TnI (Rb) (SEQ ID Identical residues are shown by dashes. Schematic representation of various recombinant TnI deletion fragments based on rabbit TnI and wild-type rabbit TnI, (SEQ ID The troponin I inhibitory region is designated and the sequences located on amino- and carboxy-terminal sides of this region are designated N' and respectively. TnI 1 120 TnI 1 l 94 TnI 96 1 TnI 122 1 8 1 contain the N' and I' and and C' regions, respectively. The number of amino acids at the beginning and end of each fragment is indicated. TnI 98 114 containing amino acid residues 98-114 is a synthetic peptide representing the I region.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to therapeutic methods and compositions based on troponin subunits. The invention provides for treatment of neovascular disorders by, for example, inhibiting angiogenesis, comprising administration of a therapeutic compound of the invention. Such therapeutic compounds (termed herein "Therapeutics") include: troponin C, WO 00/54770 PCT/US00/06667 I, and T subunits, fragments and homologs thereof (collectively "peptides of the invention"). The peptides of the invention are characterized by the property of inhibiting bovine endothelial cell (EC) proliferation in culture preferably with an ICs 5 of about 10 pM or less, more preferably with an IC 50 of about 5pM or less, most preferably with an ICs of about lpM or less. In a preferred embodiment, a Therapeutic of the invention is administered to treat a cancerous condition, for example, to inhibit the growth or reduce the volume of a solid tumor, or to prevent progression from a pre-neoplastic or non-malignant state into a neoplastic or a malignant state or to inhibit metastases. In another specific embodiment, a Therapeutic of the invention is administered to treat an ocular disorder associated with neovascularization.

In a preferred aspect, a Therapeutic of the invention is a peptide consisting of at least a fragment of troponin C, troponin I, troponin T, or combinations thereof which is effective to inhibit angiogenesis. More preferably, the Therapeutic is a peptide consisting of the inhibitory and carboxy terminal region (SEQ ID NO:14) of troponin subunit I or a fragment thereof.

In specific embodiments, the peptides of the invention are troponin C, troponin I and troponin T subunits, or fragments thereof of the fast twitch, slow twitch and cardiac isoforms from mammalian species, human, rabbit, rat, mouse, bovine, ovine and porcine.

In other embodiments, the peptides of the invention are troponin C, troponin I and troponin T subunits, or fragments thereof from nonmuscle tissues, cartilage, preferably from mammalian species, human, rabbit, rat, mouse, bovine, ovine and porcine.

Examples of the troponin subunits that can be utilized in accordance with the invention, include but are not -11- WO 00/54770 WO 0054770PCT/USOOIO6667 limited to the subunits of troponin from human fast twitch skeletal muscle, the sequences of which are given below: Fast Twitch Skeletal Muscle Troponin C (SEO ID NOt 1) 1 21 41 61 8l 101 121 141 D Q 0 A E AR S A F D M F D AD V M RM L G Q T V D E D G S G T Q M K E D A K G F D R N A D G Y S G E H V T D E N D G R ID F D Y L S E E M I G G G D IS V P T K E EL D I D F EE F L K S EE E L A I D P E EL A ElI E S L M K E F L K MM E A E K E AlI V M E C ElI D G G V FMO#- q%.rj 'OU I a. I is.. "I MR j W oc ropon AL jong LU mq:Aj 1 21 41 61 81 101 121 141 161 191 I T E K P L K I

KEH

P L H K K E M E A R E E H I D A L E R R V C R D G R Fast Skeletal Beta Troponin T (SRO ID NO:3) 1 21 41 61 e1 101 121 141 161 181 201 221 241 A Q P R I Q F E R R N R D D A K L A K A E K Q K -12- WO 00/54770 PCT/US00/06667 In another embodiment, the invention encompasses peptides which are homologous to troponin C (SEQ ID NO:1) or fragments thereof, troponin I (SEQ ID NOS:2, 10, or 15) or fragments thereof, or troponin T (SEQ ID NO:3) or fragments thereof.

In a particular embodiment, the peptides of the invention are fragments of troponin I (SEQ ID NOS:11-15) or homologous to fragments of troponin I (SEQ ID NOS:11-15).

In a specific embodiment, a Therapeutic of the invention is combined with a therapeutically effective amount of another molecule which negatively regulates angiogenesis which may be, but is not limited to, platelet factor 4, thrombospondin-l, tissue inhibitors of metalloproteases (TIMP1 and TIMP2) prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), bFGF soluble receptor, transforming growth factor P, interferon alfa, and placental proliferinrelated protein.

Paradoxically, neovascularization gradually reduces a tumor's accessibility to chemotherapeutic drugs due to increased interstitial pressure within the tumor, which causes vascular compression and central necrosis. In vivo results have demonstrated that rodents receiving angiogenic therapy show increased delivery of chemotherapy to a tumor. Teicher et al., 1994, Int. J. Cancer 57:920-925. Thus, in one embodiment, the invention provides for a pharmaceutical composition of the present invention in combination with a chemotherapeutic agent.

In another preferred aspect, a Therapeutic of the invention is combined with chemotherapeutic agents or radioactive isotope exposure.

The invention is illustrated by way of examples infra which disclose, inter alia, the inhibition of capillary endothelial cell proliferation by troponin subunits C, I, and T and the means for determining inhibition of capillary endothelial cell migration and inhibition of neovascularization in vivo by troponin subunits.

-13- WO 00/54770 PCT/US00/06667 For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections set forth below.

5.1. TROPONIN SUBUNITS, FRAGMENTS AND HOMOLOG8 The invention provides for pharmaceutical compositions comprising troponin subunits, fragments, and homologs thereof. In particular aspects, the subunits, fragments, or homologs are of fly, frog, mouse, rat, rabbit, pig, cow, dog, monkey, or human troponin subunits.

In another embodiment, the invention encompasses peptides which are homologous to troponin C (SEQ ID NO:1) or fragments thereof. In one embodiment, the amino acid sequence of the peptide has at least 80% identity compared to the troponin C from which it is derived. In another embodiment, this identity is greater than 85%. In a more preferred embodiment, this identity is greater than 90%. In a most preferred embodiment, the amino acid sequence of the peptide has at least 95% identity with the troponin C or fragment thereof. Fragments are generally at least 10 amino acids, and in alternate embodiments at least 20, 30, 40, and 100 amino acids in length.

In another embodiment, the invention encompasses a troponin submit subunit or fragment thereof encoded by a nucleic acid hybridizable to the complement of a nucleic acid encoding a troponin subunit, preferably troponin C, under low stringency, moderate stringency or high stringency conditions.

In another embodiment, the invention encompasses peptides which are homologous to troponin I(SEQ ID NOS:2, or 15) or fragments thereof. In one embodiment, the amino acid sequence of the peptide has at least 80% identity with the troponin I or fragment thereof. In another embodiment, this identity is greater than 85%. In a more preferred embodiment, this identity is greater than 90%. In a most preferred embodiment, the amino acid sequence of the peptide has at least 95% identity with the troponin I or fragment -14- WO 00/54770 PCT/US00/06667 thereof. Fragments are generally at least 4 amino acids, and in alternate embodiments at least 8, 10, 20, 30, 40, 50, and 100 amino acids in length.

In another embodiment, the invention encompasses a troponin submit subunit or fragment thereof encoded by a nucleic acid hybridizable to the complement of a nucleic acid encoding a troponin subunit, preferably troponin I, under low stringency, moderate stringency or high stringency conditions.

In another embodiment, the invention encompasses peptides which are homologous to troponin T (SEQ ID NO:3) or fragments thereof. In one embodiment, the amino acid sequence of the peptide has at least 80% identity with the troponin T or fragment thereof. In another embodiment, this identity is greater than 85%. In a more preferred embodiment, this identity is greater than 90%. In a most preferred embodiment, the amino acid sequence of the peptide has at least 95% identity with the troponin T or fragment thereof. Fragments are generally at least 10 amino acids, and in alternate embodiments at least 20, 30, 40, 50, 100, 150, and 200 amino acids in length.

In another embodiment, the invention encompasses a troponin submit subunit or fragment thereof encoded by a nucleic acid hybridizable to the complement of a nucleic acid encoding a troponin subunit, preferably troponin T, under low stringency, moderate stringency or high stringency conditions.

In a preferred embodiment, the invention encompasses peptides which are homologous to the Inhibitory and carboxy terminus region (SEQ ID NO:14).

In other embodiments, the invention encompasses peptides that are homologous to the region of human troponin I (huTnI) (SEQ ID NO:17) corresponding to amino acid residues of SEQ ID NO:17, including but not limited to residues: 94-123 (huTnI 94 123), 104-133 (huTnI 10 4 -1 33 114-143 (huTnIn 1 4 143 129-153 (huTnI1 29 153 134-173 (huTnI 134-173), 144-182 (huTnI 144 1 8 93- 112 (huTn 93 1 1 2 98-117 (huTnI 9 8 117 103-122 (huTnI 103 122 108- WO 00/54770 PCTIUSO/06667 127 (huTnI 108 127 113-132 (huTnI 11 3 1 32 and carboxy terminus region 118-137 (huTnIn,,_ 13 Additional embodiments include 94-113 (huTnI 94 13 98-117 (huTnI 98 1 17 102-121 (huTnI, 0 2- 1 2 106-125 (huTnI 1061 5 110-129 (huTnInO1 1 29 and 114-133 (huTnI, 1 4 33 Still other embodiments include carboxy terminus region of human troponin I (huTnI), 116-123 (huTnIn6 123 118-125 (huTnIns-12iz), 120-127 (huTnI1 20 12 122-129 (huTnI 122 -1 29 124-131 (huTnI 1 24 131), 126-133 (huTnI- 26 _1 33 128-135 (huTnI 128 13 130-137 (huTn 13 013 132-139 (huTnIl 3 2 3 9 134-141 (huTnI, 34 141 and 136-143 (huTnI 36143 Fragments are generally at least 4 amino acids, and in alternate embodiments at least 8, 10, 20, 50, and 75 amino acids in length.

"Homologous," as defined herein, refers to identity over an amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art or whose encoding nucleic acid is capable of hybridizing to a coding gene sequence, under high stringency, moderate stringency, or low stringency conditions.

Specifically, by way of example, computer programs for determining homology may include but are not limited to TBLASTN, BLASTP, FASTA, TEASTA, and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85(8):2444-8; Altschul et al., 1990, J. Mol. Biol. 215(3):403-10; Thompson, et al., 1994, Nucleic Acids Res. 22(22):4673-80; Higgins, et al., 1996, Methods Enzymol 266:383-402; Altschul, et al., 1990, J. Mol. Biol. 215(3):403-10). Default parameters for each of these computer programs are well known and should be utilized.

Specifically, Basic Local Alignment Search Tool (BLAST) (www.ncbi.nlm.nih.gov; It is to be understood that for determination of homology, the default parameters are set and utilized with the most recent BLAST program version available at this site.) (Altschul et al., 1990, J. of Molec. Biol., 215:403-410, "The BLAST Algorithm; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402) is a heuristic -16- WO 00/54770 PCT/US00/06667 search algorithm tailored to searching for sequence similarity which ascribes significance using the statistical methods of Karlin and Altschul 1990, Proc. Natl Acad. Sci.

USA, 87:2264-68; 1993, Proc. Nat'l Acad. Sci. USA 90:5873-77.

Five specific BLAST programs perform the following tasks: 1) The BLASTP program compares an amino acid query sequence against a protein sequence database; 2) The BLASTN program compares a nucleotide query sequence against a nucleotide sequence database; 3) The BLASTX program compares the sixframe conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database; 4) The TBLASTN program compares a protein query sequence against a nucleotide sequence database translated in all six reading frames (both strands); 5) The TBLASTX program compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

Smith-Waterman (database: European Bioinformatics Institute wwwz.ebi.ac.uk/bic_sw/) (Smith-Waterman, 1981, J.

of Molec. Biol., 147:195-197) is a mathematically rigorous algorithm for sequence alignments.

FASTA (see Pearson et al., 1988, Proc. Nat'l Acad.

Sci. USA, 85:2444-2448) is a heuristic approximation to the Smith-Waterman algorithm. For a general discussion of the procedure and benefits of the BLAST, Smith-Waterman and FASTA algorithms see Nicholas et al., 1998, "A Tutorial on Searching Sequence Databases and Sequence Scoring Methods" (www.psc.edu) and references cited therein.

It is envisioned that troponin subunits and fragments can be made by altering troponin sequences by substitutions, additions or deletions that provide for functionally equivalent molecules capable of displaying one or more functional activities associated with a full-length wild-type troponin subunit. Such functional activities include but are not limited to inhibition of angiogenesis; inhibition of metastases; inhibition of tumor growth. These include, but are not limited to, troponin subunits, -17- WO 00/54770 PCT/US00/06667 fragments, or homologs containing, as a primary amino acid sequence, all or part of the amino acid sequence of a troponin subunit including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

One embodiment of the invention provides for molecules consisting of or comprising a fragment of at least 4 (contiguous) amino acids of a troponin subunit which is capable of inhibiting endothelial cell proliferation as discussed above. In other embodiments, this molecule consists of at least 8, 10, 20 or 50 amino acids of the troponin subunit. In specific embodiments, such molecules consist of or comprise fragments of a troponin subunit that are at least 8, 10, 20, 30, 40, 50, 75, 100 and 150 amino acids in length, including but not limited to, (SEQ ID NO:14), huTnI 9412 3 huTni 104 133 huTnn 1 4 1 4 3 huTnI1 29 153 huTnI 134 173, huTnI1 4 4 182 huTn93- 1 1 2 huTn 98 1 1 7 huTnI 1 0 3 -1 2 2 huTn 1 0 8- 12 7 huTnI 13 -1 32 and carboxy terminus region huTnIn.

8 13 7 Additional embodiments include 94-113 (huTnI 9 4 n 13 98-117 (huTnI 98 102-121 (huTnI 102 121 106-125 (huTnI 1 0 6 125 110-129 (huTnIn- 0 129 and 114-133 (huTnIn.

1 4 33 Still other embodiments include carboxy terminus region 116-123 (huTnIn6- 123 118-125 (huTn, 1 8 12 120-127 (huTnI1 20 127 122- -18- WO 00/54770 PCT/US00/06667 129 (huTnI1 22 1 29 124-131 (huTnI 124 31 126-133 (huTnI 126 33 128-135 (huTnI- 2 8 1 35 130-137 (huTnI 130 o 137 132-139 (huTnI1 32 139), 134-141 (huTnI 3 4 141 and 136-143 (huTnI 1 36 143 In a preferred embodiment, the protein is a mammalian troponin subunit. In more preferred embodiments, it is a mammalian troponin C, I, or T subunit.

The troponin subunits, fragments and homologs of the invention can be derived from tissue (see, for example, Section 6, Examples 1 and 7; Ebashi et al., 1968, J. Biochem.

64:465; Yasui et al., 1968, J. Biol. Chem. 243:735; Hartshorne et al., 1968, Biochem. Biophys. Res. Commun.

31:647; Shaub et al., 1969, Biochem. J. 115:993; Greaser et al., 1971, J. Biol. Chem. 246:4226-4733; Brekke et al., 1976, J. Biol. Chem. 251:866-871; and Yates et al., 1983, J. Biol.

Chem. 258:5770-5774) or produced by various methods known in the art, for example, recombinant techniques (see, for example, Section 6, Examples 1 and 7).

Manipulations of troponin subunits can occur at the gene or protein level. For example, a cloned troponin gene sequence coding for troponin subunits C, I, or T, can be modified by any of numerous strategies known in the art.

Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. In the production of the gene encoding a fragment or homolog of a troponin subunit, care should be taken to ensure that the modified gene remains within the same translational reading frame as the troponin subunit gene, uninterrupted by translational stop signals, in the gene region where the desired troponin activity is encoded.

In a specific embodiment, a nucleic acid which is hybridizable to the complement of a troponin nucleic acid having a sequence as set forth in SEQ ID NOS:13-17), -19- WO 00/54770 PCTIUSOO/06667 or to a nucleic acid encoding a troponin fragment or derivative under conditions of low stringency is provided.

By way of example and not limitation, procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 6789-6792). Filters containing DNA are pretreated for 6'h at 0 C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 gg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 pg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 106 cpm 32 P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40°C, and then washed for h at 55 0 C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional h at 60 0 C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68 0 C and re-exposed to film. Other conditions of low stringency which may be used are well known in the art as employed for cross-species hybridizations).

In another specific embodiment, a nucleic acid which is hybridizable to a troponin nucleic acid under conditions of high stringency is provided. By way of example and not limitation, procedures using such conditions of high stringency are as follows. Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65 0 C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 gg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 650C in prehybridization mixture containing 100 Ag/ml denatured salmon sperm DNA and 5-20 X 106 cpm of 32 P-labeled probe. Washing of filters is done at 370C for 1 h in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1X SSC at 50°C for WO 00/54770 PCT/US00/06667 45 min before autoradiography. Other conditions of high stringency which may be used are well known in the art.

In another specific embodiment, a nucleic acid which is hybridizable to a troponin nucleic acid under conditions of moderate stringency is provided. Selection of appropriate conditions for such stringencies is well known in the art (see Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; see also, Ausubel et al., eds., in the Current Protocols in Molecular Biology series of laboratory technique manuals, 1987-1997 Current Protocols, 0 1994-1997 John Wiley and Sons, Inc.).

Additionally, the troponin subunit encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including, but not limited to, in vitro sitedirected mutagenesis (Hutchinson et al., 1978, J. Biol. Chem.

253:6551), use of TABO linkers (Pharmacia), etc.

Manipulations of troponin subunit C, I, or T sequence may also be made at the protein level. Included within the scope of the invention are troponin subunit fragments or other fragments or homologs which are differentially modified during or after translation, by acetylation, phosphorylation, carboxylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 acetylation, formylation, oxidation, reduction, etc.

-21- WO 00/54770 PCT/US00/06667 In addition, fragments and homologs of troponin subunits can be chemically synthesized. For example, a peptide corresponding to a portion of a troponin subunit which comprises the desired domain, or which mediates the desired activity in vitro, can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid homologs can be introduced as a substitution or addition into the troponin subunit sequence. Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, a-amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, tbutylalanine, phenylglycine, cyclohexylalanine, (-alanine, designer amino acids such as 3-methyl amino acids, Ca-methyl amino acids, and Na-methyl amino acids.

In a specific embodiment, the invention encompasses a chimeric, or fusion, protein comprising a troponin subunit or fragment thereof (consisting of at least a domain or motif of the troponin subunit that is responsible for inhibiting endothelial cell proliferation) joined at its amino or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein. Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art.

Alternatively, such a chimeric product may be made by protein synthetic techniques, by use of a peptide synthesizer.

In another embodiment, the invention encompasses combination of the troponin subunits, fragments, or homologs of the present invention to inhibit angiogenesis. Another embodiment provides for the combination of troponin subunits, fragments, or homologs with other angiogenesis inhibiting factors. Such angiogenesis inhibiting factors include, but are not limited to: angiostatic steroids, thrombospondin, platelet factor IV, transforming growth factor P, interferons, tumor necrosis factor a, bovine vitreous -22- WO 00/54770 PCT/US00/06667 extract, protamine, tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2), prolactin (16-kd fragment), angiostatin (38-kd fragment of plasminogen), bFGF soluble receptor, and placental proliferin-related protein. See, reviews by Folkman et al., 1995, N. Engl. J. Med. 333:1757-1763 and Klagsbrun et al., 1991, Annu. Rev. Physiol. 53:217-239.

5.2. ASSAYS OF TROPONIN PROTEINS FRAGMENTS AND HOMOLOGS The functional activity and/or therapeutically effective dose of troponin subunits, fragments and homologs, can be assayed in vitro by various methods. These methods are based on the physiological processes involved in angiogenesis and while they are within the scope of the invention, they are not intended to limit the methods by which troponin subunits, fragments and homologs inhibiting angiogenesis are defined and/or a therapeutically effective dosage of the pharmaceutical composition is determined.

For example, where one is assaying for the ability of troponin subunits, fragments, and homologs, to inhibit or interfere with the proliferation of capillary endothelial cells (EC) in vitro, various bioassays known in the art can be used, including, but not limited to, radioactive incorporation into nucleic acids, colorimetric assays and cell counting.

Inhibition of endothelial cell proliferation may be measured by colorimetric determination of cellular acid phosphatase activity or electronic cell counting. These methods provide a quick and sensitive screen for determining the number of endothelial cells in culture after treatment with the troponin subunit, fragment, or homolog of the invention, and an angiogenesis stimulating factor such as aFGF. The colorimetric determination of cellular acid phosphatase activity is described by Connolly et al., 1986, J. Anal. Biochem. 152:136-140. According to this method, described in Example 9, capillary endothelial cells are -23- WO 00/54770 PCT/US00/06667 treated with angiogenesis stimulating factors, such as aFGF, and a range of potential inhibitor concentrations. These samples are incubated to allow for growth, and then harvested, washed, lysed in a buffer containing a phosphatase substrate, and then incubated a second time. A basic solution is added to stop the reaction and color development is determined at 405 X. According to Connolly et al., a linear relationship is obtained between acid phosphatase activity and endothelial cell number up to 10,000 cells/sample. Standard curves for acid phosphatase activity are also generated from known cell numbers in order to confirm that the enzyme levels reflect the actual EC numbers.

Percent inhibition is determined by comparing the cell number of samples exposed to stimulus with those exposed to both stimulus and inhibitor.

Colorimetric assays to determine the effect of troponin subunits C, I, and T on endothelial cell proliferation demonstrate that all three troponin subunits interfere with bFGF-stimulated endothelial cell proliferation but have no detectable inhibitory effect on the growth of Balb/c 3T3 cells, a non-endothelial cell line. For an illustrative example, see Section 6, Examples 3 and 8, infra.

The incorporation of radioactive thymidine by capillary endothelial cells represents another means by which to assay for the inhibition of endothelial cell proliferation by a potential angiogenesis inhibitor. According to this method, a predetermined number of capillary endothelial cells are grown in the presence of 3 H-Thymidine stock, an angiogenesis stimulator such as for example, bFGF, and a range of concentrations of the angiogenesis inhibitor to be tested. Following incubation, the cells are harvested and the extent of thymidine incorporation is determined. See, Section 6, Example 3.

The ability of varying concentrations of troponin subunits, fragments or homologs to interfere with the process of capillary endothelial cell migration in response to an -24- WO 00/54770 PCT/US00/06667 angiogenic stimulus can be assayed using the modified Boyden chamber technique. See, Section 6, Example 4, infra.

Another means by which to assay the functional activity of troponin subunits, fragments and homologs, involves examining the ability of the compounds to inhibit the directed migration of capillary endothelial cells which ultimately results in capillary tube formation. This ability may be assessed for example, using an assay in which capillary endothelial cells plated on collagen gels are challenged with the inhibitor, and determining whether capillary-like tube structures are formed by the cultured endothelial cells.

Assays for the ability to inhibit angiogenesis in vivo include the chorioallantoic membrane assay and corneal pocket assays (see, Section 6, infra, Example 10, and Example 11, respectively). See also, Polverini et al., 1991, Methods Enzymol. 198:440-450. According to the corneal pocket assay, a tumor of choice is implanted into the cornea of the test animal in the form of a corneal pocket. The potential angiogenesis inhibitor is applied to the corneal pocket and the corneal pocket is routinely examined for neovascularization. See, Example 11 infra.

The therapeutically effective dosage for inhibition of angiogenesis in vivo, defined as inhibition of capillary endothelial cell proliferation, migration, and/or blood vessel ingrowth, may be extrapolated from in vitro inhibition assays using the compositions of the invention above or in combination with other angiogenesis inhibiting factors. The effective dosage is also dependent on the method and means of delivery. For example, in some applications, as in the treatment of psoriasis or diabetic retinopathy, the inhibitor is delivered in a topical-ophthalmic carrier. In other applications, as in the treatment of solid tumors, the inhibitor is delivered by means of a biodegradable, polymeric implant.

WO 00/54770 PCT/US00/06667 5.3. THERAPEUTIC USES The invention provides for compositions and methods for inhibition of angiogenesis. The invention further provides for compositions and methods for treatment or prevention of diseases or disorders associated with neovascularization by administration of a therapeutic compound of the invention. Such compounds (termed herein "Therapeutics") include troponin subunits and fragments and homologs thereof as described supra).

5.3.1. MALIGNANCIES Malignant and metastatic conditions which can be treated with the Therapeutic compounds of the present invention include, but are not limited to, the solid tumors listed in Table 1 (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia) and blood-borne tumors such as leukemias.

-26- WO 00/54770 PCT/USOO/06667 TABLE 1 MALIGNANCIES AND RELATED DISORDERS Solid tumors sarcomas and carcinomas fibrosarcoma myxosarcoma liposarcoma chondrosarcoma osteogenic sarcoma chordoma angiosarcoma endotheliosarcoma lymphangiosarcoma lymphangioendotheliosarcoma synovioma mesothelioma Ewing's tumor leiomyosarcoma rhabdomyosarcoma colon carcinoma pancreatic cancer breast cancer ovarian cancer prostate cancer squamous cell carcinoma basal cell carcinoma adenocarcinoma sweat gland carcinoma sebaceous gland carcinoma papillary carcinoma papillary adenocarcinomas cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal cell carcinoma hepatoma bile duct carcinoma choriocarcinoma seminoma embryonal carcinoma Wilms' tumor cervical cancer testicular tumor lung carcinoma small cell lung carcinoma bladder carcinoma epithelial carcinoma glioma astrocytoma medulloblastoma craniopharyngioma ependymoma -27- WO 00/54770 PCT/US00/06667 Kaposi's sarcoma pinealoma hemangioblastoma acoustic neuroma oligodendroglioma menangioma melanoma neuroblastoma retinoblastoma 5.3.2. OCULAR DISORDERS Ocular disorders associated with neovascularization which can be treated with the Therapeutic compounds of the present invention include, but are not limited to: neovascular glaucoma diabetic retinopathy retinoblastoma retrolental fibroplasia uveitis retinopathy of prematurity macular degeneration corneal graft neovascularization as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, reviews by Waltman et al., 1978, Am. J. Ophthal. 85:704-710 and Gartner et al., 1978, Surv. Ophthal. 22:291-312.

5.3.3. OTHER DISORDERS Other disorders which can be treated with the Therapeutic compounds of the present invention include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

-28- WO 00/54770 PCT/USOO/06667 5.4. DEMONSTRATION OF THERAPEUTIC OR PROPHYLACTIC UTILITY The Therapeutics of the invention can be tested in vivo for the desired therapeutic or prophylactic activity as well as for determination of therapeutically effective dosage. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc. For in vivo testing, prior to administration to humans, any animal model system known in the art may be used.

THERAPEUTIC/PROPHYLACTIC

ADMINISTRATION AND COMPOSITIONS The invention provides methods of inhibition of angiogenesis and method of treatment (and prophylaxis) by administration to a subject an effective amount of a Therapeutic of the invention. In a preferred aspect, the Therapeutic is substantially purified as set forth in Examples 1 and 7. The subject is preferably an animal, including, but not limited to, animals such as cows, pigs, chickens, etc., and is more preferably a mammal, and most preferably a human.

The invention also provides for methods of treatment and prevention by administration of an effective amount of a Therapeutic of the invention to an immunocompromised patient, for example, a patient having cancer or infected with human immunodeficiency virus (HIV).

In particular, the invention may be used to treat or prevent secondary infections or diseases associated with HIV infection or cancers.

The invention further provides methods of treatment and prevention by administration to a subject, an effective amount of a Therapeutic of the invention combined with a chemotherapeutic agent and/or radioactive isotope exposure.

The invention also provides for methods of treatment and prevention of a Therapeutic of the invention -29- WO 00/54770 PCT/US00/06667 for patients who have entered a remission in order to maintain a dormant state.

Various delivery systems are known and can be used to administer a Therapeutic of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, receptor-mediated endocytosis (see, Wu and Wu, 1987, J.

Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. It is preferred that administration is localized, but it may be systemic. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.

Pulmonary administration can also be employed, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or WO 00/54770 PCT/US00/06667 former site) of a malignant tumor or neoplastic or preneoplastic tissue.

For topical application, the purified troponin subunit is combined with a carrier so that an effective dosage is delivered, based on the desired activity ranging from an effective dosage, for example, of 1.0 MM to mM to prevent localized angiogenesis, endothelial cell migration, and/or inhibition of capillary endothelial cell proliferation. In one embodiment, a topical troponin subunit, fragment or homolog is applied to the skin for treatment of diseases such as psoriasis. The carrier may in the form of, for example, and not by way of limitation, an ointment, cream, gel, paste, foam, aerosol, suppository, pad or gelled stick.

A topical Therapeutic for treatment of some of the eye disorders discussed infra consists of an effective amount of troponin subunit, fragment, or homolog, in a ophthalmologically acceptable excipient such as buffered saline, mineral oil, vegetable oils such as corn or arachis oil, petroleum jelly, Miglyol 182, alcohol solutions, or liposomes or liposome-like products. Any of these compositions may also include preservatives, antioxidants, antibiotics, immunosuppressants, and other biologically or pharmaceutically effective agents which do not exert a detrimental effect on the troponin subunit.

For directed internal topical applications, for example for treatment of ulcers or hemorrhoids, the troponin subunit, fragment, or homolog composition may be in the form of tablets or capsules, which can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; or a glidant such as colloidal silicon dioxide. When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty -31- WO 00/54770 PCT/US00/06667 oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents.

Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.

In another embodiment, the Therapeutic can be delivered in a vesicle, in particular a liposome. See, Langer et al., 1990, Science 249:1527-1533; Treat et al., 1989, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327.

In yet another embodiment, the Therapeutic can be delivered in a controlled release system. In one embodiment, an infusion pump may be used to administer troponin subunit, such as for example, that used for delivering insulin or chemotherapy to specific organs or tumors (see Langer, supra; Sefton, CRC Crit. Ref. Biomed., 1987, Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl.

J. Med. 321:574.

In a preferred form, the troponin subunit, fragment, or homolog is administered in combination with a biodegradable, biocompatible polymeric implant which releases the troponin subunit, fragment, or homolog over a controlled period of time at a selected site. Examples of preferred polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and blends thereof. See, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball 1984, Wiley, New York; Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann.

-32- WO 00/54770 PCT/US00/06667 Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105.

In yet another-embodiment, a controlled release system can be placed in proximity of the therapeutic target, the brain, thus requiring only a fraction of the systemic dose (see, Goodson, in Medical Applications of Controlled Release, 1989, supra, vol. 2, pp. 115-138).

Other controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533).

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents.

Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be -33- WO 00/54770 PCT/US00/06667 employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents.

such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the Therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic -34- WO 00/54770 PCT/US00/06667 aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.

Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The amount of the Therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays such as those discussed in section 5.2 may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration of full-length troponin subunits are generally about 20-500 micrograms of active compound per kilogram body weight.

Suitable dosage ranges for intranasal administration of fulllength troponin subunits are generally about 0.Olpg/kg body weight to 1 mg/kg body weight. Suitable dosage ranges for intravenous administration of troponin fragments are generally about 10 micrograms to 1 milligram of active compound per kilogram body weight, preferably about 1-50 milligrams per administration, more preferably about 1-20 milligrams per human. Effective doses may be extrapolated WO 00/54770 PCT/US00/06667 from dose-response curves derived from in vitro or animal model test bioassays or systems.

Administration of the doses recited above can be repeated. In a preferred embodiment, the doses recited above are administered 2 to 7 times per week. The duration of treatment depends upon the patient's clinical progress and responsiveness to therapy.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Modifications and variations of the compositions of the present invention, and methods for use, will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to fall within the scope of the appended claims.

The following non-limiting examples demonstrate the discovery of troponin subunit inhibition of angiogenic stimulus induced endothelial cell proliferation, and means for determining the effective dosage of troponin subunit, fragment, or homolog to inhibit angiogenesis, as well as for identifying troponin subunit fragments and homologs those fragments or homologs of troponin subunit capable of inhibiting angiogenesis. The troponin subunit used in the examples is purified as described infra.

6. EXAMPLES Example 1: Purification of Troponin Subunit Components Cardiac Troponin Isolation from Tissue -36- WO 00/54770 PCT/US00/06667 The procedures of Ebashi et al., 1968, J. Biochem.

64:465-477; Yasui et al., 1968, J. Biol. Chem. 243:735-742; Hartshorne et al., 1969, Biochim. Biophys. Acta, 175:30; Schaub et al., 1969, Biochem. J. 115:993-1004; Greaser et al., 1971, J. Biol. Chem. 246:4226-4233; and Greaser et al., 1973, J. Biol. Chem. 248:2125-2133 for purifying troponin can be used. Rabbit back and leg muscles are removed, cleaned of fat and connective tissue, and ground. The ground muscle (1 kg) is stirred for 5 min. in 2 liters of a solution containing 20 mM KC1, 1 mM KHCO 3 0.1 mM CaC1 2 and 0.1 mM

DTT.

1 The suspension is filtered through cheesecloth, and the washing of the residue is repeated four times. Two liters of ethanol are then addedto the washed residue and the solution filtered after 10 min. The ethanol extraction is repeated twice. The residue is then washed 3 times with 2 liters of diethyl ether for 10 min. Finally the residue is allowed to dry at room temperature for 2 to 3 hours.

The dried powder (from 1 kg of muscle) is extracted overnight at 220 with 2 liters of a solution containing 1 M KC1, 25 mM Tris (pH 0.1 mM CaCl 2 and 1 mM DTT. After filtration through cheesecloth, the residue is once more extracted with 1 liter of 1 M KC1.

The extracts are combined and cooled to 4 0 C. Solid ammonium sulfate is added to produce approximately saturation (230 g per liter). After 30 min. the solution is centrifuged and 125 g of ammonium sulfate is then added per liter of supernatant (60% saturation). After centrifugation the precipitate is dissolved in 500 ml of a solution containing 5 mM Tris (pH 0.1 mM CaCl 2 and 0.1 mM DTT and dialyzed against 15 liters of the same solution for 6 hours and against a fresh solution overnight.

Solid KC1 is added to a final concentration of 1 M and 1 M KC1 solution is added to bring the volume to 1 liter.

The abbreviations used are: DDT, dithiothreitol; EGTA, ethylene glycol bis (-aminoethyl ether) -N,N'-tetraacetate; SDS, sodium dodecyl sulfate; SE-, sulfoethyl.

-37- WO 00/54770 PCT/US00/06667 The pH is then adjusted to 4.6 by addition of HC1, and the tropomyosin precipitate is removed by centrifugation. The pH of the supernatant is adjusted to 7.0 with KOH, and 450 g of ammonium sulfate are added per liter (70% saturation). The precipitate is dissolved in a solution containing 5 mM Tris (pH 7.5, 0.1 mM CaC1 2 and 0.1 mM DTT, and dialyzed overnight against the same solution. Solid KC1 is added to bring its concentration to 1 M, the pH adjusted to 4.6, and the precipitate which forms is removed by centrifugation. The neutralized supernatant is dialyzed against 2 mM Tris (pH 7.5) until the Nessler reaction is negative. The final yield of troponin is usually 2.5 to 3.0 g per kg of fresh muscle.

Cardiac Troponin Isolation from Tissue Bovine hearts are obtained approximately 30 min.

after death and immediately cut open, rinsed of blood, and immersed in ice. The left ventricle is removed, trimmed of excess fat and connective tissue, and ground. All subsequent extraction and preparation steps are performed at 0-30 except where noted. The ground muscle (500 g) is homogenized in a Waring Blender for 1 min. in 2.5 liters of solution containing 0.09 M KH 2

PO

4 0.06 M K 2

HPO

4 0.3 M KC1, 5 mM 2mercaptoethanol, pH 6.8. The homogenized muscle suspension is then stirred for 30 min. and centrifuged at 1000 x g for min. The precipitate is re-extracted for 30 min. and centrifuged. The residue is then washed with 2.5 liters of mM 2-mercaptoethanol and centrifuged at 1000 x g for 10 min., followed by two successive washings and centrifugations with liters of 50 mM KC1, 5mM Tris-HCl (pH and 5mM 2mercaptoethanol. The residue is then washed and centrifuged twice with 1.5 liters of 50 mM Tris-HCl (pH and 5 mM 2mercaptoethanol. The volume of the residue is measured, and the residue is mixed with 0.5 volume of 3 M KC1, 50 mM Tris- HCl (pH and 5 mM 2-mercaptoethanol. After a 16- to hour extraction at the suspension is centrifuged at 15,000 x g for 10 min. The sediment is discarded, and the supernatant is adjusted to pH 7.6 with 0.05 N HC1. The -38- WO 00/54770 PCT/US00/06667 filamentous precipitate which forms upon pH adjustment is removed by filtering the extract through nylon gauze. The protein that precipitates between 30 and 50% ammonium sulfate saturation is collected, dissolved in a solution containing 1 M KC1, and 1mM potassium phosphate (pH and 5 mM 2mercaptoethanol, and dialyzed against the same solution for 4 hours and against a fresh solution overnight. The protein solution is clarified by centrifugation at 105,000 x g for min. The troponin is then purified by chromatography on a hydroxylapatite column with the protein being eluted between 0.08 and 0.10 M phosphate. Greaser et al., 1972 Cold Spring Harbor Symp. Quant. Biol. 37:235-244. Rabbit cardiac troponin is prepared in a similar manner using a pooled batch of hearts which has been stored at -20 0 C prior to extraction.

The troponin subunits are separated by DEAE- Sephadex chromatography in 6 M urea. Bovine cardiac tropomyosin is prepared from the 50% ammonium sulfate saturation supernatant from the troponin extraction scheme (see above). Ammonium sulfate is added to 65% saturation, and the precipitate is dissolved in and dialyzed versus 1 M KC1, 1 mM potassium phosphate (pH and 5 mM 2mercaptoethanol. The protein is then purified by hydroxylapatite chromatography.

Protein Determination Protein concentrations are determined by the biuret method of Gornall et al. using bovine serum albumin as a standard. Gornall et al., 1949, J. Biol. Chem., 177:751-766.

Separation of Components A sequence of SP-Sephadex and DEAE-Sephadex chromatography gives complete separation of the three cardiac troponin components.

Recombinant Troponin Isolation and Reconstitution Protocols -39- WO 00/54770 PCT/US00/06667 Troponin I and T DNA encoding various troponin subunits and isoforms are known in the art. See, Wu et al., 1994, DNA Cell.

Biol. 13:217-233; Schreier et al., 1990, J. Biol. Chem.

265:21247-21253; and Gahlmann et al., 1990, J. Biol. Chem.

265:12520-12528.

To express a troponin subunit, DNA encoding the subunit is subcloned into a high copy number expression plasmid, such as KP3998, using recombinant techniques known in the art.

To express the cloned cDNA, E. coli transformed with the insert-containing pKP1500 vector is grown overnight at 37 0 C, then inoculated into 4 liters of Luria-Bertani broth (LB) medium and grown at 42 0 C until mid-log phase. Isopropyl- 1-thio-P-D-galactopyranoside is then added to 0.5 mM, and the culture is allowed to grow at 42 0 C overnight. Purification of expressed troponin subunit, fragment, or homolog may be adapted from published procedures (Reinach et al., 1988, J.

Biol. Chem. 250:4628-4633 and Xu et al., 1988, J. Biol. Chem.

263:13962-13969). The cells are harvested by centrifugation and suspended in 20 ml of 20 mM Tris, 20% sucrose, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 1 mg/ml lysozyme, pH After incubation on ice for 30 min., 80 ml of 20 mM Tris, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT is added and the cells broken in a French press (SLM Instruments). The cell debris is pelleted; the supernatant is made 35% in saturated (NH 4 2

SO

4 and stirred on ice for min. After sedimentation, the supernatant is made 50 mM in NaCl, 5 mM in CaC1 2 1 mM in MgCl 2 and 1 mM in DTT and then loaded onto a 1.5 X 25-cm phenyl-Sepharose (Pharmacia LKB Biotechnology Inc.) column. The column is washed first with mM Tris, 50 mM NaCl, 5 mM CaC1 2 1 mM MgCl 2 1 mM DTT, pH then with 50 mM Tris, 1 mM NaC1, 0.1 mM CaCl 2 1 mM DTT, pH 7.5, until no more protein is eluted. The crude troponin subunit is then eluted with 50 mM Tris, 1 mM EDTA, 1 mM DTT, pH 7.5. Fractions that contain troponin subunit, fragment, WO 00/54770 PCT/US00/06667 or homolog are pooled, dialyzed against 25 mM Tris, 6 M urea (United States Biochemical Corp.), 1 mM MgCl 2 1 mM DTT, pH and loaded onto a 1.5 X 25-cm DE52 (Whatman) column.

The column is eluted with a 0-0.6 M NaCl linear gradient.

Troponin subunit, fragment, or homolog which elutes from the column is dialyzed against 0.1 mM NH 4 HCO, 1 mM mercaptoethanol, lyophilized, and stored. Purity is assessed by SDS-polyacrylamide gel electrophoresis and UV spectrophotometry. Typical yields of 6 mg of purified recombinant troponin subunit, fragment, or homolog/liter of bacterial culture are expected.

The lyophilized recombinant protein is resuspended in a take up buffer consisting of 6M urea, 20 mM Hepes (pH 0.5M NaCl, 2mM EDTA, and 5mM DTT. The mixture is nutated at room temperature for 1 hour. The solution is then dialyzed at 4 0 C for six hours with 1 exchange against a dialysis buffer consisting of 0.5M NaCl, 20mM Hepes (pH and 0.5mM DTT.

Protein concentration is determined for each subunit at 280X. The extension coefficient of Troponin I is 0.40 and Troponin T is 0.50.

Troponin C The lyophilized recombinant protein is resuspended in a take up buffer consisting of 0.1 M NaCl, 20 mM Hepes (pH 2mM EDTA, and 5mM DTT. This solution is dialyzed for 6 hours at 4°C with one exchange against a dialysis buffer of 0.1 M NaCl, 20 mM Hepes (pH and 0.5 mM DTT.

Protein concentration is determined by measuring absorbance at 280\. The extension coefficient for troponin C is 0.18.

Reconstitution of Combined Subunits Protein concentrations having the same reconstitution molar ratios of troponin subunits C, I, and T are maintained for all various combinations. These concentrations of the respective proteins are combined in a -41- WO 00/54770 PCT/US00/06667 reconstitution buffer consisting of 0.1 M NaCl, 0.1 M CaC12, mM DTT, 5mM Hepes (pH Dialysis is for 20-24 hours at 4 0 C with three exchanges over a dialysis buffer consisting of 0.1 M NaCl, 0.1 m CaCl 2 0.5 mM DTT, and 5 mM Hepes (pH Protein concentration is approximated by measuring absorption at 278X. The troponin trimer has an extension coefficient of 0.45 at 278X.

Example 2: Inhibition of Endothelial Cell Proliferation measured by DNA synthesis.

The inhibitory effect of troponin subunit, fragment, or homolog on the proliferation of bFGF-stimulated EC can be measured according to the following procedure.

Endothelial Cell DNA Synthesis On day one, 5,000 bovine capillary endothelial cells in DMEM/10% CS/1% GPS are plated onto each well of a 96-well pregelatinized tissue culture plate. On day two, the cell media is changed to DMEM, 2% CS, 1% GPS, 0.5% BSA (complete medium), supplemented with 10 pl of Img/ml "cold" thymidine per 50 ml of medium. On day three, test samples in complete medium are added in duplicate. Additionally, beta Fibroblast Growth Factor (bFGF) is added to each well except for the appropriate controls, to a final concentration of 0.2 ng/well. On day four, 5 Al of 1:13 diluted 3 H-Thymidine stock is added to each well and the plate is incubated for 5-6 hours. Following incubation, the medium is aspirated, and the remainder is rinsed once with PBS, then twice for minutes each with methanol followed by two rinses each for minutes with 5% TCA. The cells are then rinsed with water three times, dried to the plate, and 100l of 0.3 N NaOH is added to each well. The contents of the well are then transfered to the scintillation counter vials and 3 mls of Ecolume added to each vial. Samples are then counted on the scintillation counter.

3T3 Cell DNA Synthesis -42- WO 00/54770 PCT/US00/06667 DNA synthesis in bFGF-stimulated 3T3 cells provides a control with which to evaluate results obtained for bFGF stimulated endothelial cell proliferation. DNA synthesis in the 3T3 cells can be determined according to the following method.

BALB/c 3T3 cells are trypsinized and resuspended at a concentration of 5 X 104 cells/ml. Aliquots of 200 gl are plated into 0.3 cm 2 microtiter wells (Microtest II tissue Culture Plates, Falcon). After reaching confluence, in a period of 2 to 3 days, the cells are further incubated for a minimum of 5 days in order to deplete the media of growth promoting factors. These growth conditions yield confluent monolayers of non-dividing BALB/c 3T3 cells. Test samples are dissolved in 50 Ml of 0.15 M NaCl and added to microtiter wells, along with 3 H]TdR. After an incubation of at least 24 hours, the media is removed and the cells are washed in PBS.

Fixation of the cells and removal of unincorporated 3 H]TdR is accomplished by the following successive steps; addition of methanol twice for periods of 5 minutes, 4 washes with H 2 0, addition of cold 5% TCA twice for periods of 10 minutes, and 4 washes with H 2 0. DNA synthesis is measured either by liquid scintillation counting or by autoradiography using a modification of the method described by Haudenschild et al., 1976, M. Exp. Cell Res. 98:175. For scintillation counting, cells are lysed in 150 Ml of 0.3 N NaOH and counted in 5 ml of Insta-Gel liquid scintillation cocktail (Packard) using a Packard Tri-Carb liquid scintillation counter.

Alternatively, autoradiography may be used to quantitate DNA synthesis by punching out the bottoms of the microtiter wells and mounting them on glass slides with silastic glue. The slides are dipped in a 1 g/ml solution of NTB2 nuclear track emulsion (Kodak) and exposed for 3-4 days. The emulsion is developed with Microdol-X solution (Kodak) for 10 minutes, rinsed with distilled H 2 0, and fixed with Rapid Fixer (Kodak) for three minutes. The autoradiographs are stained with a modified Giemsa stain. At least 1000 nuclei are counted in each well and DNA synthesis, expressed as the percentage of -43- WO 00/54770 PCT/US00/06667 nuclei labeled. Cell division is measured by counting the number of cells in microtiter wells with the aid of a grid after 40-48 hour incubations with test samples.

Example 3: Inhibition of Endothelial Cell Proliferation measured by colorimetric determination of cellular acid phosphatase activity and electronic cell counting A quick and sensitive screen for inhibition of EC proliferation in response to treatment with a troponin subunit, homolog, or derivative of the invention involves incubating the cells in the presence of varying concentrations of the inhibitor and determining the number of endothelial cells in culture based on the colorimetric determination of cellular acid phosphatase activity, described by Connolly, et al., 1986, J. Anal. Biochem.

152:136-140.

The effect of troponin on the proliferation of capillary endothelial cells (EC) was measured in an assay which measures the ability of this protein to interfere with stimulation of endothelial cell proliferation by a known angiogenesis factor (bFGF).

Capillary endothelial cells and Balb/c 3T3 cells were separately plated (2 x 10 3 /0.2 ml) onto gelatin-coated 96-well tissue culture dishes on day 1. On day 2, cells were refed with Dulbecco's modified Eagle's medium (Gibco) with calf serum (Hyclone) (DMEM/5) and bFGF (10 ng/ml) (FGF Co.) and increasing concentrations of one or more troponin subunits. These substances were added simultaneously in volumes that did not exceed 10% of the final volume. Wells containing phosphate buffered saline (PBS) (Gibco) alone and PBS bFGF were included as controls. On day 5, media was removed and cells were washed with PBS and lysed in 100 Al of buffer containing 0.1 M sodium acetate (pH 0.1% Triton X-100TM and 100 mM p-nitrophenyl phosphate (Sigma 104 phosphatase substrate). After incubation for 2 hours at 37 0

C,

the reaction was stopped with the addition of 10 Al of 1 N -44- WO 00/54770 PCT/US00/06667 NAOH. Color development was determined at 405 nm using a rapid microplate reader (Bio-Tek).

Percent inhibition was determined by comparing the cell number of wells exposed to stimulus with those exposed to stimulus and troponin subunits.

All three troponin subunits were found to inhibit bFGF-stimulated EC proliferation, as measured by the colorimetric assay.

Troponin C inhibited bFGF-stimulated endothelial cell proliferation in a dose-dependent manner in all concentrations tested (Figure Percent inhibition of bovine endothelial cell proliferation was 54%, 86%, 83%, and 100% at concentrations of 280 nM, 1.4 MM, 2.8 AM and 5.6 AM, respectively. An inhibition of 100% was observed at a concentration of 20 Ag/well (5.6 MM). IC 50 represents the concentration at which 50% inhibition of bFGF growth factorinduced stimulation was observed. The IC 50 of troponin C was determined to be 278 nM.

Troponin I inhibited bFGF-stimulated

BCE

proliferation at concentrations of 1 and 5 Ag/well, but inhibition was not observed in the sample tested at Ag/well (Figure The percent inhibition of BCE was 33% and 46% at concentrations of 240 nM and 1.2 AM, respectively.

The IC 50 of troponin I was determined to be 1.14 AM.

Troponin T inhibited bFGF-stimulated

EC

proliferation at concentrations of 10 and 20 Mg/well, but not at concentrations of 1 and 5 Mg/well (Figure

BCE

proliferation was inhibited 23% and 62% at 1.6 AM and 3.3 AM, respectively. The IC 5 0 of troponin T was determined to be 2.14 AM.

The combination of troponin subunits C and I inhibited EC at all concentrations tested (Figure The percent inhibition of proliferation of BCE was 52%, 54% 73% and 47% at 130 nM, 645 nM, 1.3 MM and 2.6 MM, respectively.

The IC 50 of this combination was determined to be 110 nM.

WO 00/54770 PCT/US00/06667 The combination of troponin subunits C, I and T was observed to inhibit bFGF-stimulated BCE proliferation by 16% at a concentration of 360 nM (5 g/well, Figure The troponin samples tested had no detectable inhibitory effect on the growth of Balb/c 3T3 cells, a nonendothelial cell type.

-46- WO 00/54770 PCT/USOO/06667 Example 4: Inhibition of Capillary Endothelial Cell Migration by Troponin Determination of the ability of the troponin subunit, fragment, or homolog to inhibit the angiogenic process of capillary EC migration in response to an angiogenic stimulus, can be determined using a modification of the Boyden chamber technique is used to study the effect of troponin subunit, fragment, or homolog on capillary EC migration. Falk et al., 1980, J. Immunol. 118:239-247 (1980). A blind-well Boyden chamber, consists of two wells (upper and lower) separated by a porous membrane. J. Exp.

Med. 115:453-456 (1962). A known concentration of growth factor is placed in the lower wells and a predetermined number of cells and troponin subunit, fragment, or homolog is placed in the upper wells. Cells attach to the upper surface of the membrane, migrate through and attach to the lower membrane surface. The membrane can then be fixed and stained for counting, using the method of Glaser et al., 1980, Nature 288:483-484.

Migration is measured using blind well chambers (Neuroprobe, no. 025-187) and polycarbonate membranes with 8 micron pores (Nucleopore) precoated with fibronectin (6.67 gg/ml in PBS) (human, Cooper). Basic FGF (Takeda Co.) diluted in DMEM with 1% calf serum (DMEM/1) is added to the lower well at a concentration of 10 ng/ml. The upper wells receive 5 x 10 5 capillary EC/ml and increasing concentrations of purified troponin subunit, fragment or homolog is used within 24 hours of purification. Control wells receive DMEM/1, either with or without bFGF. The migration chambers are incubated at 37 0 C in 10% C02 for 4 hours. The cells on the upper surface of the membrane are then wiped off by drawing the membrane over a wiper blade (Neuroprobe). The cells which have migrated through the membrane onto the lower surface are fixed in 2% glutaraldehyde followed by methanol (4 0 C) and stained with hematoxylin. Migration is quantified by counting the number of cells on the lower surface in 16 -47- WO 00/54770 PCT/US00/06667 oil immersion fields and comparing this number with that obtained for the control.

Example 5: Inhibition in vivo of Neovascularization by troponin as determined by the chick chorioallantoic membrane assay The chick chorioallantoic membrane assay (CAM), may be used to determine whether troponin subunit, fragment or homolog is capable of inhibiting neovascularization in vivo.

Taylor and Folkman, 1982, Nature (London) 297:307-312. The effect of troponin subunit, fragment or homolog on growing embryonic vessels is studied using chick embryos in which capillaries appear in the yolk sac at 48 h and grow rapidly over the next 6-8 days.

Three day post fertilization chick embryos are removed from their shells and placed in plastic petri dishes (1005, Falcon). The specimens are maintained in humidified

CO

2 at 37 0 C. On day 6 of development, samples of purified troponin subunit, fragment or homolog are mixed in methylcellulose disks and applied to the surfaces of the growing CAMs above the dense subectodermal plexus. Control specimens in which CAMs are implanted with empty methylcellulose disks are also prepared. The CAMs are injected intravascularly with India ink/Liposyn to more clearly delineate CAM vascularity. Taylor et al., 1982, Nature 297:307-312.

Following a 48 hour exposure of the CAMs to the troponin subunit, fragment, or homolog, the area around the implant is observed and evaluated. Test specimens having avascular zones completely free of India-ink filled capillaries surrounding the test implant indicate the presence of an inhibitor of embryonic neovascularization. In contrast, the control specimens show neovascularization in close proximity or in contact with the methylcellulose disks.

Histological mesodermal studies are performed on the CAMs of test and control specimens. The specimens are embedded in JB-4 plastic (Polysciences) at 4 0 C and 3 gm -48- WO 00/54770 PCT/US00/06667 sections are cut using a Reichert 2050 microtome. Sections are stained with toluidine blue and micrographs are taken on a Zeiss photomicroscope using Kodak TM x100 and a green filter.

Example 6: Inhibition in vivo of Neovascularization by troponin as determined by the rabbit corneal pocket assay Male NZW rabbits weighing 4-5 lbs. are anesthetized with intravenous pentobarbital (25 mg/kg) and 2% xylocaine solution is applied to the cornea. The eye is proptosed and rinsed intermittently with Ringer's solution to prevent drying. The adult rabbit cornea has a diameter of approximately 12 mm. An intracorneal pocket is made by an incision approximately 0.15 mm deep and 1.5 mm long in the center of the cornea with a No. 11 scalpel blade, using aseptic technique. A 5 mm-long pocket is formed within the corneal stroma by inserting a 1.5 mm wide, malleable iris spatula. In the majority of animals, the end of the corneal pocket is extended to within 1 mm of the corneal-scleral junction. In a smaller series of 22 rabbits implanted with tumor alone, pockets are placed at greater distances 2-6 mm from the corneal-scleral junction by starting the incision away from the center.

In the first assay, polymer pellets of ethylene vinyl acetate (EVAc) copolymer are impregnated with test substance and surgically implanted in a pocket in the rabbit cornea approximately 1 mm from the limbus. When this assay system is being used to test for angiogenesis inhibitors, either a piece of V2 carcinoma or some other angiogenic stimulant is implanted distal to the polymer, 2 mm from the limbus. On the opposite eye of each rabbit, control polymer pellets that are empty are implanted next to an angiogenic stimulant in the same way. In these control corneas, capillary blood vessels start growing towards the tumor implant in 5-6 days, eventually sweeping over the blank polymer. In test corneas, the directional growth of new -49- WO 00/54770 PCT/US00/06667 capillaries from the limbal blood vessels towards the tumor occurs at a reduced rate and is often inhibited such that an avascular region around the polymer is observed (Figure 1).

This assay is quantitated by measurement of the maximum vessel lengths with a stereoscopic microscope.

Example 7: Isolation of Troponin I from Cartilage Purification of Troponin I from Cartilage Troponin I was purified from bovine veal scapulae using a modification of a protocol previously described by us (Moses, et al., 1990, Science 2488, 1408-1410). Briefly, veal scapulae were vacuum frozen immediately after slaughter and stored at -20 0 C until used. Cartilage was scraped first with a periosteal elevator (Arista) and then with a scalpel blade (No. 10, Bard-Parker) until clean of all muscle and connective tissue. Cartilage slices were extracted in 2 M NaCl, precipitated with HCl and ammonium sulfate (25-20%), and fractionated using a series of chromatography steps: gel filtration on A-1.5m Sepharose (Bio-Rad) in the presence of 4M guanidine-HCl, ion exchange on a Bio-Rex 70 (Bio-Rad) cation exchange column, gel filtration on a Sephadex (superfine) (Pharmacia) column, reversed-phase highperformance liquid chromatography (HPLC) on a Hi-Pore 304 column (Bio-Rad) and gel filtration on a Progel-TSK G3000SWXL column (3.0 cm x 7.8 mm) (Supelco). Fractions obtained from each column step were tested for their ability to inhibit capillary endothelial cell (EC) proliferation which was stimulated by basic Fibroblast Growth Factor (bFGF) as described below. Fractions containing inhibitory activity were pooled and concentrated in a Savant Speed Vac concentrator for amino acid and sequence analysis. Unless otherwise stated, all reagents were obtained from Sigma.

Trypsin Digestion, HPLC Separation and Microsequencing WO 00/54770 PCT/US00/06667 Proteins were each reduced, Scarboxyamidomethylated and subjected to digestion with trypsin. The resulting peptide mixtures were fractionated by narrow-bore high performance liquid chromatography using a Zorbax C18 1.0 mm by 150 mm reverse-phase column on a Hewlett-Packard 1090 HPLC with a 1040 diode array detector.

Optimum fractions were chosen based on differential UV absorbance at 205, 277nm and 292nm, peak symmetry and resolution (Lane, et al., 1991, J. Prot; Chem. 10, 151-160).

These fractions were then further screened for length and homogeneity by matrix-assisted laser desorption time-offlight mass spectrometry (MALDI-TOF/MS) on a Thermo Bioanalysis Lasermat 2000 (Hemel, England). Tryptic peptide sequences were determined by electrospray ionization/tandem mass spectrometry on a Finnigan TSQ7000 (San Jose, CA) triple quadrupole mass spectrometer as described in Nash et al.

(Nash, et al., 1996, Curr; Biol. 6, 968-980). Alternatively, peptides were submitted to automated Edman degradation on a PE/ABD 477A (Foster City, CA) protein sequencer.

Cloning and Expression of Human Troponin I Human intercostal cartilage tissue was obtained according to bioethical guidelines pertaining to discarded clinical material. The cDNA encoding a fragment of human fast-twitch skeletal muscle troponin I was amplified by standard reverse transcriptase polymerase chain reaction (RT- PCR) from the total RNA isolated from a core sample of human cartilage using primers based on the nucleotide sequence of human fast-twitch skeletal muscle TnI (Zhu, et al., 1994, Biochim. Biophys. Acta 1217, 338-340): forward primer GCTCTGCAAACAGCTGCACGCCAAG-3' (SEQ ID NO:4) and reverse primer 5,-GCCCAGCAGGGCCTTGAGCATGGCA-3' (SEQ ID NO:5) which was cloned into PCR2.1 (Invitrogen) and sequenced in both directions. The cDNA encoding the full-length open reading frame (ORF) of human fast-twitch skeletal muscle troponin I was cloned from human skeletal muscle mRNA with Pfu -51- WO 00/54770 PCT/US00/06667 polymerase (Stratagene) under standard PCR conditions, using forward primer (5'-CTCACCATGGGAGATGAGGAGAAGC-3') (SEQ ID NO:6) and the reverse primer (SEQ ID NO:7). The PCR product was cloned into the expression vector Pet24d (Novagen) using 5'-Ncol and 3'-Xhol sites and sequenced as above.

Tissue expression of TnI was analyzed by RT-PCR as described above. Total RNA (400 ng/sample) was isolated from rat skeletal muscle, liver (Clontech), xyphoid and Swarm rat chondrosarcoma. The design of the forward GAACACTGCCCGCCTCTGCACATC-3') (SEQ ID NO:8) and reverse GAGCCCAGCAGCGCCTTCAGCATG-3') (SEQ ID NO:9) primers was based on the nucleotide sequence of rat fast-twitch skeletal muscle TnI.

Recombinant(r) human TnI was expressed according to standard protocols (Sambrook, et al., 1989, Molecular Cloning: A laboratory manual. (Cold Spring Harbor Press, New York, After 5 hrs of expression, bacteria were harvested by centrifugation. Following centrifugation at 12,000 x g for 15 min, the pellet was resuspended in 1.0 ml of Buffer A (15 mM Tris-HCl, 0.1 mM EDTA, pH The cells were disrupted by sonication. The inclusion bodies were isolated by centrifugation at 12,000 x g once for 15 min in Buffer A, followed by centrifugation once at 11,000 x g once for 15 min in Buffer A.

Purification of Recombinant Troponin I The washed pellet was dissolved in 6 M urea, 0.5 M NaC1, 5 mM HEPES, 2 mM EDTA, 5 mM DTT (pH and nutated in the above buffer for 6-8 hours at 4°C. The sample was then dialyzed against 0.5 M NaC1, 5 mM HEPES, 5 mM DTT (pH and concentrated using an Amicon concentrator (YM-10, MWCO 10,000 Da) prior to application to a Progel-TSK G3000SWXL column (30 cm x 7.8 mm). The sample was eluted using the above buffer (0.5 M NaCl, 5 mM HEPES, 5 mM DTT, pH Some of the inhibitory preparations were further fractionated on a Q-Sepharose HP column (Pharmacia Biotech) and tested as described below with no difference in biological activity.

-52- WO 00/54770 PCT/US00/06667 Purified rTnI was dialyzed against phosphate buffered saline (PBS) containing 0.5mM DTT prior to testing. Protein concentration was determined by scanning densitometric comparison (IS-1000 Digital Imaging System, Version 2.00, Alpha Innotech Corp.) with known protein standards (Novex) coelectrophoresed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie Blue.

Western Blot Analysis Immunoblotting was conducted on samples of native TnI (purified from cartilage as described above), recombinant TnI (purified as described above) and bovine chondrocyte lysates prepared as described below according to standard protocols. Cultures of primary bovine scapular chondrocytes were established and maintained as previously described by us (Moses, et al., 1990, J. Cell. Biol. 119, 474-481). Cells were rinsed with PBS and to each 10 cm culture dish was added 1 ml of boiling 2x-concentrated electrophoresis sample buffer (250 mM Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue and 2% B-mercaptoethanol). Cells were scraped from the dishes using a disposable cell scraper (Costar), transferred to a microcentrifuge tube and boiled for an additional 5 min. Following several passages though a 26 gauge needle (Becton Dickinson), the sample was clarified by centrifugation (2000 x diluted to 0.1% SDS, and the protein concentration determined using a DO Protein Assay (BioRad). All samples were separated by polyacrylamide gel electrophoresis on a 4/12% acrylamide mini-gel according to Laemmli (Laemmli, 1970, Nature 227, 680-685). Proteins were then transferred to nitrocellulose (Hybond-ECL, Amersham) using a Transblot apparatus (Biorad),incubated with a monoclonal antibody to rabbit skeletal muscle TnI (Advanced Immunochemical Inc.) and developed using the ECL western blotting system according to the manufacturer's protocol (Amersham).

-53- WO 00/54770 PCT/US00/06667 Results An in vitro assay which measures the inhibition of basic fibroblast growth factor (bFGF)-stimulated proliferation of capillary endothelial cells (EC) was used to monitor purification (Moses, et al., 1990, Science 2488, 1408-1410; Moses, et al., 1990, J. Cell. Biol. 119, 474-481; Connolly, et al., 1986, Anal. Biochem. 152; 136-140). All cartilage-derived fractions obtained from a series of chromatography steps described below were screened for this inhibitory bioactivity. Inhibitory activity eluted at an approximate molecular weight of 25,000 Da from the size exclusion column, at approximately 0.2M NaCl from the Biorex 70 cation exchange column, at approximately 23,000 Da from the Sephadex G-75 gel filtration column, at an acetonitrile concentration of approximately 38.5%, and at an approximate Mr of 22,000 Da from the Progel-TSK G3000SWXL column. Inhibitory fractions obtained from the final chromatography step were subjected to tryptic digestion and the resultant peptides were sequenced by microcapillary LC- ESI tandem mass spectrometry or automated Edman degradation.

The sequences of three peptide fragments were obtained and were identified as fragments of troponin I (Figure 6).

Since there had been no previous reports in the literature that cartilage cells, the chondrocytes, contain TnI, the cDNA encoding human cartilage TnI was cloned using a standard PCR strategy (Wu and Moses, 1996, Gene 18, 243-246) (Figure 7A). Sequencing of the PCR product revealed its identity to human fast skeletal muscle TnI (Figure 7B) (SEQ ID NO:16). TnI expression levels of rat xiphoid cartilage, Swarm rat chondrosarcoma and liver, were also determined by RT-PCR and were significantly lower than that of rat skeletal muscle, with the expression level in liver appearing to be slightly lower than that of cartilage or chondrosarcoma (Figure 7C).

In order to obtain sufficient amounts of TnI to investigate its potential as an antiangiogenic factor, a cDNA -54- WO 00/54770 PCT/US00/06667 encoding full length human fast skeletal muscle troponin I was cloned into expression vector pET-24d and transformed into E. coli BL21(DE3) p LysS strain. The expression level of recombinant human skeletal muscle troponin I was approximately 30-40% of total cellular protein. Following purification, recombinant TnI migrated as a single band, at approximately 21 kDa on SDS-PAGE (Figure 8).

Example 8: Capillary Endothelial Cell (EC) Proliferation Cell Culture Capillary EC, isolated from bovine adrenal cortex (Folkman, et al., 1979, Proc. Natl. Acad. Sci. USA 76, 5217- 5221) were obtained from Children's Hospital (Boston, MA).

These cells were demonstrated to be endothelial by staining with antisera to von Willebrand factor and by their uptake of fluoresceinated, acetylated low density lipoprotein. Cells were maintained in culture in DME (Dulbecco's Modified Eagle's Medium, Gibco Laboratories) with 10% calf serum (Hyclone) (DME/10) supplemented with 3 ng/ml bFGF or Vascular Endothelial Growth Factor (VEGF) in preparation for these assays.

BALB/c mouse 3T3 cells were maintained in L-glutamine(292 Ag/ml) as previously described (Klagsbrun, et al., 1977, Exp. Cell Res. 105, 99-108). Bovine aortic smooth muscle cells (SMC), isolated by explant from the medial layer of bovine aortas, were obtained from Children's Hospital (Boston, MA). These cells were cultured in DME/10 on uncoated tissue culture plastic as previously described (D'Amore and Smith, 1993, Growth Factors 8, 61-75).

Briefly, capillary EC (2,000 cells per well) were plated on gelatinized 96-Well culture plates in DMEM supplemented with 5% calf serum and incubated for 24 hours. On day 2, cells were treated with bFGF (Scios Nova; 1 ng/ml) and challenged with the test fractions and/or with purified TnI. For experiments in which VEGF was used as the WO 00/54770 PCT/US00/06667 mitogen, 800 cells per well were plated and allowed to incubate for 3 hours before VEGF (Biomedical Technologies Incorporated; 30 ng/ml) and TnI was added. Control wells contained cells alone and cells stimulated with bFGF or VEGF.

On day 5, growth medium was removed from the plates; cells were lysed in buffer containing the detergent Triton x-100 and the phosphatase substrate p-nitrophenyl phosphate. After incubation for 2h at 37 0 C, NaOH was added to terminate the reaction. Color development was determined using a rapid multiwell plate reader (Dynatech MR 5000) (Moses, et al., 1990, Science 2488, 1408-1410; Moses, et al., 1990, J. Cell.

Biol. 119, 474-481; Connolly, et al., 1986, Anal. Biochem.

152, 136-140). EC inhibitory activity was verified by electronic cell counting assays as previously described by us (Moses, et al., 1990, Science 2488, 1408-1410; Moses, et al., 1990, J. Cell. Biol. 119, 474-481). Tritiated thymidine incorporation assays were conducted according to the method of Shing (Shing, 1990, in Methods in Enzymolqgy, eds. Barnes, Mather, J.P. and Sato, G.H. (Academic Press, New York), pp. 91-95).

Results Purified rTnI was tested for its ability to inhibit bFGF and VEGF-stimulated capillary EC and was found to inhibit EC proliferation in a dose-dependent and saturable manner with an IC 50 (the inhibitory concentration at which one observes 50% suppression of proliferation) of approximately nM when bFGF was used as the mitogen (Figure 9A) and approximately 1.5 nM when VEGF was used (Figure 9B). Native TnI inhibited capillary EC proliferation in an equipotent manner. Tritiated thymidine assays demonstrated that recombinant TnI inhibited capillary EC DNA synthesis in a dose-dependent and saturable manner with an IC 50 of approximately 240 nM. This suppression of proliferation appears to be unique to endothelial cells given the fact that TnI did not suppress the growth of any of the non-endothelial -56- WO 00/54770 PCT/US00/06667 cells tested including bovine aortic smooth muscle cells and Balb/c 3T3 cells even when tested at doses which were over higher than that required to obtain an IC 50 value for capillary

EC.

Example 9: Cell Specificity To determine whether the proliferation of bovine aortic SMC and Balb c/3T3 cells was inhibited by TnI, the following assays were conducted. SMC were plated into multiwell dishes (2.1 cm 2 /well) at a density of 10,000 cells/well. After allowing the cells to attach overnight, fresh media was applied containing either 3 ng/ml PDGF-BB alone or in combination with increasing concentrations of purified TnI. Following incubation for 72 hrs at 37 0 C in

CO

2 the cells were rinsed in PBS, detached by trypsinization and counted electronically. The effect of TnI on quiescent BALB/c mouse 3T3 cells was assessed by measuring the incorporation of tritiated thymidine into DNA in 96-well plates as previously described (Shing, 1990, in Methods in Enzymolqgy, eds. Barnes, Mather, J.P. and Sato, G.H.

(Academic Press, New York), pp. 91-95).

Example 10: Chick Chorioallantoic Membrane (CAM) Assay All procedures were carried out in a laminar flow, hood under sterile conditions. The eggs were stored in a Favorite Egg Incubator (Leahy) at 37 0 C and 65% relative humidity. On day 3 of development, fertilized White Leghorn eggs (SPAFAS) were cracked and the embryos removed from their shells and placed in plastic petri dishes. On day 6, test substances including native rabbit TnI (Greaser and Gergely, 1971, J. Biol. Chem. 246, 4226-4233) and recombinant human TnI and appropriate buffer controls were mixed in methylcellulose, disks and applied to the surfaces of the growing CAMs above the dense subectodermal plexus. Fortyeight hours following implantation of the plastic disc, the eggs were examined for vascular reactions under a dissecting -57- WO 00/54770 PCT/US00/06667 scope (60x) and photographed (Moses, et al., 1990, Science 2488, 1408-1410; Moses, et al., 1990, J. Cell. Biol. 119, 474-481).

The CAM assay was used to determine whether rTnI was an inhibitor of angiogenesis in vivo. The results shown in Figure 10 demonstrate the significant inhibition of embryonic neovascularization as evidenced by the large avascular zone caused by 130 picomoles of rTnI. This effect was observed in 66% of the eggs tested at this dose and 100% of the eggs tested at a dose of approximately 380 picomoles.

This observation was reproduced in three separate sets of CAM assays using three different TnI preparations. Over 125 CAMs were tested in this series of experiments.

-58- WO 00/54770 PCT/US00/06667 Example 11: Mouse Corneal Pocket Assay Inhibition of angiogenesis in vivo was also demonstrated using the mouse corneal pocket assay (Chen, et al., 1995, Cancer. Res. 55, 4230-4233; 0' Reilly, et al., 1996, Nat. Med. 2, 689-692). Briefly, pellets composed of bFGF (40 ng/ml), sucrose octasulfate,and Hydron were implanted into corneal micropockets of six C57BL/6 mice as previously described Patent No. 5,837,680 to Moses et Troponin I (50 mg/kg) was administered systemically every 12 hours by subcutaneous injection. On the sixth day of treatment, corneal angiogenesis was evaluated using slit lamp microscopy and photographed.

Results In another in vivo assay, the mouse corneal pocket assay, systemic administration of rTnI significantly inhibited bFG F-induced angiogenesis (Figure 11B) when compared to corneas of control mice which received vehicle alone (Figure 11A).

Taken together, the in vivo studies described in Section 6, Examples 10 and 11 show rTnI to be a potent inhibitor of neovascularization when compared to other inhibitors tested in these same assays (Moses, et al., 1995, in International Review of Cytology, 161, 1-48).

Example 12: B16-BL6 Melanoma Model.

Murine melanoma B16-BL6 were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal calf serum (Hyclone), L-glutamine and NaHCO 3 Cells were washed with EBSS (Gibco) and trypsinized for 3 to 5 mm with 0.25% TRL/0.2% EDTA to which culture buffer was added for washing.

This preparation was then centrifuged for 10 mm at 1000 rpm, the cell pellet resuspended in fresh culture media, cell number determined using a coulter counter and cell viability determined with trypan blue (100% viability). The cell suspension was adjusted to 2.5 x 10 5 cells/ml for -59- WO 00/54770 PCT/US00/06667 implantation. B16-BL6 cells (5 x 105/0.2 ml) were .injected into the tail veins of C57BL/6 mice (approximately 6-7 weeks old). One day following tumor cell inoculation, mice were treated with rTnI systemically, twice per week, with a dose of either Img/kg (n=10) or 20 mg/kg (n=10) or vehicle (150 mM NaCl, 20 mM citrate, pH3) over, a 28 day period. On day animals were sacrificed, the number of lung surface metastases counted and the lungs weighed.

Results Recombinant TnI was tested for its ability to inhibit lung metastasis in vivo caused by a very aggressive variant of the B16 melanoma cell line, B16-BL6 (Saiki, et al., 1989 Cancer Res. 49, 3815-3822). Recombinant TnI, administered systemically, inhibited lung metastases by 52% (p<0.04 one tailed t-test) at a dose of 1 mg/kg when given only twice weekly and by 64% (p<0.02; one tailed ttest) at a dose of 20 mg/kg twice weekly [lung metastasis control SEM) 1 mg/kg (32.8+1- 4.8 SEM); 20mg/kg (25.0 7.5 SEM)] with no observed toxicity no weight or appetite loss, etc.). Lung weights were comparable in control and treated groups.

As shown by the data, TnI inhibited lung metastasis.

Example 13: Inhibition of Endothelial Cell Proliferation Using Fragments of Troponin I Recombinant peptides corresponding to fragments of rabbit (rb) TnI (SEQ ID NO:10) (Figure 12) were tested for ability to inhibit bFGF-stimulated capillary EC as described above in Section 6, Examples 2 and 8. The rbTnI fragments (SEQ ID NOS:11-15) were prepared according to Jha et al., 1996, Biochemistry 35(34):11026-11035. As shown in Table 2, various concentrations of peptides corresponding to the amino-terminal region (aa 1-94) (SEQ ID NO:11); the N' and inhibitory region (aa 1-120) (SEQ ID NO:12); the I' region (aa 98-114) (SEQ ID NO:13); the carboxy terminus and I' region (aa 96-181) (SEQ ID NO:14); the C' region (aa 122-181) (SEQ ID NO:15); and mixtures of the (SEQ ID NO:14) plus the N' (SEQ ID NO:11) fragments and the (SEQ ID NO:12) plus the C' (SEQ ID NO:15) fragments of TnI were tested for inhibition of EC proliferation.

As shown in Table 2, the fragment (SEQ ID NO:14) significantly inhibited EC proliferation. The percent inhibition of EC was 54% and 48% at concentrations of 0.1 pg/well and 0.3 Mg/well, respectively. The IC 50 was determined to be 0.1 to 0.2 fg/well (0.05MM to 0.1 MM).

Furthermore, the (SEQ ID NO:12) fragment interfered with the inhibitory activity of the C' (SEQ ID NO:15) fragment and the N' (SEQ ID NO:11) fragment interfered with the inhibitory activity of the (SEQ ID NO:14) fragment.

As shown in Section 6, Example 3, supra, fulllength TnI inhibited EC proliferation approximately 46% at a concentration of 5 Mg/well (1.2 MM). Thus, the fragment had 25 to 50-fold EC inhibitory activity compared to the full-length TnI.

These results demonstrate that fragments of troponin subunits, particularly the fragment (SEQ ID NO:14), inhibited EC proliferation in an assay that was developed to mimic the process of neovascularization. Thus, troponin subunit fragments inhibit angiogenesis.

Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

The discussion of documents, acts, materials, devices, 3 articles and the like is included in this specification solely:for oo the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed 0.0: part of the prior art base or were common general knowledge in the S. field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

*CCC

:66 •gee oooo -61- Table 2- Approx.

SEQ ID Amino Assay Assay Assay .ICS, Approx.

NO: Regiona Fragment Acids jig/well jig/ml MW nM %I g/wel1 ICS 0

ALM

11 .1 94 N' 94 0.01 0.05 10,906 5 -12 >0.3 >0.1 0.025 0.125 11 6 0.1 0.5 46 31 0.3 1.5. 138 28 12 1 -120 N' IV 120 0.01 0.05 13,923 4 .6 >l 0.025 0.025 9 0 0.1 0.5 36 12 0.3 1.5 108 17 13 98 114 I'17 4 20 1,972 10140 -12 >>40 >>100 50 25350 100 50700 -6 200 -101401 -34 14 96 181 C' it 86 0.01 0.05 9,978 5 25 0.1 to 0.05 0.025 0.125 13 28 0.2 to 0.1 0.5 50 54 0.1 0.3 1.5 1 150 48 Approx.

SEQ ID Amino Assay Assay Assay Ic 50 Approx.

NO: Regiona Fragment Acids pug/well pIg/mi MW rim %b pg/well IC 50 'pM 122 181 CO 60 0.01 0.05 6,961 7 -1 >0.3 >0.2 0.025 0.125 18 -6 0.1 0.5 .72 0.3 1.5 215 23 14,11 96-181 180 0.01 0.05 20,884 7 17 >0.3 >0.2 1-94 N* 0.025 0.125 18 0.1 0.5 72 27 0.3 1.5 215, 28 12,15 1-120 180 0.01 0.05 20,884 7 -7 >0.

122-181 C' 0.025 0.125 18 -1 0.1 0.5 72 -6 0.3 1.5 215 -1 94-113. C' it 20 20 100 2,500 40000 0 >>20 98-117 C' I, 20 20 100 2,500 40000 1 >>20 102-121 C' I, 20 20 100 2,500 40000 0 >>20 106-125 C+I'20 20 100 2,500 40000 0 >>20 110-129 C' it 20 20 100 2,500 40000 0 >>20 -114-133 C' V' 20 20 100 2,500 40000, 0 >>20 Approx.

SEQ ID Amino Assay Assay Assay

IC

50 Approx.

NO: Regiona Fragment Acids jig/well Pig/m1 MW rim %J ig/well IC 50

JIM

118-137 C' 20 20 100 2,500 40000 37 40 200 2,500 80000 116-123 c? 8 50 20900 278000 9 >>50 >>278 118-125 C' 8 50 250 900 278000 0 >>50 >>278 120-127 C' 8 50 250 900 278000 10 >>50 >278 122-129 ct8 50 250 900 278000 23 >>50 >>278 124-131 C' 8 50 250 900 278000 16 >>50 >278 126-133 C' 8 50 250 900 278000 11 >>50 >>278 128-135 C1 8 50 250 900 278000 29 150 834 130-137 c' 8 50 250 900 278000 50 50 278 132-139 C' 8 50 250 900 278000 43 75 417 134-141 C1 8 50 250 900 278000 30 150 834 136-143 C' 8 50 250 19 00 278000 0 >>50 >>278 aSEQ ID NO:2 amino acid numbers bpercent Inhibition EDITORIAL NOTE APPLICATION NUMBER 37449/00 The following Sequence Listing pages numbered 1-7 are part of the Description WO 00/54770 PCT/USOO/06667 SEQUENCE LISTING <110> Boston Life Sciences Children's Medical Center Corporation <120> PHARMACEUTICAL COMPOSITIONS COMPRISING TROPONIN SUBUNITS, FRAGMENTS AND HOMOLOGS THEREOF AND METHODS OF THEIR USE TO INHIBIT ANGIOGENESIS <130> 8657-028-228 <140> <141> <150> <151> <150> <151> <160> <170> <210> <211> <212> <213> <400> Thr Asp Ala Glu Asp Ile Thr Pro Asp Gly Arg Gin Glu Cys Glu Glu 115 Glu Glu 130 Arg Ile 09/442,099 1999-11-17 09/268,274 1999-03-15 17 FastSEQ for Windows Version 1 160

PRT

Homo sapiens Met 1 lie Gly Gin Glu Val Ala Pro Asp Gly 145 1 Gin Phe Ser Thr Ser Met Phe 100 Leu Ile Asp Gin 5 Lys Val Lys Gly Lys Arg Ala Glu Phe Ala Ala Lys Glu Thr 70 Glu Ile Glu Ser Asp 150 Glu Ala Glu Glu 55 Ile Asp Phe Ile Leu 135 Glu Ala Arg Ser Tyr Leu Ser Glu Glu Met Phe Asp 25 Leu Gly 40 Leu Asp Asp Phe Ala Lys Asp Arg 105 Phe Arg 120 Met Lys Phe Leu Phe Val Ile Glu 75 Lys Ala Ser Gly Met 155 Asp Met Ile Phe Ser Asp Gly Asp 140 Met Ala Arg Glu Leu Glu Gly Glu 125 Lys Glu Asp Met Glu Val Glu Tyr 110 His Asn Gly Gly Leu Val Met Glu Ile Val Asn Val Gly Gly Asp Met Leu Asp Thr Asp Gin 160 <210> 2 <211> 182 <212> PRT <213> Homo sapiens <400> 2 WO 00/54770 WO 0054770PCI'IUSOO/06667 Met His Glu Cys Cys Asp Asn Arg Ser Lys 145 Trp Met Gly.

Leu Giu Pro Lys Met Gin Arg Lys 130 Lys Arg Phe Asp Lys Ser Pro Gin Glu Lys Val1 115 His Glu Lys Giu Giu Ser Arg Leu Leu Val1 Leu 100 Arg Lys Asp Asn Ser 180 Glu Val Arg His His Arg Phe Met Val1 Thr Ile 165 Glu Lys Met Giu Ile Ala 70 Val Asp Ser Cys Giu 150 Glu Ser Arg Leu Ala Pro 55 Lys Gin Leu Ala Met 135 Lys Giu Asn Gin Giu 40 Giy Ile Lys Arg Asp 120 Asp Giu Lys Arg .le 25 Lys Ser Asp Thr Gly *105 Ala Leu Arg Ser Ala 10 Ala Gin Met Ala Ser 90 Lys Met Arg Asp Gly 170 Ile Al a Asn Ser Aia 75 Lys Phe Leu Aia Leu 155 Met Thr Thr Tyr Glu Giu Glu Lys Lys Asn 140 Arg Giu Aila Glu Leu Val1 Giu Leu Arg Ala 125 Leu Asp Gly Arg Leu Ala Gin Giu Giu Pro 110 Leu Lys Val1 Arg Arg Glu Glu Glu Lys Asp Pro Leu Gin Gly Lys 175 Gin Lys His Leu Tyr Met Leu Gly Val Asp 160 Lys Met 1 Glu Asp Pro Asn Ala Giu Lys Giu Lys 145 Al a Lys Giu <210> 3 <211> 21 <212> P1 <213> H <400> 3 Ser Asp G Giu Ala G Ala Giu G~ Giu Giy G: Lys Asp L~ Arg Lys L~ Lys Arg Ai ic Giu Arg GJ 115 Giu Giu AE 130 Ala Leu SE Asp Gin L Ile Leu A] 18 Asp Lys Le 195 58

RT

omo sapiens ln 0 Lu l5 I0 Giu 5 Glu Glu Lys Met Glu Ala Arg Ala Ser Arg 165 Gi u Arg Val1 Giu Lys Val1.

Gl u 70 Glu Glu Gin Lys Met 150 Gly Arg Asp Giu Giu Pro Asp 55 Leu Glu Arg Asn Arg 135 Giy Lys Arg Lys Gin Glu Arg 40 Phe Gin Glu Ala Arg 120 Arg Aia Lys Lys Ala 200 Val1 Val Pro Asp Al a Leu Glu 105 Leu Aila Asn Gin Pro 185 Lys Glu 10 Gin Lys Asp Leu Val1 90 Gin Al a Glu Tyr Thr 170 Leu Glu Giu Giu Leu Ile Ile 75 Aia Gin Giu Asp Ser 155 Aia Asn Leu Gin Asp Thr Gin Asp Leu Arg Giu Asp 140 Ser Arg Ile Trp Tyr Thr Ala Lys Ser Lys Ile Lys 125 Leu Tyr Glu Asp Glu 205 Giu Ala Pro Ly s His Giu Arg 110 Al a Lys Leu Met His 190 Thr Giu Giu Lys Arg Phe Arg Ala Arg Lys Ala Lys 175 Leu Leu Glu GiU Ile Gin Giu Ile Giu Arg Lys Lys 160 Lys Gly His WO 00/54770 PCT/US00/06667 Gin Leu Glu Ile Asp Lys Phe Glu Phe Gly Glu Lys Leu Lys Arg Gin 210 215 220 Lys Tyr Asp Ile Thr Thr Leu Arg Ser Arg Ile Asp Gin Ala Gin Lys 225 230 235 240 His Ser Lys Lys Ala Gly Thr Pro Ala Lys Gly Lys Val Gly Gly Arg 245 250 255 Trp Lys <210> 4 <211> <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 gctctgcaaa cagctgcacg ccaag <210> <211> <212> DNA <213> Artificial Sequence <220> <223> Primer <400> gcccagcagg gccttgagca tggca <210> 6 25--2-1 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 ctcaccatgg gagatgagga gaagc <210> 7 <211> <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 gcctcgagtg gcctaggact cggac <210> 8 <211> 24 <212> DNA <213> Artificial Sequence WO 00/54770 WO 0054770PCT/USOO/06667 <220> <223> Primer <400> 8 gaacactgcc cgcctctgca catc <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 gagcccagca gcgccttcag catg Gly Leu Giu Pro Lys Met Gin Arg Lys Lys 145 Arg Phe Gl Pr 50 Gl Ly Va Hi 13 Gi Ly Gi <210> <211> 181 <212> PRT <213> Oryctolagus <400> ~p Giu Glu Lys Arg 's Ser Val Met Leu .y Arg Arg Glu Ala ~o Leu Ser Leu Pro .n Leu His Ala Lys 70 .u Ile Lys Val Gin 's Leu Phe Asp Leu 100 .1 Arg Met Ser Ala 115 .s Lys Val Cys Met 0 u Asp Thr Glu Lys 150 s Asn Ile Glu Glu 165 u Ser Glu Ser 180 <210> 11 <211> 94 <212> PRT <213> Oryctolagus cuniculus As n Gin Giu Gly 55 Ile Lys Arg Asp Asp 135 Glu Lys Arg Ile Lys 40 Ser Asp Ser Gly Ala 120 Leu Arg Ser Ala Al a 25 Gin Met Al a Ser Lys 105 Met Arg Asp Gly Ile 10 Al a Asn Ala Ala Lys 90 Phe Leu Ala Leu Met 170 Thr Thr Tyr Glu Glu 75 Glu Lys Lys Asn Arg 155 Glu Ala Glu Leu Val1 Glu Leu Arg Ala Leu 140 Asp Gly Arg Leu Ala Gin Glu Glu Pro Leu 125 Lys Val Arg Arg Glu Glu Glu Lys Asp Pro 110 Leu Gin Gly Lys Gin Lys His Leu Tyr Met Leu Gly Val Asp Lys 175 His Glu Cys Cy s Asp As n Arg Se r Lys Trp 160 Met cuniculus <400> Gly Asp' Glu 1 Leu Lys Ser 11 Glu Lys Arg 5 Val Met Leu Asn Arg Ala Ile Thr Ala Arg Arg Gin His 10 Gin Ile Ala Ala Thr Glu Leu Glu Lys Glu 25 WO 00/54770 Giu Pro Lys Met Gly Arg Pro Leu Gin Leu Giu Ile Arg Ser His Lys Giu Ala Leu Pro Ala Lys 70 Val Gin Giu Lys 40 Giy Ser 55 Ile Asp Lys Ser PCTIUS0OI06667 Gin Asn Tyr Leu Ala Glu His Cys Met Ala Giu Vai Gin Glu Leu Cys Ala Aia Giu Glu Giu Lys Tyr Asp 75 Ser Lys Giu Leu Giu Asp <210.

<211- <212.

<2l3 <400, Gly Asp Gi 1 Leu Lys Sex Giu Gly Arc Pro Pro Leu Lys Gin Leu Met Glu Ile Gin Lys Leu Arg Vai Arg 115 <210> <211> <212> <213> <400> Lys Leu Phe 1 Vai Arg <210> <211> <212> <213> <400> Asn Gin Lys 1 Arg Arg Vai Ser Lys His Lys Lys Glu 12 120

PRT

Oryctolagus 12 Glu Lys Arg 5 Vai Met Leu Arg Glu Ala Ser Leu Pro His Ala Lys 70 Lys Val Gin Phe Asp Leu 100 Met Ser Ala cuniculus Asn Gin Glu Gly 55 Ile Lys Arg Asp Arg Ile Lys 40 Ser Asp Ser Gly Ala 120 Ala Al a 25 Gin Met Al a Ser Lys 105 Ile Ala Asn Ala Al a Lys 90 Phe Thr Ala Thr Glu Tyr Leu Giu Val Giu Glu 75 Giu Leu Lys Arg Arg Leu Ala Gin Glu Glu Pro Arg Glu Glu Glu Lys Asp Pro 110 Gin Lys His Leu Tyr Met Leu His Glu Cys Cys Asp Asn Arg 13 18

PRT

Oryctolagus cuniculus 13 Asp Leu Arg Gly Lys Phe Lys Arg Pro Pro Leu Arg Arg 5 10 14 86

PRT

Oryctolagus 14 Leu Phe Asp 5 Arg Met Ser Lys Val Cys Asp Thr Glu cuni cul us Leu Arg Gly Ala Asp Ala 25 Met Asp Leu 40 Lys Glu Arg 55 Lys Phe Lys Arg Pro Pro Leu 10 Met Leu Lys.Ala Leu Leu Gly Arg Ala Asn Leu Lys Gin Val Asp Leu Arg Asp Val Gly Asp WO 00/54770 pTUOI 6 6 Trp Arg Lys Asn Ile Giu Giu Lys Ser Gly met Glu Giy Arg Lys Lys 70 75 Met Phe Giu Ser Giu Ser <210> <211> <212> PRT <213> OryctolaguS cunicuius <400> 15 Leu Lys Ala Leu Leu Gly Ser Lys His Lys Val Cys Met Asp Leu Arg 1 5 10 Ala Asn Leu Lys Gin Val Lys Lys Giu Asp Thr Glu Lys Giu Arg Asp 25 Leu Arg Asp Val Gly Asp Trp Arg Lys Asfl Ile Giu G1u Lys Ser Giy 40 Met Glu Giy Arg Lys Lys Met Phe Giu Ser Glu Ser 55 <210> 16 <211> 196 <212>

DNA

<213> Homto sapiens <400> 16 gctctgcaaa cagctgcacg ccaagatcga tgcggctgaa gaggagaagt acgacatgga ggtgagggtg cagaagacca gcaaggagct ggaggacatg aaccagaagc tatttgatct gcgggccaag ttcaagcggc ccccactgcg gagggtgcgc atgtcggccg atgccatgct caaggccctg ctgggc <210> 17 <211> 182 <212>

PRT

<213> Homo sapiens <400> 17 Met Gly Asp Giu Glu Lys Arg Asn Arg Ala Ile Thr Ala Arg Arg Gin 1 5 10 His Leu Lys Ser Val Met Leu Gin Ilie Ala Ala Thr Glu Leu Glu Lys 25 Giu Glu Ser Arg Arg Giu Ala Giu Lys Gin Asn Tyr Leu Ala Giu His 40 Cys Pro Pro Leu His Ile Pro Gly Ser Met Ser Glu Val Gin Glu Leu 55 Cys Lys Gin Leu His Ala Lys Ile Asp Ala Ala Glu Giu Glu Lys Tyr 70 75 Asp Met Glu Val Arg Val Gin Lys Thr Ser Lys Glu Leu Giu Asp Met 90 Asn Gin.Lys Leu Phe Asp Leu Arg Gly Lys Phe Lys Arg Pro Pro Leu 100 105 110 Arg Arg Vai Arg Met Ser Ala Asp Ala Met Leu Lys Ala Leu Leu Gly 115 120 125 Ser Lys His Lys Val Cys Met Asp Leu Arg Ala Asn Leu Lys Gin Val 130 135 140 Lys Lys Giu Asp Thr Giu Lys Glu Arg Asp Leu Arg Asp Val Gly Asp 145 150 ,155 160 120 180 196 WO 00/54770 PCTIUS00106667 Trp Arg Lys Asn Ile Giu Glu Lys Ser Gly Met Giu Gly Arg Lys Lys 165 170 175 Met Phe Glu Ser Glu Ser 180

Claims (39)

1.A method of inhibiting angiogenesis associated with a disease or disorder which comprises administering an effective amount of a peptide in which the peptide is: a. less than 100 amino acids in length; and b. greater than 80% homology with the inhibitory and carboxy terminal region of troponin subunit I (SEQ ID NO:14).

2.The method of claim 1 in which the peptide consists of the amino acid sequence of SEQ ID NO:14.

3.The method of claim 1 or 2 in which the disease or disorder is a solid tumor. o. 15 4.The method of claim 3 in which the solid tumor is selected from the group consisting of sarcomas and carcinomas depicted in Table 1. method of claim 1 in which the disease or disorder is an 20 ophthalmic disease or disorder.

6.The method of any one of claims 1 to 5 in which the subject is a human. 25 7.The method of any one of claims 1 to 6 in which the effective amount is about 1 to about 50 milligrams per administration.

8.The method of any one of claims 1 to 7 in which the peptide is administered parenterally.

9.The method of any one of claims 1 to 7 in which the peptide is administered orally. The method of any one of claims 1 to 9 in which the troponin subunit I is a human or bovine troponin subunit I. W:\Viole\NieM51286192lgdle~deItdc

11. The method of any one of claims 1 to 10 in which the peptide is recombinantly expressed by a cell engineered to contain a nucleotide sequence encoding said protein.

12. The method of claim 1 in which the disease or disorder is lung metastases.

13. The method of any one of claims 1 to 10 in which the troponin subunit I is derived from tissue.

14. The method of claim 13 in which the tissue is connective, muscle, nerve or epithelial.

15. The method of claim 13 in which the tissue is cartilage.

16. A method for inhibiting the growth or reducing the volume of a solid tumor in a subject comprising administering an effective amount of a peptide in which the peptide is: less than 100 amino acids in length; and 20 greater than 80% homology with the inhibitory and carboxy terminal region of troponin subunit I (SEQ ID NO:14).

17. The method of claim 16 in which the peptide consists of 25 the amino acid sequence of SEQ ID NO:14.

18. The method of claim 16 or 17 in which the peptide is administered parenterally.

19. The method of claim 16 or 17 which the peptide is administered orally. The method of any one of claims 16 to 19 in which the solid tumor is selected from the group consisting of sarcomas and carcinomas depicted in Table 1. W:%ViDetVlgeM519211851921NOdeet.doc

21. The method of any one of claims 16 to 20 in which the peptide is recombinantly expressed by a cell engineered to contain a nucleotide sequence encoding said protein.

22. A method of inhibiting metastasis in a subject comprising administering an effective amount of a peptide in which the peptide is: less than 100 amino acids in length; and greater than 80% homology with the inhibitory and carboxy terminal region of troponin subunit I (SEQ ID NO:14).

23. The method of claim 22 in which the peptide consists of the amino acid sequence of SEQ ID NO:14. 15 24. The method of claim 22 or 23 in which the subject is a human having a solid tumor selected from the group consisting of sarcomas and carcinomas depicted in Table 1. The method of any one of claims 22 to 24 in which the 20 peptide is recombinantly expressed by a cell engineered to contain a nucleotide sequence encoding said protein.

26. A pharmaceutical composition comprising a peptide in which the peptide is: 25 less than 100 amino acids in length; and greater than 80% homology with the inhibitory and carboxy terminal region of troponin subunit I (SEQ ID NO:14); and a pharmaceutically acceptable carrier.

27. The composition of claim 26 in which the peptide consists of the amino acid sequence of SEQ ID NO:14.

28. The composition of claim 26 or 27 in which the troponin subunit I is a human or bovine troponin subunit I. W:IoIehiger,51921%85192 NodeletG.dOC

29. The pharmaceutical composition of any one of claims 26 to 28 in which the peptide is recombinantly expressed by a cell engineered to contain a nucleotide sequence encoding said protein. The composition of any one of claims 26 to 28 in which the troponin subunit I is derived from tissue.

31. The composition of claim 30 in which the tissue is cartilage.

32. The composition of claim 30 in which the tissue is connective, muscle, nerve, or epithelial. 15 33. A pharmaceutical composition comprising a recombinant cell containing a nucleotide sequence encoding a peptide in which the peptide is: less than 100 amino acids in length; and greater than 80% homology with the inhibitory and carboxy 20 terminal region of troponin subunit I (SEQ ID NO:14), such that said peptide is expressed in a subject.

34. A method for inhibiting the growth or reducing the volume of a solid tumor in a subject comprising administering a 25 recombinant cell containing a nucleotide sequence encoding a peptide in which the peptide is: less than 100 amino acids in length; and greater than 80% homology with the inhibitory and carboxy terminal region of troponin subunit I (SEQ ID NO:14), such that said peptide is expressed in the subject. A method of inhibiting metastases in a subject comprising administering to a subject a recombinant cell WAVioleNieh651921\ 5192 Nodelete.doc containing a nucleotide sequence encoding a peptide in which the peptide is: less than 100 amino acids in length; and greater than 80% homology with the inhibitory and carboxy terminal region of troponin subunit I (SEQ ID NO: 14), such that said peptide is expressed in the subject.

36. A method of inhibiting angiogenesis associated with a disease or disorder which comprises administering an effective amount of a peptide in which the peptide consists of a region of troponin subunit I having amino acid residues 118-137 (huTnI 118 137 of SEQ ID NO:2.

37. A method of inhibiting angiogenesis associated with a disease or disorder which comprises administering an effective amount of a peptide wherein the peptide consists of the amino acid sequence of residues 118-137 (huTn 11 8 a 1 37 of SEQ ID NO:2.

38. A method of inhibiting metastasis in a mammal comprising administering an effective amount of a peptide in which the peptide is greater than 80% homology with a region of troponin subunit I having amino acid residues 118-137 (huTnIn 8 13 7 of SEQ ID NO:2. S 25 39. The method of claim 38 in which the mammal is a human having a solid tumor selected from the group consisting of sarcomas and carcinomas depicted in Table 1.

40. The method of claim 38 or 39 in which the peptide is recombinantly expressed by a cell engineered to contain a nucleotide sequence encoding said protein. S• 41. A pharmaceutical composition comprising a recombinant cell containing a nucleotide sequence encoding a peptide in 35 which the peptide consists of the amino acid sequence of residues 118-137(huTnIne- 137 of SEQ ID NO:2. W:\Violet\Nige651921\651921 daims.doc 69

42. A method for inhibiting the growth or reducing the volume of a solid tumor in a subject comprising administering a recombinant cell containing a nucleotide sequence encoding a peptide in which the peptide is greater than 80% homology with a region of troponin subunit I having amino acid residues

118-137(huTnI 1 8 1 37 of SEQ ID NO:2, such that said peptide is expressed in the host. 43. A method of inhibiting metastases in a subject comprising administering to a subject a recombinant cell containing a nucleotide sequence encoding a peptide in which the peptide is greater than 80% homology with a region of troponin subunit I having amino acid residues 118-137(huTnIn 18 137 of SEQ ID NO:2, such that said peptide is expressed in the host. 44. A method of inhibiting angiogenesis associated with a disease or disorder which comprises administering an effective amount of a peptide in which the peptide is greater than homology with amino acid residues selected from the group consisting of 116-123 (huTnll6-1 2 3

120-127 (huTnI 20 127

122-129 (huTnI 122 -1 2 9

124-131 (huTnIl 24 131

126-133 (huTnI 2 6 1 33

128-1735 (huTnI1 2 8 13 5

130-137 (huTn 1 3 0 -1 37

132-139 (huTnI 32 1 39 and

134-141 (huTnI1 3 9 141 of the inhibitory region of troponin subunit I (SEQ ID NO:2). 45. The method of claim 44 in which the peptide consists of a region of troponin subunit I having amino acid residues selected from the group consisting of 116-123 (huTnI1 1 6- 123 30 120-127 (huTn1 1 20 -1 27 122-129 (huTnI 22 -1 29 124-131 (huTnI 24 131 I 126-133 (huTn 1 26 -1 33 128-135 (huTnI 2 8 -1 35 130-137 (huTnI 13 0 -1 3 7 132-139 (huTn1 32 -1 39 and 134-141 (huTnI 34 141 of SEQ ID NO:2. 46. The method of claim 45 in which the peptide consists of 35 a region of troponin subunit I having amino acid residues 130- 137 (huTnIo 3 0 13 7 W:\Violet\Nige651921\651921 claims.doc 47. The method of claim 45 in which the peptide consists of a region of troponin subunit I having amino acid residues 132- 139 (huTnI1 32 -1 39 48. The method of any one of claims 44 to 47 in which the disease or disorder is a solid tumor. 49. The method of claim 48 in which the solid tumor is selected from the group consisting of sarcomas and carcinomas depicted in Table 1. The method of any one of claims 44 to disease or disorder is an ophthalmic disease or 51. The method of any one of claims 44 to subject is a human. 47 in which the disorder. 50 in which the 52. The method of any one of claims 44 effective amount is about 1 to about administration. 53. The method of any one of claims 44 peptide is administered parenterally. 51 in which milligrams the per to 52 in which the 52 in which the 54. The method of any one of claims 44 to peptide is administered orally. The method of any one of claims 44 to peptide is of human or bovine origin. 54 in which the 56. The method of any one of claims 44 to 54 in which the peptide is recombinantly expressed by a cell engineered to contain a nucleotide sequence encoding said peptide. 57. The method of any one of claims 44 to 56 in which the disease or disorder is lung metastases. W:\Violet\NigeIn651921\65192 claims.doc 58. The method of any one of claims 44 to 54 in which the peptide is derived from tissue. 59. The method of claim 58 in which the tissue is connective, muscle, nerve, or epithelial. The method of claim 58 in which the tissue is cartilage. 61. A method of inhibiting angiogenesis associated with a disease or disorder which comprises administering an effective amount of a peptide having amino acid residues of the amino acid sequence selected from the group consisting of 116-123 (huTnI 1 6 123 120-127 (huTn1 20 127 122-129 (huTn 2 2 2 9 124-131 (huTnI1 24 131 126-133 (huTn1 26 1 33 128-135 (huTnI1 2 8 13 130-137 (huTnI 130 137 132-139 (huTnI 132 1 39 and 134-141 (huTnI1 3 4 14 1 of residues 118-137 (huTns 1 1 8 137 of SEQ ID NO:2 so that angiogenesis is inhibited. 62. The method of claim 61 in which the peptide consists of a region of troponin subunit I having amino acid residues 130-137 (huTnI 130 -1 37 63. The method of claim 61 in which the peptide consists of a region of troponin subunit I having amino acid residues 132-139 (huTnI1 32 -1 39 64. The method of claim 61 in which the peptide consists of the amino acid sequence of residues 130-137 (huTnI 130 137 of SEQ ID NO:2. The method of claim 61 in which the peptide consists of the amino acid sequence of residues 132-139 (huTnI1 32 -1 39 of SEQ ID NO:2. 66. A method of inhibiting metastasis in a mammal comprising administering an effective amount of a peptide in which the W:\Violet\Nigel651921\651921 daims.doc peptide is greater than 80% homology with a region of troponin subunit I selected from the group consisting of 116-123 (huTnIn 6 -1 23 120-127 (huTnI 2 0 -1 2 7 122-129 (huTn 1 22 129 124-131 (huTnI 24 131 126-133 (huTnI1 26 3 3 128-135 (huTnI1 28 -1 35 130-137 (huTnI 130 1 37 132-139 (huTnI1 32 139 and 134-141 (huTnI 34 1 4 1 having amino acid residues of SEQ ID NO:2. 67. The method of claim 66 in which the peptide consists of the amino acid sequence of residues 130-137 (huTn1 1 30 -1 37 of SEQ ID NO:2. 68. The method of claim 66 in which the peptide consists of the amino acid sequence of residues 132-139 (huTnI1 321 39 of SEQ ID NO:2. 69. The method of any one of claims 66 to 68 in which the mammal is a human having a solid tumor selected from the group consisting of sarcomas and carcinomas depicted in Table 1. 70. The method of any one of claims 66 to 69 in which the peptide is recombinantly expressed by a cell engineered to contain a nucleotide sequence encoding said protein. 71. A pharmaceutical composition for inhibiting angiogenesis associated with a disease or disorder comprising a peptide S: having amino acid residues of the amino acid sequence of residues selected from the group consisting of 116-123 (huTnI16- 123 120-127 (huTn1 20 -1 2 7 122-129 (huTnI 122-129) 124-131 (huTnI 24 31 126-133 (huTnI1 26 -1 3 3 128-135 (huTnI 2 8 -1 35 130-137 30 (huTn 1 30 1 37 132-139 (huTnI 32 -1 39 and 134-141 (huTnI1 3 4 1 41 )of SEQ ID NO:2. 72. The pharmaceutical composition of claim 71 in which the peptide is recombinantly expressed by a cell engineered to S 35 contain a nucleotide sequence encoding said peptide. W:\Violet\Nigel\651921\ c1921claims.doc 73. The composition of claim 72 in which the peptide consists of the amino acid sequence of residues 130-137 (huTnI 30 137 or 132- 139 (huTnI1 32 -1 39 of SEQ ID NO:2. 74. A pharmaceutical composition comprising a recombinant cell containing a nucleotide sequence encoding a peptide in which the peptide consists of the amino acid residues selected from the group consisting of 116-123 (huTnIn6- 123 120-127 (huTnIz 20 -1 27 i 122-129 (huTnIl 22 129 124-131 (huTnI 2413 1 126-133 (huTnI1 26 -1 33 128-135 (huTn1 1 28 13 5 130-137 (huTn1 30 o- 37 132-139 (huTn1 32 -1 39 and 134-141 (huTnI 34 141 of SEQ ID NO:2, such that said peptide is expressed in a host. The pharmaceutical composition of claim 74 in which the peptide consists of the amino acid sequence of residues 130-137 (huTnI 1 30 -137) of SEQ ID NO:2. 76. The pharmaceutical composition of claim 74 in which the peptide consists of the amino acid sequence of residues 132-139 (huTnI1 32 -1 39 of SEQ ID NO:2. 77. A method for inhibiting the growth or reducing the volume of a solid tumor in a subject comprising administering a recombinant cell containing a nucleotide sequence encoding a peptide in which the peptide is greater than 80% homology with a region of troponin subunit I having amino acid residues selected 6% from the group consisting of 116-123 (huTnIn6-1 23 120-127 0:66 (huTnI 12 0 -1 2 7 122-129 (huTnIl 2 2 1 2 9 124-131 (huTnI1 2 4 -1 3 1 126-133 (huTnI1 2 6 -1 3 3 128-135 (huTnI 2 8 -1 3 5 130-137 (huTnIi 30 -1 37 132-139 30 (huTn1 32 1 3 9 and 134-141 (huTn1 1 34 1 41 of SEQ ID NO:2, such that said peptide is expressed in the host. 78. The method of claim 77 in which the peptide consists of the amino acid sequence of residues 130-137 (huTnI 130 -1 37 of SEQ ID 35 NO:2. *W e 2 0 W:\VioletNigeI\651921\651921 claims.doc 79. The method of claim 77 in which the peptide consists of the amino acid sequence of residues 132-139 (huTnI 32 139 of SEQ ID NO:2. 80. A method of inhibiting metastases in a subject comprising administering to a subject a recombinant cell containing a nucleotide sequence encoding a peptide in which the peptide is greater than 80% homology with a region of troponin subunit I having amino acid residues selected from the group consisting of 116-123 (huTnI1 1 6- 123 120-127 (huTnI1 20 -1 27 122-129 (huTn1 1 22 -1 29 124-131 (huTn1 1 24 131 126-133 (huTnI1 26 1 33 128-135 (huTnI 28 135 130-137 (huTnI 30 o- 137 132-139 (huTnI 1 3 2 -1 3 9 and 134-141 (huTn1 1 34 14 1 )of SEQ ID NO:2, such that said peptide is expressed in the host. 81. The method of claim 80 in which the peptide consists of the amino acid sequence of residues 130-137 (huTn 1 3 0o- 1 37 of SEQ ID NO:2. 82. The method of claim 80 in which the peptide consists of the amino acid sequence of residues 132-139 (huTnI1 32 1 3 9 of SEQ ID NO:2. 83. The method according to any one of claims 1, 16, 22, 34, 25 35, 36, 37, 38, 42, 43, 44, 61, 66, 77 or 80 substantially as S: hereinbefore described with reference to the Examples. DATED: 6 May, 2004 30 PHILLIPS ORMONDE FITZPATRICK Attorneys for: 49& tA6 BOSTON LIFE SCIENCES INC. and CHILDREN'S MEDICAL CENTER CORPORATION W:1Violet'Nige \651921\651921 daims.doc