EP1165059A1 - Compositions pharmaceutiques renfermant des sous-unites de troponine, ou des fragments ou des homologues desdites sous-unites, et leur utilisation pour inhiber l'angiogenese - Google Patents

Compositions pharmaceutiques renfermant des sous-unites de troponine, ou des fragments ou des homologues desdites sous-unites, et leur utilisation pour inhiber l'angiogenese

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Publication number
EP1165059A1
EP1165059A1 EP00916329A EP00916329A EP1165059A1 EP 1165059 A1 EP1165059 A1 EP 1165059A1 EP 00916329 A EP00916329 A EP 00916329A EP 00916329 A EP00916329 A EP 00916329A EP 1165059 A1 EP1165059 A1 EP 1165059A1
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EP
European Patent Office
Prior art keywords
peptide
hutni
seq
amino acid
troponin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP00916329A
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German (de)
English (en)
Other versions
EP1165059A4 (fr
Inventor
Richard M. Thorn
Marc E. Lanser
Marsha A. Moses
Dmitri G. Wiederschain
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Childrens Medical Center Corp
Alseres Pharmaceuticals Inc
Original Assignee
Childrens Medical Center Corp
Boston Life Sciences Inc
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Priority claimed from US09/442,099 external-priority patent/US6465431B1/en
Application filed by Childrens Medical Center Corp, Boston Life Sciences Inc filed Critical Childrens Medical Center Corp
Publication of EP1165059A1 publication Critical patent/EP1165059A1/fr
Publication of EP1165059A4 publication Critical patent/EP1165059A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin

Definitions

  • the present invention provides for novel pharmaceutical compositions, and methods of use thereof for the treatment of diseases or disorders involving abnormal angiogenesis .
  • 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.
  • 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 through the newly formed capillary is initiated after the endothelial cells come into contact and connect with a preexisting capillary.
  • 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.
  • neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis.
  • 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.
  • ocular disorders include diabetic retinopathy, neovascular glaucoma, inflammatory diseases and ocular tumors (e.g., retinoblastoma) .
  • ocular tumors e.g., retinoblastoma
  • 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, e.g., reviews by Waltman et al . , 1978, Am . J. Ophthal . 85:704-710 and Gartner et al . , 1978, Surv.
  • 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.
  • agents that inhibit tumor neovascularization could play an important role in inhibiting metastatic tumor growth.
  • angiogenesis relating to vascular endothelial cell proliferation, migration and invasion, have been found to be regulated in part by polypeptide growth factors.
  • endothelial cells exposed to a medium containing suitable growth factors can be induced to evoke some or all of the angiogenic responses.
  • polypeptides with in vi tro endothelial growth promoting activity have been identified. Examples include acidic and basic fibroblast growth factors, transforming growth factors ⁇ and ⁇ , platelet-derived endothelial cell growth factor, granulocyte colony-stimulating factor, interleukin-8, hepatocyte growth factor, proliferin, vascular endothelial growth factor and placental growth factor. See, e.g., review by Folkman et al . , 1995, N. Engl . J. Med. , 333:1757-1763.
  • 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.
  • 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, Na ture, 297:307-312.
  • the reported doses for previously described angiogenesis inhibitors tested alone in the CAM assay are 50 ⁇ g of protamine (Taylor et al . (1982)), 200 ⁇ g of bovine vitreous extract (Lutty et al., 1983, Invest . Opthalmol . Vis . Sci . 24.: 53-56), and 10 ⁇ g of platelet factor IV (Taylor et al .
  • angiogenesis inhibitors effective as combinations include heparin (50 ⁇ g) and hydrocortisone (60 ⁇ g) , and B-cyclodextrin tetradecasulfate (14 ⁇ g) and hydrocortisone (60 ⁇ g) , reported by Folkman et al., 1989, Science 243:1490.
  • polymer pellets of ethylene vinyl acetate copolymer 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.
  • an angiogenesis inhibitor either a piece of carcinoma or some other angiogenic stimulant is implanted distal to the polymer 2 mm from the limbus.
  • 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.
  • 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 the actin molecule, causing the tropomyosin molecule shift, thereby exposing the myosin binding sites on actin and stimulating myosin ATPase activity.
  • 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 include: troponin subunits C, I, and T, and fragments and homologs thereof, in particular, fragments of troponin subunit I comprising the inhibitory (I') and carboxy terminal (CJ regions.
  • 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.
  • a Therapeutic of the invention is administered to treat ocular disorders associated with neovascularization.
  • 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 N', I', and C', respectively.
  • FIGURES Figure 1. Inhibition of bovine capillary Endothelial Cell (BCE) proliferation by troponin C. Percent inhibition of bFGF-stimulated BCE proliferation is shown as a function of troponin C concentration ( ⁇ g/well). Percent inhibition was determined by comparing results obtained for cells treated with stimulus alone with those obtained for samples exposed to both stimulus and inhibitor. Well volume was 200 ⁇ l.
  • Inhibition of capillary BCE proliferation by troponin T Percent inhibition of bFGF-stimulated BCE proliferation is shown as a function of troponin T concentration ( ⁇ g/well) . Percent inhibition was determined as described in Figure 1. Well volume was 200 ⁇ l . 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 ( ⁇ g/well) . Percent inhibition was determined as described in Figure 1. Well volume was 200 ⁇ l . 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 T concentration ( ⁇ g/well) . Percent inhibition was determined as described in Figure 1. Well volume was 200 ⁇ l .
  • Figure 6 Schematic representation of amino acid sequences of tryptic peptides purified from cartilage as described in Methods. Sequence similarity to human Tnl is indicated by alignment with the amino acid sequence of the human isoform.
  • FIG. 7 (A) 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 Tnl .
  • Gene-specific primers were designed based on the cDNA sequence of rat fast-twitch skeletal muscle Tnl as described in Methods.
  • Figure 8 SDS-PAGE of recombinant human Tnl before (lane A) and after (lane B) purification. In both cases, approximately 1 ⁇ g of total protein was electrophoresed, followed by silver staining as described in Methods. Recombinant Tnl migrates at a molecular weight of approximately 21,000 Da.
  • Figure 9. Inhibition of capillary EC proliferation by rTnl . Percent inhibition was determined by comparing wells exposed to the angiogenic stimulus bFGF (A) and VEGF (B) 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 Tnl preparations.
  • Figure 10 SDS-PAGE of recombinant human Tnl before (lane A) and after (lane B) purification. In both cases, approximately 1 ⁇ g of total protein was electrophoresed, followed by silver staining as described in Methods. Recombinant Tnl migrate
  • FIG. 11 Inhibition of FGF-induced angiogenesis by systemic administration of Tnl.
  • Tnl 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.
  • bFGF 40ng/ml
  • FIG. 12 Derived amino acid sequence of recombinant human Tnl (Hu) (SEQ ID NO: 17) and its sequence comparison with recombinant rabbit Tnl (Rb) (SEQ ID NO:10). Identical residues are shown by dashes.
  • B Schematic representation of various recombinant Tnl deletion fragments based on rabbit Tnl and wild-type rabbit Tnl w (SEQ ID NO: 10) .
  • the troponin I inhibitory region is designated I', and the sequences located on amino- and carboxy-terminal sides of this region are designated N' and C, respectively.
  • Tnl 1 _ 120 , Tnl- ⁇ ,,, Tnl 96 _ 181 , Tnl 122 _ 181 contain the N' and I', N', I' and C', and C' regions, respectively. The number of amino acids at the beginning and end of each fragment is indicated.
  • Tnl 98 _ 114 containing amino acid residues 98-114 is a synthetic peptide representing the I region.
  • 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.
  • therapeutic compounds include: troponin C, I, and T subunits, fragments and homologs thereof
  • peptides of the invention are characterized by the property of inhibiting bovine endothelial cell (EC) proliferation in culture preferably with an IC 50 of about 10 ⁇ M or less, more preferably with an IC 50 of about 5 ⁇ M or less, most preferably with an IC 50 of about l ⁇ M or less.
  • 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 .
  • a Therapeutic of the invention is administered to treat an ocular disorder associated with neovascularization .
  • 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 (I') and carboxy terminal (C) region ( C'+I J (SEQ ID NO: 14) of troponin subunit I or a fragment thereof.
  • 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, e.g., human, rabbit, rat, mouse, bovine, ovine and porcine.
  • the peptides of the invention are troponin C, troponin I and troponin T subunits, or fragments thereof from nonmuscle tissues, e.g., cartilage, preferably from mammalian species, e.g., human, rabbit, rat, mouse, bovine, ovine and porcine.
  • troponin subunits examples include but are not limited to the subunits of troponin from human fast twitch s keletal muscle , the sequences of which are given below :
  • the invention encompasses peptides which are homologous to troponin C (SEQ ID NO:l) 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 .
  • 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).
  • 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-1, tissue inhibitors of metalloproteases (TIMP1 and TIMP2) prolactin (16-Kd fragment) , angiostatin (38-Kd fragment of plasminogen) , bFGF soluble receptor, transforming growth factor ⁇ , interferon alfa, and placental proliferin- related protein.
  • angiogenesis may be, but is not limited to, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteases (TIMP1 and TIMP2) prolactin (16-Kd fragment) , angiostatin (38-Kd fragment of plasminogen) , bFGF soluble receptor, transforming growth factor ⁇ , interferon alfa, and placental proliferin- related protein.
  • the invention provides for a pharmaceutical composition of the present invention in combination with a chemotherapeutic agent.
  • 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.
  • examples Infra 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.
  • the invention provides for pharmaceutical compositions comprising troponin subunits, fragments, and homologs thereof.
  • the subunits, fragments, or homologs are of fly, frog, mouse, rat, rabbit, pig, cow, dog, monkey, or human troponin subunits.
  • the invention encompasses peptides which are homologous to troponin C (SEQ ID NO:l) or fragments thereof.
  • 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%.
  • this identity is greater than 90%.
  • 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, 50, 75, and 100 amino acids in length.
  • 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.
  • the invention encompasses peptides which are homologous to troponin I (SEQ ID NOS: 2, 10 or 15) or fragments thereof.
  • 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 thereof. Fragments are generally at least 4 amino acids, and in alternate embodiments at least 8, 10, 20, 30, 40, 50, 75, and 100 amino acids in length.
  • 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.
  • the invention encompasses peptides which are homologous to troponin T (SEQ ID NO: 3) or fragments thereof.
  • 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, 75, 100, 150, and 200 amino acids in length.
  • 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.
  • the invention encompasses peptides which are homologous to the Inhibitory (I') and carboxy terminus (C') region (C'+I') (SEQ ID NO: 14).
  • the invention encompasses peptides that are homologous to the C'+I' region of human troponin I (huTnl) (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 104 .
  • Additional embodiments include 94-113 (huTnI 94 _ 113 ) , 98-117 (huTnI 98 . 117 ) , 102-121 (huTnI 102 _ 121 ) , 106-125 (huTnI 106 _ 125 ) , 110-129 (huTnI 110 - ⁇ 29 ) , and 114-133 (huTnI 114 _ 133 ) .
  • Still other embodiments include carboxy terminus region (C') of human troponin I (huTnl) , 116-123 (huTnI U6 _ 123 ) , 118-125 (huTnI 118 .
  • Fragments are generally at least 4 amino acids, and in alternate embodiments at least 8, 10, 20, 30, 40, 50, and 75 amino acids in length.
  • homology 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.
  • 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.
  • BLAST BLAST
  • 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 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' 1 Acad. Sci.
  • the BLASTP program compares an amino acid query sequence against a protein sequence database
  • the BLASTN program compares a nucleotide query sequence against a nucleotide sequence database
  • the BLASTX program compares the six- frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database
  • the TBLASTN program compares a protein query sequence against a nucleotide sequence database translated in all six reading frames (both strands) ;
  • 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.
  • Smith-Waterman is a heuristic approximation to the Smith-Waterman algorithm.
  • 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, 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.
  • 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.
  • 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.
  • this molecule consists of at least 8, 10, 20 or 50 amino acids of the troponin subunit.
  • 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, C'+I' (SEQ ID NO: 14), huTnI 94 _ 123 , huTnI 104 . 133 , huTnI 114 .
  • Additional embodiments include 94-113 (huTnI 94 _ 113 ) , 98-117 (huTnI 98 . 117 ) , 102-121 (huTnI 102 . 121 ) , 106-125 (huTnI 106 . 125 ) , 110-129 (huTnI 110 _ 129 ) , and 114-133 (huTnI U4 _ 133 ) . Still other embodiments include carboxy terminus region (C') , 116-123
  • 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,
  • troponin subunits can occur at the gene or protein level.
  • 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 .
  • a nucleic acid which is hybridizable to the complement of a troponin nucleic acid e . g. , having a sequence as set forth in SEQ ID NOS:13-17
  • a nucleic acid encoding a troponin fragment or derivative under conditions of low stringency is provided.
  • 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 40°C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 ⁇ g/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 ⁇ g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10 6 cpm 32 P-labeled probe is used.
  • Filters are incubated in hybridization mixture for 18-20 h at 40°C, and then washed for 1.5 h at 55°C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60°C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68°C and re-exposed to film. Other conditions of low stringency which may be used are well known in the art (e.g., as employed for cross-species hybridizations) .
  • a nucleic acid which is hybridizable to a troponin nucleic acid under conditions of high stringency is provided.
  • 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°C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA.
  • Filters are hybridized for 48 h at 65°C in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X 10 6 cpm of 32 P-labeled probe. Washing of filters is done at 37°C for 1 h in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and
  • 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 e.g., Sambrook et al., 1989, Molecular Cloning,
  • 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 site- directed mutagenesis (Hutchinson et al., 1978, J . Biol . Chem .
  • 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, e .g. , 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.
  • fragments and homologs of troponin subunits can be chemically synthesized.
  • 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.
  • 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, -amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, designer amino acids such as ⁇ -methyl amino acids, C -methyl amino acids, and N ⁇ -methyl amino acids.
  • 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.
  • 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.
  • such a chimeric product may be made by protein synthetic techniques, e . g. , by use of a peptide synthesizer.
  • 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.
  • angiogenesis inhibiting factors include, but are not limited to: angiostatic steroids, thrombospondin, platelet factor IV, transforming growth factor ⁇ , interferons, tumor necrosis factor ⁇ _ , bovine vitreous 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, e.g., reviews by: angiostatic steroids, thrombospondin, platelet factor IV, transforming growth factor ⁇ , interferons, tumor necrosis factor ⁇ _ , bovine vitreous extract, protamine, tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) , prolactin (16-kd fragment) , angiostatin (38-kd fragment of plasminogen) , bFGF soluble receptor
  • 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.
  • EC capillary endothelial cells
  • 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.
  • capillary endothelial cells are 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 ⁇ .
  • angiogenesis stimulating factors such as aFGF
  • 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.
  • 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.
  • angiogenesis stimulator such as for example, bFGF
  • 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, e.g., Section 6, infra , Example 10, and Example 11, respectively). See also, Polverini et al., 1991, Methods Enzymol . 198:440-450.
  • 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, e.g., Example 11 infra .
  • the therapeutically effective dosage for inhibition of angiogenesis in vivo 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.
  • the inhibitor is delivered in a topical-ophthalmic carrier.
  • the inhibitor is delivered by means of a biodegradable, polymeric implant.
  • 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 include troponin subunits and fragments and homologs thereof (e.g., as described supra) .
  • 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.,
  • 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, e.g., reviews by Waltman et al.,
  • 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, Osier-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions. 5.4. DEMONSTRATION OF THERAPEUTIC
  • 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.
  • 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.
  • suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc.
  • any animal model system known in the art may be used.
  • 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.
  • 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) .
  • a Therapeutic of 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 for patients who have entered a remission in order to maintain a dormant state.
  • a Therapeutic of the invention e.g., encapsulation in liposomes, microparticles, microcapsules, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J " .
  • 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 (e . g. , 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.
  • 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, e . g. , by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • compositions of the invention 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, e.g., 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.
  • administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre- neoplastic tissue.
  • the purified troponin subunit is combined with a carrier so that an effective dosage is delivered, based on the desired activity (i.e., ranging from an effective dosage, for example, of 1.0 ⁇ M to 1.0 mM to prevent localized angiogenesis, endothelial cell migration, and/or inhibition of capillary endothelial cell proliferation.
  • 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.
  • 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, Pri ogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; or a glidant such as colloidal silicon dioxide.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Pri ogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide.
  • dosage unit form can contain, in addition to material of the above type, a liquid carrier such as a fatty
  • Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
  • the Therapeutic can be delivered in a vesicle, in particular a liposome.
  • 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 (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid . , pp. 317-327.
  • the Therapeutic can be delivered in a controlled release system.
  • 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 ;
  • 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.
  • a biodegradable, biocompatible polymeric implant which releases the troponin subunit, fragment, or homolog over a controlled period of time at a selected site.
  • 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
  • a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, 1989, supra , vol. 2, pp. 115-138).
  • compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • 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 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.
  • composition 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.
  • 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
  • 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.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • 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.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • 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 full- length troponin subunits are generally about 0.01 pg/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 from dose-response curves derived from in vitro or animal model test bioassays or systems.
  • the doses recited above can be repeated.
  • 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.
  • 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 (i.e., those fragments or homologs of troponin subunit capable of inhibiting angiogenesis.
  • the troponin subunit used in the examples is purified as described infra .
  • the dried powder (from 1 kg of muscle) is extracted overnight at 22° with 2 liters of a solution containing 1 M KCl, 25 mM Tris (pH 8.0), 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 KCl.
  • the extracts are combined and cooled to 4°C. Solid ammonium sulfate is added to produce approximately 40% 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 7.5), 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.
  • 5 mM Tris pH 7.5
  • 0.1 mM CaCl 2 0.1 mM DTT
  • Solid KCl is added to a final concentration of 1 M and 1 M KCl solution is added to bring the volume to 1 liter.
  • DDT dithiothreitol
  • EGTA ethylene glycol bis ( ⁇ -aminoethyl ether) -N, N'-tetraacetate
  • SDS sodium dodecyl sulfate
  • SE- sulfoethyl.
  • the pH is then adjusted to 4.6 by addition of HCl, 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 CaCl 2 , and 0.1 mM DTT, and dialyzed overnight against the same solution.
  • Solid KCl 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.
  • 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 0-3° 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 P0 4 , 0.06 M K 2 HP0 4 , 0.3 M KCl, 5 mM 2- mercaptoethanol, pH 6.8.
  • the homogenized muscle suspension is then stirred for 30 min. and centrifuged at 1000 x g for 20 min. The precipitate is re-extracted for 30 min. and centrifuged.
  • the residue is then washed with 2.5 liters of 5 mM 2-mercaptoethanol and centrifuged at 1000 x g for 10 min. , followed by two successive washings and centrifugations with 1.5 liters of 50 mM KCl, 5mM Tris-HCl (pH 8.1), and 5mM 2- ercaptoethanol.
  • the residue is then washed and centrifuged twice with 1.5 liters of 50 mM Tris-HCl (pH 8.1), and 5 mM 2- mercaptoethanol .
  • the volume of the residue is measured, and the residue is mixed with 0.5 volume of 3 M KCl, 50 mM Tris- HCl (pH 8.1), and 5 mM 2-mercaptoethanol.
  • 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 HCl.
  • the 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 KCl, and ImM potassium phosphate (pH 6.8), and 5 mM 2- mercaptoethanol, 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 30 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
  • Rabbit cardiac troponin is prepared in a similar manner using a pooled batch of hearts which has been stored at -20°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 KCl, 1 mM potassium phosphate (pH 7.0), and 5 mM 2- mercaptoethanol. 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.
  • DNA encoding various troponin subunits and isoforms are known in the art. See, e.g., Wu et al., 1994, DNA Cell .
  • 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.
  • a high copy number expression plasmid such as KP3998
  • E. coli transformed with the insert-containing pKP1500 vector is grown overnight at 37°C, then inoculated into 4 liters of Luria-Bertani broth (LB) medium and grown at 42°C until mid-log phase. Isopropyl- 1-thio- ⁇ -D-galactopyranoside is then added to 0.5 mM, and the culture is allowed to grow at 42°C overnight. Purification of expressed troponin subunit, fragment, or homolog may be adapted from published procedures (Reinach et al., 1988, J " .
  • 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 7.5. 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) .
  • SLM Instruments French press
  • the cell debris is pelleted; the supernatant is made 35% in saturated (NH 4 ) 2 S0 4 and stirred on ice for 30 min. After sedimentation, the supernatant is made 50 mM in NaCl, 5 mM in CaCl 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 50 mM Tris, 50 mM NaCl, 5 mM CaCl 2 , 1 mM MgCl 2 , 1 mM DTT, pH 7.5, then with 50 mM Tris, 1 mM NaCl, 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.
  • Troponin subunit, fragment, or homolog Fractions that contain troponin subunit, fragment, or homolog are pooled, dialyzed against 25 mM Tris, 6 M urea (United States Biochemical Corp.), 1 mM MgCl 2 , 1 mM DTT, pH 8.0, 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 HC0 3 , l 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 7.5), 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°C for six hours with 1 exchange against a dialysis buffer consisting of 0.5M NaCl, 20mM Hepes (pH 7.5), and 0.5mM DTT.
  • Protein concentration is determined for each subunit at 280 ⁇ .
  • the extension coefficient of Troponin I is 0.40 and Troponin T is 0.50.
  • the lyophilized recombinant protein is resuspended in a take up buffer consisting of 0.1 M NaCl, 20 mM Hepes (pH 7.5), 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 7.5), and 0.5 mM DTT.
  • Protein concentration is determined by measuring absorbance at 280 ⁇ .
  • the extension coefficient for troponin C is 0.18.
  • 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 reconstitution buffer consisting of 0.1 M NaCl, 0.1 M CaC12, 5 mM DTT, 5mM Hepes (pH 7.5). Dialysis is for 20-24 hours at 4°C with three exchanges over a dialysis buffer consisting of 0.1 M NaCl, 0.1 CaCl 2 , 0.5 mM DTT, and 5 mM Hepes (pH 7.5). Protein concentration is approximated by measuring absorption at 278 ⁇ . The troponin trimer has an extension coefficient of 0.45 at 278 ⁇ .
  • Proliferation measured bv 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 ⁇ l of lmg/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.
  • bFGF beta Fibroblast Growth Factor
  • 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 10 4 cells/ml.
  • Aliquots of 200 ⁇ l 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.
  • test samples are dissolved in 50 ⁇ l 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.
  • 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 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 .
  • EC capillary endothelial cells
  • 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.
  • cells were refed with Dulbecco's modified Eagle's medium (Gibco) with 5% 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.
  • 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 1) .
  • Percent inhibition of bovine endothelial cell proliferation (“BCE") was 54%, 86%, 83%, and 100% at concentrations of 280 nM, 1.4 ⁇ M, 2.8 ⁇ M and 5.6 ⁇ M, respectively. An inhibition of 100% was observed at a concentration of 20 ⁇ g/well (5.6 ⁇ M) .
  • IC 50 represents the concentration at which 50% inhibition of bFGF growth factor- induced 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 ⁇ g/well, but inhibition was not observed in the sample tested at 10 ⁇ g/well ( Figure 2) .
  • the percent inhibition of BCE was 33% and 46% at concentrations of 240 nM and 1.2 ⁇ M, respectively.
  • the IC 50 of troponin I was determined to be 1.14 ⁇ M.
  • Troponin T inhibited bFGF-stimulated EC proliferation at concentrations of 10 and 20 ⁇ g/well, but not at concentrations of 1 and 5 ⁇ g/well (Figure 3) .
  • BCE proliferation was inhibited 23% and 62% at 1.6 ⁇ M and 3.3 ⁇ M, respectively.
  • the IC 50 of troponin T was determined to be 2.14 ⁇ M.
  • the combination of troponin subunits C and I inhibited EC at all concentrations tested ( Figure 4) .
  • the percent inhibition of proliferation of BCE was 52%, 54% 73% and 47% at 130 nM, 645 nM, 1.3 ⁇ M and 2.6 ⁇ M, respectively.
  • the IC 50 of this combination was determined to be 110 nM.
  • 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 5) .
  • the troponin samples tested had no detectable inhibitory effect on the growth of Balb/c 3T3 cells, a non- endothelial cell type.
  • 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
  • a blind-well Boyden chamber consists of two wells (upper and lower) separated by a porous membrane. J. Exp.
  • 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
  • Migration is measured using blind well chambers (Neuroprobe, no. 025-187) and polycarbonate membranes with 8 micron pores (Nucleopore) precoated with fibronectin (6.67 ⁇ g/ml in PBS) (human, Cooper).
  • Basic FGF Takeda Co.
  • DMEM 1% calf serum
  • 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°C in 10% C0 2 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°C) and stained with hematoxylin. Migration is quantified by counting the number of cells on the lower surface in 16 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 may be used to determine whether troponin subunit, fragment or homolog is capable of inhibiting neovascularization in vivo .
  • troponin subunit, fragment or homolog 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.
  • 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.
  • 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°C and 3 ⁇ m sections are cut using a Reichert 2050 microtome. Sections are stained with toluidine blue and micrographs are taken on a Zeiss photo-microscope using Kodak TM xlOO and a green filter.
  • Example 6 Inhibition in vivo of Neovascularization by troponin as determined by the rabbit corneal pocket assay
  • mice 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.
  • the end of the corneal pocket is extended to within 1 mm of the corneal-scleral junction.
  • pockets are placed at greater distances - 2-6 mm from the corneal-scleral junction by starting the incision away from the center.
  • 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.
  • EVAc ethylene vinyl acetate copolymer
  • test substance either a piece of V2 carcinoma or some other angiogenic stimulant is implanted distal to the polymer, 2 mm from the limbus.
  • control polymer pellets that are empty are implanted next to an angiogenic stimulant in the same way.
  • capillary blood vessels start growing towards the tumor implant in 5-6 days, eventually sweeping over the blank polymer.
  • 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°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 G-75 (superfine) (Pharmacia) column, reversed-phase high- performance 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) .
  • HPLC reversed-phase high- performance liquid chromatography
  • 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 Tnl (Zhu, et al., 1994, Biochim . Biophys .
  • 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 polymerase (Stratagene) under standard PCR conditions, using forward primer (5 ' -CTCACCATGGGAGATGAGGAGAAGC-3 ' ) (SEQ ID NO: 6) and the reverse primer (5 ' -GCCTCGAGTGGCCTAGGACTCGGAC- 3') (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 Tnl was analyzed by RT-PCR as described above.
  • Total RNA 400 ng/sample
  • the design of the forward (5'- GAACACTGCCCGCCTCTGCACATC-3 ' ) (SEQ ID NO: 8) and reverse (5'- GAGCCCAGCAGCGCCTTCAGCATG-3 ' ) (SEQ ID NO: 9) primers was based on the nucleotide sequence of rat fast-twitch skeletal muscle Tnl.
  • the washed pellet was dissolved in 6 M urea, 0.5 M NaCl, 5 M HEPES, 2 mM EDTA, 5 mM DTT (pH 7.5), and nutated in the above buffer for 6-8 hours at 4°C.
  • the sample was then dialyzed against 0.5 M NaCl, 5 mM HEPES, 5 mM DTT (pH 7.5) 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 7.5).
  • inhibitory preparations were further fractionated on a Q-Sepharose HP column (Pharmacia Biotech) and tested as described below with no difference in biological activity.
  • Purified rTnl 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.
  • Tnl (purified from cartilage as described above) , recombinant Tnl (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% bro ophenol blue and 2% B-mercaptoethanol) .
  • 2x-concentrated electrophoresis sample buffer 250 mM Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 0.006% bro ophenol blue and 2% B-mercaptoethanol
  • Proteins were then transferred to nitrocellulose (Hybond-ECL, Amersham) using a Transblot apparatus (Biorad), incubated with a monoclonal antibody to rabbit skeletal muscle Tnl (Advanced Immunochemical Inc.) and developed using the ECL western blotting system according to the manufacturer's protocol (Amersham) . Results
  • Inhibitory activity eluted at an approximate molecular weight of 25,000 Da from the A-1.5m 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 Ed an degradation. The sequences of three peptide fragments were obtained and were identified as fragments of troponin I ( Figure 6) .
  • FIG. 7A Sequencing of the PCR product revealed its identity to human fast skeletal muscle Tnl ( Figure 7B) (SEQ ID NO: 16). Tnl 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) .
  • a cDNA 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.
  • recombinant Tnl migrated as a single band, at approximately 21 kDa on SDS-PAGE ( Figure 8) .
  • Capillary EC isolated from bovine adrenal cortex (Folkman, et al., 1979, Proc . Natl . Acad . Sci . USA 76, 5217-
  • BALB/c mouse 3T3 cells were maintained in DME/10, L-glutamine(292 ⁇ g/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).
  • capillary EC (2,000 cells per well) were plated on gelatinized 96-Well culture plates in DMEM supplemented with 5% (v/v) 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 Tnl. For experiments in which VEGF was used as the mitogen, 800 cells per well were plated and allowed to incubate for 3 hours before VEGF (Biomedical Technologies Incorporated; 30 ng/ml) and Tnl was added. Control wells contained cells alone and cells stimulated with bFGF or VEGF.
  • 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,
  • rTnl 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 65 nM when bFGF was used as the mitogen ( Figure 9A) and approximately 1.5 nM when VEGF was used ( Figure 9B) .
  • IC 50 the inhibitory concentration at which one observes 50% suppression of proliferation
  • SMC bovine aortic SMC and Balb C/3T3 cells were inhibited by Tnl.
  • 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 Tnl. Following incubation for 72 hrs at 37°C in 10% C0 2 , the cells were rinsed in PBS, detached by trypsinization and counted electronically. The effect of Tnl 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
  • Tnl and appropriate buffer controls were mixed in methylcellulose, disks and applied to the surfaces of the growing CAMs above the dense subectodermal plexus. Forty- eight hours following implantation of the plastic disc, the eggs were examined for vascular reactions under a dissecting scope (60x) and photographed (Moses, et al., 1990, Science
  • the CAM assay was used to determine whether rTnl 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 rTnl. 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 Tnl preparations. Over 125 CAMs were tested in this series of experiments.
  • pellets composed of bFGF (40 ng/ml), sucrose octasulfate,and Hydron were implanted into corneal micropockets of six C57BL/6 mice as previously described (U.S. Patent No. 5,837,680 to Moses et al.).
  • Troponin I 50 mg/kg was administered systemically every 12 hours by subcutaneous injection.
  • corneal angiogenesis was evaluated using slit lamp microscopy and photographed.
  • Example 12 B16-BL6 Melanoma Model.
  • Murine melanoma B16-BL6 were cultured in RPMI 1640 (Gibco) supplemented with 10% (v/v) fetal calf serum (Hyclone) , L-glutamine and NaHC0 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 implantation.
  • B16-BL6 cells (5 x 10 5 /0.2 ml) were injected into the tail veins of C57BL/6 mice (approximately 6-7 weeks old) .
  • animals were sacrificed, the number of lung surface metastases counted and the lungs weighed.
  • Recombinant peptides corresponding to fragments of rabbit (rb) Tnl (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 rbTnl fragments (SEQ ID NOS: 11-15) were prepared according to Jha et al., 1996, Biochemistry 35 (34) :11026-11035.
  • the percent inhibition of EC was 54% and 48% at concentrations of 0.1 ⁇ g/well and 0.3 ⁇ g/well, respectively.
  • the IC 50 was determined to be 0.1 to 0.2 ⁇ g/well (0.05 ⁇ M to 0.1 ⁇ M) .
  • the N'+I' (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 C'+I' (SEQ ID NO: 14) fragment.
  • Tnl inhibited EC proliferation approximately 46% at a concentration of 5 ⁇ g/well (1.2 ⁇ M) .
  • the C'+I' fragment had 25 to 50-fold EC inhibitory activity compared to the full-length Tnl.
  • troponin subunits particularly the C'+I' fragment (SEQ ID NO: 14), inhibited EC proliferation in an assay that was developed to mimic the process of neovascularization.
  • troponin subunit fragments inhibit angiogenesis.

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Abstract

L'invention concerne des compositions pharmaceutiques renfermant des quantités efficaces sur le plan thérapeutique de sous-unités C, I ou T de troponine, ou bien de fragments ou d'homologues desdites sous-unités, qui peuvent être utilisées pour le traitement de maladies ou de troubles mettant en jeu une angiogenèse anormale. L'invention concerne également le mode d'utilisation desdites compositions.
EP00916329A 1999-03-15 2000-03-14 Compositions pharmaceutiques renfermant des sous-unites de troponine, ou des fragments ou des homologues desdites sous-unites, et leur utilisation pour inhiber l'angiogenese Withdrawn EP1165059A4 (fr)

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FELDMAN LAURIE ET AL: "Troponin I inhibits capillary endothelial cell proliferation by interaction with the cell's bFGF receptor." MICROVASCULAR RESEARCH, vol. 63, no. 1, January 2002 (2002-01), pages 41-49, XP002218582 ISSN: 0026-2862 *
JHA PRAKASH K. ET AL: 'Interaction of deletion mutants of troponins I and T: COOH-terminal truncation of troponin T abolishes troponin I binding and reduces Ca-2+ sensitivity of the reconstituted regulatory system' BIOCHEMISTRY vol. 35, no. 51, 1996, pages 16573 - 16580 *
JHA PRAKASH K. ET AL: 'Photo-cross-linking of rabbit skeletal troponin I deletion mutants with troponin C and its thiol mutants: The inhibitory region enhances binding of troponin I fragments to troponin C' BIOCHEMISTRY vol. 35, no. 34, 1996, pages 11026 - 11035 *
MOSES M A ET AL: "Troponin I is present in human cartilage and inhibits angiogenesis" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 96, 16 March 1999 (1999-03-16), pages 2645-2650, XP002131230 ISSN: 0027-8424 *
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VALLINS W J ET AL: "MOLECULAR CLONING OF HUMAN CARDIAC TROPONIN I USING POLYMERASE CHAIN REACTION" FEBS LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 270, no. 1/2, 17 September 1990 (1990-09-17), pages 57-61, XP000470204 ISSN: 0014-5793 *

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