DE29724845U1 - Use of troponin subunits as angiogenesis inhibitors - used for treating e.g. tumours, ocular neovascularisation, arthritis, psoriasis, atherosclerotic plaques or nonunion fractures - Google Patents

Abstract

A novel pharmaceutical composition comprises a carrier and an angiogenesis inhibiting amount of a peptide which is: (a) an inhibitor of basic fibroblast growth factor (bFGF)-stimulated bovine endothelial cell proliferation having an IC50 of at least 10 mu M; (b) greater than 75 amino acids in length; and (c) greater than 80% homologous with a subunit selected from human fast-twitch troponin subunit C, subunit I or subunit T having the amino acid sequences given in the specification.

Description

1 INTRODUCTION

The Invention provides a pharmaceutical composition for Treatment of diseases or disorders that have abnormal angiogenesis require.

2. BACKGROUND

angiogenesis, the process of developing and forming new blood vessels is playing an important role in numerous physiological events, in both normal and pathological events. Angiogenesis is in response to specific signals and involves a complex process that is characterized by the infiltration of basal lamina by vascular endothelial cells in response on angiogenic growth signals (e), the migration of endothelial cells to the source of the signal or signals and the subsequent proliferation and formation of the capillary tube. The blood flow through the newly formed capillary is started after the endothelial cells in contact with a pre-existing capillary have come and connect with it.

at of course occurring balance between endogenous stimulators and inhibitors predominate in angiogenesis the inhibitory influences. Rastinejad et al., 1989, Cell 56: 345-355. In such rare cases, at which the neovascularization under normal physiological conditions takes place like wound healing, organ regeneration, embryonic development and reproductive processes in women, angiogenesis becomes strict regulated and spatial and limited in time. Under the conditions of pathological angiogenesis, such as. one that characterizes the solid tumor growth fail these regulatory controls.

The unregulated angiogenesis becomes pathological and stops the progress of many neoplastic and non-neoplastic diseases. A number of heavier ones Diseases is dominated by abnormal neovascularization, such as u. a. solid tumor growth and metastases, arthritis, some types of eye diseases and psoriasis. See, for example, the overviews 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, ed. Klein and Weinhouse, Academic Press, New York, pp. 175-203; Patz, 1982, Am. J. Ophthalmol. 94: 715-743; and Folkman et al., 1983, Science 221: 719-725. The process contributes to a number of pathological conditions angiogenesis to the disease state. For example, there is always more data suggesting that the growth of solid tumors depends on angiogenesis. Folkman and Klagsbrun, 1987, Science 235: 442-447.

The Maintaining corneal, lens and avascularity of the Trabekel network is for seeing as well as for the eye physiology is crucial. There are different eye diseases many of which lead to blindness, with eye neovascularization in response to the diseased Condition. These eye diseases include diabetic retinopathy, neovascular Glaucoma, inflammatory diseases and eye tumors (e.g. retinoblastoma). There are also a number of other eye disorders associated with neovascularization go hand in hand, including retrolental fibroplasia, uveitis, premature retinopathy, macular degeneration, and about 20 Eye disorders associated with choroidal neovascularization, and about 40 eye diseases associated with neovascularization the iris go along. See, for example, overviews by Waltman et al, 1978, Am. J. Ophthal. 85: 704-710 and Gartner et al., 1978, Surv. Ophthalmolo. 85: 704-710 and Gartner et al., 1978, Surv. Ophthalmolo. 22: 291-312. The treatment of these diseases is currently ongoing, especially as soon Neovascularization has occurred, is inappropriate, and often comes it to blindness. Studies have shown that vasoinhibitory Factors present in normal eye tissue (cornea and vitreous) are lost in the disease state.

On Inhibitor of angiogenesis can play an important therapeutic role with the restriction of contributions this process for have the pathological progress of the underlying disease states, as well as providing a valuable measure to study their etiology. Means, for example, tumor neovascularization can inhibit play an important role in inhibiting metastatic tumor growth play.

The components of angiogenesis that affect vascular endothelial cell proliferation, migration and entry Urgency are found to be partially regulated by polypeptide growth factors. Experiments in culture show that endothelial cells that have been exposed to a medium with suitable growth factors can be induced to elicit some or all of the angiogenic responses. Several polypeptides with endothelial growth promoting activity in vitro have been identified. Examples include acidic and basic fibroblast growth factors, transforming growth factors α and β, platelet-derived endothelial cell growth factor, granulocyte colony stimulating factor, interleukin 8, hepatocyte growth factor, proliferin, vascular endothelial growth factor and placenta growth factor. See, for example, overview by Folkman et al., 1995, N. Engl. J. Med., 333: 1757-1763.

extracts from several different tissue sources contain antiangiogenic Activity, several molecules, like platelet factor-4, Thrombospondin, protamine, and transforming growth factor B, are found to regulate various aspects of angiogenesis negative, like tent proliferation or cell migration, there was none only tissue-derived macromolecule in the booth identified the technique that could inhibit angiogenesis. Please refer e.g. overviews by Folkman, J., 1995, N. Engl., J. Med. 333; 1757-1763 and D'Amore. 1985, Prog. Clin. Biol. Res. 221: 269-283. There is therefore a strong need for further identification and characterization of chemical agents that continued prevent deregulated spread of vascularization, and the potentially broad applicability as a therapy for such diseases in which neovascularization plays a major role.

capillary endothelial ("EC") proliferate in Response to an angiogenic stimulus during neovascularization. Ausprunk and Folkman 1977, J. Microvasc. Res. 14: 153-65. On In vitro assay that responds to endothelial tent proliferation known angiogenesis-stimulating factors, such as acidic ones or basic fibroblast growth factor (aFGF or bFGF) designed to mimic the neovascularization process in vitro becomes. This type of test is the test of choice with which the stimulation of the Capillary EC proliferation due to various angiogenic factors will be shown. Shing et al., 1984, Science 223; 1296 to 1298.

The Process of capillary EC migration through the extracellular matrix to an angiogenic stimulus is also a crucial event that for angiogenesis is required. See, for example, overview by Ausprunk et al., 1977, J. Microvasc. Res. 14: 53-65. This process offers an additional one Test that mimics the neovascularization process in vitro becomes. A modification of the Boyden chamber technique was developed using who monitors the EC hike has been. Boyden et al., 1962, J. Exptl. Med. 115: 453-456, example 4. There are currently only a few tissue-derived EC cell migration inhibitors. See, for example, overview von Langer et al., 1976, Science 193: 70-72.

Beginning In the 1970s, a number of in vivo angiogenesis model bioassays were widely used used. These model systems included rabbit cornea pocket, Chicken chorioallantoic membrane ("CAM"), dorsal air sac test in rats and rabbit air chamber bioassays. For an overview see Blood et al., 1990, Biochem. et Biophys Acta 1032: 89-118. The development of controlled Release polymers, the big one molecules can release such as angiogenesis stimulators and inhibitors, were for the use of these Tests crucial. Langer et al., 1976, Nature 263: 797-800.

at The CAM bioassay uses fertilized chicken embryos in peptri dishes bred. On day 6 of development, a slice of a release polymer, such as methyl cellulose with the test sample or with a suitable control substance the vascular Membrane housed on its front edge. On day 8 of development becomes the area observed and evaluated around the implant. Avascular zones, surrounding the test implant indicate the presence of an inhibitor for the Embryo neovascularization. Moses et al., 1990, Science, 248: 1408-1410 and Taylor et al., 1982, Nature, 297: 307-312. The cans described for the previously described angiogenesis inhibitors used alone in the CAM assay are examined are 50 μg Protamine (Taylor et al., (1982)), 200 μg bovine vitreous extract (Lutty et al., 1983, Invest Ophthalmol. Vis. Sci. 24: 53-56) and 10 µg platelet factor IV (Taylor et al., (1982). The lowest described doses of Angiogenesis inhibitors that act as combinations include Heparin (50 μg) and hydrocortisone (60 μg) and B-cyclodextrin tetradecasulfate (14 µg) and hydrocortisone (60 µg) by Folkman et al., 1989, Science 243: 1490.

According to the rabbit cornea test, polymer pellets made of ethylene vinyl acetate copolymer ("EVAC") are impregnated with the test substance and surgically implanted in a pocket in the rabbit cornea about 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 another angiogenic stimulant is implanted distal to the polymer 2 mm from the limbus. Empty control polymer pellets are found in the other eye of the rabbit implanted in the same way near an angiogenic stimulant. With these control corneas, the capillary blood vessels begin to grow to the tumor implant in 5-6 days and eventually overgrow the pure polymer. In these koreas, the directional growth of the new capillaries from the limbal blood vessel to the tumor occurs at a reduced rate and is often inhibited in such a way that an avascular area around the polymer is observed. This test is quantified by measuring the maximum tube length with a stereospecific microscope.

Troponin, a complex of 3 polypeptides, is an accessory protein that is closely associated with actin filaments in the vertebrate muscle. The troponin complex works in conjunction with the muscle form of tropomyosin to mediate the Ca ²⁺ dependence of myosin ATPase activity and thereby control muscle contraction. The troponin polypeptides are designated T, I, and C according to their tropomyosin binding, inhibitory and calcium binding activities. Troponin T binds to tropomyosin and is probably responsible for the positioning of the troponin complex on the thin muscle filament. Troponin I binds to actin, and the complex formed by troponin I and T and tropomyosin inhibits the interaction of actin and myosin. Troponin C can bind up to 4 calcium molecules. Studies suggest that increasing the calcium level in the troponin C muscle causes troponin I to lose its hold on the actin molecule, causing a tropomyosin molecule shift, exposing the myosin binding sites on the actin and the myosin -ATPase activity is stimulated. Before this invention, it was not known that troponin subunits inhibited the process of endothelial cell proliferation.

3. SUMMARY THE INVENTION

The Invention relates to pharmaceutical compositions with the troponin subunits C, I or T or their fragments, in therapeutically effective amounts, that can inhibit endothelial cell proliferation. The invention relates also pharmaceutical compositions, the analogues of the troponin subunits C, I or T and analogs of their fragments contain in therapeutic effective amounts that can inhibit endothelial cell proliferation. The pharmaceutical according to the invention The composition or the therapeutic compound is suitable for the treatment of neovascular Disorders. These therapeutic compounds (referred to herein as "therapeutics") include: troponin subunits C, I, and T, and fragments and analogues thereof. The therapeutic agent according to the invention is administered to treat a cancerous condition, or with it Progression from a preneoplastic or pre-malign Prevents condition in a neoplastic or malignant condition becomes. One can use the therapeutic agent according to the invention also administer to eye disorders, that are associated with neovascularization.

3.1. definitions

The Definitions used here:

The Term "troponin subunit" means when this is not preceded by the terms C, I or T, any the troponin subunits C, I or T.

4. SHORT DESCRIPTION THE FIGURES

1 , Inhibition of bovine capillary endothelial cell (BCE) proliferation by troponin C. The percentage inhibition of bFGF-stimulated BCE proliferation is shown as a function of the troponin C concentration (nM). Percent inhibition was determined by comparing the results for cells treated with stimulus alone to those obtained for samples exposed to both stimulus and inhibitor. The sample volume was 200 μl.

2 , Inhibition of capillary BCE proliferation by troponin I. The percentage inhibition of bFGF-stimulated BCE proliferation is shown as a function of the troponin I concentration (nM). The percentage inhibition was as in 1 described determined. The sample volume was 200 μl.

3 , Inhibition of capillary BCE proliferation by troponin T. The percentage inhibition of bFGF-stimulated BCE proliferation is shown as a function of troponin T concentration (nM). The percentage inhibition was as in 1 described determined. The sample volume was 200 μl.

4 , Inhibition of BCE proliferation by troponin C and I. The percentage inhibition of bF GF-stimulated BCE proliferation is shown as a function of troponin I and C concentration (nM). The percentage inhibition was as in 1 described determined. The sample volume was 200 μl.

5 , Inhibition of capillary BCE proliferation by troponin C, I and T. The percentage inhibition of bFGF-stimulated BCE proliferation is shown as a function of the troponin C, I and T concentration (nM). The percentage inhibition was as in 1 described determined. The sample volume was 200 μl.

6 , Inhibition of tumor growth in SCID mice.

5. INCOMING DESCRIPTION OF THE INVENTION

The invention relates to compositions based on troponin subunits. The compositions allow the treatment of neovascular disorders by administering a therapeutic amount of the therapeutic compound of the invention. The therapeutic compounds (referred to herein as "therapeutics") include: troponin C, I and T subunits, fragments and analogues thereof (collectively, "Peptides of the Invention"). The peptides according to the invention are characterized by the property of inhibiting bovine endothelial cell proliferation in culture with an IC ₅₀ of 10 μM or less. The therapeutic agent according to the invention is preferably administered in order to treat a cancerous state or to prevent the progression from a pre-neoplastic or non-malignant state into a neoplastic or a malignant state. The therapeutic agent according to the invention can also be administered to treat an eye disease associated with neovascularization.

In In a preferred aspect, a therapeutic agent according to the invention is a peptide from at least one fragment of troponin C, troponin I, troponin T or the troponins C and I, which is the endothelial cell proliferation can effectively inhibit.

Examples for the Troponin subunits that can be used in the present invention include Subunits of troponin of the human fast-twitch skeletal muscle, the sequences of which are given below.

In a further embodiment, the invention comprises peptides which are homologous to the human fast-twitch skeletal troponin C (Seq. ID No. 1) or to their fragments. In one embodiment, the amino acid sequence of the peptide has at least 80% identity compared to the fragment of the human fast-twitch skeletal troponin C from which it is derived (the "prototype fragment"). In another embodiment, this identity is greater than 85%. In a more preferred embodiment, this is Id tity greater than 90%. In a most preferred embodiment, the amino acid sequence of the peptide has at least 95% identity with the prototype fragment. Fragments can be at least 10 amino acids and, in preferred embodiments, at least 50, 75, 100 and 120 amino acids.

at another embodiment The invention encompasses peptides homologous to human fast twitch Skeletal troponin I (SEQ ID NO: 2) or its fragments. In one embodiment has the amino acid sequence of the peptide at least 80% identity with the prototype of the human fast-twitch skeletal troponin I fragment. In another embodiment is this identity larger than 85%. With a stronger one preferred embodiment is this identity larger than 90%. One is the strongest preferred embodiment has the amino acid sequence the peptide has at least 95% identity with the prototype fragment. Fragments can at least 10 amino acids comprise, and in preferred embodiments at least 50, 75, 100 and 120 amino acids.

at a further embodiment The invention encompasses peptides homologous to human fast twitch Skeletal troponin T (SEQ ID NO: 3) or fragments thereof. In one embodiment has the amino acid sequence of the peptide at least 80% identity with the prototype of the human fast-twitch skeletal beta-troponin-I. In another embodiment is this identity larger than 85%. With a stronger one preferred embodiment is this identity larger than 90%. One is the strongest preferred embodiment has the amino acid sequence the peptide has at least 95% identity with the prototype fragment. fragments can at least 10 amino acids comprise, and in preferred embodiments at least 50, 75, 100, 120 and 200 amino acids.

at other specific embodiments are the peptides according to the invention Troponin C, Troponin I and Troponin T subunits of the fast-twitch, slowtwitch and heart isoforms from other mammalian species, e.g. humans, Rabbit, rat, mouse, beef, sheep and pork.

at a specific embodiment becomes a therapeutic agent according to the invention combined with a therapeutically effective amount of another molecule, that negatively regulates angiogenesis, for example, but not limited to platelet factor 4, thrombospondin-1, tissue inhibitors of the metal proteases (TIMP1 and TIMP2), prolactin (16 kd fragment), angiostatin (38 kd fragment of plasminogen), more soluble in bfGf Receptor, transforming growth factor β, interferon alpha, and placenta proliferin-related protein.

Paradoxically Neovascularization gradually reduces accessibility the tumors opposite Chemotherapy drugs due to increased interstitial pressure inside the tumor that causes vascular compression and a central neurosis is brought about. In vivo results have shown that negroes receiving angiogenic therapy increased Show chemotherapy delivery to a tumor. Teicher et al., 1994, Int. J. Cancer 57: 920-925. Thus, in one embodiment, the invention provides a pharmaceutical according to the invention Composition ready in combination with a chemotherapy drug.

at A preferred aspect is a therapeutic agent according to the invention with chemotherapeutic agents or with exposure to combined with a radioactive isotope.

The Invention is illustrated by means of examples, see below, among other things, the inhibition of capillary endothelial cell proliferation through the troponin subunits C, I and T and the measures to determine the inhibition of capillary endothelial cell migration and Inhibition of neovskularization in vivo by troponin subunits reveal.

The detailed description of the invention is intended to illustrate the Revelation and not as a limitation in the following Divided subsections.

5.1. Troponin subunits, FRAGMENTS AND ANALOG

The Invention provides pharmaceutical compositions containing troponin subunits, their fragments and analogs include. With certain aspects come from the subunits, fragments or analogs of troponin subunits from Fly, frog, mouse, rat, rabbit, pig, cow, dog, monkey or Man.

The aim is that the troponin subunit fragments can be produced by the Tro ponin sequences can be changed by substitutions, additions or deletions, which thus provide functionally equivalent molecules. These include, but are not limited to, troponin subunits, fragments, or analogs that contain, as the primary amino acid sequence, all or part of the amino acid sequence of a troponin subunit, including the altered sequences in which the functionally equivalent amino acid residues are against residues in the sequence exchanged that lead to a silent change. For example. For example, one or more amino acid residues in the sequence can be exchanged for another amino acid with a similar polarity, which acts as a functional equivalent, resulting in a silent change. Replacements for an amino acid in the sequence can be selected from other members of the class to which the amino acid belongs. For example. include the non-polar (hydrophobic) amino acids 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.

A embodiment The invention provides molecules ready which is a fragment with at least 10 (continuous) amino acids one Include or consist of troponin subunit, which the Can inhibit endothelial cell proliferation. In other embodiments this molecule exists from at least 20 or 50 amino acids the troponin subunit. These exist in specific embodiments Fragments from a troponin subunit with at least 75, 120 or 200 amino acids or include them.

at a preferred embodiment the protein is a troponin subunit from a mammal. In alternative embodiments is a troponin C, I or T subunit a mammal.

The Troponin subunit fragments according to the invention and analogues can derived from tissue (see, for example, Example 1, 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 made by various prior art methods become. The treatments that lead to their production can be based on Gene or protein level. A cloned troponin gene sequence which encodes the troponin subunits C, I or T, for example. modified by one of many prior art strategies become. Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. The sequence may be one or more at appropriate locations Restriction endonucleases are cleaved, followed by another enzymatic modification, if desired, isolated and ligated in vitro become. When producing the gene, which is a derivative or analogue a troponin subunit, you should make sure that the modified gene remains safely in the same reading frame for translation like the gene for the troponin subunit, and not in the gene region where the desired Troponinaktivität is encoded, is interrupted by translation stop signals.

The nucleic acid sequence encoding the troponin subunit can be mutated in vitro or in vivo, so that translation, initiation and / or termination sequences are generated and / or destroyed, or variations in the coding regions are generated and / or new restriction endonuclease sites are formed or beforehand existing ones are destroyed so that an in vitro modification is further simplified. Any technique for mutagenesis of the prior art can be used, including but not limited to filters Erich preparing mutagenesis in vitro (Hutchinson et al, 1978, J. Biol Chem. 253:.. 6551), use of TAB ^® linkers (Pharmacia) etc.

Treatments of the sequence for the troponin subunit C, I or T can also be done at the protein level. Included in the scope of the invention are fragments of the troponin subunit or other fragments or anaologa which are differentially modified during or after translation, for example by acetylation, phosphorylation, carboxylation, amidation, derivatization by known protective or blocking groups, proteolytic cleavage , Binding to an antibody molecule or other cell ligand, etc. Any of numerous chemical modifications can be made using known techniques, including but not limited to specific cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH ₄ , acetylation, formylation , Oxidation, reduction, etc.

In addition, fragments and analogues of troponin subunits can be chemically synthesized. For example. can be a peptide corresponding to a portion of a troponin subunit and the desired one Domain or the desired activity mediated in vitro can be synthesized using a peptide synthesizer. In addition, if desired, non-classical amino acids or chemical amino acid analogs can be introduced into the sequence of the troponin subunit as a substitution or addition. Non-classical amino acids include, but are not limited to, the D-isomers of the usual amino acids α-aminobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, Designer amino acids, such as β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids.

at a specific embodiment the invention includes a chimeric or fusion protein comprising a troponin subunit or a fragment thereof (that from at least one domain or a motif of the troponin subunit that exists for inhibition of endothelial cell proliferation is responsible), at its amino or carboxy terminus via a peptide bond to one amino acid sequence of another protein is bound. Such chimeric product ice can be made by adding the appropriate nucleic acid sequences, the the desired amino acid sequences encode, using methods of the prior art in the right Reading frames ligated together, and the chimeric product by method of the prior art. Alternatively, such chimeric Product by protein synthesis techniques, for example by using a Peptide synthesizer, getting produced.

5.2. TROPONINE PROTEIN ASSAYS, FRAGMENTS AND ANA-LOGA

The functional activity and / or therapeutically effective dose of troponin subunits, fragments and analogs examine themselves in vitro by various methods. This procedure are based on the physiological processes involved in angiogenesis involved. Although they are within the scope of the invention, they are is not intended to be the process by which the troponin subunits, Defines fragments and analogs that inhibit angiogenesis and / or through which a therapeutically effective dose of the pharmaceutical Restrict the composition.

at examining the ability of troponin subunits, fragments and analogues for inhibition or interaction with the proliferation of capillary endothelial cells (EC) in vitro, can different bioassays of the prior art are used, including, but not restricted on, incorporation of radioactive substances into nucleic acids, colorimetric tests and counting of cells.

The Inhibition of endothelial cell proliferation can be caused by colorimetric Determination of cellular acid Phosphatase activity or electronic cell counting be measured. These procedures offer quick and sensitive Screening to determine the number of endothelial cells in culture after treatment with the troponin subunit according to the invention, the derivative or analogue and one that stimulates angiogenesis Factor like aFGF. Colorimetric determination of cellular acid phosphatase is by Connolly et al., 1986, J. Anal. Biochem. 152: 136-140. According to this The method described in Example 3 becomes the capillary endothelial cells with angiogenesis-stimulating factors, such as aFGF, and a range treated by potential inhibitor concentrations. These samples are incubated to allow growth and then harvested washed, lysed in a buffer containing a phosphatase substrate, and then incubated a second time. A basic solution will be added to stop the reaction and color development is determined at 405 λ. According to Connolly et al. becomes a linear relationship between the activity of the acidic Phosphatase and endothelial cell count up to 10,000 cells / sample obtained. Standard curves for the activity The acidic phosphatase are also made from known cell numbers generated, thus confirmed is that the enzyme levels reflect the actual EC numbers. The percentage Inhibition is determined by looking at the number of cells in the specimens Were exposed to stimulus compared to those who both Stimulus as well as inhibitor were exposed.

colorimetric Tests to determine the effect of troponin subunits C, I and T on endothelial cell proliferation show that all 3 troponin subunits stimulated bFGF Disrupt endothelial cell proliferation.

Troponin C inhibited bFGF-stimulated endothelial cell proliferation in a dose-dependent manner at all concentrations tested ( 1 ). The percent inhibition of bovine endothelial cell proliferation ("BCE") was 54%, 86%, 83% and 100% at concentrations of 280 nm, 1.4 μM, 2.8 μM and 5.6 μM, respectively. 100% inhibition was observed at a concentration of 20 μg / well (5.6 μM). The IC ₅₀ represents the concentration at which 50% inhibition of aFGF growth factor-induced stimulation was observed. Troponin C IC ₅₀ was determined to be 278 nM.

Troponin I inhibited bFGF-stimulated BCE proliferation at concentrations of 1 and 5 μg / well, but no inhibition was observed in the sample tested at 10 μg / well ( 2 ). The percent inhibition of BCE was 33% and 46% at concentrations of 240 nM and 1.2 μM, respectively. The IC ₅₀ of Troponin I was determined to be 1.14 μM.

Troponin T inhibited bFGF-stimulated EC proliferation at concentrations of 10 and 20 μg / well, but not at concentrations of 1 and 5 μg / well ( 3 ). BCE proliferation was inhibited by 23% and 62% at 1.6 μM and 3.3 μM, respectively. The Troponin T IC ₅₀ was determined to be 2.14 μM.

The combination of troponin subunits C and I inhibited EC at all concentrations tested ( 4 ). The percent inhibition of BCE was 52%, 54%, 73% and 47% at 130 nM, 645 nM, 1.3 μM and 2.6 μM, respectively. The IC _{50 of} this combination was determined to be 110 nM.

The combination of the troponin subunits C, I and T was observed to inhibit aFGF-stimulated BCE proliferation by 16% at a concentration of 360 nM (5 μg / well, 5 ).

The investigated troponin samples had no detectable effect the growth of Balb / c 3T3 cells, a non-endothelial cell type.

The incorporation of radioactive thymidine by capillary endothelial cells represents a further measure with which the inhibition of endothelial cell proliferation by a potential angiogenesis inhibitor is examined. According to this method, a predetermined number of capillary endothelial cells are grown in the presence of ³ H-thymidine stock solution, an angiogenesis stimulator such as bFGF and a region of the angiogenesis inhibitor to be examined. After the incubation, the cells are harvested and the extent of thymidine incorporation is determined. See example 2.

The ability different concentrations of troponin subunits, fragments or analogues, the process of capillary endothelial cell migration in Interference with an angiogenic stimulus can be disrupted with the modified Boyden chamber technique to be examined. See section 2 and example 4, see below.

A further consequences, through which the functional activity of the troponin subunits, fragments and Analogs being investigated include the ability of the compounds to Inhibition of directed migration of capillary endothelial cells what finally for capillary tube formation leads. That ability can be determined, for example, with a test based on collagen gels plated capillary endothelial cells challenged with the inhibitor and by determining whether capillary tube structures through cultured endothelial cells have arisen.

Testing on the ability to inhibit angiogenesis in vivo include the chicken chorionic allantoin membrane test (see section 2 and example 5, see below) and rat or Rabbit Korneataschentests. See Polverini et al., 1991, Methods Enzymol. 198: 440-450. According to the Korneat ash tests a tumor of choice in the cornea of the test animal in the form of a Grain pocket implanted. The potential angiogenesis inhibitor will applied to the corneal pocket, and the corneal pocket is routinely opened Neovascularization examined. See section 2 and example 6, below.

A Embodiment according to the invention offers the combination of the troponin subunits according to the invention, Fragments or analogues to inhibit angiogenesis. Another Embodiment offers the combination of troponin subunit, Fragments, or analogs with other angiogenic inhibiting factors. These angiogenesis inhibiting factors include, but are not limited on: angiostatic steroids, thrombospondin, platelet factor IV, transforming Growth factor β, Interferons, tumor necrosis factor α, Bovine vitreous extract. Protamine, tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2), Prolactin (16 kd fragment), angiostatin (38 kd fragment from plasminogen), bFGFlöslicher Receptor, and placenta proliferin-related protein. See, for example, overviews by Folkman et al., 1995, N. Engl. J. Med. 333: 1757-1763 and Klagsbrun et al., 1991, Annu. Rev. Physiol. 53: 217-239.

The therapeutically effective dosage for inhibiting angiogenesis in vivo, defined as inhibiting capillary endothelial cell proliferation, migration and / or ingrowth of blood vessels, can be extrapolated from in vitro inhibition tests with the above-mentioned compositions according to the invention or in combination with other angiogenesis-inhibiting factors. The effective dosage depends likewise on the procedure and the measures of the supply. In some applications, such as treating psoriasis or diabetic retinopathy, the inhibitor is delivered in a topical ophthalmic vehicle. In other applications, such as in the treatment of solid tumors, the inhibitor is delivered with a biodegradable polymer implant. The protein can also be modified by polyethylene glycol treatment.

5.3. THERAPY USES

The Invention allows the treatment of diseases or disorders associated with neovascularization by administration a therapeutic according to the invention Connection. These therapeutic compounds (referred to here as "therapeutics") include troponin subunits and their fragments and analogs (e.g. as described below).

5.3.1 MALIGNITIES

Malignant and metastatic states, which deal with the therapeutic Get connections treated, include, but are not limited to those listed in Table 1 solid tumors (for an overview of these disorders, see Fishman et al., 1985, Medicine, 2nd Edition, J.B. Lippincott Co., Philadelphia):

TABLE 1

MALIGNITIES AND RELATED FAULTS

firm tumors

sarcomas and carcinomas

• fibrosarcoma
• myxosarcoma
• liposarcoma
• chondrosarcoma
• osteogenic sarcoma
• chordoma
• angiosarcoma
• endotheliosarcoma
• lymphangiosarcoma
• lymphangioendotheliosarcoma
• synovioma
• mesothelioma
• Ewing's tumor
• leiomyosarcoma
• rhabdomyosarcoma
• colon cancer
• pancreatic cancer
• breast cancer
• ovarian cancer
• Prostate Cancer
• Squamous cell carcinoma
• Basal cell carcinoma
• adenocarcinoma
• sweat glands carcinoma
• sebaceous gland carcinoma
• papillary carcinoma
• Papillaradenokarzinome
• cystadenocarcinoma
• medullary carcinoma
• bronchogenic carcinoma
• Renal cell carcinoma
• hepatoma
• Cholangiocarcinoma
• choriocarcinoma
• seminoma
• embryonal carcinoma
• Wilms' tumor
• cervical cancer
• testicular tumor
• lung cancer
• Small cell lung cancer
• bladder cancer
• sEpithelkarzinom
• sGliom
• astrocytoma
• medulloblastoma
• craniopharyngioma
• ependymoma
• sKaposi's sarcoma
• pinealoma
• hemangioblastoma
• acoustic neuroma
• oligodendroglioma
• Menangiom
• melanoma
• neuroblastoma
• retinoblastoma

5.3.2. EYE DISORDERS

• neovaskuläres Glaukom
• diabetische Retinopathie
• Retinoblastom
• retrolentale Fibroplasie
• Uveitis
• Frühgeborenen-Retinopathie
• makulöse Degeneration
• Hornhauttransplantat-Neovaskularisation

sowie anderen Augenentzündungserkrankungen, Augentumoren und Erkrankungen, die mit einer Chorion- oder Iris-Neovaskularisierung einhergehen. Siehe bspw. Übersichten von Waltman et al., 1978, Am. J. Ophthal. 85: 704–714 und Gartner et al., 1978, Surv. Opthal. 22: 291–312.Eye disorders associated with neovascularization can be treated with the therapeutic agents of the present invention, including but not limited to

• neovascular glaucoma
• diabetic retinopathy
• retinoblastoma
• retrolental fibroplasia
• uveitis
• Retinopathy of prematurity
• macular degeneration
• Corneal graft neovascularization

as well as other eye inflammation diseases, eye tumors and diseases associated with chorionic or iris neovascularization. See, for example, reviews by Waltman et al., 1978, Am. J. Ophthal. 85: 704-714 and Gartner et al., 1978, Surv. Opthal. 22: 291-312.

5.3.3. Other disorders

Other disorders, which deal with the therapeutic agents according to the invention get treated, include, but are not limited to hemangioma, Arthritis, psoriasis, angiofibroma, atherosclerosis plaques, delayed wound healing, Granulations, hemophilic Joints, hypertrophic scars, non-uniform fractures, Osler-Weber syndrome, pyogenes Granuloma, scleroderma, trachoma and vascular adhesions.

5.4. EVIDENCE OF USEFULNESS FOR THERAPY AND PROPHY LAX

The Therapeutics according to the invention can to the desired in vivo therapeutic or prophylactic activity as well as on the determination the therapeutically effective dosage are examined. These connections can For example, before testing in humans in suitable animal model systems to be examined, including, but not restricted on rats, mice, Chicken, Cows, monkeys, Rabbits, etc. For in vivo testing, before administration used any animal model system of the prior art on humans become.

5.5. THERAPEUTIC OR PROPHYLACTIC ADMINISTRATION AND COMPOSITIONS

The Invention provides treatment (and prophylaxis) methods Administration of an effective amount of a therapeutic agent according to the invention ready for an individual. In a preferred aspect, this is The therapeutic agent is essentially purified as illustrated in Example 1 is. The individual is preferably an animal, including but not limited on animals like cows, Pigs, chickens etc. and it is preferably a mammal, and most preferred a human.

The The invention also provides methods of treatment by administration to an individual of an effective amount of a therapeutic agent according to the invention in combination with a chemotherapy drug and / or with exposure across from a radioactive isotope.

The Invention also provides methods of treating patients who have entered remission with a therapeutic agent according to the invention ready to maintain an inactive state.

It are various delivery systems that are suitable for the administration of a therapeutic agent according to the invention Let it be used, e.g. encapsulation in liposomes, microparticles, Microcapsules, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432). Submission procedures include but are not restricted on topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic and oral routes. The connections can be administered by any suitable route for example by infusion or bolus injection, by absorption through Epithelial or mucous membrane linings (e.g. oral mucosa, rectal and intestinal mucosa, etc.) and can be biological along with others active agents who administered. Administration is preferred locally, but it can also be done systemically. In addition, the pharmaceutical Composition desirably via a appropriate pathway into the central nervous system, such as u. a. intraventricular and intrathecal injection; the intraventricular injection can be by a intraventricular Catheter, for example attached to a reservoir, such as an Ommaya reservoir, be relieved. The pulmonary Administration can also be used, for example, through use an inhaler or nebulizer, and by formulation with an aerosol-forming agent.

at a certain embodiment, one can desirably the pharmaceutical according to the invention Administer compositions locally to the site requiring treatment required; this can be achieved, for example, by local infusion during the operation, topical application, for example in connection with wound dressing after the operation, through a catheter, with a suppository, or by means of an implant, the implant being a porous, non-porous or is gelatinous material, including membranes such as silicone rubber membranes or fibers, but is not limited to this. In one embodiment can be administered by direct injection at the site (or earlier Site) of a malignant tumor or neoplastic or pre-neoplastic Fabric.

For the topical Application, the purified troponin subunit is combined with a carrier, so that an effective dosage is delivered based on the desired activity (i.e. in the range of an effective dosage, e.g. from 1.0 μM to 1.0 mM), thus local angiogenesis, endothelial cell migration and / or Inhibition of capillary endothelial cell proliferation is prevented. In one embodiment a topical troponin subunit, fragment or analog for the treatment of diseases such as psoriasis, applied to the skin. The carrier can be in the form of, for example, ointment, cream, gel, paste, foam, aerosol, suppositories, Plaster or gel stick is present, but is not limited to this.

On topical therapeutic to treat some of the eye disorders, which are discussed below consists of an effective amount of one Troponin subunit of a fragment or analogue in an ophthalmologically acceptable Excipients such as buffered saline, mineral oil, vegetable oils such as corn or peanut oil, Crude oil gel Miglyol 182, alcohol solutions, or liposomes or liposome-like products. Any of these compositions can also contain preservatives, antioxidants, antibiotics, Immunosuppressive and other biologically or pharmaceutically active Contain agents that have no harmful effects on the troponin subunit.

For targeted internal topical applications, e.g. for the treatment of ulcers or hemorrhoids, the troponin subunit, the fragment or the analogue composition can 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, tragacanth or gelatin; an excipient such as starch or lactose, a disintegrant such as alginic acid, Primogel or corn starch; a glide agents such as magnesium stearate or sterotes; or a lubricant such as colloidal silica. If the unit dosage form is a capsule, it may contain a liquid carrier such as fatty acid oil in addition to the material of the previous type. In addition, the unit dosage forms can also contain various other materials that modify the physical form of the dosage unit, for example coatings made of sugar, shellac or other enteric agents.

Suppositories included usually the active ingredient in the range from 0.5 to 10% by weight; oral formulations included preferably 10 to 95 active ingredient.

at a further embodiment the therapeutic agent can be in a vesicle, in particular a liposome, be delivered. See Langer, et al., 1990, Science 249: 1527-1533, Treat et al., 1989, in "Liposomes and in the Therapy of Infectious Diseases and Cancer, "Lopez-Berestein and Fidler (ed.), Liss., New York, pp. 363-365; Lopez-Berestein, ibid., Pp. 317-327.

at a further embodiment The therapeutic agent can be used in a controlled release system be delivered. In one embodiment can use an infusion pump to deliver the troponin subunit, such as the one for the delivery of insulin is used, or chemotherapy for specific Organs or tumors are used (see Langer, see above; Sefton CRC Crit. Ref. Biomed., 1987, Eng. 14: 2301; Buchwald et al., 1980, Surgery 88: 507; Saudek et al., 1989, N. Eng. J. Med. 321: 574.

at a preferred form is the troponin subunit, the fragment or analogue in combination with a biodegradable, biocompatible Polymer implant administered to the troponin subunit Fragment or analogue over releases a controlled period at a selected location. Examples for preferred Polymer materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate and their copolymers and mixtures. See Medical Applications of Controlled Release, Langer and Wise (ed.), 1974, CRC Pres., Boca Raton, Florida; Controlled drug bioavailability, drug product design and Performance, Smolen and Ball (ed.), 1984, Wiley, New York; Raner 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. Neurol. 25: 351; Howard et al., 1989, J. Neurosurg. 71: 105. In another embodiment can a controlled release system near a therapeutic target, i.e. of the brain, like this that only a part of the synthetic dose is required (see e.g. Goodson, in Medical Applications of Controlled Release, 1989, see above, vol. 2, pp. 115-138).

Other Controlled release systems are in the overview discussed by Langer (1990, Science, 249: 1527-1523).

The The invention also provides pharmaceutical compositions. These compositions comprise a therapeutically effective amount a therapeutic, and a pharmaceutically acceptable carrier.

The Formulations according to the invention can be formulated as neutral or salt forms. pharmaceutical compatible Salts include those formed with free amino groups as from hydrochloric, phosphoric, vinegar, oxalic, tartaric acids etc. are derived, and those formed with free carboxyl groups like those of sodium, potassium, ammonium, calcium, iron hydroxides, Isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine etc. are derived.

In one specific embodiment, the term "pharmaceutically acceptable" means approved by the federal or state government regulatory agency, or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and particularly in humans. The term "carrier" refers to a diluent, excipient, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be steep liquids, such as water and oils, for example those of crude oil, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like, polyethylene glycols, glycerol, 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 used as liquid carriers, in particular 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, dry skimmed milk, glycerin, propylene, glycol, water, ethanol and the like. The composition may also be in small amounts if desired Contain humectants or emulsifiers 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 tonicity agents such as sodium chloride or dextrose can also be used. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose containers made of glass or plastic.

This Compositions can the form of solutions, Suspensions, emulsions, tablets, pills, capsules, powders, formulations to the delayed Have release and the like. The composition can be as suppository with conventional Binders and carriers, such as triglycerides, microcrystalline cellulose, tragacanth or Gelatin can be formulated. The oral formulation can contain standard carriers, like pharmaceutical grades of mannitol, lactose, starch, Magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. examples of suitable pharmaceutical carriers are in "Remington's Pharmaceutical Sciences "by E.W. Martin described. These compositions contain a therapeutic effective amount of therapeutic agent, preferably in a purified form together with an appropriate amount of a carrier so that the shape is correct for a Administration to the patient is provided. The wording should for the route of administration may be suitable.

at a preferred embodiment is the composition according to routine procedures formulated as a pharmaceutical composition for intravenous administration is adapted to people. The compositions for intravenous administration are common solutions in sterile isotonic aqueous Buffer. The composition can also be a solubilizing agent if necessary and a local anesthetic contain, like lignocaine, so the pain at the injection site is alleviated. The ingredients are usually either separated or mixed together in unit dosage form, for example dry lyophilized powder or as an anhydrous concentrate in a hermetically sealed container, such as an ampoule or a sachet that show the amount of active ingredient. Should the composition through Infusion can be administered with an infusion bottle be distributed using sterile water or sterile saline pharmaceutical grade contains. If the composition is administered by injection, a Ampoule provided with sterile water for injection or saline so that the ingredients are mixed before administration become.

The Amount of the therapeutic agent according to the invention, who are for the treatment of a particular disorder or a particular one Suitable state depends on the nature of the disorder or condition, and can be done through standard clinical techniques be determined. In vitro assays, such as those described in Section 5.2. discussed can be may be used so that optimal dosage ranges can be found. The exact dose to be used in the formulation hangs too the route of administration and the severity of the disease or disorder, and should depend on the judgment of the practitioner and the respective circumstances the patient's decision. Suitable dosage ranges for intravenous administration however, are usually at about 20–500 μg of active ingredient per kg body weight. Suitable dosage ranges for intranasal administration is usually about 0.01 pg / kg body weight at 1 mg / kg body weight. Effective cans can extrapolated from dose response curves derived from model test bioassays or systems are derived in vitro or in animals.

The Invention provides a pharmaceutical package or kit comprising one or more containers, those with one or more of the ingredients of the pharmaceutical according to the invention Compositions filled are. One or more such containers can optionally be used Enclose the package insert in the form prescribed by the government agency is the manufacture, use or sale of pharmaceutical or regulated organic products, the package insert the Approval by the manufacturing authority for manufacture, use or reflects the sale for human administration.

modifications and modifications of the compositions and methods of the invention to be used by those skilled in the art from the foregoing Description visible. These modifications and variations should be within the scope of the accompanying claims.

The following non-limiting Examples show the discovery of inhibition by the troponin subunit the endothelial cell proliferation induced by an angiogenic stimulus and measures to determine the efficient dosage of the troponin subunit, Fragment or analogue for inhibition of angiogenesis and for identification of fragments and analogues of the troponin subunit (i.e. such fragments or analogues of the troponin subunit that inhibit angiogenesis can). The troponin subunit used in the examples is as described below cleaned.

Example 1: Purification the components of the troponin subunit

Cardiac troponin isolation from tissue

The methods 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 5 min in 2 l of a solution with 20 mM KCl, 1 mM KHCO ₃ , 0.1 mM CaCl ₂ , and 0.1 mM DTT touched. The suspension is filtered through gauze and the washing of the residue is repeated four times. Two liters of 95% ethanol are then added to the washed residue and the solution is filtered after 10 min. The ethanol extraction is repeated twice. The residue is then washed three times with 2 liters of diethyl ether for 10 minutes. Finally, the residue is left to dry at room temperature for 2 to 3 hours.

The dried powder (from 1 kg of muscle) is extracted overnight at 22 ° with 2 liters of a solution with 1 M KCl, 25 mM Tris (pH 8.0), 0.1 mM CaCl ₂ and 1 mM DTT. After filtration through gauze, the residue is extracted again with 1 liter of 1 M KCl.

The extracts are combined and cooled to 4 ° C. Solid ammonium sulfate is added to produce about 40% saturation (230 g per liter). After 30 minutes, the solution is centrifuged and 125 g of ammonium sulfate are 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 against 15 liters of the same solution 6 Hours and dialyzed against a fresh solution overnight.

Solid KCl is added to a final concentration of 1 M and 1 M KCl solution is added so that the volume is brought to 1 liter. The pH is then adjusted to 4.6 by adding 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 were added per liter (70% saturation). The precipitate is dissolved in a solution of 5 mM Tris (pH 7.5, 0.1 mM CaCl ₂ and 0.1 mM DTT ₎ and dialyzed against the same solution overnight. Solid KCl is added to its concentration bring to 1 M, adjust the pH to 4.6, remove the precipitate by centrifugation and neutralize the neutralized supernatant 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 fresh muscle.

Cardiac troponin isolation from tissue

Beef hearts are removed about 30 minutes after death, cut open immediately and, after rinsing the blood, immersed in ice. The left ventricle is removed and, after removing excess fat and connective tissue, ground. All subsequent extraction and preparation steps are carried out at 0 to 3 ° unless otherwise stated. The ground muscle (500 g) is in a Waning blender for 1 min in 2.5 liters of a solution with 0.09 M KH ₂ PO ₄ , 0.06 MK ₂ HPO ₄ , 0.3 M KCl, 5 mM 2- Mercaptoethanol, pH 6.8 homogenized. The homogenized muscle suspension is then stirred for 30 minutes and centrifuged at 1000 × g for 20 minutes. 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 × g for 10 min, followed by 2 successive washes and centrifugation with 1.5 liters of 50 mM KCl, 5 mM Tris-HCl (pH 8, 1) 5 mM 2-mercaptoethanol. 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. After a 16-20 hour extraction at 0 °, the suspension is centrifuged at 15,000 × g for 10 minutes. The sediment is discarded and the supernatant is adjusted to pH 7.6 with 0.05N HCl. The fibrous precipitate that forms when the pH is adjusted is removed by filtering the extract through nylon gauze. The protein, which precipitates between 30 and 50% ammonium sulfate saturation, is collected, dissolved in a solution with 1 M KCl and 1 mM potassium phosphate (pH 6.8) and 5 mM 2-mercaptoethanol and against the same solution for 4 hours and dialyzed against a fresh solution overnight. The protein solution is clarified by centrifugation at 105,000 x g for 30 min. The troponin is then chromatographed on a Hy droxyapatite column, 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 heart troponin is similarly prepared with a pooled heart approach which was stored at -20 ° C prior to extraction.

The Troponin subunits are determined by DEAE-Sephadex chromatography separated in 6 M urea. Bovine heart tropomyosin is produced from the 50% ammonium sulfate saturation supernatant from the troponin extraction scheme (see above). Ammonium sulfate for 65% saturation added and the precipitate is dissolved in 1 M KCl, 1 mM potassium phosphate (pH 7.0) and 5 mM 2-mercaptoethanol dissolved and dialyzed. The protein is then purified by hydroxyapatite chromatography.

Protein determination - The protein concentrations are through the biuret process by Gornall et al. determined as standard using bovine serum albumin. Gornall et al., 1949, J. Biol. Chem. 177: 751-766.

separation of components - one Sequence of SP-Sephadex and DEAE-Sephadex chromatography results a complete Separation of the three cardiac troponin components.

Recombinant Troponin Isolation and reconstitution protocols for Troponin I and T

DNA, encoding different troponin subunits and isoforms, exists in the state of the art. See, e.g., 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 Expression of the troponin subunit becomes DNA, which is the subunit encoded into a high copy number expression plasmid such as KP3998, subcloned using prior art recombination techniques.

For expression of the cloned cDNA, E. coli, which was 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 then until the medium Log phase grown at 42 ° C. Isopropyl-1-thio-β-D-galactopyranoside is then added to 0.5 mM and the culture is grown overnight at 42 ° C. Purification of the expressed troponin subunit, fragment or analog can be adjusted according to the 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 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 are added, and the cells are broken up in a French Press (SLM Instruments). The cell debris is pelleted; the supernatant is adjusted to 35% with saturated (NH _{4 2} SO ₄ and stirred on ice for 30 min. After sedimentation, the supernatant is adjusted to 50 mM NaCl, 5 mM CaCl ₂ , 1 mM MgCl ₂ and 1 mM DTT and then on a 1.5 × 25 cm phenyl-Sepharose (Pharmacia LKB Biotechnology Inc.) column is loaded and the column is first loaded with 50 mM Tris, 50 mM NaCl, 5 mM CaCl ₂ , 1 mM MgCl ₂ , 1 mM DTT, pH value 7.5, then washed until no more protein eluted with 50 mM Tris, 1 mM NaCl, 0.1 mM CaCl _2, 1 mM DTT, pH 7.5. The crude troponin subunit is then reacted with 50 mM Tris, 1mM EDTA, 1mM DTT, pH 7.5 eluted. Fractions containing troponin subunit, fragment, or analogs are pooled against 25mM Tris, 6M urea (United Staters Biochemical Corp.), 1 mM MgCl _2, 1 mM DTT, pH 8.0, dialyzed and loaded onto a 1.5 x 25 cm DE52 (Whatman) column. The column is eluted with a linear 0-0.6 M NaCl gradient The troponin subunit, fragment or An alogon, which elutes from the column, is dialyzed against 0.1 mM NH ₄ HCO ₄ , 1 mM β-mercaptoethanol, lyophilized and stored. The purity is determined by means of SDS polyacrylamide gel electrophoresis and UV spectrophotometry. Typical yields of 6 mg purified recombinant troponin subunit, fragment or analog / liter bacterial culture are expected.

The lyophilized recombinant protein is placed in an uptake buffer resuspended from 6 M urea, 20 mM Hepes (pH 7.5), 0.5 M NaCl, 2 mM EDTA and 5 mM DTT. The mixture is at Room temperature swiveled for 1 hour. The solution is then 6 hours with 1 Exchange for a dialysis buffer consisting of 0.5 M NaCl, 20 mM Hepes (pH 7.5) and 0.5 mM DTT, dialyzed.

The Protein concentration is for each subunit is determined at 280 λ. The extension coefficient of troponin I is 0.40 and that of troponin T 0.50.

Troponin C

The lyophilized recombinant protein is in an uptake buffer, consisting of 0.1 M NaCl, 20 mM Hepes (pH 7.5), 2 mM EDTA, and 5 mM DTT, resuspended. This solution is 6 hours at 4 ° C with a Exchange for a dialysis buffer made of 0.1 M NaCl, 20 mM Hepes (pH 7.5) and 0.5 mM DTT dialyzed.

The Protein concentration is determined by measuring the absorbance at 280 λ. The Extension coefficient for Troponin C is 0.18.

Reconstitution of the united Subunits:

Protein concentrations with the same reconstitution molar ratios of the troponin subunits C, I and T are maintained for all different combinations. These concentrations of the respective proteins are combined in a reconstitution buffer with 0.1 M NaCl, 0.1 M CaCl ₂ , 5 mM DTT, 5 mM Hepes (pH 7.5). Dialysis is carried out for 20 to 24 hours at 4 ° C. with three exchanges against a dialysis buffer consisting of 0.1 M NaCl, 0.1 mM CaCl ₂ , 0.5 mM DTT and 5 mM Hepes (pH 7 , 5).

The Protein concentration is approximated by measuring the absorbance at 278 λ. The Troponin trimer has an extension coefficient of 0.45 at 278 λ.

Example 2: Inhibition of Endothelial cell proliferation measured by DNA synthesis

The inhibitory effect of the troponin subunit, the fragment or the analogue to the proliferation of the bFGF-stimulated EC can according to the following Procedure procedures can be measured.

Endothelial cell DNA Synthesis:

On day 1, 5000 bovine capillary endothelial cells in DMEM / 10% CS / 1% GPS are plated on each well of a 96-well pre-gelatinized tissue culture plate. On day 2, the medium is changed to DMEM, 2% CS, 1% GPS, 0.5% BSA (complete medium), which is supplemented with 10 μl 1 mg / ml "cold" thymidine per 50 ml medium. On day 3, the test samples are given in complete medium in duplicate. In addition, bFGF is added to each well, except for the appropriate controls at a final concentration of 0.2 ng / well. On day 4, 5 ul of 1:13 dilute ³ H-thymidine stock solution is added to each well and the plate is incubated for 5-6 hours. After the incubation, the medium is aspirated and the residue is rinsed once with PBS, then twice for 5 min each with methanol, followed by two rinses with 5% TCA for 10 min. The cell contents of the wells are then rinsed three times with water, dried to the plate, and 100 ul 0.3 N NaOH are added to each well. The contents of the well are then transferred to scintillation counters and 3 ml of Ecolume added to each tube. The samples are then counted in the scintillation counter.

3T3 cell DNA synthesis

The DNA synthesis in bFGF-stimulated 3T3 cells creates a control which is used to evaluate the results obtained for bFGF-stimulated endothelial cell proliferation expected. DNA synthesis in the 3T3 cells can be determined according to the following procedure become.

BALB / c-3T3 cells are trypsinized at a concentration of 5 × 10 ⁴ cells / ml and resuspended. Aliquots of 200 μl are plated in 0.3 cm ² microtiter wells (Microtest II tissue culture plates, Falcon). After confluence has been reached within a period of 2 to 3 days, the cells are further incubated for at least 5 days so that the media is depleted in terms of growth-promoting factors. These growth conditions result in confluent monolayers of non-dividing Balb / c-3T3 cells. Test samples are dissolved in 50 μl 0.15 M NaCl and placed in microtiter wells together with [ ³ H] TdR. After an incubation of at least 24 hours, the medium is removed and the cells are washed in PBS. The fixation of the cells and the removal of unincorporated [ ³ H] -TdR is carried out by the following successive steps; Add methanol twice for 5 min periods, 4 washes with H ₂ O, add cold 5% TCA twice for 10 min periods, and 4 washes with H ₂ O. DNA synthesis is performed either by liquid scintillation counting or by autoradiography a modification of the method described by Haudenschild et al., 1976, M. Exp. Cell Res. 98: 175. For scintillation counting, the cells are lysed in 150 ul 0.3 N NaOH and in 5 ml insta gel liquid scintillati ons cocktail (Packard) counted with a Packard Tri-Carb liquid scintillation counter. Alternatively, autoradiography can be used to quantify the DNA synthesis by punching out the bottoms of the microtiter wells and attaching them to glass slides with silicone rubber adhesive. The slides are dipped in a 1 g / ml solution of NTB2 core detection emulsion (Kodak) and exposed for 3 to 4 days. The emulsion is developed with Microdol-X solution (Kodak) for 10 min, rinsed with distilled H ₂ O, and fixed with Rapid Fixer (Kodak) for 3 min. The autoradiograms are stained with a modified Giemsa stain. At least 1000 nuclei are counted in each well and DNA synthesis is expressed as a percentage of the labeled nuclei. Cell division is measured by counting the number of cells in microtiter wells using a grid after 40 to 48 hours of incubation with test samples.

Example 3: Inhibition of Endothelial cell proliferation, measured by colorimetric determination the activity of cellular acid phosphatase and electronic cell counting.

A rapid and sensitive screening for inhibition of EC proliferation in Response to treatment with a troponin subunit according to the invention, analog or derivative involves incubating the cells in the presence of various Concentration inhibitor and determination of the number of endothelial cells in culture based on the colorimetric determination of cellular acid activity Phosphatase, described by Connolly et al., 1986, J. Anal. Biochem. 152: 136-140.

The Inventors measured the effect of troponin on the proliferation of capillary endothelial cells (EC) in a test of ability this protein to disrupt stimulation of endothelial cell proliferation by a known Angiogenesis factor (bFGF) measures.

Capillary endothelial cells and Balb / c-3T3 cells were separately plated on gelatin-coated tissue culture plates on day 1 (2 x 10 ³ / 0.2 ml). On day 2, the cells were resuspended 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 the troponin subunit. These substances were added simultaneously in volumes that do not exceed 10% of the final volume. Wells containing phosphate buffered saline (PBS) (Gibco) and PBS + bFGF alone were used as controls. On day 5, the medium was removed and the cells were washed with PBS and placed in 100 μl buffer with 0.1 M sodium acetate (pH 5.5), 0.1% Triton X-100 ^TM and 100 mM p-nitrophenyl phosphate ( Sigma 104 phosphatase substrate) lysed. After incubation for 2 hours at 37 ° C., the reaction was stopped with the addition of 10 μl of 1 N NaOH. The color development was determined at 405 nm with a fast microplate reader (Bio-Tek).

The percent inhibition was determined by comparing the cell number of the wells that were exposed to a stimulus with those who were exposed to stimulus and troponin units.

All 3 troponin subunits inhibited bFGF-stimulated EC proliferation, such as it was determined by the colorimetric test.

Troponin C inhibited bFGF-stimulated endothelial cell proliferation in a dose-dependent manner at all concentrations examined ( 1 ). The percent inhibition of bovine endothelial cell proliferation ("BCE") was 54%, 86%, 83% and 100% at concentrations of 280 nm, 1.4 uM, 2.8 uM and 5.6 uM, respectively. 100% inhibition was observed at a concentration of 20 μg / well (5.6 μM). The IC ₅₀ represents the concentration at which 50% inhibition of the 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 μg / well, but no inhibition was observed in the sample that was examined at 10 μg / well ( 2 ). The percent inhibition of BCE was 33% and 46% at concentrations of 240 nM and 1.3 μM, respectively. The IC ₅₀ of Troponin I was determined to be 1.14 μM.

Troponin T inhibited bFGF-stimulated EC proliferation at concentrations of 10 and 20 μg / well, but not at concentrations of 1 and 5 μg / well ( 3 ). BCE proliferation was inhibited 23% and 62% at 1.6 μM and 3.3 μM, respectively. Troponin's IC ₅₀ was determined to be 2.14 μM.

The combination of troponin subunits C and I inhibited EC at all concentrations tested ( 4 ). The percent inhibition of BCE proliferation was 52%, 54%, 73% and 47% at 130 nM, 645 nM, 1.3 μM and 2.6 μM, respectively. The IC _{50 of} this combination was determined to be 110 nM.

The combination of the troponin subunits C, I and T has been observed to inhibit bFGF-stimulated BCE proliferation by 16% at a concentration of 360 nM (5 μg / well, 5 ).

The investigated troponin samples had no detectable inhibitory effect on the growth of Balb / c-3T3 cells, a non-endothelial cell type.

Example 4: Inhibition of Capillary endothelial cell migration through troponin

The Determination of ability the troponin subunit, the derivative or analogue for inhibition of the angiogenic processes of capillary EC migration in response for an angiogenic stimulus can be determined with a modification of the Boyden chamber technique be used to study the effect of the troponin subunit, the Derivatives or the analogue used on the capillary EC migration becomes. Falk et al., 1980, J. Immunol. 118: 239-247 (1980). A Boyden Chamber of Blinds consists of two depressions (an upper and a lower one), through a porous Membrane are separated. J. Exp. Med. 115: 453-456 (1962). An acquaintance Concentration of growth factor is in the lower wells housed, and a predetermined number of cells and troponin subunit, derivative or analogue is housed in the upper wells. cell bind to the top surface the membrane, migrate through the lower membrane surface and adhere to it. The membrane can then be fixed and stained for counting using the method of Glaser et al., 1980 Nature 288: 483-484 becomes.

The hike is carried out with blind-well chambers (Neuroprobe, No. 025-187) and polycarbonate membranes with 8 micron pores (nucleopore), which were precoated with fibronectin (6.67 μg / ml in PBS) (Mensch, Cooper) , measured. 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 top wells are given 5 × 10 ⁵ capillary EC / ml, and increasing concentrations of purified troponin subunit, derivative, or analog are used within 24 hours of cleaning. Control wells are given DMEM / 1, either with or without bFGF. The migration chambers are incubated for 4 hours at 37 ° C in 10% CO ₂ . The cells on the upper surface of the membrane are then wiped off by pulling the membrane over a wiper blade (neuroprobe). The cells that had migrated through the membrane to the lower surface are fixed in 2% glutaraldehyde, followed by methanol (4 ° C) and stained with hematoxylin. The migration is quantified by counting the number of cells on the bottom surface in 16 oil immersion fields and comparing this number with that for the control.

Example 5: Inhibition of Tumor growth determined by a SCID mouse model system.

The effects of recombinant troponin I on the growth of human PC-3 prostate cancer cells were determined in immunodeficient (SCID) mice in a treatment and control group of 4 mice each. Dorsal subcutaneous implantation of 10 ⁶ PC-3 cells was made and observed until a volume of 100 to 400 mm ^{3 was} achieved. After the tumor reached a threshold volume, subcutaneous injections of 50 mg / kg of recombinant troponin I were started in the treatment group twice daily.

6 shows that 28 days of treatment resulted in an approximately 50% reduction in tumor volume in the treatment group compared to the tumor volume in the control group.

Example 6: In vivo inhibition neovascularization by troponin, determined by the mouse corneal pocket test

A pellet of sucrose octasulfate, Hydron ^™ and basic fibroblast growth factor (40 ng / pellet) was placed in a corneal micro-pocket of a mouse. The mouse received subcutaneous injections of recombinant troponin I, 50 mg / kg every 12 hours, starting 48 hours before implantation

The Corneal angiogenesis was determined by slit lamp microscopy. Leading up to day 6 angiogenesis in control eyes to vessels that entered the pellet on day 6 ranged. At this point there was a 50% reduction in blood vessel density and 30% inhibition of blood vessel length in the treated animals observed.

Claims (17)

A pharmaceutical composition comprising an amount of at least one peptide and a pharmaceutically acceptable carrier therefor, characterized in that the peptide is an analog or a fragment of a troponin subunit selected from the group consisting of subunits C, I and T, and Can effectively inhibit angiogenesis. Pharmaceutical composition according to claim 1, characterized in that the composition additionally a effective amount of one of the analog or fragment of the troponin subunit different molecule, which negatively regulates angiogenesis. Pharmaceutical composition according to claim 1 or 2, characterized in that the peptide a. is an inhibitor of bFGF-stimulated bovine endothelial cell proliferation with an IC ₅₀ of 10 μM or less; b. has a length between 10 and 120 amino acids; and 'c. a homology of more than 80% to a corresponding fragment of a subunit, selected from the human fast-twitch troponin subunit C (Seq.-ID.No. 1), the human fast-twitch troponin subunit I (Seq.- ID No. 2) and the human fast twitch troponin subunit T (SEQ ID NO. 3). Composition according to claim 3, characterized in that the peptide is an analog or a fragment of a troponin subunit from a mammal is selected from the group consisting of human troponin subunits, Beef, rabbit, mouse and rat. Composition according to claim 3 or 4, characterized in that that the peptide has a homology of more than 80% to a corresponding one Fragment of the human fast twitch troponin subunit C or human fast twitch troponin subunit I has. Composition according to claim 3, characterized in that the peptide has a homology of more than 95% to a human Has troponin subunit. Composition according to claim 6, characterized in that, the human troponin subunit the human fast-twitch troponin subunit C or the human fast twitch troponin subunit I. Pharmaceutical composition according to one of claims 1 to 7, characterized in that the peptide is a fragment of at least 10 amino acids is. Pharmaceutical composition according to one of claims 1 to 7, characterized in that the peptide is a fragment of at least 50 amino acids is. Pharmaceutical composition according to one of claims 1 to 7, characterized in that the peptide is a fragment of at least 75 amino acids is. Composition according to one of claims 1 to 10, characterized in that the carrier for topical application the eye or skin is acceptable. Composition according to one of claims 3 to 10, characterized in that the angiogenesis inhibitor in one biodegradable, biocompatible polymer dispenser is. Peptide for use as an inhibitor drug of angiogenesis, characterized in that the peptide is an analog or fragment of a troponin subunit, selected from the group consisting of the subunits C, I and T, and it is effective for inhibiting angiogenesis. Peptide according to claim 10, characterized in that the peptide a. is an inhibitor of bFGF-stimulated bovine endothelial cell proliferation with an IC ₅₀ of 10 μM or less; b. has a length between 10 and 120 amino acids; and c. a homology of more than 80% to a corresponding fragment of a subunit, selected from the human fast-twitch troponin subunit C (Seq.-ID.No. 1), the human fast-twitch troponin subunit I (Seq.- ID No. 2) and the human fast twitch troponin subunit T (SEQ ID NO. 3). Peptide according to claim 13 or 14, characterized in that the peptide is a fragment of at least 10 amino acids. Peptide according to claim 13 or 14, characterized in that the peptide is a fragment of at least 50 amino acids. Peptide according to claim 13 or 14, characterized in that the peptide is a fragment of at least 75 amino acids.