WO1999001150A1 - Novel composition for treating, preventing and/or delaying ischemic cell death - Google Patents

Abstract

Described is the modulation of ischemic cell death. In particular, pharmaceutical compositions are provided comprising a protein having the biological function of acidic fibroblast growth factor (aFGF) and/or a nucleic acid molecule encoding said protein having the biological function of aFGF and/or an activator of stress - activated protein kinases (SAPK) and/or a nucleic acid molecule encoding said activator of SAPK which are particularly useful for treating, preventing and/or delaying ischemic cell death. Furthermore, methods for treating, preventing and/or delaying ischemic cell death comprising contacting organs, tissue or cells with a protein having the biological function of aFGF and/or a nucleic acid molecule encoding said protein having the biological function of aFGF and/or an activator of SAPKs and/or a nucleic acid molecule encoding said activator of SAPK are described.

Description

Title of the invention

Novel composition for treating, preventing and/or delaying ischemic cell death

Field of the invention

The present invention relates generally to the modulation of ischemic cell death. In particular, the present invention provides pharmaceutical compositions comprising a protein having the biological activity of acidic fibroblast growth factor (aFGF) and/or a nucleic acid molecule encoding said protein having the biological activity of aFGF and/or an activator of stress-activated protein kinases (SAPK) and/or a nucleic acid molecule encoding said activator of SAPK which are particularly useful for treating, preventing and/or delaying ischemic cell death. The present invention also relates to a method for treating, preventing and/or delaying ischemic cell death comprising contacting organs, tissue or cells with a protein having the biological activity of aFGF and/or an activator of SAPK and/or a nucleic acid molecule encoding said aFGF and/or activator of SAPKs.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated herein by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.

Background of the invention

Organs which depend on postmitotic cells for proper function (e.g. cardiomyocytes in the heart and neurons in the brain) are irreversibly destroyed by long lasting ischemia leading to infarction and stroke. Blood vessels occluded by atherosclerotic processes or thrombi cause these life threatening diseases. The self-defense and reparative processes of the body involving new blood vessel (collateral) formation, vessel dilatation and plaque removal are too slow to protect cardiomyocyte and neuronal cell death. Heart cardiac infarction, stroke and peripheral artery disease are the most common diseases of the Western world. The functional integrity and formation (angiogenesis) of blood vessels is regulated by tissue hormones and growth factors which themselves are activated by local hypoxia, ischemia or injury. In the treatment of subjects with arterial occlusive diseases most of the current treatment strategies aim at ameliorating their effects. The only curative approaches involve angioplasty (balloon dilatation) or bypassing surgery. The former carries a high risk of restenosis and can only be performed in certain arterial occlusive diseases, like ischemic heart disease. The latter is invasive and also restricted to certain kinds of arterial occlusive diseases. Repetitive short-term coronary occlusions have a cardioprotective effect against a subsequent long period of ischemia (Murry, Circ. 74 (1986), 1124-1136). This is called ischemic preconditioning and is considered as an endogenous protection against myocardial infarction for the in situ beating heart. Recent reports suggest the involvement of the tyrosine receptor kinase (TRK) system as a mechanism of protection (Sellke, Am. J. Physiol. 271 (1996), H713-720), and expression of TRK-ligands following brief ischemia-reperfusion has been demonstrated (Vogt, Basic Res. Cardiol. 91 (1997), 389-400). In a further study (Schaper, Ann. NY Acad. Sci. 723 (1994), 284-291 ), changes in myocardial gene expression following brief coronary occlusions under open chest conditions in the pig were found. Amongst other upregulated genes an increased mRNA of aFGF, VEGF and Insulin-like growth factor (IGF-II) could be shown. However, while a cardioprotective effect of IGF-II could be demonstrated (Vogt (1997), supra), VEGF did not display any cardioprotective effects. Thus, the role of different growth factors in modulation of cellular responses of the myocardium to ischemia and ischemia/reperfusion is yet unclear and there is no established treatment for ischemic cell death. Thus, the technical problem of the present invention is to provide compositions for treating, preventing and/or delaying ischemic cell death.

The solution to this technical problem is achieved by providing the embodiments characterized in the claims.

Description of the invention

Accordingly, the invention relates to a pharmaceutical composition comprising a protein having the biological activity of acidic fibroblast growth factor (aFGF) and/or a nucleic acid molecule encoding said protein having the biological activity of aFGF and/or an activator of stress-activated protein kinases (SAPK) and/or a nucleic acid molecule encoding said activator of SAPK and optionally a pharmaceutically acceptable carrier.

In the context of the present invention the term "acidic fibroblast growth factor" or '"aFGF" refers to proteins and peptides which can act on FGF transmembrane signaling receptors with intrinsic protein tyrosine kinase activity and lead to initiating the mitogenic activity responsible for the cytoprotective effect. Thus, according to the present invention, any aFGF or other substances which are functionally equivalent to an aFGF, namely which are capable of excelling cytoprotective effects can be used for the purpose of the present invention. These substances include compounds that have been obtained by peptide mimetics or compounds that are derived from natural aFGF and modified by recombinant DNA technology but essentially retain their biological function. The action of the aFGFs employed in the present invention may not be limited to the above-described property but they may also activate, for example, PLCγ and/or PKC.

Acidic fibroblast growth factor (aFGF) is a member of the FGF family, that consists of nine structurally related polypeptides that play a key role in numerous aspects of embryogenesis, growth, angiogenesis and cell survival (Gospodarowicz, Cell Differ. Dev. 91 (1986), 1-17; Baird, Recent Prog. Horm. Res. 42 (1986), 143-205; Clegg, J. Cell Biol. 105 (1987), 949-956; Slack, Nature 326 (1987), 197-200; Liu, Endocrinology 123(4) (1988), 2027-2031). In neuronal models and in the skeletal musculature a trophic and protective effect in the setting of ischemia and reperfusion has been suggested (Cuevas, Neuroscience Letters 197 (1995), 183-186; Cuevas, Neural. Res. 16 (1994), 365-369; Fu, Chin. Medical Journal 108 (1995), 209-214). In order to elucidate the role of aFGF in ischemia, the present inventors investigated whether aFGF has a cardioprotective effect via infusion of the compound directly into the myocardium. The advantage of this method is that systemic hemodynamic effects of the compound that could affect the infarct size measurement can be excluded (Vogt, Basic Res Cardiol. 91 (1997), 389-400).

In accordance with the present invention, it has surprisingly been found that aFGF is cardioprotective and capable of mimicking ischemic preconditioning. Experiments performed in accordance with the present invention demonstrate that local infusion of aFGF significantly decreases myocardial infarction compared to the region at risk. Thus, aFGFs or nucleic acid molecules encoding aFGFs can be used to prevent, delay or treat ischemic cell death, which is needed for the cure of several occlusive diseases and particularly useful for bypass-operations and heart transplantations. The same holds true for other compounds essentially retaining the biological function of aFGF that have been described hereinabove. The aFGFs to be employed in the pharmaceutical compositions, methods and uses of the present invention may be obtained from various commercial sources or produced as described in the prior art. The potential exists, in the use of recombinant DNA technology, for the preparation of various derivatives of aFGF comprising a functional part thereof or proteins which are functionally equivalent to aFGF as described above. In this context, as used throughout this specification "functional equivalent" or "functional part" of an aFGF means a protein having part or all of the primary structural conformation of an aFGF possessing at least the biological property of excelling cardioprotective effects or as a product obtained by peptidomimetics. The functional part of said protein or the functionally equivalent protein may be a derivative of an aFGF by way of amino acid deletion(s), substitution(s), insertion(s), addition(s) and/or replacement(s) of the amino acid sequence, for example by means of site directed mutagenesis of the underlying DNA. Recombinant DNA technology is well known to those skilled in the art and described, for example, in Sambrook et al. (Molecular cloning; A Laboratory Manual, Second Edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour NY (1989)). For example, it was found in accordance with the present invention that the cytoprotective effect is caused by the mitogenic part of the protein, since infusing of a truncated version of aFGF did not induce cardioprotection. aFGF or functional parts thereof or compounds such as proteins which are functionally equivalent to aFGFs, may be produced by known conventional chemical and semi-chemical means or by recombinant techniques employing the amino acid and DNA sequences described in the prior art (Crumley, Biochem. Biophys. Res.

Comm. 171 (1990), 7-13; Tischer, Biochem. Biophys. Res. Comm. 164 (1989), 1121-

1129; Warns, Oncogene 6 (1991), 1521-1529). For example, aFGF may be produced by culturing a suitable cell or cell line which has been transformed with a DNA sequence encoding upon expression under the control of regulatory sequences an aFGF or a functional part thereof or a protein which is functionally equivalent to aFGF.

Suitable techniques for the production of recombinant DNA and proteins are described in, e.g., Sambrook, supra. The nucleic acid molecule encoding aFGF or a functional derivative thereof can be operably linked to regulating sequences allowing the expression of said aFGF or functional derivative thereof in the cell, tissue or organ of the patient. Suitable regulatory sequences and vectors which may be employed to express the nucleic acid molecule encoding aFGF or a functional derivative thereof are known in the art and are described, for example, in Kaneda, Rinsho Byori 45

(1997), 99-105; Bank, Bioessays 18 (1996), 999-1007; Calos, Trends Genet. 12

(1996), 463-466; Malosky, Curr. Opin. Cardiol. 11 (1996), 361-368; Walther, J. Mol.

Med. 74 (1996), 379-392 and Sun, J. Invest. Dermatol. 108 (1997), 313-318.

The term "activator of stress-activated protein kinases (SAPK)" within the meaning of the present invention refers to compounds, for example organic compounds, nucleic acid molecules, (poly)peptides, etc. capable of inducing at least one member of the stress-activated protein kinases (SAPK) which is a subfamily of the mitogen-activated protein kinases (MAPK). Again, said activatiors may be obtained by peptidomimetics or by recombinat DNA techniques described above. Advantageously said member of SAPK is p46 and/or p55. It could be shown in accordance with the present invention that activation of stress-activated protein kinases (SAPK) surprisingly results in reduction of infarct size probably by the same mechanism that acts in ischemic preconditioning. All terminally differentiated cells including cardiac myocytes activate adaptative responses to stress, e.g. ischemia, which are designed to help the cell survive future insults. Recently, the stress-activated protein kinases (SAPKs) a novel subfamily of the mitogen-activated protein kinases (MAPK) have been identified to be up-regulated in ischemia/reperfusion (Canman, Nature 384 (1996), 213-214; Knight, Biochem. Biophys. Res. Comm. 218 (1996), 83-88). These kinases named after their substrate c-jun, are also known as c-jun NH2-terminal kinases (JNKs). Since SAPKs are activated in response to various cellular stresses, including UV light (Hibi, Genes Dev. 7 (1993), 2135-2148), heat shock and to inflammatory cytokines (Kyriakis, Nature 369 (1994), 156-160) the present inventors decided to study the consequences of activation of the SAPKs pathway in the pathophysiological state of ischemia. The protein synthesis inhibitor anisomycin, a known SAPK activator, was used to stimulate the kinases in an in vivo pig model. The results obtained in accordance with the present invention surprisingly revealed that anisomycin is capable of inducing cardioprotective effects due to the activation of SAPKs. Thus, it is to be expected that other activators of SAPKs can be used as well for inducing cardioprotection and thus ischemic preconditioning. Further suitable compounds capable of inducing SAPKs are described in the appended examples and/or can be easily identified through search in the literature and appropriate databases.

In a further embodiment the invention relates to a method for treating, preventing and/or delaying ischemic cell death comprising contacting organs, tissue or cells with a protein having the biological function of acidic fibroblast growth factor (aFGF) and/or a nucleic acid molecule encoding said protein having the biological function of aFGF and/or an activator of stress-activated protein kinases (SAPK) and/or a nucleic acid molecule encoding said activator of SAPK. For example, in organ transplantation the respective organ may be kept alive in the presence of the above described compounds.

In another embodiment, the invention relates to the use of a protein having the biological function of acidic fibroblast growth factor (aFGF) and/or a nucleic acid molecule encoding said protein having the biological function of aFGF and/or an activator of stress-activated protein kinases (SAPK) and/or a nucleic acid molecule encoding said activator of SAPK for the preparation of a pharmaceutical composition for preventing, treating and/or delaying ischemic cell death. Said pharmaceutical compositions can be used, for example, with or instead of the compounds commonly used for the treatment of heart stroke, such as aspirin and/or streptokinase.

The pharmaceutical composition of the invention comprises at least one protein having the biological activity of aFGF as defined above and/or at least one activator of SAPK as defined above and/or their enconding nucleic acid molecules, respectively, and optionally a pharmaceutically acceptable carrier or exipient. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, it should aso be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 10⁶ to 10¹⁶ copies of the DNA molecule. The compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery.

In a preferred embodiment, the pharmaceutical compositions, methods and uses of the invention may be employed for diseases wherein said cell death is caused by a vascular disease or a cardiac infarct or a stroke. In a particularly preferred embodiment, the pharmaceutical compositions, methods and uses of the invention are for the treatment of subjects suffering from arteriosclerosis, a coronary artery disease, a cerebral occlusive disease, a peripheral occlusive disease, a visceral occlusive disease, a mesenterial arterial insufficiency or an ophthamic or retenal occlusion.

In a further preferred embodiment, the pharmaceutical compositions, methods and uses of the invention are for the treatment of subjects before, during or after exposure to an agent or radiation or surgical treatment which damage or destroy arteries.

In a most preferred embodiment, the application of the pharmaceutical compositions, methods and the uses of the invention result in ischemic preconditioning and/or ischemic tolerance of organs and/or tissues.

In a preferred embodiment, the protein having the biological function of aFGF used in the pharmaceutical compositions, methods and uses of the invention is a recombinant aFGF. DNA sequences encoding aFGFs which can be used in the methods and uses of the invention are described in the prior art. Moreover, DNA and amino acid sequences of aFGFs are available in the GenBank database. As described above, methods for the production of recombinant proteins are well-known to the person skilled in the art; see, e.g., Sambrook et al., supra.

In a preferred embodiment of the pharmaceutical composition, methods or uses described above, the SAPK is (are) p46 and/or p55. As will be shown in the appended examples the cardioprotective effect of upregulated SAPKs is predominantly due to activation of the SAPKs p46 and p55.

In a particularly preferred embodiment of the invention, the activator of the SAPK comprised in the pharmaceutical compositions, methods or uses is anisomycin or a functional derivative or analogue thereof which may be obtained, e.g., by peptidomimetics. As described in the appended examples anisomycin was found to induce cytoprotective effects due to SAPK activation. Anisomycin (1 ,4,5-trideoxy-1 ,4-imino-5-(4-methoxyphenyl)-D-xylo-pentitol 3-acetate; [2R-(2α,3α,4β)]-2-[(4-methoxyphenyl)methyl]-3,4-pyrrolidinediol 3-acetate; 2-p- methoxyphenylmethyl-3-acetoxy-4-hydroxypyrrolidine; Flagecidin. C₁₄H₁₉NO₄; mol wt 265.30. C 63.38%, H 7.22%, N 5.28%, O 24.12%) is a protein synthesis inhibiting antibiotic originally isolated from Streptomyces griseolus and S. roseo-chromogenes; Sobin, J. Am. Chem. Soc. 76 (1954), 4053; U.S. pat. 2,691 ,618. The structure, stereochemistry and synthesis is described in Beerebom, J. Org. Chem. 30 (1965), 2334, Schaefer, J. Org. Chem. 33 (1968), 166; Butler, J. Org. Chem. 33 (1968), 2136; Oida, Chem. Pharm. Bull. 16 (1968), 2086 and Chem. Pharm. Bull. 17 (1969), 1405; Felner, Helv. Chim. Acta 53 (1970), 754; Verheyden, Pure Appl. Chem. 50 (1978), 1363 and Schumacher, Am. Chem. Soc. 104 (1982), 6076. The potential exists in the use of chemical derivatization for the preparation of various functional derivatives and analogues of anisomycin; see, e.g., Hall, J. Med. Chem. 26 (1983), 469-475. For the purpose of the present invention "functional derivative and analogue" of anisomycin means molecules the chemical structure of which is based on that of anisomycin and which are capable of inducing cardioprotective effects. The cardioprotective effects of the anisomycin-derived compounds may even be enhanced as compared to the natural antibiotic. Methods for the preparation of such derivatives and analogues are well known to those skilled in the art and are described in, for example, Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, New York, USA. Furthermore, said derivatives and analogues can be tested for cytoprotective effects according to methods known in the art or as described, for example, in the appended examples.

In a further preferred embodiment, the pharmaceutical composition is designed for administration in conjugation with growth factors, preferably fibroblast growth factor such as basic fibroblast growth factor (bFGF), insulin-like growth factor-ll (IGF-II) or vascular endothelial growth factor (VEGF). The latter embodiment is particularly suited for enhancing angiogenesis as well as ischemic preconditioning. Pharmaceutical compositions comprising, for example, aFGF and/or anisomycin, and another growth factor such as VEGF may be used for the treatment of peripheral vascular diseases or coronary artery disease. In another preferred embodiment, the method of the invention comprises

(a) obtaining cells, tissue or an organ from a subject;

(b) introducing a nucleic acid molecule encoding the protein having the biological function of aFGF or the activator of SAPK into said cells, thereby conferring expression of the protein having the biological function of aFGF or activator in a form suitable for the interaction of said protein having the biological function of aFGF or said activator of SAPK with its receptor; and

(c) reintroducing the cells, tissue or organ obtained in step (b) into the same or a different subject.

It is envisaged by the present invention that the proteins having the biological activity of aFGF, the activators of SAPK and the nucleic acid molecules encoding said proteins or activators of SAPK are administered either alone or in combination, and optionally together with a pharmaceutically acceptable carrier or exipient. Said nucleic acid molecules may be stably integrated into the genome of the cell or may be maintained in a form extrachromosomally, see, e.g., Calos, Trends Genet. 12 (1996), 463-466. On the other hand, viral vectors described in the prior art and cited above may be used for transfecting certain cells, tissues or organs.

Furthermore, it is possible to use a pharmaceutical composition of the invention which comprises a nucleic acid molecule encoding aFGF in gene therapy. Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenoviruses, and adeno-associated viruses, among others. Delivery of nucleic acid molecules to a specific site in the body for gene therapy may also be accomplished using a biolistic delivery system, such as that described by Williams (Proc. Natl. Acad. Sci. USA 88 (1991 ), 2726-2729).

Standard methods for transfecting cells with nucleic acid molecules are well known to those skilled in the art of molecular biology, see, e.g., WO 94/29469. Gene therapy to prevent or decrease the development of ischemic cell death may be carried out by directly administering the nucleic acid molecule encoding aFGF to a patient or by transfecting cells with said nucleic acid molecule ex vivo and infusing the transfected cells into the patient. Furthermore, research pertaining to gene transfer into cells of the germ line is one of the fastest growing fields in reproductive biology. Gene therapy, which is based on introducing therapeutic genes into cells by ex-vivo or in- vivo techniques is one of the most important applications of gene transfer. Suitable vectors and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., WO94/29469, WO 97/00957 or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and references cited therein. The nucleic acid molecules comprised in the pharmaceutical composition of the invention may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) containing said nucleic acid molecules into the cell. Preferably, said cell is a germ line cell, embryonic cell, or egg cell or derived therefrom.

It is to be understood that the introduced nucleic acid molecules encoding the protein having the biological activity of aFGF or activator of SAPK express said protein or activator after introduction into said cell and preferably remain in this status during the lifetime of said cell. For example, cell lines which stably express said protein having the biological activity of aFGF or said activator of SAPK may be engineered according to methods well known to those skilled in the art. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the recombinant DNA molecule or vector of the invention and a selectable marker, either on the same or separate vectors. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows for the selection of cells having stably integrated the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the protein having the biological activity of aFGF or said activator of SAPK.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, Cell 11 (1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska, Proc. Natl. Acad. Sci. USA 48 (1962), 2026), and adenine phosphoribosyltransferase (Lowy, Cell 22 (1980), 817) in tk^", hgprt^" or aprt^" cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, Proc. Natl. Acad. Sci. USA 77 (1980), 3567; O'Hare, Proc. Natl. Acad. Sci. USA 78 (1981 ), 1527), gpt, which confers resistance to mycophenolic acid (Mulligan, Proc. Natl. Acad. Sci. USA 78 (1981 ), 2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, J. Mol. Biol. 150 (1981 ), 1 ); hygro, which confers resistance to hygromycin (Santerre, Gene 30 (1984), 147); or puromycin (pat, puromycin N-acetyl transferase). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047); and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.).

Thus, in a preferred embodiment, the nucleic acid molecule comprised in the pharmaceutical composition, preferably for the use of the invention is designed for the expression and secretion of the aFGF or activator of SAPK by cells in vivo in a form suitable for the interaction with its receptor by, for example, direct introduction of said nucleic acid molecule or introduction of a plasmid, a plasmid in liposomes, or a viral vector (e.g. adenoviral, retroviral) containing said nucleic acid molecule.

In a preferred embodiment, the pharmaceutical composition in the use of the invention is designed for administration by intracoronar, intramuscular, intravenous, intraperitoneal or subcutenous routes. In the examples of the present invention the human form of the aFGF protein was administered locally via osmotic minipump.

In a particular preferred embodiment of the present invention, said protein having the bilogical activity of aFGF is aFGF.

In a further embodiment, the present invention relates to the use of any one of the beforedescribed nucleic acid molecules in gene therapy, for example, for curing inborn or aquired ischemic diseases. These and other embodiments are disclosed or are obvious from and encompassed by the description and examples of the present invention. Further literature concerning any one of the methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries, using for example electronic devices. For example the public database "Medline" may be utilized which is available on internet, e.g. under http://www.ncbi.nlm.nih.gov/ PubMed/medline.html. Further databases and addresses can be obtained using http://www.lycos.com.

The pharmaceutical compositions, uses, methods of the invention can be used for the treatment of all kinds of diseases hitherto unknown as being related to or dependent on the modulation of ischemic cell death. The pharmaceutical compositions, methods and uses of the present invention may be desirably employed in humans, although animal treatment is also encompassed by the methods and uses described herein.

Brief description of the figures

Figure 1 : Experimental groups for testing with aFGF and bFGF. Six groups of animals were studied:

Control animals (group I) were subjected to 60 min LAD-occlusion (CO) and 120 min reperfusion (REP). The groups II and III received various compounds: aFGF (0.5-1 μg/ml) and bFGF (2 μg/ml) by means of intramyocardial microinfusion (IM) for 60 min prior to the LAD occlusion. Groups IV and V were treated with the growth factor antagonist suramin (0.4 μg/ml) or the tyrosine kinase inhibitor genisteine (0.35 μg/ml) prior to FGF infusion. Group VI was treated with the truncated aFGF (0.5-1 μg/ml).

Figure 2: Infarct areas: Treatment with the growth factor antagonist suramin and the tyrosine kinase inhibitor genisteine in comparison with the aFGF and bFGF induced cardioprotection. Control: 83.4±2.8%, aFGF: 51.8±7.7, bFGF: 57.2±6.5%, suramin: 77.0±1.2%, genistein: 77.2±2.4%, truncated aFGF:78.3±0.73%; Figure 3: Hemodynamic data during IM. Systemic hemodynamics (mean±SEM, error bars hidden behind the used symbols) remained unchanged during intramyocardial microinfusion. Compared to baseline values (t=0), none of the registered parameters was changed significantly as determined by Bonferroni-adjustment. LVP: left ventricular pressure, AOP: aortic pressure, HR: heart rate, dP/dt: first derivative of left ventricular pressure.

Figure 4: Intramyocardial microinfusion of aFGF. The needles for IM (arrows, 26 gauge) were placed in pairs into the subsequent ischemic part of the left ventricle. The fluorescent microspheres demarcate the none fluorescent area of risk. After TTC-staining myocardial protection was defined as stained tissue surrounding the microinfusion-needles in transmurally infarcted myocardium.

Figure 5: Reduction of infarcted areas by bFGF. Treatment with bFGF significantly reduced infarcted area (IA) normalized to ischemic area (RA) as determined by TTC-staining and planimetry, (shown in double exposure technique).

Figure 6: Prevention of cardioprotection. Infusion of A: Suramin, B: Genistein prior to the aFGF/bFGF treatment prevented cardioprotection. Fluorescent microspheres demarcate the none fluorescent risk area, whereas TTC- staining shows the infarcted area (shown in double exposure technique). The area around the needles does not show any cardioprotection.

Figure 7: Localization of aFGF (green), counterstaining with phalloidin (red).

A: In control tissue endogenous aFGF was detected in the extracellular matrix and in perinuclear localization of myocytes. B: Accumulation of exogenous aFGF is found mainly in a perinuclear localization within numerous myocytes. Figure 8: Localization of bFGF (green).

A: In control tissue bFGF was found in endothelial and interstitial cells in a perinuclear localization.

B: Accumulation of exogenous bFGF in the perinuclear space of interstitial cells. This localization is comparable to that seen in control tissue.

Figure 9: Experimental groups for anisomycin testing. Two groups of animals were studied: control animals (group 1 , n=6) were subjected to 60 min LAD occlusion and 120 min reperfusion. Group 2 (n=6) was treated with anisomycin (500 μmol) for 60 min prior to the index ischemia. Animals of group 3 (n=4) received AN/KHL and were biopsied at 0, after 10 and 30 min of infusion.

Figure 10: Hemodynamic data. Systemic hemodynamic data from all experiments shown as mean±standard error of mean. Minutes -60 to 0: intramyocardial microinfusion. Minutes 0 to 60 min: LAD occlusion. Minutes 61-180 min: reperfusion. AOP = aortic pressure, AOP syst. = aortic systolic pressure, AOP diast. = aortic diastolic pressure, HR = heart rate.

Figure 11 : Infarct areas. Treatment with the SAPK activator anisomycin reduces infarct sizes significantly from 83.4+2.8% (control) to 48.1+5.1% (p<0.01).

Figure 12: Intramyocardial microinfusion of anisomycin. The needles for IM (arrows, 26 gauge) were placed in pairs into the subsequent ischemic part of the left ventricle. The fluorescent microspheres demarcate the none fluorescent area of risk. After TTC-staining myocardial protection was defined as stained tissue surrounding the microinfusion-needles in transmurally infarcted myocardium. Figure 13: Graphs showing the quantitative changes in activities of SAPKs p46 and p55 after 10 and 30 minutes of infusion. Quantitative analysis of gels was performed using Phosphorimage SF (Molecular Dynamics). Data are expressed as a percentage of control value (control nonischemic tissue) and KHL treated tissue, each bar represents the mean ± S.E.M.

Figure 14: Stimulation of SAPKs in the cytosolic fractions isolated from biopsies obtained from control tissue (C), at different time points of anisomycin and KHL infused tissue (10, 30 min). The in gel GST-c-jun kinase assay showed the activation of 46- and 55 kDa protein kinases (p-46, p-55). The maximal activation of anisomycin infused tissue was reached after 30 min, KHL treated tissue induced an insignificant increase of JNKs activity at both time points.

A better understanding of the present invention and of its many advantages will be had from the following examples, given by way of illustration.

Examples

Example 1 : The in vivo animal test system

The experimental protocol described in this study was approved by the Bioethical Committee of the District of Darmstadt, Germany. Furthermore, all animals in this study were handled in accordance with the guiding principles in care and use of animals as approved by the American Physiological Society and the investigation conformed with the Guide for care and use of laboratory animals published by the US National Institutes of Health. Animal preparation

Twenty-eight male castrated German landrace-type domestic pigs with body weights between 32.8 kg and 38,6 kg (35.7+3.3 kg) were premedicated with 2 mg-kg^"1 BW i.m. azaperone and 2 mg-kg^-'' BW s.c. piritramid 30 minutes prior to the initiation of anaesthesia with 10 mg-kg^-1 BW metomidate. After endotracheal intubation a bolus of 25 mg-kg^-1 BW of α-chloralose was given intravenously. Anaesthesia was maintained by a continuous intravenous infusion of 25 mg-kg^-1 α-chloralose. The animals were ventilated artificially with a pressure-controlled respirator (Stephan Respirator ABV, F.

Stephan GmbH, Quickborn, Germany) with room air enriched with 2 I min^-1 oxygen. Arterial blood gases were analyzed frequently to guide adjustment of the respirator settings. Additional doses of piritamid (10 mg) were given i.v. every 60 minutes. Both internal jugular veins were cannulated with polyethylene tubes for administration of saline, piritramid and α-chloralose. Arterial sheath catheters (7F) were inserted into both common carotid arteries. To measure aortic blood pressure, the left sheath was advanced into the aortic arch and connected with a Statham transducer (P23XL, Statham, Puerto Rico). A 5F high fidelity catheter tipped manometer (Millar Instruments, Houston, Texas, USA) was inserted via the right common carotid artery into the left ventricle to measure left ventricular pressure and to calculate its first derivative (LV dP/dt). The chest was opened by a midsternal thoracotomy and the heart was suspended in a pericardial cradle. A loose ligature was placed halfway around the left anterior descending coronary artery (LAD), and was subsequently tightened to occlude the vessel. In the pigs subjected to intramyocardial microinfusion, eight 26 gauge needles were connected by tubing with a peristaltic pump (Minipulse, Gilson, Germany) were placed in pairs along the LAD into the myocardium perpendicular to the epicardial surface. After preparation a stabilization period of 30 minutes was allowed and the different experimental protocols were started.

For anisomycin testing sixteen castrated male landrace-type domestic pigs (34.6+2.7 kg) were used and treated as described above. One channel was reserved for the infusion of the Krebs buffer which served as an additional control. Chemicals

Azaperone, metomidate and piritamid were purchased from Janssen Pharmaceutica, Neuss, Germany. Anisomycin is purchased from Biomol Feinchemikalien GmbH, Hamburg, Germany. Anisomycin was dissolved in Krebs-Henseleit buffer (pH: 7.4). Myelin basic protein, PKI, EGTA, PMSF, bovine serum albumin, ATP, dithiotreitol, SDS-PAGE reagents and polyclonal anti-ERK1/2, α-chloralose, TTC and Genistein (0.3 μg/ml) were obtained from Sigma Chemical Co. aFGF (0.5 μg/ml) and bFGF (2.0 μg/ml) were purchased from Biotrend. Suramin (0.5 μg/ml) was obtained from Research Biochemicals Inc., Natick, MA, USA. All test compounds were dissolved in Krebs Henseleit buffer (pH 7.4).TTC was dissolved in 100 mmol/L phosphate buffer (pH 7.0).The fluorescent zinc-cadmium sulfide microspheres (diameter 2-15 mm) were purchased from Duke Scientific Corporation, Palo Alto, California, USA. Horseradish peroxidase-l inked goat anti-rabbit immunoglobulin, the enhanced chemiluminescence (ECL) reagents, the nitrocellulose membrane, the rainbow molecular weight markers, autoradiography films and γ³²P-ATP were obtained from Amersham International. Tween 20 was purchased from Serva. The specific polyclonal antibodies against JNK and p38 kinase were purchased from Santa Cruz Biotechnology. Recombinant c-jun containing the N-terminal regulatory region of amino acids 1-135 and recombinant MAPKAP2 (GST-MAPKAP ₄₆^₀₀) were expressed as glutathione S-transferase fusion protein in Escheria coli and purified by glutathione-Sepharose (Pharmacia) chromatography.

Exclusion criteria

Perfusion sites were excluded from evaluation if systolic-diastolic cardiac movements caused dislocation of the needles or if the TTC-staining areas of protected and control tissue were not clearly demarcated by necrotic tissue inbetween. Succesfull countershock defibrillation was not a criterium for exclusion. In one animal treated with aFGF countershocking caused dislocation of some microinfusion needles. These infusion sites were excluded from evaluation. Experimental groups

The present study consisted of five experimental groups for aFGF (Fig 1 ) and two experimental groups for anisomycin (Fig. 9). Group I (the control group) was subjected to 60 minutes of occlusion and two hours of reperfusion. In group 2 (aFGF or Fig. 1 and anisomycin Fig. 9) and group 3 (bFGF, Fig. 1) the peptides were administered 60 min prior to the index ischemia of 60 minutes and the following reperfusion period of 2 hours. For aFGF experiments Group 4 was treated with Suramin, an nonspecific FGF antagonist 60 min prior to the 60 min microinfusion of aFGF or bFGF. Group 5 was treated with Genistein for 60 minutes prior to the aFGF respectively bFGF microinfusion. Animals of Group 3 (Fig. 9) received AN/KHL and were biopsied at 0, after 10 and 30 min of infusion. Cyclohexyladenoslne was locally infused as a positive control respectively Krebs-Henseleit as a negative control. Additionally a small group of three animals was treated with truncated aFGF devoid of the mitogenic part of the protein; the experimental conditions were the very same as with aFGF/bFGF.

Determination of infarct size

At the end of the experimental protocol concentrated fluoresceine (1%) was injected i.v. as a reperfusion marker (hearts with non-reperfused risk regions were excluded). Thereafter the left anterior descending (LAD) coronary artery and the aorta were occluded and 200 mg of zinc cadmium fluorescent microspheres (Duke Scientific) in 20 ml Ringer^'s solution were injected into the ascending aorta, while the descending aorta was clamped. After uptake of the microspheres (at about 2 minutes after injection), the animals were sacrificed with an intravenous bolus of 20% potassium chloride to achieve cardiac arrest. The heart was excised and both atria and the right ventricle were removed. The left ventricle was cut into slices along the pairwise inserted microinfusion-needles perpendicular to the LAD. Heart slices were weighed and afterwards incubated at 37°C in triphenyltetrazolium chloride (TTC) (1 %) in PBS, pH 7.0 for 15 min. Myocardium at risk of infarction was identified by the presence of fluoresceine and by the absence of fluorescent microspheres at a wavelenght of 366 nm. The infarcted area was de-marcated by the absence tetrazolium precipitation. The slices were photographed under UV- and tungsten lamp light by double exposure and the color slides were used for further planimetric evaluation.

An index ischemia of 60 minutes followed by a 120 minutes of reperfusion leads to a confluent area of transmural infarction. Non-infarcted myocardium near the needle tip in an index ischemia position is always a definite sign of protection, because it is never observed spontaneously or with KHB-infusions.

Example 2: Infusion of aFGF decreases myocardial infarction

Human recombinant aFGF (0.5 μg/ml) and bFGF (2 μg/ml) were applied by means of direct intramyocardial infusion (Figure 4) (IM) as described in Example 1 for 60 min prior to a 60 min LAD-occlusion and 120 min reperfusion. Direct infusion maximizes local concentrations and minimizes systemic side effects. As shown in Figure 3 the hemodynamic parameters remained unchanged during intramyocardial microinfusion. Compared to control values no significant change was observed in any measured parameter. In addition, no ventricular premature beats were detected during the infusion. Hemodynamic data are expressed as mean ± standard error of mean (SEM). The test of statistical differences of the hemodynamic measurements was done by Boneferroni-adjustments and by pairwise mean differences (ANOVA, Scheffe-test). A p value smaller than 0.05 was considered statistically significant. Figure 2 depicts the effect of intramyocardial microinfusion of aFGF and bFGF compared to the control group. Both compounds were administered for 60 min before index ischemia. aFGF induced an infarct size reduction (Figure 4) of 51.8± 7.7% vs. control 83.4±2.8%, p<0.05. To induce cardioprotection (Figure 5) by bFGF a fourfold higher concentration was needed (57.3±6.5% vs 83.4±2.8%, p<0.05). Truncated aFGF did not induce cardioprotection (78.3±0.73%). Treatment with suramin (77.0±1.2% vs 83.4±2.8%) or genistein (77.2±2.4% vs 83.4±2.8%) in the presence of FGF abolished cardioprotection (Figure 2 and 6).

In contrast to VEGF both aFGF and bFGF showed a cytoprotective effect which significantly reduced infarct size. This allows us to conclude that not all tyrosine kinase receptor ligands afford protection, or that perhaps the receptor for VEGF was inactive in myocardium prior to index ischemia. A cytoprotective effect for bFGF has been described previously in various animal models of neuronal ischemia (Fisher, Journal of Cerebral Blood Flow and Metabolism 15 (1995), 953-959; Jiang, Journal of the neurological Sciences 149 (1996), 173-179; Bethel, Stroke 28 (1997), 609-615). Since neurons and cardiac myocytes are terminally differentiated cells that cannot compensate cell loss by cell division there seems to be a concordance in their need for cytoprotection. In the present study a delay of the necrotizing effect of a 60 minutes index ischemia was found. Increasing the index ischemia from 60 minutes to 90 minutes showed that the cardioprotection vanished. Since a 45 minutes occlusion is sufficient to produce a transmural infarction (previous data), then the time benefit of cardioprotection is more than 15 but probably somewhat less than 45 minutes. Thus a+bFGF infusion has increased the time required for complete infarction. Investigating the signal transduction suramin, a growth factor antagonist, was infused prior to the aFGF or bFGF infusion to determine whether the observed effects are receptor mediated. Suramin prevented FGF-induced cardioprotection. Until now two classes of FGF receptors have been identified. One of them consists of a group of transmembrane signaling receptors with intrinsic protein tyrosine kinase activity. These bind FGF with high affinity and are responsible for initiating the biological (i.e., mitogenic) activity (Dionne, EMBO J. 9 (1990), 2685-2692). The second group of receptors includes a family of cell surface heparan sulfate proteoglycans that bind FGF with low affinity but high capacity. These receptors do not transmit a biological response (Ruoslahti, Cell 64 (1991 ), 867-869) and are regarded as a continuous source of bound FGF to the high affinity receptors (Klagsbrun, Cell 67 (1991 ), 229-231). In vitro experiments showed that they function as accessory molecules for high affinity FGF receptors (Spivak-Kroizman, Cell 79 (1994), 1015-1024). Furthermore exogenously added heparin/heparin-like molecules can function as a soluble form of low affinity FGF receptor, obviating the need for cell surface heparan sulfate molecules to act as low affinity receptors (Burgess, Annu. Rev. Biochem. 58 (1989), 575-606). Digestion of cell surface heparan sulfate or prevention of its sulfation has been shown to abolish FGF binding to its high affinity receptors and thus to inhibit the biological activity of FGF (Rapraeger, Science 252 (1991), 1705-1708). So far the present findings showed that the observed cardioprotection is receptor-mediated but this does not necessarily imply a receptor activation, since various pathways do exist. FGF ligand binding induces receptor dimerization, resulting in autophosphorylation of the receptor intracellular tyrosine residues {Coughlin, J. Biol. Chem. 263 (1988), 988-993) and initiation of downstream signaling via the MAPKinases p42 and p44 ending in the activation of transcription factors. To elucidate the role of tyrosine kinases it was tried to biock the signal cascade by applying genistein, a tyrosine kinase inhibitor prior to the FGF infusion. The result of this study showed that tyrosine kinase inhibition prevented the cardioprotection by aFGF and that receptor activation is involved as well. The results of these experiments suggest that the cytotrophic effect of aFGF is due to the growth factor^'s mitogenic activity and that the hemodynamic component plays no part in it. This assumption was verified in a series of experiments infusing a truncated aFGF i.e. the non-mitogenic but hemodynamically active epitope (Cuevas (1991 ), supra), under the same conditions as the fully active growth factor. The results of these experiments showed no cardioprotection, excluding that the effect might be due to vasod ilatation.

Example 3: Infused FGF is taken up by myocytes (aFGF) and non-myocytes (bFGF) and is translocated to the nucleus

Immunohistochemical studies showed the presence of aFGF in control and experimental tissue in the extracellular matrix of the myocardium. Tissue samples were mounted with Tissue Tek (OCT compound, Miles Inc., U.S.A.) and cryosections with a thickness of 5 mm were cut with a cryostat (Leica, Benzheim, Germany). The sections were collected on gelatine covered glass slides, air dried and fixed with 4% paraformaldehyd for 10 minutes. Thereafter the slides were incubated in a BSA (0.5%) and glycin (0.5%)/Triton (0.05%) solution for 20 minutes each. Rinsing in phosphate buffered saline (PBS, pH:7.4) was followed by incubation with diluted primary antibodies (mouse monoclonal antibodies against human recombinant aFGF, respectively mouse monoclonal antibodies against human recombinant bFGF, both obtained from Sigma) for 12 hours at 4°C temperature. The sections were washed in PBS three times (3 minutes each) and incubated with the biotinylated second antibody (Biotin SP-conjugated antimouse, Dianova) for 1 hour. After repeated rinsing the third incubation was carried out with Cy-2-conjugated streptavidin (Rockland) for 30 minutes. Nuclei were stained with Aminoactinomycin D (Molecular Probes, Eugene, U.S.A.) diluted 1 :100; contractile proteins were stained with Phalloidin (Sigma, Chemical Co) diluted 1 :200 for 30 minutes. After rinsing in PBS, the sections were covered with Mowiol (Hoechst A.G., Frankfurt, Germany) and coverslipped. Omission of the first antibody served as negative control to check for nonspecific binding of the second antibody system.

As shown in Figure 7, perinuclear localization was detected after administration of exogenous aFGF, which was markedly more intense compared to that of endogenous aFGF. bFGF was found mainly in endothelial and interstitial cells, and, like aFGF, in a perinuclear localization (Figure 8); Accumulation of bFGF in cardiac myocytes could not be detected. The peptide localizations were aided by counterstaining of the nuclei with aminoactinomycin and of the cytoplasm with phalloidin. Previous immunhistochemical investigations in-vitro experiments revealed a further pathway described as the translocation of added FGF as FGF-FGFR complex to the nucleus (Prudovsky, J. Biol. Chem. 269 (1994), 31720-31724). This seems to be cycle dependent since it was preferably found during the transition from GO to G1 phase of the cell cycle (Baldin, EMBO J. 9 (1990), 1511-1517) and leads to an increase in the transcription of ribosomal genes (Bouche, Proc. Natl. Acad. Sci. U.S.A. 84 (1987), 6770-6774. Wiedlocha (Cell 76 (1994), 1039-1051 ) showed that stimulation of DNA synthesis correlates with the transport of aFGF to the nucleus and is independent of the tyrosine phosphorylation. In the present experiments aFGF becomes redistributed from an extracellular to a (peri)nuclear localizaton in myocytes. These show a brighter staining pattern compared to the endogenous aFGF and that in the extracellular matrix (Weiner, Proc. Natl. Acad. Sci. USA 86 (1989), 2683-2687). Since the uptake and intracellular localization of exogenous aFGF after tyrosine kinase inhibition by genistein was still evident it is unlikely that this pathway is important for the cardioprotection. Although nuclear uptake of aFGF by myocytes was observed it is unlikely that these nuclei had entered the cell cycle.

These results are in contrast with Wiedlocha^'s work but an explanation for this discrepancy, beside the fact that in the present studies an in vivo model is used, is that cardiac myocytes are terminally differentiated cells which do not undergo any cell cycle changes and unlike cultured NIH 3T3-cell lines, they do not proliferate. It should also be noted that aFGF has a multitude of effects in addition to induction of cell proliferation and/or DNA synthesis (Cuevas (1991 ), supra; Nurcombe, Science 260 (1993), 103-106; Tamm, Proc. Natl. Acad. Sci. USA 88 (1991 ), 3372-3376) and that various pathways might be involved in these processes.

As has been shown in accordance with the present invention, fibroblast growth factors can ameliorate ischemia induced cell death. A question that may arise is by which means does the cardioprotection occur? One attractive hypothesis, since MAPKs are assumed to be involved, would be the phosphorylation of cytoplasmic and nuclear proteins (Clerk, J. Biol. Chem. 269 (1994), 32848-32857; Davis, J. Biol. Chem. 268 (1993), 14553-14556). MAPKs are important mediators of signal transduction from the cell surface to the nucleus being involved not only in the regulation of cell hypertrophy but also in the response to cellular stresses such as hypoxia or ischemia. Short ischemia followed by reperfusion have been shown to induce transcriptional changes in the heart associated with increased levels of proto-oncogenes (Brand, Circ. res. 71 (1992), 1351-1360; Webster, J. Biol. Chem. 268 (1993), 16852-16858). So far the differential activation of cytoplasmic MAPKs during ischemia/reperfusion implicates the possible role of MAPKs in modulation of cellular responses of the myocardium to the ischemia and ischemia/reperfusion. But the knowledge about the effector side of the protective effect still remains to be determined.

Example 4: Intramyocardial infusion of anisomycin induces cardioprotective effects due to SAPK activation

To test the inventors' hypothesis that the protective effect of ischemic preconditioning is due to the activation of SAPKs, they were stimulated by infusing anisomycin (500 μmol) directly into the mycardium prior to the index ischemia. The hemodynamic parameters remained unchanged during intra-myocardial microinfusion. Compared to control values no significant change was observed in any measured parameter (Fig. 10). In addition, no ventricular premature beats were detected during the infusion. Figure 11 depicts the effect of intramyocardial microinfusion of anisomycin and the control group. The compound was administered for 60 minutes prior to index ischemia. Local infusion of anisomycin induced an infarct size reduction (see Fig. 12) of 48.1±5.1% vs. control 83.4±2.8%, p<0.01 (Fig. 11 ). The underlying mechanism is due to the activation of the SAPKs p46 and p55 as will be shown in the following examples.

Example 5: Activation of stress activated protein kinases (SAPK)

The protein synthesis inhibitor anisomycin was used to activate the SAPKs p46 and p55 as described in example 4. Since anisomycin is also mentioned to activate the p38 kinase (Nahas, Biochem. J. 318 (1996), 247-253; Stein, J. Biol. Chem. 271 (1996), 11427-11433), GST-MAPKAP2, the specific substrate for p38, was included to differentiate the kinase activities. Furthermore MBP, the substrate for the extracellular-signal regulated protein kinases (ERKs) p42 and p44, was included in the present studies. Mechanical stretch is known to induce kinase activity, therefore we used KHL treated tissue as a comparative control value. The time points were 0, 10, 30 min.

The left ventricular biopsies were resuspended in 5 vol of ice-cold buffer A containing 20 mM Tris-HCI, 0.25 sucrose, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, 0.5 mM PMSF, 100 μM sodium orthovavadate and 10 mM sodium fluoride (pH 7.4) and homogenized with a Teflon-glas homogenizer. The homogenate was centrifuged at 14000 x g for 30 min at 4°C. After this the pellet was resuspended in buffer. The supernatant represented the cytosolic fraction, the resuspended pellet was designated as the particulate fraction.

Proteins from cytosolic fractions (20 μg) were separated in 10% SDS polyacrylamide gels containing 0.25 mg/ml of c-jun protein. After electrophoresis the gels were washed for 1 hour with 20% (v/v) 2-propanol in 50 mM Tris HCI (pH 8.0), then for 1 hour with 5 mM mercaptoethanol in 50 mM Tris-HCI, pH 8.0. The proteins were denatured by incubation for 1 hour with 50 mM Tris-HCI, pH 8.0, containing 6 M guanidine-HCI. Renaturation was achieved by incubation with 50 mM Tris HCI, pH 8.0, containing 0.1 % (v/v) Nonidet p-40 and 5 mM β-mercaptoethanol for 16 hours. After preincubation of gels in 40 mM Hepes, pH 8.0, containing 5 mM mercaptoethanol and 10 mM magnesium chloride, the in-gel phosphorylation of c-jun was performed in 40 mM Hepes, 0.5 mM EGTA, 10 mM magnesium chloride, 1.0 μM PKI, 25 mM (γ³³P)-ATP (5mCi/ml), pH 8.0, at 25°C for 4 hours. After extensive washing in 5% (w/v) trichloracetic acid and 5% (w/v) sodium pyrophosphate, the gels were dried and quantitative analysis was performed using a Phosphorlmage SF analyzer (Molecular Dynamics). To test the specificity of SAPK phosphorylation control experiments withour c-jun were performed. Data are given as a mean±SEM. Statistical analysis was performed using unpaired Student's test. Significance was accepted at p<0.05.

Table I:

Measurement of the various kinase activities after AN microinfusion compared to KHL values as control values (%).

Using c-jun as a substrate for the SAPK p46 and p55; MBP for the ERKs p42 and p44 and MAPKAP2 as a substrate for the p38 kinase.

The p55 kinase activity reached its peak at the 30 min infusion showing a 4.2 fold increase compared to the control value and a 3.2 fold rise compared to the KHL infusion. The 10 min time point showed an initial but not significant 2.3 fold- or a 1.7 fold increase compared to the KHL data. The p46 SAPK showed a significant 7.1 fold increase compared to the control after 30 min and a 4 fold rise compared to the KHL data. Data similar to p55 was obtained after 10 min infusion, a 2.9 fold or a 1.8 fold increase respectively compared to the KHL value; see also Figure 13. Measuring the p38 kinase activity after anisomycin treatment showed the following results: a 3.8 fold increase after 30 min compared to the control value, but only a 1.32 fold rise compared to the KHL data; the 10 min data were a 4.4 fold, respectively a non significant change, see Table I.

Using MAPKAP2 as a substrate for SAPKs, showed a 3.0 fold increase for p55 compared to KHL treated tissue; similar results were obtained for p46 a 2.2 fold increase (both 30 min data). Investigating the ERKs it could be demonstrated at the 30 min time point for p42 a 1.74 fold and for p44 a 1.98 fold increase compared to KHL data. Since various groups maintain (Nahas (1996), supra; Stein (1996), supra) that anisomycin stimulates the p38 kinase as well, we verified this point by measuring the p38 kinase activity with its specific substrate GST-MAPKAP2. Using specific substrates for the different kinases; myelin basic protein (MBP) for the ERKs p42 and p44, GST-MAPKAP2 for the p38 kinase and GST-c-jun as the substrate for the SAPKs, data were obtained for all three kinase families. It could be proven by in-gel phosphorylation that anisomycin activated mainly p46 and p55 in comparison to the insignificant activity increase of the p38 kinase and the ERKs.

Sadoshima (Sadoshima, EMBO J. 12 (1993), 1681-1692; Komuro, FASEB J. 10 (1996), 631-636) could demonstrate that mechanical forces like stretch can cause a rapid activation of multiple second messenger systems, including the various MAP kinases. These in turn could contribute to induction of early gene expression, which in turn might have an impact on cardioprotection. These aspects were included in the present investigations by infusing Krebs-Henseleit solution under the very same conditions like anisomycin into the myocardium, obtaining drill biopsies from these treated areas and measuring the SAPKs activity by in-gel phosphorylation. The p46 and p55 were activated but not to that extend as with anisomycin treatment (Fig. 14). It is therefore concluded that the observed cardioprotection is mainly due to activation of the SAPKs. Furthermore, since an increase in p46 and p55 activity could be demonstrated using MAPKAP 2 as a SAPKs substrate, the conclusion can be drawn that MAPKAP2 can serve as a substrate as well.

Cardiac myocytes activate adaptive responses to ischemia/ reperfusion which are designed to help the cell survive future insults. This can be mimicked by pharmacological stimulation of the responsible pathway in this case the SAPKs. The SAPK pathway involves sequential activation of the proteins MEKK1 and SEK1 , but the upstream regulators or signaling events remain unresolved. Diverse signals including inflammation, protein synthesis inhibitors, ischemic reperfusion and osmotic stress can activate the SAPKs family (Kyriakis (1994), supra; Derijard, Cell. 76 (1994), 1025-1037; Knight (1996), supra; Pombo, J. Biol. Chem. 76 (1994), 26546- 26551 ). In contrast the activation of the ERKs is better understood since it is mediated through receptor protein kinases or G-protein-coupled receptors (Malarkey, Biochem. J. 309 (1995), 361-375). Furthermore Bogoyevitch et al (Bogoyevitch, J. Biol. Chem. 269 (1994), 1110-1119) reported that neither PMA nor phenylephrine could activate the SAPK in the same extent as ERKs. There is evidence that in cell lines the SAPK can additionally be activated by a Gaq-dependent pathway activated by the mi-muscarinic agonist carbachol (Koch, Proc. Natl. Acad. Sci. USA 91 (1994), 12706-12710; Coso, J. Biol. Chem. 270 (1995), 5620-5624). However it is still not clear how the molecular basis for this interaction between agonists with heterotrimeric G-protein-linked receptors and SAPKs works. The time course study revealed that the SAPKs reached their activity peak after 30 min of continuous infusion and declined at the 60 min time point. In in vivo experiments a time period of 30 min of microinfusion did not induce any cytoprotection, this aspect implies that in the remaining 30 min until the onset of the index ischemia a process or a product responsible for the protection is being induced.

Recent investigations performed in embryonic kidney cell line 293 detected a further distal kinase substrate, the MAPK-activated protein kinase named 3pK (Ludwig, Mol. Cell Biol. 16 (1996), 6687-6697). 3pK can be targeted by all three kinase family members, ERK, p38 and SAPK. Since it is abundantly present in the heart and skeletal muscle (Sithanandam, Mol Cell Biol. 16 (1996), 868-876) one might suggest a tissue-specific function. Thus, 3pK being a convergence point of mitogenic and stress signalling, one can assume an involvement in cardiac protection.

The findings of the present invention are particularly interesting, since it introduces the new aspect of cytoprotection of activated of SAPKs. Various groups claim the hypothesis that activation of the SAPK induce apoptotic cell death (Ham, Neuron. 14 (1995), 927-939; Verheij, Nature 380 (1996), 75-79) or like Xia et al (Xia, Science 270 (1995), 1326-1331) who hypothesizes that the balance between ERKs and SAPKs decides cell survival or death. All known investigations were performed in-vitro in cell- lines under non physiological conditions, e.g. withdrawal of growth factors. The present experiments with brief occlusions performed in vivo provided no evidence for apoptosis, on the contrary the results of the present invention clearly show that stimulating the SAPK cascade pharmacologically induces cardioprotection. The present invention is not to be limited in scope by its specific embodiments described which are intended as single illustrations of individual aspects of the invention and any proteins, nucleic acid molecules, or compounds which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described therein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Said modifications intended to fall within the scope of the appended claims. Accordingly, having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the appended claims is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims

Claims

1. A pharmaceutical composition comprising a protein having the biological function of acidic fibroblast growth factor (aFGF) and/or a nucleic acid molecule encoding said protein having the biological function of aFGF and/or an activator of stress- activated protein kinases (SAPK) and/or a nucleic acid molecule encoding said activator of SAPK and optionally a pharmaceutically acceptable carrier.

2. A method for treating, preventing and/or delaying ischemic cell death comprising contacting organs, tissue or cells with a protein having the biological function of aFGF and/or a nucleic acid molecule encoding said protein having the biological function of aFGF and/or an activator of stress-activated protein kinases (SAPK) and/or a nucleic acid molecule encoding said activator of SAPK.

3. Use of a protein having the biological function of aFGF and/or a nucleic acid molecule encoding said protein having the biological function of aFGF and/or an activator of stress-activated protein kinases (SAPK) and/or a nucleic acid molecule encoding said activator of SAPK for the preparation of a pharmaceutical composition for preventing, treating and/or delaying cell death.

4. The method of claim 2 or the use of claim 3, wherein said cell death is caused by a vascular disease or a cardiac infarct or a stroke.

5. The method or the use of claim 4, wherein said vascular disease is arteriosclerosis, a coronary artery disease, a cerebral occlusive disease, a peripheral occlusive disease, a visceral occlusive disease, a mesenterial arterial insufficiency or an ophthamic or retenal occlusion.

6. The method of claim 2, 4 or 5 or the use of any one of claims 3 to 5 for the treatment of subjects before, during or after exposure to an agent or radiation or surgical treatment which damages or destroys arteries.

7. The method of any one of claims 2 or 4 to 6 or the use of any one of claims 3 to 6, wherein said treating, preventing and/or delaying of cell death results in iscfiemic preconditioning and/or ischemic tolerance of organs and/or tissues.

8. The pharmaceutical composition of claim 1 , the method of any one of claims 2 or 4 to 7, or the use of any one of claims 3 to 7, wherein the the protein having the biological function of aFGF is a recombinant a protein.

9. The pharmaceutical composition of claim 1 or 8, the method of any one of claims 2 or 4 to 8 or the use of any one of claims 3 to 8, wherein the SAPK is (are) p46 and/or p55.

10. The pharmaceutical composition of claim 1 , 8 or 9, the method of any one of claims 2 or 4 to 9 or the use of any one of claims 3 to 9, wherein the activator is anisomycin or a functional derivative thereof.

11. The method of any one of claims 2 or 4 to 10, further comprising contacting the organ, tissue or cell with a growth factor.

12. The pharmaceutical composition of any one of claims 1 or 8 to 10 or the use of any one of claims 3 to 10, wherein the pharmaceutical composition

(a) further comprises a growth factor; and/or

(b) is designed for administration in conjunction with a growth factor.

13. The method of any one of claims 2 or 4 to 11 , comprising the following steps

(a) obtaining cells, tissue or an organ from a subject;

(b) introducing a nucleic acid molecule encoding the a protein having the biological function of aFGF or the activator of SAPK into said cells, thereby conferring expression of the a protein having the biological function of aFGF or activator in a form suitable for the interaction of said a protein having the biological function of aFGF or said activator of SAPK with its receptor; and (c) reintroducing the cells, tissue or organ obtained in step (b) into the same or a different subject.

14. The pharmaceutical composition of claim 1 , 8 to 10 or 12 or the use of any one of claims 3 to 10 or 12, wherein the nucleic acid molecule in the pharmaceutical composition is designed for the expression of the protein having the biological function of aFGF or the activator of SAPK by cells in vivo in a form suitable for the interaction of said a protein having the biological function of aFGF or said activator of SAPK with its receptor.

15. The pharmaceutical composition of any one of claims 1 , 8 to 10, 12 or 14 or the use of any one of claims 3 to 10, 12 or 14, wherein the pharmaceutical composition is designed for administration by intracoronary, intramuscular, intravenous, intraperitoneal, intraarterial or subcutaenous routes.

16. The pharmaceutical composition of any one of claims 1 , 8 to 10, 12 , 14 or 15, method of any one of claims 2 or 4 to 11 or 13 or the use of any one of claims 3 to 10, 12, 14 or 15, wherein the protein having the biological activity of aFGF is aFGF.

17. Use of a nucleic acid molecule encoding a protein having the biological function of aFGF and/or of an activator of stress-activated protein kinases (SAPK) in gene therapy.