WO1999061039A2 - Novel composition for modulating ischemic cell death - Google Patents

Abstract

Described is the modulation of ischemic cell death. In particular, pharmaceutical compositions are provided comprising an inhibitor of p38 kinase and/or a nucleic acid molecule encoding said inhibitor 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 an inhibitor of p38 kinase and/or a nucleic acid molecule encoding said inhibitor are described. In addition, methods and uses employing agents that activate p38 kinase for the treatment of tumors are provided.

Description

Title of the invention

Novel composition for modulating 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 an inhibitor of p38 kinase and/or a nucleic acid molecule encoding said inhibitor 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 an inhibitor of p38 kinase and/or a nucleic acid molecule encoding said inhibitor. Furthermore, the present invention relates to a method for the treatment of tumors comprising contacting tissue, cells or organs with an agent which activates p38 kinase. The present invention further involves the use of an agent which activates p38 kinase for the preparation of pharmaceutical compositions for the treatment of tumors.

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

One of the major mechanisms responsible for regulation of cellular functions in response to physiological and pathological stimuli is protein phosphorylation. Recent evidence suggests that some protein kinase could play an important role also in the response of the myocardial cells to ischemia and/or reperfusion (1-3). Mitogen- activated protein kinase (MAPK) cascades represent important signaling pathways involved in the regulation of several cellular processes (4, 5). MAPK cascades transduce signals from diverse receptor types (receptor protein tyrosine kinase, G protein coupled receptors, "stress" receptors) and thus act as intracellular signaling pathways distal to receptor stimulation. The MAPKs represent a superfamily of at least three subfamilies of protein kinase (ERK, SAPKs/JNKs, p38-MAPKs) that differ in mechanisms of upstream stimulation and probably also in their substrate specificities. The best characterized is extracellular-signal regulated protein kinase (ERK) cascade. The ERKs (ERK-1 and ERK-2) are activated by several trophic and mitogenic factors in heart (6, 7) and also by mechanical loading (8, 9). By phosphorylating transcription factors, they participate in the transmission of trophic extracellular signals to the nucleus and so initiate various cellular responses involved in growth and differentiation. The SAPKs/JNKs and the p38-MAPK cascades operate in parallel to the ERK cascade. Unlike the ERKs, SAPKs/JNKs and p38-MAPK are only weakly activated by trophic stimuli but are strongly activated by a variety of cellular stresses (hyperosmotic shock, low concentrations of protein synthesis, UV, heat shock, etc. (10-13). Recently, p38-MAPK has been shown to be activated in isolated rat hearts by ischemia and ischemia/reperfusion (1). Furthermore, ischemia/reperfusion (but not ischemia alone) induced also a significant activation of SAPKs/JNKs (1). Another study showed that ischemia/reperfusion stimulated the SAPKs/JNKs and ERKs in the isolated perfused heart (2). Furthermore, in cultured neonatal rat cardiomyocytes, hypoxia/reoxygenation has been shown to activate components of ERK cascade and the SAPKs/JNKs (14). Another study had demonstrated that hypoxia and hypoxia/reoxygenation activate the Raf-1 , MAPK kinase and MAPKs and S6 kinase in cultured rat cardiomyocytes (15). These results not only demonstrate the stimulation of MAPKs in myocardial cells by ischemia and reperfusion but also indicate the occurrence of differential regulation. After exposure of myocardium to episodes of ischemia by brief coronary occlusions, each followed by reperfusion, the heart becomes more resistant to a subsequent potentially lethal long-term ischemia. This phenomenon, known as ischemic preconditioning, increases the tolerance to ischemic myocardial injury in different animal models (16, 17). Some pharmacological substances that act at different receptor levels (A- receptors, muscarinic, α1 -receptors) are known to mimic the effect of ischemic preconditioning. However, the precise mechanisms underlying both ischemic and pharmacological preconditioning are still not known. Recently, WO 97/35855 described the use of 2,4,5-substituted imidazole compounds and compositions in the treatment of CNS injuries to the brain, such as head tumor and ischemic stroke to the brain area. While the mode of action was described as inhibition of the cytokine specific binding protein (CSBP)/p38/RK kinase pathway, the treatment of diseases related to ischemic cell death other than ischemic brain injuries were not envisaged.

Thus, the technical problem of the present invention is to provide compositions for modulating cell death, in particular 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 for modulating ischemic cell death of non-cerebral organ, tissue or cells comprising an inhibitor of p38 kinase and/or a nucleic acid molecule encoding said inhibitor and optionally a pharmaceutically acceptable carrier.

The term "ischemic cell death" as used herein denotes a disorder of the body that results from insufficient blood supply to a particular area of a tissue or an organ, such as the heart, usually as a consequence of an embolus, thrombi, or local atheromatous closure of the blood vessel.

The term "p38 kinase" refers to a protein kinase of mammalian origin that displays, dependent on the phosphorylation status of the protein, an apparent molecular weight of 38 kD as determined, for example, by SDS-PAGE. Said protein is usually to be found in the heart, the brain and in most other organs as well. p38 kinase, also called p38-MAPK (mitogen-activated protein kinase) or RK represents the mammalian homologue of the yeast HOH kinase and participates in a cascade controlling cellular responses to cytokines and stress (Rouse, Cell. 78 (1994), 1027- 1037; Han, Science 265 (1994), 808-811 ; Lee, Nature 372 (1994), 793-746; Freshney, Cell 78 (1994), 1039-1049). Like the SAPK/JNK pathway, p38-MAPK is activated by a variety of cellular stresses including osmotic shock, inflammatory cytokines, lipopolysaccharides, UV lights stress. The upstream located protein kinases MKK3 and SEK phosphorylate p38-MAPK on tyrosine and threonine at the sequence T^*GY^* resulting in p38-MAPK activation. Activated p38-MAPK has been shown to phosphorylate and activate MAPKAP kinase-2 (12) and to phosphorylate the transcription factors such as ATF-2 (31). The activity of this protein kinase may be selectively and potent inhibited by SB203580 (Cuenda A. et al., FEBS Lett. 364 (1995), 229-233). This compound helps to distinguish functional activities mediated by SAPK/JNKs and by p38 kinase. The specificity of said kinase may vary to some extent in different species.

In the context of the present invention the term "an inhibitor of p38 kinase" refers to compounds, for example, organic compounds, nucleic acid molecules, (poly)peptides, etc., capable of inhibiting at least one member of the p38 kinase mediated signal transduction pathway either on the gene expression or protein level. Preferably, the inhibitor acts directly on p38 kinase or antagonizes the biological activity of p38 kinase. However, inhibitors or antagonists exerting their effect on other members of the p38 kinase pathway are also encompassed by the present invention.

MAPKs represent a superfamily of Pro-directed Ser-/Thr protein kinase that are involved in the transduction of signals from the plasma membrane to nuclear and other intracellular targets (4, 5). The three MAPK cascades (the ERK, SAPK/JNK and p38-MAPK cascades) represent parallel pathways which have different substrate specificities and are regulated by distinct stimuli (4, 18). In the heart, as in many other tissues, ERKs are activated by peptide growth factors, phorbol esters and Gq protein-coupled receptor agonists (6, 7). These agonists are hypertrophic in cardiac myocyte (6, 7), and on this correlative basis a role of ERKs in cardiac hypertrophy was proposed (7, 22, 23). In contrast to the ERKs, the SAPKs/JNKs and p38-MAPKs are activated by cellular stresses in the heart (including ischemia and ischemia/reperfusion (1 , 2, 10). The biological role of these "stress-regulated" MAPKs is unclear. However, a common feature of all MAPKs is their ability to phosphorylate the transactivation domains of numerous transcription factors and thus modulate transcriptional activity/specificity. Characteristic features of the hypertrophic phenotype include changes at the level of gene transcription and increased cell size. An early event is the increased expression of immediate-early genes encoding nuclear transcription factors (c-jun, c-fos, Egr-1) (24). It has been shown that stresses such as hypoxia or short ischemia followed by reperfusion also induce transcriptional changes in the heart that are associated with increased abundances of c-fos, c-jun, jun B and Egr-1 mRNA (25-27). In addition to immediate early genes, genes encoding calcium binding proteins, heat shock proteins and growth factors are upregulated (25-27). Immediate early genes are defined as genes whose transcription does not require de novo protein synthesis. It should be noted that the regulatory regions of many intermediate early genes contain consensus sequences for the binding of transcription factors phosphorylated by MAPKs. Thus transcription of c-fos can be upregulated by an ERK or a SAPK/JNK-catalyzed phosphorylation of transcription factors of the ternary complex factor family such as Elk-1 (28, 29). Similar mechanisms of upregulation probably exist for c-jun, the promoter region of which contains two sites (jun 1 and jun 2) to which c-Jun/ATF2 heterodimers bind. Both c-Jun and ATF2 are substrates of SAPKs/JNKs and p38- MAPK (30, 31).

Recent studies observed the activation of p38-MAPK only when the heart was preconditioned or during reperfusion in contrast to the findings of the present invention.

However, most of the previous studies on cardiac MAPKs have used isolated hearts or myocytes and/or assay systems that were not suitable to detect alterations of MAPK activity. In accordance with the present invention the activation of MAPKs was studied during ischemia and ischemia/reperfusion in porcine myocardium in vivo. In contrast to the previous studies it has been surprisingly found that p38- MAPK can be used to mediate cellular responses of the myocardium to ischemia and ischemia/reperfusion, i.e. in cardioprotection elicited by short coronary occlusions (ischemic preconditioning). The most important findings of the study of the present invention are that ischemia and ischemia/reperfusion differentially activated the three distinct protein kinase cascades (ERKs, SAPKs/jnks and p38- MAPK) in pig myocardium in vivo. The ERKs were activated during ischemia and this was increased during reperfusion (Fig. 2A, Fig. 3A and B). The SAPks/JNKs were markedly activated but only during reperfusion (Fig. 4A, Fig. 5A and B). p38- MAPK was activated during ischemia but the activity decreased during reperfusion and was markedly attenuated during the second period of ischemia (Fig. 6A, Fig. 7A). In accordance with the present invention it was furthermore found that direct infusion of the p38 kinase antagonist SB203580 into normal non-ischemic myocardium had a marked anti-ischemic effect during a subsequent period of index ischemia without markedly reducing the p38 activity; see Figures 8 to 11. Thus, an inhibitor of p38 kinase or nucleic acid molecules encoding such an inhibitor 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. As the results obtained in accordance with the present invention surprisingly revealed that an inhibitor of the p38 kinase signal transduction pathway is capable of inducing cardioprotective effects due to its inhibitory action on p38 kinase, it is to be expected that other inhibitors of p38 kinase or of proteins acting downstream or upstream of said pathway can be used as well for inducing cardioprotection and thus ischemic preconditioning. Further suitable compounds capable of inhibiting p38 kinase are known to the person skilled in the art, e.g., pyridinyl-imidazole compounds such as described in, e.g., WO 97/35855, and can be easily identified through search in the literature and appropriate databases, and/or ascertained according to the methods described in the appended examples. Thus, according to the present invention, any inhibitor of p38 kinase as defined above, namely which is capable of exerting cytoprotective effects can be used for the purpose of the present invention. The inhibitors of p38 kinase to be employed in the pharmaceutical compositions, methods and uses of the present invention such as SB203580 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, e.g., proteinaceous inhibitors of p38 kinase or for the production of antisense nucleic acid molecules capable of interfering with p38 kinase gene expression. 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 Harbor Laboratory Press, Cold Spring Harbor NY (1989)). Furthermore, inhibitors of p38 kinase include compounds that have been obtained by peptidomimetics or compounds that are derived from natural, e.g., peptide inhibitors and modified by, e.g., chemical means and/or recombinant DNA technology but essentially retain their inhibitory function. The action of the inhibitors employed in accordance with the present invention may not be limited to the above-described property but they may also inactivate, for example, other protein kinase.

In a further embodiment the invention relates to a method for treating, preventing and/or delaying cell death comprising contacting non-cerebral organs, tissue or cells with an inhibitor of p38 kinase and/or a nucleic acid molecule encoding said inhibitor. 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 inhibitors of p38 kinase and/or a nucleic acid molecule encoding said inhibitor for the preparation of a pharmaceutical composition for preventing, treating and/or delaying cell death of non-cerebral organs, tissue or cells. 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 inhibitor of p38 kinase and/or their encoding nucleic acid molecules, respectively, and optionally a pharmaceutically acceptable carrier or excipient. 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 also 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 10¹² 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 or any other ischemic cause or disease.

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 peripheral occlusive disease, a visceral occlusive disease, renal 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 of any kind of high risk intervention in ischemic disease treatment.

In a preferred embodiment, an inhibitor of p38 kinase used in the pharmaceutical compositions, methods and uses of the invention is a(n) antibody, (poly)peptide, nucleic acid, small organic compound, ligand, hormone, PNA or peptidomimetic. Nucleic acid molecules specifically hybridizing to p38 kinase encoding genes and/or their regulatory sequences may be used for repression of expression of a gene encoding p38 kinase, for example due to an antisense or triple helix effect or for the construction of appropriate ribozymes (see, e.g., EP-B1 0 291 533, EP-A1 0 321 201 , EP-A2 0 360 257) which specifically cleave the (pre)-mRNA of a gene encoding a p38 kinase. The nucleic and amino acid sequences encoding p38 kinase are known in the art and described, for example, in Wang, J. Biol. Chem. 272 (1997), 23668-23674; Stein, J. Biol. Chem. (1997), 19509-19517; Raingeaud, Mol. Cell. Biol. 16 (1996), 1247-1255; Han, Biochem. Biophys. Acta 1265 (1995), 224-227; Kumar, Biophys. Res. Commun. 235 (1997), 533-538. Selection of appropriate target sites and corresponding ribozymes can be done as described for example in Steinecke, Ribozymes, Methods in Cell Biology 50, Galbraith et al. eds. Academic Press, Inc. (1995), 449-460.

Nucleic acids comprise DNA or RNA or a hybrid thereof. Furthermore, said nucleic acid may contain, for example, thioester bonds and/or nucleotide analogues, commonly used in oligonucleotide anti-sense approaches. Said modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the cell. Furthermore, the so-called "peptide nucleic acid" (PNA) technique can be used for the detection or inhibition of the expression of a nucleic acid. For example, the binding of PNAs to complementary as well as various single stranded RNA and DNA nucleic acid molecules can be systematically investigated using, e.g., thermal denaturation and BIAcore surface-interaction techniques (Jensen, Biochemistry 36 (1997), 5072-5077). The synthesis of PNAs can be performed according to methods known in the art, for example, as described in Koch, J. Pept. Res. 49 (1997), 80-88; Finn, Nucleic Acids Research 24 (1996), 3357- 3363. Furthermore, folding simulations and computer redesign of structural motifs of the p38 kinase or its substrates can be performed using appropriate computer programs (Olszewski, Proteins 25 (1996), 286-299; Hoffman, Comput. Appl. Biosci. 11 (1995), 675-679). Computer modeling of p38 kinase folding can be used for the conformational and energetic analysis of detailed protein models (Monge, J. Mol. Biol. 247 (1995), 995-1012; Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45). In particular, the appropriate programs can be used for the identification of interactive sites of a putative inhibitor and with the p38 kinase by computer assistant searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 114-120). Further appropriate computer systems for the computer aided design of protein and peptides are described in the prior art, for example in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. The results obtained from the above-described computer analysis can be used for, e.g., the preparation of peptidomimetics of SB203580 or other known inhibitors of p38 kinase. Such pseudopeptide analogues of the natural amino acid sequence of the peptide substrates may very efficiently mimic the parent molecule (Benkirane, J. Biol. Chem. 271 (1996), 33218-33224). Superactive peptidomimetic analogues of small peptide hormones in other systems are described in the prior art (Zhang, Biochem. Biophys. Res. Commun. 224 (1996), 327-331). Appropriate peptidomimetics of SB203580 and other inhibitors of p38 kinase can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive amide alkylation and testing the resulting compounds, e.g., according to the methods described hereinafter and in the appended examples. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715. Furthermore, a three-dimensional and/or crystallographic structure of SB203580 and other inhibitors of p38 kinase or of p38 kinase can be used for the design of peptidomimetic inhibitors of p38 kinase (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558). Furthermore, antibodies specifically recognizing p38 kinase or parts thereof, i.e. specific fragments or epitopes of such p38 kinase and thereby inactivating the p38 kinase may be employed. These antibodies can be monoclonal antibodies, polyclonal antibodies or synthetic antibodies as well as fragments of antibodies, such as Fab, Fv or scFv fragments etc. Antibodies or fragments thereof can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988 or EP-B1 0 451 216 and references cited therein. For example, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage display antibodies which bind to an epitope of the p38 kinase (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).

Putative inhibitors which can be used in accordance with the present invention including peptides, proteins, nucleic acids, antibodies, small organic compounds, ligands, hormones, peptidomimetics, PNAs and the like capable of inhibiting p38 kinase may be identified according to the methods known in the art, for example as described in EP-A-0 403 506 or in the appended examples. In a particularly preferred embodiment of the invention, the inhibitor of p38 kinase comprised in the pharmaceutical compositions, methods or uses is SB203580 or a functional pyridinyl-imidazole derivative or analogue thereof which may be obtained by, e.g., peptidomimetics. As described in the appended examples SB203580 was found to induce cytoprotective effects due to inhibition of p38 kinase. The structural formula of SB203580 is C₂ιHι₆FN₃OS; see also, e.g., Lee, Nature 372 (1994), 739. For the purpose of the present invention "functional derivative or analogue" of SB203580 means molecules the chemical structure of which is based on that of SB203580 and which are capable of inducing cardioprotective effects. The cardioprotective effects of the SB203580-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. Furthermore, peptidomimetics and/or computer aided design of appropriate derivatives and analogues can be used, for example, according to the methods described above.

Attenuation of p38 activity by repeated ischemia may be a key element in the molecular mechanism of ischemic preconditioning. This new model is strengthened by pharmacological observations presented in the appended examples where the SAPKs/JNKs agonist anisomycin and the p38-MAPK inhibitor SB 203580 significantly prolong survival. In these experiments "pharmacological preconditioning" was induced by tool drugs instead of brief occlusions. The prevention of the consequences of p38 activation induced cardioprotection but without significant reduction of kinase activity. This is not surprising because SB203580 is known to act downstream in the p38-triggerd cascade. A protective effect was also observed in the case of the tyrosine kinase receptor agonists that activate ERKs (IGF-2 and FGF-1) (35). The protective effect of brief occlusions may also be limited by the action of protein phosphatases and inhibition of these phosphatases by okadaic acid during reperfusion also increases survival as could be shown recently (36). In order to provide for a synergistic effect it is thus further envisaged to include the above- described compounds or other compounds known to induce cytoprotective effects in the pharmaceutical compositions, methods and uses of the present invention.

Thus, in a further preferred embodiment, the pharmaceutical composition uses and methods of the invention are designed to be applied in conjugation with anisomycin, okadaic acid or growth factors, preferably fibroblast growth factor such as acidic or basic fibroblast growth factor (aFGF, bFGF), insulin-like growth factor-II (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, SB203580 and/or another inhibitor of p38 kinase, and a growth factor such as aFGF may advantageously 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 inhibitor of p38 kinase into said cells, thereby conferring expression of said inhibitor; and

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

Suitable cells, tissue and organs are known to the person skilled in the art and comprise, for example, smooth muscle cells, pericytes, endothelial cells as well as blood cells, e.g., monocytes or circulating precursor cells of vessel cells. It is envisaged by the present invention that an inhibitor of p38 kinase and the nucleic acid molecules encoding said inhibitor are administered either alone or in combination, and optionally together with a pharmaceutically acceptable carrier or excipient. 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 may be used for transfixing certain cells, tissues or organs. Furthermore, it is possible to use a pharmaceutical composition of the invention which comprises a nucleic acid molecule encoding an inhibitor of p38 kinase 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 an inhibitor of p38 kinase 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., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911 -919; Anderson, Science 256 (1992), 808-813; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; 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 molecule 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 inhibitor of p38 kinase express said inhibitor 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 inhibitor of p38 kinase 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 nucleic acid 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 an inhibitor of p38 kinase. Such cells may be also be administered in accordance with the pharmaceutical compositions, methods and uses of the invention.

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 aprf 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 transf erase). 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-omithine, 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 of an inhibitor of p38 kinase by cells in vivo in 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. The differential activation of MAPKs that utilize different effector pathways and that exhibit contrasting effects like protective and facilitative effects on programmed cell death (32) is interesting and may provide a new addition to existing models for the molecular mechanism of ischemic preconditioning. As has been surprisingly revealed by the experiments performed in accordance with the present invention, activation of SAPKs/JNKs and ERKs is cardioprotective and activation of p38-MAPK is cardiotoxic. Lasting ischemia of "naive" tissue leads to the activation of both the ERKs and p38-MAPK. It leads to cell death because the protective influence of the ERKs is cancelled by the increased activity of p38-MAPK. In this condition, the protective effect of SAPKs/JNKs is absent because these are only activated by reperfusion following a brief occlusion. Thus, if the p38 kinase was activated in neoplastic cells of a tumor, suppression and/or inhibition of tumor growth is to be expected. Accordingly, the present invention also relates to a method for the treatment of tumors comprising contacting organs, tissue or cells with an agent which activates p38 kinase. Agents which activate p38 kinase may be peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, neural transmitters, peptidomimetics, or PNAs (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198). Examples of such agents include IL-1β, TNF-alpha, UV-light, osmose and pyridinyl-imidazole derivatives.

The present invention further relates to the use of an agent which activates p38 kinase for the preparation of a pharmaceutical composition for the treatment of tumors.

In accordance with the foregoing, agents that activate p38 kinase also include those which exert their activity down- or upstream of the p38 kinase mediated signal. For example, compounds that act on MAPKAP-2.

In a preferred embodiment of the present invention, the agent used in the methods and uses of the invention as described above inhibits or neutralizes or antagonizes the biological activity of an inhibitor of p38 kinase. Various compounds are described in the prior art that counteract the effective treatment of tumors due to their inhibitory effect on p38 kinase signal transduction pathway. These adverse side effects may be overcome by (co)administering of an agent that abolishes or neutralizes the inhibitory activity of such compounds.

In a preferred embodiment of the methods and uses of the invention, said inhibitor to be neutralized is SB203580 or a functionally equivalent pyridinyl-imidazple compound such as one of those described above, including, e.g., 4-(4-Fluorophenyl)- 2-(4-hydroxyphenyl)-5-(4-pyridyl)1 H-imidazole; FHPI) and derivatives thereof.

In another preferred embodiment, the agent which activates p38 kinase is an antibody and/or a stimulatory form of p38 kinase.

In a preferred embodiment, the methods and uses of the invention are employed for the treatment of a tumor which is a vascular tumor, preferably selected from the group consisting of Colon Carcinoma, Sarcoma, Carcinoma in the breast, Carcinoma in the head/neck, Mesothelioma, Glioblastoma, Lymphoma and Meningeoma.

In another preferred embodiment, the pharmaceutical composition in the use of the invention is designed for administration by intracoronary, intramuscular, intravenous, intraperitoneal, intraarterial or subcutaneous routes. In the examples of the present invention the inhibitor of p38 kinase, i.e. SB203580 was administered locally via osmotic minipump.

The said p38 kinase inhibitors and agents which active p38 kinase or, if possible and appropriate, their encoding nucleic acid molecule may be used for therapeutical purposes in various forms. Either as in the experiments described herein, locally via implanted pumps, or as arterial or venous boluses either systemically or locally via specially designed catheters or other device. They may also be injected intramuscularly or into any other tissues in which cell death needs to be modulated. Alternatively they can be bound to microcapsules or microspheres before injection. Another approach would be to use a gene-transfer approach as outlined above, either using, e.g., a plasmid, or a plasmid embedded in liposomes, or viral vectors. One may either use an in vivo gene-transfer approach for which multiple devices, like double balloon or other catheters have been designed or via direct injection into the targeted tissue as described above. Alternatively it is possible to use an ex vivo approach isolating cells which are known to lodge in tissues, for example, in which vessel growth needs to be promoted or inhibited from the body which are then transfected using one of the above mentioned methods and reinjected.

In a further embodiment, the present invention relates to the use of any one of the before described nucleic acid molecules in gene therapy, for example, for curing inborn or acquired 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, such as http://www.ncbi.nlm.nih.gov/, http://www.infobiogen.fr/, http://www.fmi.ch/biology/research_tools.html, http://www.tigr.org/, are known to the person skilled in the art and can also be obtained using, e.g., http://www.lycos.com. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.

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.

Summary of the Invention

The present invention is based on the ability to prevent or delay ischemic cell death. In a first embodiment, the invention provides a pharmaceutical composition comprising an inhibitor of p38 kinase and/or a nucleic acid molecule encoding said inhibitor, and optionally a pharmaceutically acceptable carrier.

In yet another embodiment, the invention provides a method for treating, preventing and/or delaying ischemic cell death comprising contacting organs, tissue or cells with an inhibitor of p38 kinase and/or a nucleic acid molecule encoding said inhibitor, and optionally a pharmaceutically acceptable carrier.

In yet another embodiment, the invention provides a method for treating, preventing and/or delaying ischemic cell death comprising contacting organs, tissue or cells with an inhibitor of p38 kinase and/or a nucleic acid molecule encoding said inhibitor.

In still another embodiment, the present invention relates to uses and methods employing agents which activate p38 kinase for the treatment of tumors.

Brief description of the figures

Figure 1

Experimental groups. The present study consisted of two different experimental blocks. The first block (A) consisted of a group of animals which were subjected to ischemia/reperfusion protocol. Left ventricular biopsies were taken from control, ischemic and reperfused tissue (time points indicated by arrows). The second block (B) consisted of six different groups. Three groups of animals were studied in in vivo experiments: group I- animals were subjected to 60 min LAD occlusion and 120 min reperfusion (control group); group II- animals received KHB by means of intramyocardial infusion for 60 min prior to 60 min ischemia; group III- animals received anisomycin (AN) by means of intramyocardial infusion for 60 min prior to 60 min ischemia; group IV- the myocardium was treated with SB203580 for 60 min prior to the LAD occlusion. Animals of groups V and VI received the microinfusion of AN, SB 203580 or KHB for 60 min and were biopsied at the time points indicated by the arrows.

Figure 2

Effect of ischemia and ischemia/reperfusion on activity of ERKs in porcine heart. The soluble fractions were isolated from biopsies obtained from control tissue, at different time points of the first ischemia (ISCH 1 , 2 and 10 min), reperfusion (REP 1 , 5, 15 and 30 min) and the subsequent second ischemia (ISCH 1 , 2 and 10 min). In (A), the in gel phosphorylation of MBP was performed as described in Example 2. The gel is representative of three independent experiments. The arrows on the right indicate the positions of ERK-1 (p44-MAPK) and ERK-2 (p42-MAPK). (B) Western blotting of ERKs with anti-ERK specific antibody. The arrow on the right indicates the position of 46 kDa molecular mass marker. (C), Western blotting with phospho-specific antibody that recognizes a Tyr-204 phosphorylated ERKs. The arrow on the right indicates the position of 46 kDa molecular mass marker.

Figure 3

Quantification of ERK-1 (A) and ERK-2 (B) activation by ischemia (ISCH1), reperfusion (REP) and the subsequent second ischemic episode (ISCH2). Data were derived from in gel kinase assays (see Fig. 2) and are expressed as a percentage of control value (control nonischemic tissue). Each bar represents the mean ± S.E.M. of three separate experiments (^*p<0.05, ^**p<0.01 vs. control). Quantitative analysis of gels was performed using Phospho Image SF (Molecular Dynamics).

Figure 4

Effect of ischemia and ischemia/reperfusion on activity of SAPKs/JNKs in porcine heart. The soluble fractions were isolated from biopsies obtained from control tissue, at different time points of the first ischemia (ISCH 1 , 2 and 10 min), reperfusion (REP 1 , 5, 15 and 30 min) and the subsequent second ischemia (ISCH 1 , 2 and 10 min). In (A), the in gel phosphorylation of c-Jun-μ-135 was performed as described in Example 2. The gel is representative of four independent experiments. The arrows on the right indicate the positions of JNK-46 and JNK-55. (B) The in gel phosphorylation of c-JUN 1-135 after immunoprecipitation with anti JNK antibody. The arrow on the right indicates the position of 46 kDa molecular mass marker. (C), Western blotting of SAPK/JNK. The arrow on the right indicates the position of 46 kDa molecular mass marker (SAPK/JNK-1).

Figure 5

Quantification of JNK-46 (A) and JNK-55 (B) activation by ischemia (ISCH1), reperfusion (REP) and the subsequent second ischemia (ISCH2). Data were derived from in gel kinase assays (see Fig. 4) and are expressed as a percentage of control value (control nonischemic tissue). Each bar represents the mean ± S.E.M. of four separate experiments (^**p<0.01 vs control). Quantitative analysis of gels was performed using Phospho Image SF (Molecular Dynamics).

Figure 6

Effect of ischemia and ischemia reperfusion on activity of p38-MAPK and a 45 kDa protein kinase by ischemia and ischemia/reperfusion in porcine heart. The soluble fractions were isolated from biopsies obtained from control tissue, at different time points of the first ischemia (ISCH 1 , 2 and 10 min), reperfusion (REP 1 , 5, 15 and 30 min) and the subsequent second ischemia (ISCH 1 , 2 and 10 min). In (A), the in gel phosphorylation of MAPKAP-K246-400 was performed as described in the examples. The gel is representative of four independent experiments. The arrow on the right indicates the position of p38-MAPKERK-1 (p44-MAPK) and ERK-2 (p42- MAPK). (B) After immunoprecipitation of p38-MAPK from soluble fraction using anti- p38-MAPK antibody, the in gel kinase assay with MAPKAP-K2 46-400 as a substrate was performed. Left part of the gel (lanes 1-4) shows the results of the in gel phosphorylation of MAPKAP-K2 46-400 with probes after immunoprecipitation. Right part of the gel shows the in gel phosphorylation of MAPKAP-K2 46-400 with original probes (without immunoprecipitation). (C), Western blotting of p38-MAPK with anti- p38-MAPK antibody. The arrow on the right indicates the position of p38-MAPK. (D) Western blotting with phospho-specific antibody that recognizes Tyr-182 and Thr-180 dual phosphoryiated p38-MAPK. P- positive control. The arrow on the right indicates the position of phospho-p38-MAPK.

Figure 7

Quantification of p38-MAPK and 46kDa protein kinase activation by ischemia (ISCH1), reperfusion (REP) and the subsequent second ischemia (ISCH2). Data were derived from in gel kinase assays (see Fig. 6) and are expressed as a percentage of control value (control nonischemic tissue). Each bar represents the mean ± S.E.M. of four separate experiments (^*p<0.05, **p<0.01 vs control; #p<0.05 vs ISCH1-10). Quantitative analysis of gels was performed using Phospho Image SF (Molecular Dynamics). Figure 8

Hemodynamic data: (A,B) anisomycin, (C,D) SB 203580

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

Figure 9

Effect of local infusion of KHB (KH), anisomycin (AN) and SB 203580 (SB) for 60 min prior to the 60 min LAD on the infarct size in pig myocardium.

Figure 10

Intramyocardial infusion of anisomycin and protein kinase inhibitor SB 203580. The needles for intramyocardial infusion were placed into the subsequent ischemic part of the left ventricle. The fluorescent microspheres demarcate the non fluorescent area of risk. After TTC staining the myocardial protection was defined as stained tissue surrounding the microinfusion needles in transmurally infarcted myocardium. (A) microinfusion anisomycin. (B) microinfusion of KHB (needle on the right) and anisomycin (needle on the left). (C) microinfusion of SB 203580.

Figure 11

Quantification of p38-MAPK activation after local infusion of protein kinase inhibitor SB203580. Data were derived from in gel phosphorylation of MAPKAP-K2 and are expressed as a percentage of value obtained for corresponding KHB-treated tissue. Each bar represents the mean ±S.E.M. SB- infusion of SB 203580 for 60 in; SB-I5- infusion for 60 min followed by 5 min of ischemia; SB-I10- infusion for 60 min followed by 10 min of ischemia. Quantitative analysis of gels was performed using Phosphorlmage SF (Molecular Dynamics)

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.

Chemicals

Horseradish peroxidase-linked goat anti-rabbit immunoglobulin, the enhanced chemiluminescence (ECL) reagents, nitrocellulose membranes, rainbow molecular mass markers, autoradiography films and (γ³²P)-ATP were from Amersham International. The polyclonal antibodies against p38-MAPK and protein A-agarose were from Santa Cruz Biotechnology. The polyclonal antibodies against phospho- p38-MAPK were from New England Biolabs. Recombinant MAPKAP-K2 (residues 46-400 encompassing the catalytic domain) were expressed in E. coli as glutathione- S-transferase proteins and were purified by glutathione-Sepharose (Pharmacia) chromatography. Azaperone and piritramide were purchased from Janssen Pharmaceutica. SB 203580 was from Calbiochem, the fluorescent zinc-cadmium sulfide microspheres (diameter 2-15 μm) were purchased from Duke Scientific Corporation.

Animal preparation

24 male castrated German landrace-type domestic pigs weighing about 30-35 kg were premedicated with azapeone (2 mg/kg of body weight i.m.) and 2 mg/kg BW piritamide s.c. 30 min prior to the initiation of general anaesthesia with a bolus of chloraose (25 mg/kg), maintained by a continuos intravenous infusion of 25 mg/kg x h 1. The animals were artificially ventilated with room air enriched with 11/min of oxygen. The rate of ventilation was controlled on the basis of blood gas analyses. After a median sternotomy, the heart was suspended in a pericardial cradle and a 30 min stabilization period was allowed. Arterial pressure, heart rate and the ECG were continuously monitored and recorded on the hard disk of a MacLab computer, regional ischemia was achieved by occluding the left anterior descending coronary artery (LAD) midway along its course between base and apex. Left ventricular drill biopsies were taken from control tissue and at 2, 5, and 10 minutes of preconditioning ischemia at 5, 15 and 30 minutes of reperfusion and at 2 and 10 minutes into a second period of ischemia (index ischemia). Biopsies weighed about 80 mg and were about 4 mm long, i.e. they reached from epi- to mid-myocardium. In the pigs subjected to intramyocardial microinfusion 8 26 gauge needles were connected by tubing with a peristaltic pump (Miniplus, Gilson, Germany) and were placed in pairs along the LAD into the myocardium perpendicular to the epicardial surface. The compounds we tested were infuse with an infusion rate of 20 μl/min for a period of 60 min (further details see Vogt et al.) prior to the index ischemia (see below).

Experimental groups

The present study consisted of two different experimental settings (Fig.1). The first block consisted of a group of 4 animals which were subjected to regional ischemia by ligating the LAD. Left ventricular biopsies were taken from control (nonischemic) tissue and at 2, 5 and 10 minutes of preconditioning at 5, 15 and 30 minutes of reperfusion and at 2 and 10 minutes into the second period of ischemia (index ischemia). The obtained biopsies were used for the kinase assays.

The second experimental block consisted of 4 groups. Animals in group 1 (n=6, the control group for infarct size studies) were subjected to 60 min occlusion and 60 min of reperfusion. In group 2 (n=6) KHB was administered by local infusion 60 min prior index ischemia of 60 min and the following reperfusion period of 60 min. Animals in group 3 (n=6) were treated with the specific p38-MAPK inhibitor SB 203580 (60 mmol diluted in KHB containing 1 promille DMSO) under the same regimen like KHB.

The following experimental group was used for kinase assay studies. Animals of group 4 (n=2) received local infusion of SB 203580 (60 mmol) or KHB for 60 min which was followed by an ischemic period. The drill biopsies were obtained after the microinfusions were stopped and at 5 and 10 min of the following ischemia.

Determination of infarct size

At the end of the experimental protocol the LAD and the aorta were occluded respectively clamped and 200 mg of zinc cadmium fluorescent microspheres in 20 ml Ringer^'s solution was injected into the ascending aorta. After the distribution of the microspheres 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 as the nonfluorescent area by UV-light (366nm) examination. The infarcted area was demarcated by the absence of the characteristic red TTC-stain. The slices were photographed by double exposure with UV-light and normal artificial day light and the color transparencies were used for further planimetric evaluation.

Statistics

Ischemic and ischemic/reperfused tissue biopsy material was always compared with control tissue from the same heart. Differences were evaluated using a paired Student t-test. The accepted level of significance was p=0.05. For the infarct size quantification we used the unpaid Student t-test, p<0.05 was accepted as significant.

Example 2: Stimulation of p38-MAPK by ischemia and ischemia/reperfusion

Unlike the other group of "stress-regulated"(the SAPKs/JNKs), ischemia alone significantly stimulated the activity of p38-MAPK. Biopsies were obtained from the pigs' myocandium and suspended in ice-cold buffer containing in mmol/L: 20 Tris- HCI, 250 sucrose, 1.0 EDTA, 1.0 EGTA, 1.0 DTT, 0.1 sodium orthovandate, 10 NaF and 0.5 PMSF, pH 7.4 and were homogenized with a Teflon-glas homogenizer. The homogenate was centrifuged at 14,000xg for 30 min at 4°C. Laemmli sample buffer was added to the supernatant and the proteins were denatured by heating at 100°C for 5 min. Proteins (20 μg) were separated on 10 % SDS-polyacrylamide gels containing 0.5 mg/ml of GST-MAPKAP-K2 (for p38-MAPK). After electrophoresis, the gels were washed for 1 h with 20% (v/v) 2-propanol in 50 mmol/Tris.HCI (pH 8.0), then for 1 h with 5 mmol/L 2-mercaptoethanol in 50 mmol/L Tris.HCI, pH 8.0, and the proteins in the gels were denatured by incubation for 2 h with 50 mmol/L Tris.HCI, pH 8.0, containing 6 mol/L guanidine-HCI. Renaturation was achieved by incubation with 50 mmol/L Tris.HCI, pH 8.0 containing 0.1 % (v/v) Nonidet P-40 and 5 mmol/L 2- mercaptoethanol for 16 h. After preincubation of gels in 40 mmol/L Hepes (pH 8.0) containing 5 mmol/L 2-mercaptoethanol and 10 mmol/L magnesium chloride, the in gel phosphorylation of substrates was performed in 40 mmol/L Hepes (pH 8.0), 0.5 mmol/L EGTA, 10 mmol/L magnesium chloride, 1.0 mmol/L protein kinase A inhibitory peptide, 25 mmol/L (γ³²P)-ATP (5 μCi/ml) at 25°C for 4 hours. After extensive washing in 5% (w/v) trichloracetic acid containing 2% (w/v) sodium pyrophosphate, the gels were dried and quantitative analysis was performed using a Phosphorimager SF (Molecular Dynamics).

Activation was more marked than for ERKs. The maximum activation (8.1 -fold) occurred 10 min after onset of ischemia (Fig. 6A, Fig. 7A). Unlike the ERKs (Fig. 3A and B) or the SAPKs/JNKs (Fig. 5A and B), the p38 activity had decreased to 6.5- fold after 5 min of reperfusion, 4.6-fold after 15 min and to 3.7 fold after 30 min of reperfusion. During the subsequent second (index) ischemia the p38-MAPK activity was significantly (p<0.05) attenuated at 10 min compared with 10 min preconditioning ischemia (Fig. 7A). This is not the case for the ERKS (Fig. 3A and B) or the SAPKs/JNKs (Fig. 5A and B) where were no significant differences. In addition to p38-MAPK additional protein kinase activities were detected that were able to phosphorylated MAPKAP-K2. During ischemia and ischemia/reperfusion it was found that a 45 kDa protein kinase increased in activity (Fig. 6A, Fig. 7B). The activity of this protein increased 2.1 fold after 10 min of ischemia and attained a maximum of 4.5-fold activation at 15 min of reperfusion (Fig. 7B). To confirm the identity of the p38-MAPK, the in gel kinase phosphorylation assay was performed with probes obtained after immunoprecipitation with anti-p38-MAPK antibody. To confirm the specificity of reaction by the in gel kinase assays MAPKs from soluble fractions (200 μg protein) were immunoprecipitated with antisera against p38-MAPK. After incubation for 4h at 4°C the immune complexes were incubated for further 4h with protein A agarose beads. The resulting complexes were collected by centrifugation, resuspended in Laemmli sample buffer, boiled and in gel kinase assays were performed with the appropriate substrate.

It was found that the antibody precipitated the 38 kDa protein kinase (p38-MAPK) but not the 45 kDa protein kinase (Fig. 6B). The activity of p38-MAPK was also significantly increased in probes after immunoprecipitation after 10 min of the first ischemia. During reperfusion the activity decreased and the activity at 10 min of the second period of ischemia was lower than at 10 min of the first ischemia (Fig. 6B). Western blot assay confirmed that there were no changes in p38-MAPK abundance during the experimental protocol (Fig. 6C). Soluble fractions of the heart were subjected to SDS-PAGE in 10% polyacrylamide gels and proteins were transferred onto nitrocellulose membranes. Anti-p38-MAPK (0.5 μg/ml), anti-phospho-p38-MAPK (1 :300) antibodies were used for primary immunodetection. The secondary antibody directed against all antibodies was peroxidase labeled anti-rabbit immunoglobulin (1 :3000). Bound antibodies were detected by the ECL Western blot detection method. With a specific anti-phospho-p38-MAPK antibody it was investigated also the amount of the phosphorylated form of p38-MAPK and found that maximal levels of phospho-p38-MAPK occurred at 10 min of the first ischemia. During reperfusion and the second period of ischemia the levels of phosphoprotein decreased (Fig. 6D).

Example 3: Influence of SB 203580 on infarct size in the pig myocardium

The local infusion of the specific p38-MAPK inhibitor SB 203580 for 60 min prior to index ischemia significantly reduced the infarct size from 83.4±2.8% (control, group I) to 36.8±3.7% (p<0.001) (Fig. 9 and 10). This result points to the negative role of p38-MAPK activation in myocardial ischemia. Important is also the finding that the local infusion of KHB (negative control) for 60 min prior to index ischemia did not influence the infarct size as compared to the control.

Example 4: Effect of local infusion of SB 203580 on p38-MAPK activities

The inhibitor of p38-MAPK, SB 203580 was infused into the pig myocardium for different time periods. As a negative control we used KHB-infusions under identical conditions as for SB 203580. Biopsies were obtained at the end of the local infusion (total duration 60 min) and at 5 and 10 min of ischemia after the microinfusion. The p38-MAPK activities were investigated by in gel phosphorylation of MAPKAP-K2 as a substrate. We found that the infusion of SB 203580 for 60 min did not change the p38-MAPK activity. The study of p38-MAPK activity during subsequent ischemia after SB-infusion revealed a decreased activity at 10 min of ischemia but only at borderline statistical significance (p<0.06) (Fig. 11). This is not surprising because the compound inhibits the cascade further downstream.

As has been demonstrated in accordance with the present invention, the p38 kinase is activated primarily in ischemia and declines in reperfusion, in contrast to the SAPKs which are upregulated only in reperfusion. This has been shown by inhibiting the p38 kinase pathway by direct intramyocardial infusion of its specific inhibitor SB 203585. The experimental group consisted of five pigs (general anesthesia, artificial ventilation, open chest conditions) which were treated with SB 203585 during 60 min prior to the index ischemia (60 min) and the following reperfusion period (180 min). One channel was used for KHB infusion and served as a negative control. A local cardioprotective effect was observed around the needles, reducing the infarct size significantly (36.8±3.7% vs. 83.4+2.8%, p<0.001) compared to the control group (n=6), KHB infusion sites were negative. Hemodynamic side effects (AOP, HR) during drug application were not observed. These results provide for the dualistic principle of the p38 kinase and the SAPKs, assigning the detrimental of ischemia effect to the p38 kinase, inhibition of these delay cell death.

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.

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Claims

1. A pharmaceutical composition for treating, preventing and/or delaying cell death of non-cerebral organs, tissue or cells comprising an inhibitor of p38 kinase and/or a nucleic acid molecule encoding said inhibitor and optionally a pharmaceutically acceptable carrier.

2. A method for treating, preventing and/or delaying cell death comprising contacting non-cerebral organs, tissue or cells with an inhibitor of p38 kinase and/or a nucleic acid molecule encoding said inhibitor.

3. Use of an inhibitor of p38 kinase and/or a nucleic acid molecule encoding said inhibitor for the preparation of a pharmaceutical composition for preventing, treating and/or delaying cell death of non-cerebral organs, tissue or cells.

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 peripheral occlusive disease, a visceral occlusive disease, a renal 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, wherein said method or said pharmaceutical composition is designed to be applied to a subject 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 ischemic 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 inhibitor is a(n) antibody, (poly)peptide, nucleic acid, small organic compound, ligand, hormone, PNA or peptidomimetic.

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 inhibitor is SB203580 or a functional pyridinyl-imidazole derivative or analogue thereof.

10. The method of any one of claims 2 or 4 to 9, further comprising contacting the organ, tissue or cell with a growth factor and/or an activator of ERKs or SAPKs/JNKs.

11. The pharmaceutical composition of any one of claims 1 , 8 or 9 or the use of any one of claims 3 to 9, wherein the pharmaceutical composition

(a) further comprises a growth factor and/or an activator of ERKs or SAPKs/JNKs; and/or

(b) is designed for administration in conjunction with a growth factor and/or an activator of ERKs or SAPKs/JNKs.

12. The method of any one of claims 2 or 4 to 10, comprising the following steps

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

(b) introducing a nucleic acid molecule encoding the inhibitor of p38 kinase into said cells, thereby conferring expression of said inhibitor; and

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

13. The pharmaceutical composition of claim 1 , 8 or 11 or the use of any one of claims 3 to 8 or 11 , wherein the nucleic acid molecule in the pharmaceutical composition is designed for the expression of said inhibitor by cells in vivo.

14. A method for the treatment of tumors comprising contacting organs, tissue or cells with an agent which activates p38 kinase.

15. Use of an agent which activates p38 kinase for the preparation of a pharmaceutical composition for the treatment of tumors.

16. The method of claim 14 or the use of claim 15 wherein the agent neutralizes or inhibits the biological activity of an inhibitor of p38 kinase.

17. The method or the use of claim 16, wherein the inhibitor is SB203580 or a functionally equivalent pyridinyl-imidazole compound.

18. The method or the use of claim 16 or 17, wherein the agent is an antibody or a stimulatory form of p38 kinase.

19. The method of any one of claims 14 or 16 to 18 or the use of any one of claims 15 to 18, wherein the tumor is a vascular tumor.

20. The method or the use claim 19, wherein the tumor is selected form the group consisting of Colon Carcinoma, Sarcoma, Carcinoma in the breast, Carcinoma in the head/neck, Mesothelioma, Glioblastoma, Lymphoma and Meningeoma.

21. The pharmaceutical composition of any one of claims 1 , 8, 9, 11 or 13 or the use of any one of claims 3 to 9, 11 , 13 or 15 to 20, wherein the pharmaceutical composition is designed for administration by intracoronary, intramuscular, intravenous, intraperitoneal, intraarterial or subcutaneous routes.

22. Use of a nucleic acid molecule encoding an inhibitor of p38 kinase for the preparation of a pharmaceutical composition for use in gene therapy.