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WO1999016457A2 - Nouvelle composition pour traiter, prevenir et/ou retarder la mort cellulaire ischemique - Google Patents

Nouvelle composition pour traiter, prevenir et/ou retarder la mort cellulaire ischemique Download PDF

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Publication number
WO1999016457A2
WO1999016457A2 PCT/EP1998/006269 EP9806269W WO9916457A2 WO 1999016457 A2 WO1999016457 A2 WO 1999016457A2 EP 9806269 W EP9806269 W EP 9806269W WO 9916457 A2 WO9916457 A2 WO 9916457A2
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inhibitor
mkp
pharmaceutical composition
nucleic acid
protein
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WO1999016457A3 (fr
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Wolfgang Schaper
Patrik Htun
Miroslav Barancik
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Max Planck Gesellschaft zur Foerderung der Wissenschaften
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Max Planck Gesellschaft zur Foerderung der Wissenschaften
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/005—Enzyme inhibitors

Definitions

  • Novel composition for treating, preventing and/or delaying ischemic cell death
  • the present invention relates generally to the modulation of ischemic cell death.
  • the present invention provides pharmaceutical compositions comprising an inhibitor of mitogen protein phosphatases (MKP) and/or a nucleic acid molecule encoding said inhibitor which are particularly useful for treating, preventing and/or delaying ischemic cell death.
  • MKP mitogen protein phosphatases
  • 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 mitogen protein phosphatases (MKP) and/or a nucleic acid molecule encoding said inhibitor.
  • Organs which depend on postmitotic ceils for proper function e.g. cardiomyocytes in the heart and neurons in the brain
  • 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.
  • ischemic preconditioning 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.
  • TRK tyrosine receptor kinase
  • IGF-II Insulin-like growth factor
  • MAPKs mitogen-activated protein kinases
  • kinases transduce signals from diverse receptor types (receptor protein tyrosine kinases, G protein coupled receptors, "stress” receptors) and thus act as intracellular signaling pathways distal to receptor stimulation.
  • the subfamilies of MAPK cascade differ in mechanisms of upstream stimulation and probably also in their substrate specificities (Robinson, Curr. Opin. Cell Biol. 9 (1997), 180-186).
  • the MAPKs are phosphoproteins and require increased phosphorylation for their activation. The degree of phosphorylation depends on the balance between phosphorylation mediated by upstream located kinases and dephosphorylation mediated by the action of protein phosphatases.
  • control mechanisms involved in regulation of MAPK activities include both increased protein kinase phosphorylation as well as their dephosphorylation and the enhanced phosphorylation of cellular proteins might represent a fine balance between activated protein kinases and repressed phosphoprotein phosphatases.
  • the technical problem of the present invention is to provide compositions for treating, preventing and/or delaying ischemic cell death.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an inhibitor of mitogen protein phosphatases (MKP) and/or a nucleic acid molecule encoding said inhibitor and optionally a pharmaceutically acceptable carrier.
  • MKP mitogen protein phosphatases
  • an inhibitor of mitogen protein phosphatases refers to compounds, for example organic compounds, nucleic acid molecules, (poly)peptides, etc. capable of inhibiting at least one member of the mitogen protein phosphatases (MKP) either on the gene expression or protein level.
  • MKP is a family of protein phosphatases that can be devided into two subgroups: the mitogen kinase phosphatase 1 (MKP-1 , PP-1 ) and MKP-2 (Cohen, J. Biol. Chem. 264 (1989), 21435-21438).
  • the MKP-2 group can be distinguished into three groups: 2A (PP-2A), divalent cation dependent, 2B, calcium and calmodulin dependent, and 2C, Mg 2+ dependent. Both subfamilies have been found in cardiac tissue abundantly, suggesting a significant physiological role (Misra-Press, J. Biol. Chem. 270 (1995), 14587-14596; Gupta, Am. J. Physiol. 270 (1996), H1159-H1164). It has been also shown for both MKPs to be immediate early gene products (Sun, Cell 75 (1993), 487-493) and to be transcriptionally regulated by a variety of agents.
  • oxidative stress/hypoxia was shown to induce a human gene encoding the protein phosphatase (Keyse, Nature 359 (1992), 644-647). Furthermore they are involved in various signaling pathways, suggesting to provide a feedback loop for activated protein kinases.
  • the regulatory mechanism is understood as follows: Activated mitogen-activated protein kinases (MAPK) due to an initiated cascade of sequential phosphorylated steps, can phosphorylate other Ser/Thr kinases (Cobb, Sem. Cancer Biol. 5 (1994), 261-268) or transcription factors such as TCF/Elk-1 with the result of promoting gene transcription (Sun, Cell 75 (1993), 487-493).
  • MK mitogen-activated protein kinases
  • One of the genes that is induced by mitogen stimulation is the murine 3CH134 gene (the human homolog CL 100), which encodes the dual-specificity MKP-1 (Keyse, Nature 359 (1992), 644- 647).
  • an inhibitor of mitogen protein phosphatases is cardioprotective and capable of mimicking ischemic preconditioning.
  • Experiments performed in accordance with the present invention demonstrate that local infusion of okadaic acid (OA) which is known as an inhibitor of mitogen protein phosphatases (MKP) significantly decreases myocardial infarction compared to the region at risk.
  • OA okadaic acid
  • an inhibitor of SerThr mitogen protein phosphatases (MKP) 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.
  • SAPKs stress-activated protein kinases
  • MAPKs mitogen-activated protein kinases
  • JNKs c-jun NH2- terminal kinases
  • 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). Suggestive effector proteins for the kinase are proto-oncogenes but the responsible effector protein for cardioprotection is still unknown.
  • the present inventors decided to study the consequences of inhibiting phosphatases of the above-described signal cascade. Okadaic acid, a known mitogen protein phosphatase inhibitor was used and found by the present inventors to enhance the activity of the upregulated JNK/SAPKs in a pig model; see the appended examples.
  • okadaic acid is capable of inducing cardioprotective effects due to its inhibitory action on mitogen protein phosphatases (MKP).
  • MKP mitogen protein phosphatases
  • okadaic acid (OA) a marine toxin from the black sponge Halichondria okadaii, an inhibitor of Ser/Thr phosphatases, protects pig myocardium against ischemic injury in an in vivo model.
  • the molecular mode of action is the prevention of inactivation of the Stress Activated Protein Kinase (SAPK/JNK) branch of the Mitogen-Activated- Protein-Kinases (MAPK) by Mitogen Protein (Kinase) Phosphatase (MKP) PP2A.
  • SAPK/JNK Stress Activated Protein Kinase
  • MAPK Mitogen-Activated- Protein-Kinases
  • MKP Mitogen Protein Phosphatase
  • okadaic acid 600 nmol/L or solvent were locally infused into left ventricular myocardium and biopsies from in situ beating hearts (anesthetized animals, open chest) were obtained from solvent- and OA-treated tissue after 10, 30 and 60 min of infusion.
  • OA infusion increased the activity (determined by in gel phosphorylation of N-terminal JUN) of both 46 and 55 kDa SAPK/JNKs 2-3 fold at 30 and 60 min of infusion.
  • Western blot analysis with phospho-specific SAPK/JNK (Thr 183/Tyr 185) antibodies showed an increased content of the phosphorylated forms of both SAPK/JNKs.
  • any inhibitor of mitogen protein phosphatases namely which are capable of excerting cytoprotective effects can be used for the purpose of the present invention.
  • the inhibitors of mitogen protein phosphatases (MKP) to be employed in the pharmaceutical compositions, methods and uses of the present invention such as okadaic acid may be obtained from various commercial sources or produced as described in the prior art.
  • inhibitors of MKP 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 kinases.
  • the invention relates to a method for treating, preventing and/or delaying ischemic cell death comprising contacting organs, tissue or cells with an inhibitor of mitogen protein phosphatases (MKP) and/or a nucleic acid molecule encoding said inhibitor.
  • MKP mitogen protein phosphatases
  • the respective organ may be kept alive in the presence of the above described compounds.
  • the invention relates to the use of inhibitors of mitogen protein phosphatases (MKPs) and/or a nucleic acid molecule encoding said inhibitor for the preparation of a pharmaceutical composition for preventing, treating and/or delaying ischemic cell death.
  • MKPs mitogen protein phosphatases
  • 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 mitogen protein phosphatases (MKP) and/or their encoding nucleic acid molecules, respectively, and optionally a pharmaceutically acceptable carrier or exipient.
  • suitable pharmaceutical carriers 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 ⁇ g to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 ⁇ g 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 10 6 to 10 12 copies of the DNA molecule.
  • 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.
  • Okadaic acid is known as a specific and potent inhibitor of Ser/Thr phosphatases PP-1 and PP-2A. 50% inhibition of the catalytic subunit of PP- 1 and PP-2A occurs at about 10 and 0.1 nmol/L, respectively. Total inhibition of protein phosphatase 2A occurs at 1 nmol/L okadaic acid (Hardie, Methods in Enzymology 201 (1991), 469-476). In the examples of the present invention a higher concentration was infused. However, the space of distribution of OA, once infused into the tissue, will significantly decrease the actual concentration toward the periphery.
  • 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 ischemic cause or disease.
  • 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, renal occlusive disease, a mesenterial arterial insufficiency or an ophthamic or retenal occlusion.
  • 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.
  • 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.
  • an inhibitor of mitogen protein phosphatases (MKP) 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 MKP encoding genes and/or their regulatory sequences may be used for repression of expression of a gene encoding MKP, 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 MKP.
  • the nucleic and amino acid sequences encoding MKPs are known in the art and described, for example, in Misra-Press, J. Biol. Chem.
  • 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.
  • PNA peptide nucleic acid
  • the so-called "peptide nucleic acid” (PNA) technique can be used for the detection or inhibition of the expression of a nucleic acid.
  • PNA peptide nucleic acid
  • 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.
  • the appropriate programs can be used for the identification of interactive sites of a putative inhibitor and with the MKP 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 okadaic acid or other known inhibitors of MKP.
  • pseudopeptide analogues of the natural amino acid sequence of the peptide 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 okadaic acid and other inhibitors of MKPs 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.
  • antibodies specifically recognizing MKPs or parts, i.e. specific fragments or epitopes, of such MKPs and thereby inactivating the MKP 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.
  • surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the MKP (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 MKP 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.
  • the inhibitor of mitogen protein phosphatases (MKP) comprised in the pharmaceutical compositions, methods or uses is okadaic acid or a functional derivative or analogue thereof which may be obtained by, e.g., peptidomimetics.
  • okadaic acid was found to induce cytoprotective effects due to inhibition of MKP.
  • Okadaic acid (9,10-Deepithio-9,10-didehydroacanthifolicin; halochondrine A.
  • C H ⁇ f a; mol wt 805.02.C 65.65%, H 8,51 %, O 25.84%) is a ionophoric polyether first identified in marine organisms and isolated from marine black sponges, Halichondria (okadai or melanodocia). The total synthesis of okadaic acid was described in, e.g., Isobe, Tetrahedron 43 (1987), 4767, and its ability to inhibit protein phosphatases was described in, e.g., Takai, FEBS Letters 217 (1987), 81 and Bialojan, Biochem. J. 256 (1988), 283.
  • okadaic acid means molecules the chemical structure of which is based on that of okadaic acid and which are capable of inducing cardioprotective effects.
  • the cardioprotective effects of the okadaic acid-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.
  • 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.
  • peptidomimetics and/or computer aided design of appropriate derivatives and analogues can be used, for example, according to the methods described above.
  • the pharmaceutical composition uses and methods of the invention are designed to be applied in conjugation with growth factors, preferably fibroblast growth factor such as acidic or basic fibroblast growth factor (aFGF, bFGF), insulin-like growth factor-ll (IGF-II) or vascular endothelial growth factor (VEGF).
  • growth factors preferably fibroblast growth factor such as acidic or basic fibroblast growth factor (aFGF, bFGF), insulin-like growth factor-ll (IGF-II) or vascular endothelial growth factor (VEGF).
  • aFGF acidic or basic fibroblast growth factor
  • IGF-II insulin-like growth factor-ll
  • VEGF vascular endothelial growth factor
  • Pharmaceutical compositions comprising, for example, okadaic acid and/or another inhibitor of mitogen protein phosphatases (MKP), and a growth factor such as aFGF may advantageously be used for the treatment of peripheral vascular diseases or coronary artery disease.
  • MKP mitogen
  • the method of the invention comprises
  • step (c) reintroducing the cells, tissue or organ obtained in step (b) into the same or a different subject.
  • an inhibitor of mitogen protein phosphatases (MKP) and the nucleic acid molecules encoding said inhibitor 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.
  • viral vectors described in the prior art may be used for transfecting certain cells, tissues or organs.
  • a pharmaceutical composition of the invention which comprises a nucleic acid molecule encoding an inhibitor of mitogen protein phosphatases (MKP) 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).
  • 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 mitogen protein phosphatases (MKP) to a patient or by transfecting cells with said nucleic acid molecule ex vivo and infusing the transfected cells into the patient.
  • MKP mitogen protein phosphatases
  • 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.
  • said cell is a germ line cell, embryonic cell, or egg cell or derived therefrom.
  • the introduced nucleic acid molecules encoding the inhibitor of mitogen protein phosphatases express said inhibitor after introduction into said cell and preferably remain in this status during the lifetime of said cell.
  • cell lines which stably express said inhibitor of mitogen protein phosphatases (MKP) 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 an inhibitor of mitogen protein phosphatases (MKP).
  • MKP mitogen protein phosphatases
  • 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.
  • 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).
  • 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.
  • 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 mitogen protein phosphatases (MKP) 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.
  • MKP mitogen protein phosphatases
  • the pharmaceutical composition in the use of the invention is designed for administration by intracoronary, intramuscular, intravenous, intraperitoneal, intraarterial or subcutaneous routes.
  • the inhibitor of MKP i.e. okadaic acid was administered locally via osmotic minipump.
  • 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 acquired ischemic diseases.
  • 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.
  • the present invention is based on the ability to prevent or delay ischematic cell death.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an inhibitor of mitogen protein phosphatases (MKP) and/or a nucleic acid molecule encoding said inhibitor, and optionally a pharmaceutically acceptable carrier.
  • MKP mitogen protein phosphatases
  • the invention provides a method for treating, preventing and/or delaying ischemic cell death comprising contacting organs, tissue or cells with an inhibitor of mitogen protein phosphatases (MKP) and/or a nucleic acid molecule encoding said inhibitor, and optionally a pharmaceutically acceptable carrier.
  • MKP mitogen protein phosphatases
  • the invention provides a method for treating, preventing and/or delaying ischemic cell death comprising contacting organs, tissue or cells with an inhibitor of mitogen protein phosphatases (MKP) and/or a nucleic acid molecule encoding said inhibitor.
  • MKP mitogen protein phosphatases
  • the present study consisted of five experimental groups. Group I was subjected to 60 min of occlusion and 60 min of reperfusion (control group I). In group II the animals were subjected to 5 min of occlusion and 45 min of reperfusion followed by index ischemia (60 min) and reperfusion (60 min) (control group II). In group III okadaic acid (600 nmol/L) or KHB were administered 60 min prior to the index ischemia of 60 min and the following reperfusion period of 60 min. In group IV okadaic acid (600 nmol/L) or KHB were infused for 10 min prior and for 45 min after the 5 min of occlusion.
  • Panel A shows a full size transmural infarct after local infusion of the solvent (Krebs Henseleit buffer.
  • Panel B shows the infusion of okadaic acid (OA) with a slavaged region around the needle tip and along the needle track. KHB infusion via the left needle shows no salvage.
  • Panel C shows small patchy infarcts and large salvaged regions caused by the combination of OA infusion with a 5 minute period of coronary occlusion followed by 45 minute of reperfusion prior to the 60 minute period of index ischemia followed by reperfusion.
  • the needles for intramyocardial microinfusion were placed into the subsequent ischemic part of the left ventricle.
  • the fluorescent microspheres demarcate the non fluorescent area of risk.
  • TTC staining the myocardial protection was defined as stained tissue surrounding the microinfusion needles in transmurally infarcted myocardium.
  • JNK-46 B and JNK-55 (C) activation after local infusion of okadaic acid. Data were derived from in gel kinase assays and are expressed as a percentage of values for corresponding KHB-treated tissue. Each bar represents the mean ⁇ S.E.M. (*p ⁇ 0.05 vs corresponding KHB control). Quantitative analysis of gels was performed using Phosphorimage SF (Molecular Dynamics). (D) Western blotting with phospho- specific antibody that recognizes phosphorylated SAPK/JNKs.
  • Example 1 The in vivo animal test system
  • Azaperone, metomidate and piritramid were purchased from Janssen Pharmaceutica, Neuss, Germany.
  • Okadaic acid was purchased from Calbiochem or Biomol Feinchemikalien GmbH, Hamburg, Germany.
  • Okadaic acid 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, ⁇ -chloralose and TTC were obtained from Sigma, Deisenhofen, Germany. 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.
  • Protein Ser/Thr phosphatase (PSP) assay system, phospho-ERK, phospho-SAPKJNKs, phospho-p38/MAPK antibodies, horseradish peroxidase-linked goat anti-rabbit immunoglobulin and enhanced chemiluminescence (ECL) reagents were from New England Biolabs. Prestained molecular mass markers were from Bio-Rad.
  • Horseradish peroxidase-linked goat anti-rabbit immunoglobulin, the enhanced chemiluminescence (ECL) reagents, the nitrocellulose membrane, the rainbow molecular weight markers, autoradiography films and ⁇ 32 P-ATP were obtained from Amersham International. Tween 20 was purchased from Serva. The specific polyclonal antibodies against ERKs, SAPK/JNK and p38/MAPK kinase were purchased from Santa Cruz Biotechnology.
  • Recombinant c-jun containing the N-terminal regulatory region (encompassing the N- terminal transactivating domain) of amino acids 1-135 and recombinant MAPKAP2 (GST-MAPKAP 46 ⁇ 00 encompassing the catalytic domain) were expressed as glutathione S-transferase fusion protein in Escherichia coli and purified by glutathione-Sepharose (Pharmacia) chromatography; see Bogoyevitch, supra.
  • the chest was opened by midstemal thoracotomy and the heart was suspended in a pericardial cradle.
  • a loose ligature was placed halfway around the left anterior descending artery (LAD), and was subsequently tightened to occlude the vessel.
  • LAD left anterior descending artery
  • eight 26 gauge needles were connected by tubing with a peristaltic pump (Miniplus, 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.
  • Okadaic acid was diluted in KHB and KHB infusion served as a negative control.
  • the infusion rate was 8 microliters per minute.
  • 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 in between. Successful countershock defibrillation was not a criteria for exclusion.
  • Fig. 1 The present study consisted of five experimental groups (Fig. 1 ). Group I was subjected to 60 min of occlusion and 60 min of reperfusion (control group 1 ). In group II the animals were subjected to 5 min of occlusion and 45 min of reperfusion followed by index ischemia (60 min) and reperfusion (60 min) (control group 2). In group III okadaic acid (600 nmol/L) or KHB were administered 60 min prior to the index ischemia and the following reperfusion period. In group IV okadaic acid (600 nmol/L) or KHB were infused for 10 min prior and for 45 min after the 5 min occlusion period which was followed by the index ischemia and reperfusion.
  • 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.
  • 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 triphenyltetrazoiium chloride (TTC) (1%) in PBS, pH: 7.0 for 15 min.
  • TTC triphenyltetrazoiium chloride
  • 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 with UV-light and normal artificial day light and the pictures were used for further planimetric evaluation.
  • Example 2 Infusion of okadaic acid decreases myocardial infarction
  • the left ventricular biopsies from pig myocardium were resuspended in 5 vol of ice-cold buffer A containing 20 mM Tris-HCI, 0.25 M sucrose, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, 0.5 mM PMSF, 100 mM sodium orthovavadate and 10 mM sodium fluoride (pH 7.4) and homogenized with a Teflon-glas homogenizer.
  • NaF and PMSF were omitted from the homogenization buffer. The homogenate was centrifugated at 14000 x g for 30 min at 4°C.
  • the pellet was resuspended in buffer.
  • the supernatant after this centrifugation represented the cytosolic fraction, the resuspended pellet was designed as a particulate fraction.
  • the Laemmli sample buffer was added to the supernatant, and the proteins were denatured by heating. The denatured probes were applied to SDS-PAGE and used for assays of MAPKs by in gel phosphorylation and for Western blot analysis.
  • proteins from cytosolic fractions (20 mg) were separated in 10 % SDS polyacrylamide gels containing 0.25 mg/ml of c-jun protein. Furthermore, equivalent amounts of proteins were separated on SDS-polyacrylamide gels containing 0.5 mg/ml of myelin basic protein (for ERKs), 0.5 mg/ml of GST-c-jun ⁇ (for SAPKs/JNKs) or 0.5 mg/ml of GST-MAPKAP-K2 46 . 400 (for p38-MAPK).
  • 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 denaturated by incubation for 1 or 2 hours 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 ⁇ -mercaptoethanol for 16 hours.
  • the p55 kinase activity reached 2.4 fold rise compared to the KHB infusion.
  • the p46 SAPK showed a significant 2.8 fold increase compared to the KHB data.
  • Measuring the p38 kinase activity after okadaic acid treatment showed a non significant 1.5 fold increase compared to the control value.
  • Investigating the ERKs (p42 and p44) for both a non significant 1.5 fold increase could be demonstrated compared to KHB.
  • the amount of the phosphorylated form of SAPK/JNKs was investigated with a phospho-specific SAPK/JNK (Thr 183/Tyr 185) antibody in control, KHB- and OA- treated tissue. It was found that the local infusion of okadaic acid induced a significant increase of the phosphorylated SAPK/JNKs (Fig. 5D). Western blot assay with a specific SAPK/JNK antibody showed that there were no changes in SAPK/JNKs abundance when cytosolic fractions from untreated, KHB- and OA- treated tissue were compared.
  • the p38-MAPK activities were investigated by in gel phosphorylation of MAPKAP-K2 as a substrate. Local infusion of OA did not stimulate the p38-MAPK activity as compared to the corresponding KHB controls (Fig. 7A and B).
  • the phospho-specific p38-MAPK (Thr180/Tyr 182) antibody was used to determine the content of phosphorylated p38-MAPK but no significant changes during OA-treatment were observed.
  • Example 5 Analysis of protein Ser/Thr phosphatase activities
  • the activities of protein Ser/Thr phosphatases were investigated using the protein Ser/Thr phosphatase (PSP) assay system.
  • Radiolabeled myelin basic protein (MBP) was prepared by phosphorylation on serine and threonine residues with protein kinase A in the presence of [ ⁇ - 32 P]-ATP.
  • the protein Ser/Thr phosphatase activities were then determined by measuring the release of inorganic phosphate (TCA- soluble radioactivity) from labeled MBP.
  • the reaction mixture used for the assay of total protein Ser/Thr phosphatases contained assay buffer (50 mmol/L Tris.HCI, 0.1 mmol/L Na2EDTA, 5 mmol/L DTT, 0.01 % Brij 35, pH 7.0) and diluted protein sample.
  • assay buffer 50 mmol/L Tris.HCI, 0.1 mmol/L Na2EDTA, 5 mmol/L DTT, 0.01 % Brij 35, pH 7.0
  • the protein samples were diluted in assay buffer and we used approximately 1.5 mg of protein per reaction.
  • the reaction was started by adding the
  • protein phosphatase inhibitor-2 (I-2, NEB), which inhibits specifically the catalytic subunits of protein phosphatase 1 (PP-1 ) and we used 0.4 mg of I-2 per reaction (the resulting concentration in the assay system was 8 mg/ml).
  • the protein phosphatase activity obtained in the presence of protein phosphatase inhibitor I-2 represents the protein phosphatase 2 (PP-2A) activity.
  • the protein phosphatase activities were then expressed relative to the protein content (specific activity). Protein concentrations were determined by using BSA as a standard (Bradford Analyt Biochem 72 (1976), 248-254).
  • the Ser/Thr phosphatase activities were investigated after local infusion of 600 nmol/L okadaic acid at 10, 30 and 60 min. Okadaic acid was diluted in KHB and local infusion of KHB served as a negative control. It was found that the infusion of OA for 30 and 60 min significantly attenuated the Ser/Thr protein phosphatase activities as compared to corresponding KHB controls (Fig. 4A). There were no significant differences in protein phosphatase activities between the infusion of OA for 30 and 60 min.
  • the protein phosphatase 2A (PP-2A) activities were measured in the presence of inhibitor 1-2 to neutralize PP1. In this case a significant reduction of PP- 2A activities after 60 min infusion of OA was observed (Fig. 4B). The PP-2A activities in this case represented about 79% of control KHB activity. In all experimental situations the protein phosphatase activities after KHB infusion did not differ from phosphatase activities determined in untreated control tissue.
  • Soluble fractions of heart were subjected to SDS-PAGE in 10% polyacrylamide gels and proteins were transferred onto nitrocellulose membranes.
  • Anti-ERK, anti- phospho-ERK, anti-SAPK/JNK, anti-phospho-SAPK/JNK, anti-p38/MAPK and anti- phospho-p38/MAPK antibodies were used for primary immunodetection.
  • the secondary antibody directed against all antibodies was peroxidase labeled anti-rabbit immunoglobulin. Bound antibodies were detected by the ECL Western blot detection method.
  • okadaic acid causes the inhibition of Ser/Thr protein phosphatases in the intact myocardium where it increases phosphorylation and activation of SAPK/JNKs as a consequence of decreased dephosphorylation.
  • the other MAPKinase pathways (ERKs and p38) appear unaffected by the drug.
  • the results suggest that the SAPK/JNK activity is tightly controlled by the protein phosphatase PP2A.
  • OA treatment exhibited a powerful cardioprotective effect as judged by the infarct size reduction to 30% of its control value.
  • the protective effect of okadaic acid indicates the involvement of protein phosphorylation in the adaptation of the myocardium to ischemic stress.
  • Ser/Thr phosphatase activities after the inhibitor treatment suggest that the effect of the inhibitor, exemplified by okadaic acid, is mediated through these enzymes, in particular through the SAPK/JNK pathway. It is thus expected that other compounds which are capable of inhibiting Ser/Thr phosphatase activities exhibit similar protective effects.
  • 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.

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Abstract

L'invention concerne la modulation de la mort cellulaire ischémique. L'invention traite, en particulier, de compositions pharmaceutiques comprenant un inhibiteur de phosphatases de protéines de mitogène (MKP) et/ou une molécule d'acide nucléique codant cet inhibiteur, qui présentent une grande utilité pour traiter, prévenir et/ou retarder la mort cellulaire ischémique. En outre, l'invention concerne des procédés pour traiter, prévenir et/ou retarder la mort cellulaire ischémique consistant à placer des organes, des tissus ou des cellules en contact avec un inhibiteur de phosphatases de protéines de mitogènes (MKP) et/ou une molécule d'acide nucléique codant cet inhibiteur.
PCT/EP1998/006269 1997-10-01 1998-10-01 Nouvelle composition pour traiter, prevenir et/ou retarder la mort cellulaire ischemique Ceased WO1999016457A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999061039A3 (fr) * 1998-05-22 2000-02-17 Max Planck Gesellschaft Nouvelle composition de modulation de la mort cellulaire ischemique
WO2002009680A3 (fr) * 2000-08-02 2002-07-18 Michael Walter Substance a activite pharmacologique, destinee au traitement de maladies cardio-vasculaires

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0725786A (ja) * 1990-05-16 1995-01-27 Univ Rockefeller アルツハイマー病を伴うアミロイドーシスの治療
EP0551200A1 (fr) * 1992-01-07 1993-07-14 National University Of Singapore Protéine phosphatase inhibiteur pour l'utilisation en thérapeutique
WO1997003660A2 (fr) * 1995-07-21 1997-02-06 The Rockefeller University Modulation de l'activite de la phosphatase de la proteine et utilisation concernant la fecondite et la contraception

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999061039A3 (fr) * 1998-05-22 2000-02-17 Max Planck Gesellschaft Nouvelle composition de modulation de la mort cellulaire ischemique
WO2002009680A3 (fr) * 2000-08-02 2002-07-18 Michael Walter Substance a activite pharmacologique, destinee au traitement de maladies cardio-vasculaires

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