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WO1999001150A1 - Nouvelle composition servant a traiter, a prevenir ou a retarder la mort cellulaire ischemique - Google Patents

Nouvelle composition servant a traiter, a prevenir ou a retarder la mort cellulaire ischemique Download PDF

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Publication number
WO1999001150A1
WO1999001150A1 PCT/EP1998/004134 EP9804134W WO9901150A1 WO 1999001150 A1 WO1999001150 A1 WO 1999001150A1 EP 9804134 W EP9804134 W EP 9804134W WO 9901150 A1 WO9901150 A1 WO 9901150A1
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Prior art keywords
afgf
protein
sapk
activator
biological function
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PCT/EP1998/004134
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English (en)
Inventor
Wolfgang Schaper
Patrik Htun
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Priority to AU88557/98A priority Critical patent/AU8855798A/en
Publication of WO1999001150A1 publication Critical patent/WO1999001150A1/fr

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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
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

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 a protein having the biological activity of acidic fibroblast growth factor (aFGF) and/or a nucleic acid molecule encoding said protein having the biological activity of aFGF and/or an activator of stress-activated protein kinases (SAPK) and/or a nucleic acid molecule encoding said activator of SAPK which are particularly useful for treating, preventing and/or delaying ischemic cell death.
  • aFGF acidic fibroblast growth factor
  • SAPK stress-activated protein kinases
  • the present invention also relates to a method for treating, preventing and/or delaying ischemic cell death comprising contacting organs, tissue or cells with a protein having the biological activity of aFGF and/or an activator of SAPK and/or a nucleic acid molecule encoding said aFGF and/or activator of SAPKs.
  • Organs which depend on postmitotic cells 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
  • VEGF Insulin-like growth factor
  • IGF-II Insulin-like growth factor
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a protein having the biological activity of acidic fibroblast growth factor (aFGF) and/or a nucleic acid molecule encoding said protein having the biological activity of aFGF and/or an activator of stress-activated protein kinases (SAPK) and/or a nucleic acid molecule encoding said activator of SAPK and optionally a pharmaceutically acceptable carrier.
  • aFGF acidic fibroblast growth factor
  • SAPK stress-activated protein kinases
  • the term "acidic fibroblast growth factor” or '"aFGF” refers to proteins and peptides which can act on FGF transmembrane signaling receptors with intrinsic protein tyrosine kinase activity and lead to initiating the mitogenic activity responsible for the cytoprotective effect.
  • any aFGF or other substances which are functionally equivalent to an aFGF, namely which are capable of excelling cytoprotective effects can be used for the purpose of the present invention.
  • These substances include compounds that have been obtained by peptide mimetics or compounds that are derived from natural aFGF and modified by recombinant DNA technology but essentially retain their biological function.
  • the action of the aFGFs employed in the present invention may not be limited to the above-described property but they may also activate, for example, PLC ⁇ and/or PKC.
  • Acidic fibroblast growth factor is a member of the FGF family, that consists of nine structurally related polypeptides that play a key role in numerous aspects of embryogenesis, growth, angiogenesis and cell survival (Gospodarowicz, Cell Differ. Dev. 91 (1986), 1-17; Baird, Recent Prog. Horm. Res. 42 (1986), 143-205; Clegg, J. Cell Biol. 105 (1987), 949-956; Slack, Nature 326 (1987), 197-200; Liu, Endocrinology 123(4) (1988), 2027-2031).
  • aFGF is cardioprotective and capable of mimicking ischemic preconditioning.
  • Experiments performed in accordance with the present invention demonstrate that local infusion of aFGF significantly decreases myocardial infarction compared to the region at risk.
  • aFGFs or nucleic acid molecules encoding aFGFs can be used to prevent, delay or treat ischemic cell death, which is needed for the cure of several occlusive diseases and particularly useful for bypass-operations and heart transplantations. The same holds true for other compounds essentially retaining the biological function of aFGF that have been described hereinabove.
  • aFGFs to be employed in the pharmaceutical compositions, methods and uses of the present invention may be obtained from various commercial sources or produced as described in the prior art.
  • functional equivalent or “functional part” of an aFGF means a protein having part or all of the primary structural conformation of an aFGF possessing at least the biological property of excelling cardioprotective effects or as a product obtained by peptidomimetics.
  • the functional part of said protein or the functionally equivalent protein may be a derivative of an aFGF by way of amino acid deletion(s), substitution(s), insertion(s), addition(s) and/or replacement(s) of the amino acid sequence, for example by means of site directed mutagenesis of the underlying DNA.
  • Recombinant DNA technology is well known to those skilled in the art and described, for example, in Sambrook et al. (Molecular cloning; A Laboratory Manual, Second Edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour NY (1989)).
  • the cytoprotective effect is caused by the mitogenic part of the protein, since infusing of a truncated version of aFGF did not induce cardioprotection.
  • aFGF or functional parts thereof or compounds such as proteins which are functionally equivalent to aFGFs may be produced by known conventional chemical and semi-chemical means or by recombinant techniques employing the amino acid and DNA sequences described in the prior art (Crumley, Biochem. Biophys. Res.
  • aFGF may be produced by culturing a suitable cell or cell line which has been transformed with a DNA sequence encoding upon expression under the control of regulatory sequences an aFGF or a functional part thereof or a protein which is functionally equivalent to aFGF.
  • nucleic acid molecule encoding aFGF or a functional derivative thereof can be operably linked to regulating sequences allowing the expression of said aFGF or functional derivative thereof in the cell, tissue or organ of the patient.
  • Suitable regulatory sequences and vectors which may be employed to express the nucleic acid molecule encoding aFGF or a functional derivative thereof are known in the art and are described, for example, in Kaneda, Rinsho Byori 45
  • activator of stress-activated protein kinases within the meaning of the present invention refers to compounds, for example organic compounds, nucleic acid molecules, (poly)peptides, etc. capable of inducing at least one member of the stress-activated protein kinases (SAPK) which is a subfamily of the mitogen-activated protein kinases (MAPK).
  • SAPK stress-activated protein kinases
  • MAPK mitogen-activated protein kinases
  • said activatiors may be obtained by peptidomimetics or by recombinat DNA techniques described above.
  • said member of SAPK is p46 and/or p55.
  • SAPK stress-activated protein kinases
  • SAPKs c-jun NH2-terminal kinases
  • JNKs c-jun NH2-terminal kinases
  • the invention relates to a method for treating, preventing and/or delaying ischemic cell death comprising contacting organs, tissue or cells with a protein having the biological function of acidic fibroblast growth factor (aFGF) and/or a nucleic acid molecule encoding said protein having the biological function of aFGF and/or an activator of stress-activated protein kinases (SAPK) and/or a nucleic acid molecule encoding said activator of SAPK.
  • aFGF acidic fibroblast growth factor
  • SAPK stress-activated protein kinases
  • SAPK stress-activated protein kinases
  • the invention relates to the use of a protein having the biological function of acidic fibroblast growth factor (aFGF) and/or a nucleic acid molecule encoding said protein having the biological function of aFGF and/or an activator of stress-activated protein kinases (SAPK) and/or a nucleic acid molecule encoding said activator of SAPK for the preparation of a pharmaceutical composition for preventing, treating and/or delaying ischemic cell death.
  • aFGF acidic fibroblast growth factor
  • SAPK stress-activated protein kinases
  • Said pharmaceutical compositions can be used, for example, with or instead of the compounds commonly used for the treatment of heart stroke, such as aspirin and/or streptokinase.
  • the pharmaceutical composition of the invention comprises at least one protein having the biological activity of aFGF as defined above and/or at least one activator of SAPK as defined above and/or their enconding nucleic acid molecules, respectively, and optionally a pharmaceutically acceptable carrier or exipient.
  • 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.
  • 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.
  • 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 aso be in the range of 1 ⁇ g to 10 mg units per kilogram of body weight per minute, respectively.
  • 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.
  • 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.
  • the pharmaceutical compositions, methods and uses of the invention are for the treatment of subjects suffering from arteriosclerosis, a coronary artery disease, a cerebral occlusive disease, a peripheral occlusive disease, a visceral occlusive disease, a mesenterial arterial insufficiency or an ophthamic or retenal occlusion.
  • 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.
  • the protein having the biological function of aFGF used in the pharmaceutical compositions, methods and uses of the invention is a recombinant aFGF.
  • DNA sequences encoding aFGFs which can be used in the methods and uses of the invention are described in the prior art.
  • DNA and amino acid sequences of aFGFs are available in the GenBank database.
  • methods for the production of recombinant proteins are well-known to the person skilled in the art; see, e.g., Sambrook et al., supra.
  • the SAPK is (are) p46 and/or p55.
  • the cardioprotective effect of upregulated SAPKs is predominantly due to activation of the SAPKs p46 and p55.
  • the activator of the SAPK comprised in the pharmaceutical compositions, methods or uses is anisomycin or a functional derivative or analogue thereof which may be obtained, e.g., by peptidomimetics. As described in the appended examples anisomycin was found to induce cytoprotective effects due to SAPK activation.
  • Anisomycin (1 ,4,5-trideoxy-1 ,4-imino-5-(4-methoxyphenyl)-D-xylo-pentitol 3-acetate; [2R-(2 ⁇ ,3 ⁇ ,4 ⁇ )]-2-[(4-methoxyphenyl)methyl]-3,4-pyrrolidinediol 3-acetate; 2-p- methoxyphenylmethyl-3-acetoxy-4-hydroxypyrrolidine; Flagecidin. C 14 H 19 NO 4 ; mol wt 265.30. C 63.38%, H 7.22%, N 5.28%, O 24.12%) is a protein synthesis inhibiting antibiotic originally isolated from Streptomyces griseolus and S.
  • the pharmaceutical composition is designed for administration in conjugation with growth factors, preferably fibroblast growth factor such as basic fibroblast growth factor (bFGF), insulin-like growth factor-ll (IGF-II) or vascular endothelial growth factor (VEGF).
  • growth factors preferably fibroblast growth factor such as basic fibroblast growth factor (bFGF), insulin-like growth factor-ll (IGF-II) or vascular endothelial growth factor (VEGF).
  • bFGF basic fibroblast growth factor
  • IGF-II insulin-like growth factor-ll
  • VEGF vascular endothelial growth factor
  • Pharmaceutical compositions comprising, for example, aFGF and/or anisomycin, and another growth factor such as VEGF may be used for the treatment of peripheral vascular diseases or coronary artery disease.
  • 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.
  • the proteins having the biological activity of aFGF, the activators of SAPK and the nucleic acid molecules encoding said proteins or activators of SAPK are administered either alone or in combination, and optionally together with a pharmaceutically acceptable carrier or exipient.
  • Said nucleic acid molecules may be stably integrated into the genome of the cell or may be maintained in a form extrachromosomally, see, e.g., Calos, Trends Genet. 12 (1996), 463-466.
  • viral vectors described in the prior art and cited above may be used for transfecting certain cells, tissues or organs.
  • a pharmaceutical composition of the invention which comprises a nucleic acid molecule encoding aFGF in gene therapy.
  • Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenoviruses, and adeno-associated viruses, among others. Delivery of nucleic acid molecules to a specific site in the body for gene therapy may also be accomplished using a biolistic delivery system, such as that described by Williams (Proc. Natl. Acad. Sci. USA 88 (1991 ), 2726-2729).
  • Standard methods for transfecting cells with nucleic acid molecules are well known to those skilled in the art of molecular biology, see, e.g., WO 94/29469.
  • Gene therapy to prevent or decrease the development of ischemic cell death may be carried out by directly administering the nucleic acid molecule encoding aFGF to a patient or by transfecting cells with said nucleic acid molecule ex vivo and infusing the transfected cells into the patient.
  • 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.
  • nucleic acid molecules comprised in the pharmaceutical composition of the invention may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) containing said nucleic acid molecules into the cell.
  • said cell is a germ line cell, embryonic cell, or egg cell or derived therefrom.
  • the introduced nucleic acid molecules encoding the protein having the biological activity of aFGF or activator of SAPK express said protein or activator after introduction into said cell and preferably remain in this status during the lifetime of said cell.
  • cell lines which stably express said protein having the biological activity of aFGF or said activator of SAPK may be engineered according to methods well known to those skilled in the art. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the recombinant DNA molecule or vector of the invention and a selectable marker, either on the same or separate vectors. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows for the selection of cells having stably integrated the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the protein having the biological activity of aFGF or said activator of SAPK.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, Cell 11 (1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska, Proc. Natl. Acad. Sci. USA 48 (1962), 2026), and adenine phosphoribosyltransferase (Lowy, Cell 22 (1980), 817) in tk “ , hgprt " or aprt " cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, Proc. Natl.
  • trpB which allows cells to utilize indole in place of tryptophan
  • hisD which allows cells to utilize histinol in place of histidine
  • ODC ornithine decarboxylase
  • DFMO 2-(difluoromethyl)-DL-ornithine
  • the nucleic acid molecule comprised in the pharmaceutical composition preferably for the use of the invention is designed for the expression and secretion of the aFGF or activator of SAPK by cells in vivo in a form suitable for the interaction with its receptor by, for example, direct introduction of said nucleic acid molecule or introduction of a plasmid, a plasmid in liposomes, or a viral vector (e.g. adenoviral, retroviral) containing said nucleic acid molecule.
  • aFGF or activator of SAPK by cells in vivo in a form suitable for the interaction with its receptor by, for example, direct introduction of said nucleic acid molecule or introduction of a plasmid, a plasmid in liposomes, or a viral vector (e.g. adenoviral, retroviral) containing said nucleic acid molecule.
  • the pharmaceutical composition in the use of the invention is designed for administration by intracoronar, intramuscular, intravenous, intraperitoneal or subcutenous routes.
  • the human form of the aFGF protein was administered locally via osmotic minipump.
  • said protein having the bilogical activity of aFGF is aFGF.
  • the present invention relates to the use of any one of the beforedescribed nucleic acid molecules in gene therapy, for example, for curing inborn or aquired ischemic diseases.
  • 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.
  • public database "Medline” may be utilized which is available on internet, e.g. under http://www.ncbi.nlm.nih.gov/ PubMed/medline.html. Further databases and addresses can be obtained using http://www.lycos.com.
  • 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.
  • Figure 1 Experimental groups for testing with aFGF and bFGF. Six groups of animals were studied:
  • Control animals (group I) were subjected to 60 min LAD-occlusion (CO) and 120 min reperfusion (REP).
  • the groups II and III received various compounds: aFGF (0.5-1 ⁇ g/ml) and bFGF (2 ⁇ g/ml) by means of intramyocardial microinfusion (IM) for 60 min prior to the LAD occlusion.
  • Groups IV and V were treated with the growth factor antagonist suramin (0.4 ⁇ g/ml) or the tyrosine kinase inhibitor geisseine (0.35 ⁇ g/ml) prior to FGF infusion.
  • Group VI was treated with the truncated aFGF (0.5-1 ⁇ g/ml).
  • Figure 2 Infarct areas: Treatment with the growth factor antagonist suramin and the tyrosine kinase inhibitor genisteine in comparison with the aFGF and bFGF induced cardioprotection. Control: 83.4 ⁇ 2.8%, aFGF: 51.8 ⁇ 7.7, bFGF: 57.2 ⁇ 6.5%, suramin: 77.0 ⁇ 1.2%, genistein: 77.2 ⁇ 2.4%, truncated aFGF:78.3 ⁇ 0.73%; Figure 3: Hemodynamic data during IM. Systemic hemodynamics (mean ⁇ SEM, error bars hidden behind the used symbols) remained unchanged during intramyocardial microinfusion.
  • LVP left ventricular pressure
  • AOP aortic pressure
  • HR heart rate
  • dP/dt first derivative of left ventricular pressure
  • FIG. 4 Intramyocardial microinfusion of aFGF.
  • the needles for IM (arrows, 26 gauge) were placed in pairs into the subsequent ischemic part of the left ventricle.
  • the fluorescent microspheres demarcate the none fluorescent area of risk. After TTC-staining myocardial protection was defined as stained tissue surrounding the microinfusion-needles in transmurally infarcted myocardium.
  • Figure 5 Reduction of infarcted areas by bFGF.
  • Treatment with bFGF significantly reduced infarcted area (IA) normalized to ischemic area (RA) as determined by TTC-staining and planimetry, (shown in double exposure technique).
  • FIG. 6 Prevention of cardioprotection. Infusion of A: Suramin, B: Genistein prior to the aFGF/bFGF treatment prevented cardioprotection. Fluorescent microspheres demarcate the none fluorescent risk area, whereas TTC- staining shows the infarcted area (shown in double exposure technique). The area around the needles does not show any cardioprotection.
  • Figure 7 Localization of aFGF (green), counterstaining with phalloidin (red).
  • A In control tissue endogenous aFGF was detected in the extracellular matrix and in perinuclear localization of myocytes.
  • B Accumulation of exogenous aFGF is found mainly in a perinuclear localization within numerous myocytes.
  • Figure 8 Localization of bFGF (green).
  • Figure 11 Infarct areas. Treatment with the SAPK activator anisomycin reduces infarct sizes significantly from 83.4+2.8% (control) to 48.1+5.1% (p ⁇ 0.01).
  • Figure 12 Intramyocardial microinfusion of anisomycin.
  • the needles for IM (arrows, 26 gauge) were placed in pairs into the subsequent ischemic part of the left ventricle.
  • the fluorescent microspheres demarcate the none fluorescent area of risk.
  • TTC-staining myocardial protection was defined as stained tissue surrounding the microinfusion-needles in transmurally infarcted myocardium.
  • Figure 13 Graphs showing the quantitative changes in activities of SAPKs p46 and p55 after 10 and 30 minutes of infusion. Quantitative analysis of gels was performed using Phosphorimage SF (Molecular Dynamics). Data are expressed as a percentage of control value (control nonischemic tissue) and KHL treated tissue, each bar represents the mean ⁇ S.E.M.
  • Figure 14 Stimulation of SAPKs in the cytosolic fractions isolated from biopsies obtained from control tissue (C), at different time points of anisomycin and KHL infused tissue (10, 30 min).
  • the in gel GST-c-jun kinase assay showed the activation of 46- and 55 kDa protein kinases (p-46, p-55).
  • the maximal activation of anisomycin infused tissue was reached after 30 min, KHL treated tissue induced an insignificant increase of JNKs activity at both time points.
  • Example 1 The in vivo animal test system
  • a 5F high fidelity catheter tipped manometer (Millar Instruments, Houston, Texas, USA) was inserted via the right common carotid artery into the left ventricle to measure left ventricular pressure and to calculate its first derivative (LV dP/dt).
  • the chest was opened by a midsternal thoracotomy and the heart was suspended in a pericardial cradle.
  • a loose ligature was placed halfway around the left anterior descending coronary artery (LAD), and was subsequently tightened to occlude the vessel.
  • LAD left anterior descending coronary artery
  • Azaperone, metomidate and piritamid were purchased from Janssen Pharmaceutica, Neuss, Germany.
  • Anisomycin is purchased from Biomol Feinchemikalien GmbH, Hamburg, Germany.
  • Anisomycin was dissolved in Krebs-Henseleit buffer (pH: 7.4).
  • Myelin basic protein, PKI, EGTA, PMSF, bovine serum albumin, ATP, dithiotreitol, SDS-PAGE reagents and polyclonal anti-ERK1/2, ⁇ -chloralose, TTC and Genistein were obtained from Sigma Chemical Co.
  • aFGF 0.5 ⁇ g/ml
  • bFGF 2.0 ⁇ g/ml
  • Tween 20 was purchased from Serva.
  • the specific polyclonal antibodies against JNK and p38 kinase were purchased from Santa Cruz Biotechnology.
  • Recombinant c-jun containing the N-terminal regulatory region of amino acids 1-135 and recombinant MAPKAP2 (GST-MAPKAP 46 ⁇ 00 ) were expressed as glutathione S-transferase fusion protein in Escheria coli and purified by glutathione-Sepharose (Pharmacia) chromatography.
  • Perfusion sites were excluded from evaluation if systolic-diastolic cardiac movements caused dislocation of the needles or if the TTC-staining areas of protected and control tissue were not clearly demarcated by necrotic tissue inbetween. Succesfull countershock defibrillation was not a criterium for exclusion. In one animal treated with aFGF countershocking caused dislocation of some microinfusion needles. These infusion sites were excluded from evaluation. Experimental groups
  • the present study consisted of five experimental groups for aFGF (Fig 1 ) and two experimental groups for anisomycin (Fig. 9).
  • Group I the control group
  • group 2 aFGF or Fig. 1 and anisomycin Fig. 9
  • group 3 bFGF, Fig. 1
  • the peptides were administered 60 min prior to the index ischemia of 60 minutes and the following reperfusion period of 2 hours.
  • Group 4 was treated with Suramin, an nonspecific FGF antagonist 60 min prior to the 60 min microinfusion of aFGF or bFGF.
  • Group 5 was treated with Genistein for 60 minutes prior to the aFGF respectively bFGF microinfusion.
  • Animals of Group 3 (Fig. 9) received AN/KHL and were biopsied at 0, after 10 and 30 min of infusion. Cyclohexyladenoslne was locally infused as a positive control respectively Krebs-Henseleit as a negative control. Additionally a small group of three animals was treated with truncated aFGF devoid of the mitogenic part of the protein; the experimental conditions were the very same as with aFGF/bFGF.
  • 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.
  • TTC triphenyltetrazolium chloride
  • Myocardium at risk of infarction was identified by the presence of fluoresceine and by the absence of fluorescent microspheres at a wavelenght of 366 nm.
  • the infarcted area was de-marcated by the absence tetrazolium precipitation.
  • the slices were photographed under UV- and tungsten lamp light by double exposure and the color slides were used for further planimetric evaluation.
  • Example 2 Infusion of aFGF decreases myocardial infarction
  • FIG. 2 depicts the effect of intramyocardial microinfusion of aFGF and bFGF compared to the control group. Both compounds were administered for 60 min before index ischemia. aFGF induced an infarct size reduction (Figure 4) of 51.8 ⁇ 7.7% vs. control 83.4 ⁇ 2.8%, p ⁇ 0.05. To induce cardioprotection ( Figure 5) by bFGF a fourfold higher concentration was needed (57.3 ⁇ 6.5% vs 83.4 ⁇ 2.8%, p ⁇ 0.05).
  • aFGF and bFGF showed a cytoprotective effect which significantly reduced infarct size. This allows us to conclude that not all tyrosine kinase receptor ligands afford protection, or that perhaps the receptor for VEGF was inactive in myocardium prior to index ischemia.
  • a cytoprotective effect for bFGF has been described previously in various animal models of neuronal ischemia (Fisher, Journal of Cerebral Blood Flow and Metabolism 15 (1995), 953-959; Jiang, Journal of the neurological Sciences 149 (1996), 173-179; Bethel, Stroke 28 (1997), 609-615).
  • FGF receptors Two classes of FGF receptors have been identified. One of them consists of a group of transmembrane signaling receptors with intrinsic protein tyrosine kinase activity. These bind FGF with high affinity and are responsible for initiating the biological (i.e., mitogenic) activity (Dionne, EMBO J. 9 (1990), 2685-2692).
  • the second group of receptors includes a family of cell surface heparan sulfate proteoglycans that bind FGF with low affinity but high capacity.
  • Example 3 Infused FGF is taken up by myocytes (aFGF) and non-myocytes (bFGF) and is translocated to the nucleus
  • Nuclei were stained with Aminoactinomycin D (Molecular Probes, Eugene, U.S.A.) diluted 1 :100; contractile proteins were stained with Phalloidin (Sigma, Chemical Co) diluted 1 :200 for 30 minutes. After rinsing in PBS, the sections were covered with Mowiol (Hoechst A.G., Frankfurt, Germany) and coverslipped. Omission of the first antibody served as negative control to check for nonspecific binding of the second antibody system.
  • Aminoactinomycin D Molecular Probes, Eugene, U.S.A.
  • contractile proteins were stained with Phalloidin (Sigma, Chemical Co) diluted 1 :200 for 30 minutes. After rinsing in PBS, the sections were covered with Mowiol (Hoechst A.G., Frankfurt, Germany) and coverslipped. Omission of the first antibody served as negative control to check for nonspecific binding of the second antibody system.
  • aFGF becomes redistributed from an extracellular to a (peri)nuclear localizaton in myocytes. These show a brighter staining pattern compared to the endogenous aFGF and that in the extracellular matrix (Weiner, Proc. Natl. Acad. Sci. USA 86 (1989), 2683-2687). Since the uptake and intracellular localization of exogenous aFGF after tyrosine kinase inhibition by genistein was still evident it is unlikely that this pathway is important for the cardioprotection. Although nuclear uptake of aFGF by myocytes was observed it is unlikely that these nuclei had entered the cell cycle.
  • fibroblast growth factors can ameliorate ischemia induced cell death.
  • a question that may arise is by which means does the cardioprotection occur?
  • MAPKs are assumed to be involved, would be the phosphorylation of cytoplasmic and nuclear proteins (Clerk, J. Biol. Chem. 269 (1994), 32848-32857; Davis, J. Biol. Chem. 268 (1993), 14553-14556).
  • MAPKs are important mediators of signal transduction from the cell surface to the nucleus being involved not only in the regulation of cell hypertrophy but also in the response to cellular stresses such as hypoxia or ischemia.
  • FIG. 10 depicts the effect of intramyocardial microinfusion of anisomycin and the control group. The compound was administered for 60 minutes prior to index ischemia. Local infusion of anisomycin induced an infarct size reduction (see Fig. 12) of 48.1 ⁇ 5.1% vs. control 83.4 ⁇ 2.8%, p ⁇ 0.01 (Fig. 11 ).
  • the underlying mechanism is due to the activation of the SAPKs p46 and p55 as will be shown in the following examples.
  • SAPK stress activated protein kinases
  • the protein synthesis inhibitor anisomycin was used to activate the SAPKs p46 and p55 as described in example 4. Since anisomycin is also mentioned to activate the p38 kinase (Nahas, Biochem. J. 318 (1996), 247-253; Stein, J. Biol. Chem. 271 (1996), 11427-11433), GST-MAPKAP2, the specific substrate for p38, was included to differentiate the kinase activities. Furthermore MBP, the substrate for the extracellular-signal regulated protein kinases (ERKs) p42 and p44, was included in the present studies. Mechanical stretch is known to induce kinase activity, therefore we used KHL treated tissue as a comparative control value. The time points were 0, 10, 30 min.
  • the left ventricular biopsies were resuspended in 5 vol of ice-cold buffer A containing 20 mM Tris-HCI, 0.25 sucrose, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, 0.5 mM PMSF, 100 ⁇ M sodium orthovavadate and 10 mM sodium fluoride (pH 7.4) and homogenized with a Teflon-glas homogenizer. The homogenate was centrifuged at 14000 x g for 30 min at 4°C. After this the pellet was resuspended in buffer. The supernatant represented the cytosolic fraction, the resuspended pellet was designated as the particulate fraction.
  • Proteins from cytosolic fractions (20 ⁇ g) were separated in 10% SDS polyacrylamide gels containing 0.25 mg/ml of c-jun protein. After electrophoresis the gels were washed for 1 hour with 20% (v/v) 2-propanol in 50 mM Tris HCI (pH 8.0), then for 1 hour with 5 mM mercaptoethanol in 50 mM Tris-HCI, pH 8.0. The proteins were denatured by incubation for 1 hour with 50 mM Tris-HCI, pH 8.0, containing 6 M guanidine-HCI.
  • Renaturation was achieved by incubation with 50 mM Tris HCI, pH 8.0, containing 0.1 % (v/v) Nonidet p-40 and 5 mM ⁇ -mercaptoethanol for 16 hours.
  • the in-gel phosphorylation of c-jun was performed in 40 mM Hepes, 0.5 mM EGTA, 10 mM magnesium chloride, 1.0 ⁇ M PKI, 25 mM ( ⁇ 33 P)-ATP (5mCi/ml), pH 8.0, at 25°C for 4 hours.
  • c-jun as a substrate for the SAPK p46 and p55; MBP for the ERKs p42 and p44 and MAPKAP2 as a substrate for the p38 kinase.
  • the p55 kinase activity reached its peak at the 30 min infusion showing a 4.2 fold increase compared to the control value and a 3.2 fold rise compared to the KHL infusion.
  • the 10 min time point showed an initial but not significant 2.3 fold- or a 1.7 fold increase compared to the KHL data.
  • the p46 SAPK showed a significant 7.1 fold increase compared to the control after 30 min and a 4 fold rise compared to the KHL data.
  • Data similar to p55 was obtained after 10 min infusion, a 2.9 fold or a 1.8 fold increase respectively compared to the KHL value; see also Figure 13.
  • Measuring the p38 kinase activity after anisomycin treatment showed the following results: a 3.8 fold increase after 30 min compared to the control value, but only a 1.32 fold rise compared to the KHL data; the 10 min data were a 4.4 fold, respectively a non significant change, see Table I.
  • MAPKAP2 as a substrate for SAPKs, showed a 3.0 fold increase for p55 compared to KHL treated tissue; similar results were obtained for p46 a 2.2 fold increase (both 30 min data).
  • Sadoshima Sadoshima, EMBO J. 12 (1993), 1681-1692; Komuro, FASEB J. 10 (1996), 631-636
  • Sadoshima Sadoshima, EMBO J. 12 (1993), 1681-1692; Komuro, FASEB J. 10 (1996), 631-636
  • These in turn could contribute to induction of early gene expression, which in turn might have an impact on cardioprotection.
  • These aspects were included in the present investigations by infusing Krebs-Henseleit solution under the very same conditions like anisomycin into the myocardium, obtaining drill biopsies from these treated areas and measuring the SAPKs activity by in-gel phosphorylation.
  • the p46 and p55 were activated but not to that extend as with anisomycin treatment (Fig. 14).
  • SAPKs Cardiac myocytes activate adaptive responses to ischemia/ reperfusion which are designed to help the cell survive future insults. This can be mimicked by pharmacological stimulation of the responsible pathway in this case the SAPKs.
  • the SAPK pathway involves sequential activation of the proteins MEKK1 and SEK1 , but the upstream regulators or signaling events remain unresolved. Diverse signals including inflammation, protein synthesis inhibitors, ischemic reperfusion and osmotic stress can activate the SAPKs family (Kyriakis (1994), supra; Derijard, Cell. 76 (1994), 1025-1037; Knight (1996), supra; Pombo, J. Biol. Chem. 76 (1994), 26546- 26551 ).
  • 3pK MAPK-activated protein kinase
  • ERK kinase family members
  • SAPK SAPK-activated protein kinase
  • the findings of the present invention are particularly interesting, since it introduces the new aspect of cytoprotection of activated of SAPKs.
  • Various groups claim the hypothesis that activation of the SAPK induce apoptotic cell death (Ham, Neuron. 14 (1995), 927-939; Verheij, Nature 380 (1996), 75-79) or like Xia et al (Xia, Science 270 (1995), 1326-1331) who hypothesizes that the balance between ERKs and SAPKs decides cell survival or death. All known investigations were performed in-vitro in cell- lines under non physiological conditions, e.g. withdrawal of growth factors.

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Abstract

L'invention concerne la modulation de la mort cellulaire ischémique. Elle concerne, en particulier, des compositions pharmaceutiques contenant une protéine possédant la fonction biologique du facteur acide de croissance de fibroblastes (aFGF) et/ou une molécule d'acide nucléique codant ladite protéine possédant la fonction biologique de aFGF et/ou un activateur de protéine kinases activées par le stress (SAPK) et/ou une molécule d'acide nucléique codant ledit activateur de SAPK, lesdites compositions étant particulièrement utiles pour traiter, prévenir ou retarder la mort cellulaire ischémique. Elle concerne, de plus, des procédés servant à traiter, à prévenir ou à retarder la mort cellulaire ischémique et consistant à mettre en contact des organes, des tissus ou des cellules avec une protéine possédant la fonction biologique de aFGF et/ou une molécule d'acide nucléique codant ladite protéine possédant la fonction biologique de aFGF et/ou un activateur de SAPKs et/ou une molécule d'acide nucléique codant ledit activateur de SAPK.
PCT/EP1998/004134 1997-07-03 1998-07-03 Nouvelle composition servant a traiter, a prevenir ou a retarder la mort cellulaire ischemique WO1999001150A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6826969B1 (en) 1999-10-11 2004-12-07 Abas, Incorporated Torque measurement apparatus
WO2004108167A1 (fr) * 2003-06-05 2004-12-16 Gencell Sas Plasmide codant un facteur de croissance des fibroblastes pour le traitement de l'hypercholesterolemie ou du diabete associe a des anomalies angiogeniques
US7305882B1 (en) 1999-10-08 2007-12-11 Abas, Incorporated Accelerometer using magnetic transducer technology
WO2009048119A1 (fr) * 2007-10-12 2009-04-16 National Institute Of Advanced Industrial Science And Technology Composition médicinale contenant une protéine chimère extrêmement fonctionnalisée

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EP0505811A2 (fr) * 1991-03-26 1992-09-30 BOEHRINGER INGELHEIM INTERNATIONAL GmbH Facteur de croissance des fibroblastes (aFGF) amélioré et sa préparation
WO1994012201A1 (fr) * 1992-11-23 1994-06-09 Boehringer Ingelheim Espana S.A. Utilisation de facteurs de croissance des fibroblastes comme agents neuro-protecteurs et neuro-modulateurs
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7305882B1 (en) 1999-10-08 2007-12-11 Abas, Incorporated Accelerometer using magnetic transducer technology
US6826969B1 (en) 1999-10-11 2004-12-07 Abas, Incorporated Torque measurement apparatus
WO2004108167A1 (fr) * 2003-06-05 2004-12-16 Gencell Sas Plasmide codant un facteur de croissance des fibroblastes pour le traitement de l'hypercholesterolemie ou du diabete associe a des anomalies angiogeniques
WO2009048119A1 (fr) * 2007-10-12 2009-04-16 National Institute Of Advanced Industrial Science And Technology Composition médicinale contenant une protéine chimère extrêmement fonctionnalisée
JPWO2009048119A1 (ja) * 2007-10-12 2011-02-24 独立行政法人産業技術総合研究所 高機能化キメラ蛋白質を含有する医薬組成物
JP2012143234A (ja) * 2007-10-12 2012-08-02 National Institute Of Advanced Industrial Science & Technology 高機能化キメラ蛋白質を含有する細胞増殖促進用培地
JP5004250B2 (ja) * 2007-10-12 2012-08-22 独立行政法人産業技術総合研究所 高機能化キメラ蛋白質を含有する医薬組成物

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