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WO2008034623A2 - Médicament destiné à la prophylaxie, au traitement ou au diagnostic de maladies ischémiques - Google Patents

Médicament destiné à la prophylaxie, au traitement ou au diagnostic de maladies ischémiques Download PDF

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Publication number
WO2008034623A2
WO2008034623A2 PCT/EP2007/008215 EP2007008215W WO2008034623A2 WO 2008034623 A2 WO2008034623 A2 WO 2008034623A2 EP 2007008215 W EP2007008215 W EP 2007008215W WO 2008034623 A2 WO2008034623 A2 WO 2008034623A2
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mice
use according
adenosine
ischemia
nucleotide
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English (en)
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WO2008034623A3 (fr
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Holger Eltzschig
Tobias Eckle
Almut Grenz
Melanie Hart
Hartmut Osswald
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Eberhard Karls Universitaet Tuebingen
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Eberhard Karls Universitaet Tuebingen
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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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03005—5'-Nucleotidase (3.1.3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/01—Hydrolases acting on acid anhydrides (3.6) in phosphorus-containing anhydrides (3.6.1)
    • C12Y306/01005—Apyrase (3.6.1.5), i.e. ATP diphosphohydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

Definitions

  • the present invention relates to a medicament for the prophylaxis or treatment or diagnosis of ischemic diseases, methods for diagnosing a predisposition to or disease of an ischemic disease in a subject, and a pharmaceutical or diagnostic composition.
  • Ischemia is understood to mean the reduction or interruption of the perfusion of an organ, organ or tissue due to a lack of arterial blood supply.
  • the associated lack of oxygen which is referred to as hypoxia, leads to the long-term death of cells, tissues or parts of organs.
  • the diseases attributable to such reduced blood flow are called ischemic diseases. They are currently the most common diseases associated with the vascular system.
  • the current therapeutic approaches to the treatment of ischemic diseases are based, inter alia, on the administration of anti-inflammatory substances, such as glucocorticoids.
  • vasodilators such as. Nitric oxide donors, and antibodies to adhesion molecules and increasing the ischemic tolerance by reducing oxygen radicals by glutathione is taken.
  • IP ischemic preconditioning
  • ischemic diseases such as myocardial ischemia
  • myocardial ischemia are one of the major health problems of Western industrialized countries. Renal ischemia also contributes significantly to the morbidity and mortality of patients, such as after surgery or invasive diagnostics. For example, during surgical procedures that require clamping of the aorta or renal vessels, for example, during the surgical correction of aneurysms or the treatment of peripheral vascular disease, renal failure due to ischemia occurs in 15 to 30% of cases. After surgical intervention in the heart, at least 1 to 10% of cases of acute renal failure due to ischaemia occur during normal development.
  • nucleotide phosphohydrolase for the manufacture of a medicament for the prophylaxis and / or treatment and / or diagnosis of ischemic diseases.
  • nucleotide phosphohydrolase the symptoms of ischemic diseases, which were induced in mice artificially induced, can be greatly reduced and the affected animals compared to untreated animals show a significantly higher survival. It was also found that the administered nucleotide phosphohydrolase causes a protection of areas of risk (AARs), so that not only an acute treatment of ischemic diseases, but also a prophylaxis is possible.
  • AARs areas of risk
  • a nucleotide phosphohydrolase or else nucleotide or nucleotide phosphatase is an enzyme which catalyzes the hydrolytic cleavage of phosphate groups of nucleoside monophosphates and thus the degradation of mononucleotides to the corresponding nucleosides.
  • Substrate specificity for 5'-phosphate groups is also referred to as 5'-nucleotidase or 5'-nucleotide phosphohydrolase.
  • the invention can also be used diagnostically. For example, in a high-risk patient who is believed to have a predisposition to ischemic disease because of a family background, or who is already showing initial symptoms of ischemic disease, it may be easy to determine if this is indeed the case. Thus, in such a case, after administering the nucleotide phosphohydrolase, the symptoms will measurably improve, so that a positive diagnosis can then be made.
  • the object underlying the invention is hereby completely solved.
  • the inventors make use of molecular mechanisms that are recognized for the first time, which lead to the development of ischemic diseases or are based on protection by IP, in order to create a potent drug or diagnostic agent.
  • nucleotide phosphohydrolase it is preferred according to the invention if 5'-nucleotidase (CD73) is used as nucleotide phosphohydrolase.
  • the 5 'nucleotidase is also referred to as CD 73 (EC 3.1.3.5) according to the nomenclature of the CD markers.
  • CD73 in its native form, is an ecto-enzyme, i. it unfolds its activity in the interstitium or in the extracellular space. It hydrolyzes 5'-AMP to adenosine, which in turn acts from the extracellular side to specific membrane receptors of the cells.
  • 5'-nucleotidase which is not taken up in the cells, increases the degradation of 5'AMP to adenosine in the interstitium and has the advantage that such a nucleotide phosphohydrolase is used, which is particularly suitable according to findings of the inventors for the prophylaxis or treatment of ischemic diseases. It was shown that cd73 A - mice, which are genetically deficient in CD73 and show the experimental symptoms of ischemic diseases, can be successfully treated by the administration of 5'-nucleotidase. Specifically, protection of the risk area comparable to that observed with IP treatment could be achieved.
  • nucleotide phosphohydrolase alternatively or additionally apyrase (CD39) is used.
  • Apyrase is an enzyme that is activated by calcium or magnesium and is present in biological systems - as apyrase - in plasma membrane-bound form as an ecto-enzyme (EC 3.6.1.5).
  • Apyrase is also referred to as CD39 according to the nomenclature of CD markers.
  • Other synonyms for apyrase are NTPDasel, ATP diphosphohydrolase, ATPDase and lymphoid cell activation antigen.
  • the apyrase as a nucleotide phosphohydrolase, catalyzes the hydrolysis of ATP to form AMP and orthophosphate.
  • the ecto-apyrase can also convert ADP and other nucleoside diphosphates and triphosphates.
  • the apyrase is also particularly well, according to findings of the inventors for the prophylaxis or treatment of ischemic diseases. For example, it has been shown that an inhibition of endogenous apyrase in experimental animals too an increased susceptibility to ischemic diseases, whereas exogenous administration of apyrase has demonstrated therapeutic efficacy against ischemic diseases.
  • nucleotide phosphohydrolase or 5'-nucleotidase is present in soluble form.
  • nucleotide phosphohydrolase need not be in membrane-bound form for efficacy against ischemic diseases. Also, the soluble form shows efficacy, which brings advantages in particular in the formulation of the drug.
  • nucleotide phosphohydrolase is in micronized form.
  • nucleotide phosphohydrolase understood, which is reduced in size compared to the natural or wild-type variant.
  • the size reduction is to a size at which the enzyme is still enzymatically active, i. still contains the active center, but other, for example, structuring sections are missing.
  • This reduction may be enzymatic or by recombinant DNA technology.
  • This measure has the advantage that such a nucleotide phosphohydrolase is used, which can leave the vascular system and unfold their effect in the interstitium. Experience has shown that this is only possible with difficulty in the natural or wild-type variant.
  • the ischemic disease is selected from the group consisting of: myocardial ischemia, renal ischemia, intestinal ischemia, and liver ischemia.
  • This measure has the advantage that a drug is provided for the treatment of particularly significant ischemic diseases.
  • the inventors were able to establish that the effect of nucleotide phosphohydrolase is particularly advantageous in the case of ischemia-related organ failure in which the organs heart, kidney, intestine or liver were affected.
  • the nucleotide phosphohydrolase can furthermore be present in the form of a coding sequence in an expression vector.
  • nucleotide phosphohydrolases are the NCBI
  • the coding sequence of human 5'-nucleotidase is available under the accession number NM_001776
  • the coding sequence of human ecto-apyrase is available under accession number NP_001767
  • the expression vector may have other portions, such as promoters, enhancers, etc., which ensure expression of the nucleotide phosphohydrolase in the patient.
  • the drug has an activity enhancer.
  • any compound or composition is suitable which is suitable for the prophylaxis or treatment of ischemic diseases and does not adversely affect the therapeutic effect of nucleotide phosphohydrolase against ischemic diseases or possibly even enhanced.
  • These may, for example, be substances which have anti-inflammatory or immunomodulatory action.
  • This measure has the advantage that the effect of nucleotide phosphorous, which has been recognized for the first time by the inventors and is used therapeutically, hydrolase additionally reinforced and the drug is thereby further improved in its potency.
  • the action enhancer used is preferably an adenosine A 2 B receptor agonist, for example BR 4887.
  • BR4887 also referred to as BAY 60-6583, is a specific adenosine A 2 B receptor agonist manufactured by Bayer HealthCare.
  • BAY 60-6583 is a specific adenosine A 2 B receptor agonist manufactured by Bayer HealthCare.
  • an inhibitor of the nucleoside transporter is furthermore preferred as an enhancer, wherein preferably such an inhibitor is used which inhibits the nucleoside transporter ENT-I or ENT-2.
  • vascular adenosine is predominantly absorbed by the equilibrative nucleoside transporters (ENT) -I and -2. These transporters are involved in the regulation of adenosine signaling. It has been shown that the repression of ENT-I as a function of hypoxyinducible factor 1 (HIF 1) during hypoxia results in decreased vascular adenosine uptake and in enhanced adenosine signaling effects; see. Eltzschig HK et al., "HIF-1-dependent repression of equilibrative nucleoside transporter (ENT) in hypoxia", /. Exp. Med. 202, 1493-1505 (2005). These observations make inhibitors of ENT-I and ENT-2 particularly suitable enhancers of the invention. It is inventively preferred if dipyridamole is used as inhibitor of ENT-I or ENT-2.
  • HIF 1 hypoxyinducible factor 1
  • another aspect of the present invention relates to a method for diagnosing a predisposition or disease to an ischemic disease in an animal, comprising the steps of: (1) providing a biological sample derived from the subject, (2) (3) optionally determining the operability and / or activity of the nucleotide phosphohydrolase, (4) correlating the absence of nucleotide phosphohydrolase in the biological sample or one in the biological sample to the biological sample for the presence of a nucleotide phosphohydrolase; the wild type reduced functionality and / or activity of the nucleotide phosphohydrolase in the biological sample with a positive diagnosis.
  • a biological sample is any sample that can be used to determine if the animal is expressing a functional nucleotide phosphohydrolase.
  • a biological sample are tissues at risk areas (AAR), for example myocardial, renal, intestinal or liver tissue, a salivary or blood, hair sample, etc.
  • AAR tissues at risk areas
  • suitable biological samples may also be used Preferably, these samples contain representative genetic material, such as total DNA, which is why nucleated cells are generally suitable.
  • step (2) Examination for the presence of a nucleotide phosphohydrolase according to step (2) is carried out by methods known in the art, for example under Use of specific antibodies. It is also preferred if in step (2) a mutation screening is carried out, in which it is determined whether in the coding sequence for the nucleotide phosphohydrolase a mutation is present which leads to a complete failure of the protein or to a loss of activity over the Wild type leads.
  • the operability or activity of the nucleotide phosphohydrolase is determined in step (3) according to methods well known in the art. It is preferable that the adenosine formation rate of the nucleotide phosphohydrolase in the biological sample be determined in comparison with the adenosine formation rate of wild-type nucleotide phosphohydrolase or fully functional nucleotide phosphohydrolase, respectively.
  • the biological sample does not contain an active nucleotide phosphohydrolase, for example, due to a polymorphism, it can be diagnosed that the subject subject has or is predisposed to a decreased ischemic tolerance and therefore predisposed to the development of ischemic disease ischemic disease has manifested.
  • the implementation of the method according to the invention has the advantage that for the first time the molecular causes of an ischemic disease are used diagnostically. So far, the diagnosis of ischemic diseases based largely on empirically determined parameters, such as the oxygen partial pressure in the arterial blood and the use of imaging techniques, such as echocardiography, MRI, SPECT, etc. These methods are, however, associated with a high risk of error, so that the inventive method contributes to a safe diagnosis and enables a targeted treatment of ischemic diseases.
  • Another object of the present invention relates to a pharmaceutical and / or diagnostic composition, the nucleotide phosphohydrolase in a therapeutically effective amount as well as a pharmaceutically acceptable carrier and optionally an enhancer.
  • nucleotide phosphohydrolase and the effect enhancer reference is made to the above statements in connection with the use according to the invention, which apply equally here.
  • Pharmaceutically acceptable carriers are well known in the art, cf. Kibbe A.H., Handbook of Pharmaceutical Excipients. American Pharmaceutical Association and Pharmaceutical Press, 3rd Edition (2000). It is understood that the pharmaceutical or diagnostic composition may comprise other ingredients or adjuvants, such as binding, disintegrants, lubricants and salts, etc., to provide a galenically suitable form and to ensure a sufficient concentration of the active ingredient at the site of action ,
  • Another object of the invention is a method for diagnosing a predisposition or disease to an ischemic disease in an animal, comprising the steps of: (1) administering the above diagnostic composition to a subject showing symptoms of ischemic disease; (2) determining whether the symptoms are improving, and (3) correlating an improvement in symptoms with a positive diagnosis.
  • this test can be used to examine patients in whose family heart disease or other diseases associated with ischemic conditions have occurred relatively frequently, and it is to be determined whether a predisposition to ischemic intolerance or a reduced ischemia tolerance exists.
  • Another object of the present invention is the use of nucleotide phosphohydrolase for the preparation of an ischemic preconditioning (IP) drug.
  • Ischemic preconditioning is understood to mean a mechanism in which short and repetitive episodes of ischemia and reperfusion with respect to a particular organ result in protection of that organ, for example against an infarct.
  • the preconditioning stimulus of oxygen deprivation induces remodeling and genetic changes in the organ that protect the organ for a short time. In the case of the heart, this protection is manifested by the fact that, in the case of a heart attack, less heart muscle tissue dies and fewer cardiac arrhythmias occur.
  • nucleotide phosphohydrolase ie with 5'-nucleotidase (CD73) or apyrase (CD39)
  • CD73 5'-nucleotidase
  • CD39 apyrase
  • Another object of the present invention relates to the use of an adenosine A2B receptor agonist, preferably of BR4887 / BAY 60-6583, for the manufacture of a medicament for the prophylaxis and / or treatment and / or diagnosis of ischemic diseases, preferably of myocardial ischemia and / or renal ischemia.
  • an adenosine A2B receptor agonist preferably of BR4887 / BAY 60-6583
  • the inventors have surprisingly found that administration of a specific adenosine A ⁇ B receptor agonist after induction of hypoxia results in a markedly reduced infarct or improved renal function, respectively, than in untreated control animals.
  • Corresponding results were found for the other organs (liver, intestine); Data not shown.
  • the inventors have further developed a method for the prophylaxis and / or treatment of ischemic diseases in a patient comprising the steps of: (1) administering the above-described pharmaceutical composition of the present invention to a patient, and (2) optionally repeating the administration according to Step 1).
  • the inventors have further developed a method for the prophylaxis and / or treatment of ischemic diseases in a patient comprising the steps of: (1) activating endogenous nucleotide phosphohydrolase, such as ecto-5'-nucleotidase (CD73) and or ecto-apyrase (CD39) in a patient, and (2) if necessary, repeat the activation according to step (1).
  • This method is based on increasing the activity of the endogenous nucleotide phosphohydrolase. This could be done, for example, by increasing the extracellularly provided 5'-AMP, which in turn is possible, for example, by CD39.
  • the inventors also provide activators that increase the activity of the endogenous nucleotide phosphohydrolase. These activators are suitable for the preparation of a medicament for the prophylaxis and / or treatment and / or diagnosis of ischemic diseases.
  • FIG. 1 CD73 is induced by cardiac ischemic preconditioning (IP), (a) mouse model of cardiac IP.
  • IP cardiac ischemic preconditioning
  • Age-, gender-, and weight-matched mice were anesthetized with pentobarbital and mechanically ventilated.
  • Ischemic preconditioning was performed in situ using a suspended weight system for atraumatic occlusion of the left coronary artery.
  • the IP protocol consisted of four cycles of I Sham / Reperfusion (5 minutes each). The animals were sacrificed at the times indicated and the risk area was excised. Transcriptional responses to IP were examined after isolating RNA from the risk area, DNase digestion, and a real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR), starting from cDNA.
  • real-time RT-PCR real-time reverse transcriptase polymerase chain reaction
  • the CD73 mRNA is induced by the IP. After the indicated time points, the risk areas were excised, the total RNA was isolated and the CD73 mRNA levels were determined by real-time RT-PCR. The data were calculated in relation to an internal housekeeping gene ( ⁇ -actin) and expressed as x-fold change compared to control (no IP) ⁇ SD at the times indicated. The results are from 6 experiments on each condition, (c) The CD73 protein is induced by the IP. The risk area was excised, flash frozen, lysed at the indicated time points, and the proteins were separated by SDS-PAGE. The membranes were incubated with an anti-CD73 antibody.
  • mice were subjected to IP.
  • the cardiac tissue from the risk area was collected 90 minutes after IP, cut, stained with polyclonal anti-CD73 antibody, visualized with a confocal laser scanning microscope (90 min, WT) and compared to the non-preconditioned tissue (control).
  • the secondary antibody was used alone ("negative") or the primary and secondary antibodies were used on previously characterized mice with targeted gene deletion of cd73 (90 min, KO) WT: wild-type mice, KO: cd73 / mice
  • WT wild-type mice
  • KO cd73 / mice
  • enzyme activity was evaluated by measuring the conversion of [ 14 C] IMP to [ 14 C] inosine
  • APCP has been used as a CD73 inhibitor and CD73 activity is shown as APCP-inhibited IMP-hydrolyzing activity. pressed in nmol IMP hydrolyzed / h / mg protein and compared with non-preconditioned myocardium (C).
  • CD73 is induced by renal ischemic preconditioning (IP),
  • IP renal ischemic preconditioning
  • (a) mouse model of renal IP Age-, sex- and weight-matched mice were anesthetized with pentobarbital, subjected to a right nephrectomy, followed by in situ ischemic preconditioning with a hanging weight system for atraumatic occlusion of the left renal artery.
  • the IP protocol consisted of four cycles of ischaemia / reperfusion (4 minutes each) followed by the indicated times of reperfusion.
  • (b) The CD73 mRNA is induced by the IP. After the indicated time periods, the kidneys were excised, the total RNA isolated and the CD73 mRNA levels determined by real-time RT-PCR.
  • the kidneys were recovered after the indicated periods after IP, cut, stained with an anti-CD73 antibody and visualized on a confocal laser scanning microscope. Tissue from a perfused but unpre-conditioned wild type mouse served as a control. The staining of wild-type tissue with a secondary antibody alone is shown in the upper left panel and labeled as "negative.”
  • E The CD 73 enzyme activity is induced by IP Kidneys were excised after IP treatment, snap frozen and the extracts wur- prepared as described in the Materials and Methods section. The 5'-nucleotidase enzyme activity was evaluated by measuring the conversion of [ 14 C] IMP to [ 14 C] inosine in the presence and absence of APCP. Enzyme activity is expressed as nmol / h / mg protein of APCP-inhibitable IMP hydrolysis activity and is compared to non-preconditioned kidneys (-IP).
  • Fig. 3 The inhibition of CD73 by APCF 'treatment abolishes cardioprotection by ischemic preconditioning (IP).
  • IP ischemic preconditioning
  • AMP-induced bradycardia is blocked by APCP infusion.
  • a bolus of 5'-adenosine monophosphate (AMP) was administered (50 ⁇ l of an 8 mg / ml solution of AMP) and AMP-induced bradycardia was determined. It should be noted that treatment with APCP blocks AMP-inducible bradycardia, (c) APCP treatment attenuates cardioprotection by IP.
  • Fig. 4 Inhibition of CD73 by APCP treatment abolishes renal protection by ischemic preconditioning (IP).
  • IP ischemic preconditioning
  • Renal CD73 enzyme activity is inhibited by APCP infusion.
  • Age-, weight- and sex-matched C57BL / 6J mice were anesthetized and the specific CD73 inhibitor APCP (2 mg per kg) ip or sterile saline was administered.
  • the kidneys were excised 30 min after the administration and the enzyme activity was evaluated by measuring the conversion of [ 14 C] IMP to [ 14 C] inosine. The results are expressed as nmol / h / mg protein of APCP-inhibitable IMP hydrolysis activity.
  • the APCP treatment prevents renal protection by the IP.
  • Fig. 5 Cardioprotection by ischemic preconditioning (IP) is abrogated in cd73 'mice, (a) Measurement of CD /' 3 activity in hearts from cd73 'mice. Cd73 / mice (black bars) and age-, weight-, and sex-matched littermate controls (white bars) were anesthetized, intubated, and bleed through a catheter placed in the carotid artery. to let. The hearts were isolated, snap frozen, and the CD73 enzyme activity was measured as part of the APCP-inhibited IMP hydrolysis activity.
  • Cd73 / ⁇ mice (e) and age-, weight- and sex-matched littermate controls (f) received intra-arterial infusion of adenosine (200 ⁇ l / h, adenosine 8 mg / ml), ie a dose previously determined to be one that does not induce hypotension or bradycardia. 16 min after the start of the infusion, the ischemic preconditioning was carried out as described above, the infarct size was determined by double staining with Evan's Blue and 2,3,5-triphenyltetrazolium chloride.
  • IP ischemia was reconstituted to 1 WT level with 1 U of soluble 5'-nucleotidase by infusion via a carotid artery catheter, (g, h) reconstitution of cd.73 f mice with soluble S'-nucleotidase.
  • Infarct size was determined by double staining with Evan's Blue and 2,3,5-triphenyltetrazolium chloride. The results are presented as mean ⁇ SD from 6 animals per condition.
  • Fig. 6 Renal protection from ischemic preconditioning (IP) is in cd73 ' ⁇ - mice canceled (a) Measurement of CD73 activity in kidneys cd? '3 ⁇ ' mice. Cd73 ⁇ mice and age-, weight- and sex-matched littermate controls were anesthetized and the kidneys were excised. Kidneys were snap frozen and CD73 enzyme activity was measured as a proportion of APCP-inhibited hydrolysis activity. The results are expressed in nmol of IMP hydrolyzed / h / mg protein. The renal protection by IP is abolished in cd73 ⁇ 'mice.
  • Pre-conditioning was performed in situ with 4 cycles of IP (4 min ischemia, 4 min reperfusion) followed by 30 min of ischemia. Renal function tests were performed after 24 hours of recovery, (b) measurement of plasma creatinine concentration. (c) Measurement of potassium content in plasma (plasma K + ). (d) Measurement of creatinine clearance. (e) measurement of urinary flow rate, (f) measurement of urinary sodium excretion (urinary Na + excretion), (g) urinary potassium excretion (urinary K + excretion), (h) quantitative determination of PMN tissue accumulation by measurement of myeloperoxidase (MPO) - tissue level. The data are from 6 to 8 mice per condition and the results are expressed as means ⁇ SD.
  • FIG. 7 The adenosine A2 ⁇ receptor is selectively induced by ischemic preconditioning (IP), (a) modulation of the level of adenosine receptor transcript by IP.
  • IP ischemic preconditioning
  • Age-, weight- and sex-matched littermates were anesthetized with pentobarbital and mechanically ventilated. Hemodynamic preconditioning was performed in situ using a suspended weight system for atraumatic occlusion of the left coronary artery.
  • the IP protocol consisted of 4 cycles of ischemia / reperfusion (each 5 minutes). The animals were sacrificed at the indicated times and the risk areas were excised.
  • the transcriptional responses of all 4 adenosine receptors (Ai, AZA, A 2 B, and A3) to IP were determined after isolation of RNA from the risk area, DNase digestion, and a real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). examined by the cDNA. The data were calculated relative to an internal household gene ( ⁇ -actin) and expressed as x-fold change compared to simulated animals without IP treatment (controls, C ⁇ SD).
  • results are from 6 experiments per condition, (b) To compare the relative levels of transcript of the various adenosine receptors in preconditioned (black bar, 90 min after IP) or non-preconditioned tissue (white bars, control) from the risk area, transcriptional levels were normalized relative to the adenosine receptor with the lowest transcription levels (A 2 A), (c) the adenosine A 2 B receptor protein is induced by IP. Age-, weight- and sex-matched littermates were anesthetized with pentobarbital and mechanically ventilated. There was an in situ ischemic preconditioning as described above.
  • mice were sacrificed 90 min after IP and tissue from the at-risk area was excised, stained with antibodies to the adenosine A2B receptor, visualized on a confocal laser scanning microscope and compared to cardiac tissue from non-preconditioned animals (control). Additionally, the secondary antibody was used alone ("negative") and the primary and secondary antibodies were used in mice with targeted deletion of the adenosine A2B receptor (90 min KO) WT: wild-type mice, KO: A 2 BAR ⁇ A mice.
  • Figure 8 IP is abolished in adenosine A2B receptor knockout mice, (a) characterization of adenosine A2B receptor mutant mice. Measurements of transcript levels of the adenosine receptors by reverse transcriptase real-time polymerase chain reaction in cardiac tissue from mice mutated for the adenosine A 2 B receptor were performed by Deltagene (WT: wild-type, HZ: heterozygous deletion the adenosine A2B receptor, KO: homozygous adenosine A2B receptor deletion).
  • WT wild-type
  • HZ heterozygous deletion the adenosine A2B receptor
  • KO homozygous adenosine A2B receptor deletion
  • FIG. 9 Histologic signs of renal protection by IP are absent in cd73 'mice.
  • PPM polymorphonuclear neutrophils
  • Fig. 10BR4887 / BAY 60-6583 (a) Chemical structure and (b) ECso values of BR4887 / BAY 60-6583 on the adenosine receptors AIAAR, A ⁇ AAR and A 2 BAR.
  • Fig. 11 Treatment with adenosine A ⁇ receptor agonist protects against myocardial ischemia.
  • E-van's Blue concentration of E-van's Blue in the organ was quantified after formamide extraction (55 C for 2 h) by measuring the absorbances at 610 nm after subtracting the reference absorbance at 450 nm. Data are expressed as mean ⁇ SD E-van's Blue OD / mg wet tissue and are pooled from 6 animals per condition. Note: Lower cardiac Evan's Blue retention in mice treated with BR4887.
  • a treatment with adenosine A 2 B receptor agonist protects against renal ischaemia.
  • (c) The effect shown in (b) is abolished in mice deficient in the adenosine A 2 B receptor (A2B- / -).
  • Fig. 13 Reconstitution of cd? 3 'mice with soluble 5'-nucleotidase.
  • cd / S 'mice were treated with 1 U of soluble 5'-nucleotidase from Crotalus atrox veniplex ip (+ 5'-NT) or with vehicle control (-5'-NT).
  • Experiments were performed with or without preconditioning in situ with 4 cycles of IP (4 min ischemia, 4 min reperfusion), with or without treatment with soluble 5'-nucleotidase. Renal functional tests were performed after 24-hour reperfusion.
  • FIG. 14 Treatment with soluble 5'-nucleotidase mimics the protective effect of IP in WT mice.
  • Cd73 + / + mice received 1 U of soluble 5'-nucleotidase purified from C. atrox venom 30 min prior to renal ischemia.
  • the mice were treated with or without ischemic preconditioning (4 min ischemia, 4 min reperfusion, 4 cycles) followed by 30 min of renal ischemia, with or without soluble 5'-nucleotidase.
  • the renal function tests were performed after 24 hours of reperfusion, (a) measurement of plasma creatinine concentration. (b) Measurement of potassium in plasma (plasma K + ). (c) Measurement of creatinine clearance.
  • Fig. 15 Histological signs of renal protection by treatment of wild-type mice with soluble 5'-Nudeotidase.
  • PMN polymorphonuclear neutrophils
  • the kidneys were isolated ip 24 h after 30 minutes of ischemia with or without 5'-nucleotidase treatment, (a) Representative sections (x400, after H & E staining).
  • c Representative sections with chloroacetate esterase staining (x400).
  • quantification of granulocyte infiltration The data are from 6 to 7 mice per condition, and the results are expressed as means ⁇ SD. embodiments
  • mice bearing a mutation in cd73, in the adenosine Ai or A 3 receptors on the BL6 strain mice bearing a mutation in the adenosine A2A receptor on the CDI line were prepared, validated and characterized as described above; see. Thompson LF et al. (2004), "Crucial RoIe for Ecto-5'-Nucleotidase (CD73) in Vascular Leakage During Hypoxia", /. Exp. Med. 200, 1395-1405.
  • Adenosine A 2B receptor mutants were made by Deltagen Inc., (San Carlos, CA). A 112 bp fragment (from base 156 to base 267) from the 1076 bp protein coding region of A 2 B was replaced by a 9.6 kb IRES-lacZ reporter and neomycin resistance cassette. The characterization and validation was performed by Deltagen and in the laboratories of the inventors in Tübingen. Thereafter, PCT genotyping according to the manufacturer's instructions, real-time RT-PCR to determine adenosine receptor expression patterns, and Western blotting confirmed the successful deactivation of adenosine A2B receptor transcript and protein levels in the tissues examined.
  • mice were placed on a temperature controlled heat plate (RT, Effenberg, Munich, Germany) with a rectal thermometer probe connected to a thermal feedback controller to maintain a body temperature of 37 ° C.
  • the infarcts were determined by calculating the percentage of myocardial infarction compared to the risk area, using the previously described double staining technique with Evan's Blue and triphenyltetrazolium chloride (TTC); see. Eckle, T. et al. (Supra).
  • TTC triphenyltetrazolium chloride
  • the risk area and infarct size were determined by planimetry using NIH Image 1.0 software and the extent of myocardial damage was calculated as percent of infarcted myocardium in relation to the risk area.
  • mice were anesthetized with sodium pentobarbital (70 mg / kg) and placed on a temperature-controlled hot plate (RT, Effenberg, Munich, Germany) in a left lateral position.
  • the operations were performed under an upright dissecting microscope (Leica, MZ95, Bensheim, Germany).
  • an incision was made on the right side of the body and the right kidney was removed.
  • the animals were placed in a right lateral position and an incision was made in the left side for access to the left kidney.
  • the left kidney was carefully removed from adjacent connective tissue and placed with its ventral side down in an acrylic dish.
  • the renal artery was dissected and a nylon wig (Ethicon, Norderstedt, Germany) was wrapped around the artery. Occlusion of the renal artery for ischaemia (30 min) and IP (4 cycles of 5 min ischemia and 4 min reperfusion 30 min before ischemia) was done using a hanging weight system as previously described; see. Eckle, T. et al. (Supra). Successful occlusion of the renal artery was associated with immediate staining. Change from red to white connected. According to the experimental procedure (ischemia with or without previous IP), the left kidney was returned to its anatomical position in the retroperitoneal cavity and the wound closed.
  • mice received 0.3 ml of normal saline ip and recovered for 2 h under a heat lamp. Subsequently, these were placed in metabolic cages (Tecniplast, Hohenpeissenberg, Germany) for the determination of the renal functional parameters.
  • Cardialtroponins replaced creatine kinase and lactate dehydrogenase isoenzymes as standard criteria for the diagnosis of myocardial injury. Since there is a correlation of cardial troponin with infarct sizes, blood was removed for cTnI measurement via a central venous puncture. Plasma cTnI concentrations were measured using a quantitative cTnI rapid assay (Life Diagnostics, Inc., Westchester, PA, USA).
  • mice were kept in metabolic cages two hours after the experimental procedure (Tecniplast, Hohenpeissenberg, Germany). Renal function was determined by measuring plasma and urine creatinine 24 hours after renal ischemia using a commercially available colorimetric method according to the manufacturer's protocol (LT-SYS, Labor +technik, Berlin, Germany). Plasma and urine concentrations of Na + , K + were determined with a flame emission photometer (ELEX 6361, Eppendorf AG, Hamburg, Germany). Renal excretory and hemodynamic values were calculated using standard formulas. Mice were screened after 24 hours of observation. killed in the metabolic cage and recovered the plasma samples. Furthermore, the kidneys were isolated and stored at -8O 0 C for further analysis.
  • the content of CD73 and adenosine A 2 B receptor protein was determined from the risk area.
  • C57BL / 6J mice (Charles River, Sulzfeld, Germany) were killed after cardiac IP as described above. The remaining blood was removed, the risk area was at the indicated time points (30, 60, 90 and 120 min after IP) excised and immediately frozen at -80 0 C inserted.
  • Tissues were homogenized and incubated for 10 min in ice-cold lysis buffer (10 7 PMN / 500 ⁇ l, 150 mM NaCl, 25 mM Tris pH 8.0, 5 mM EDTA, 2% Triton X-100, and 10% mammalian tissue protease inhibitor cocktail, sigma Aldrich) and collected in microfuge tubes. After centrifugation at 14,000 g to remove cell debris, the pellet was discarded. The proteins were grown in reducing Lämmli sample buffer and for 5 min Heated to 90 ° C. The samples were separated on a 12% polyacrylamide gel and transferred to nitrocellulose membranes.
  • the membranes were blocked for 1 h at room temperature in PBS supplemented with 0.2% Tween 20 (PBS-T) and 4% BSA.
  • the membranes were amplified in 10 ⁇ g / ml CD73 rabbit polyclonal antibody against amino acids 275-574 (Santa Cruz, Danvers, USA) or A 2 B goat polyclonal antibody against the C-terminus (Santa Cruz, Danvers, USA) for 1 h at room temperature followed by 10 min washes in PBS.
  • the membranes were then incubated in 1: 3000 goat anti-rabbit HRP in the case of CD73 (Perbio Science, Bonn, Germany) or donkey anti-goat HRP in the case of A 2 BAR (Santa Cruz, Danvers, USA) , The washes were repeated and the proteins were detected by enhanced chemiluminescence.
  • IP was performed in C57BL / 6J mice (Charles River, Sulzfeld, Germany) as described in detail above. Mice were killed and hearts were then removed at the indicated time points (30, 60, 90 min after IP) and fixed in Tissue-Tek (Sakura) for 24 hours. Cryostat sections were embedded on glass coverslips, air-dried and then air-dried in acetone / methanol (1: 1) for 3 min at room temperature.
  • Sections were prepared by incubation in 5% [w / v (weight per volume)] skim milk and 0.1% (w / v) Triton X-100 (Sigma, Kunststoff, Germany) in Tris-buffered saline (TBS) for 30 min blocked.
  • CD73 rabbit polyclonal antibody to amino acids 275-574 (Santa Cruz, Danvers, USA) or A2B goat polyclonal antibody to the C-terminus (Santa Cruz, Danvers, USA) were 1: 200 in the same solution and the sections were incubated for 1 h at room temperature.
  • the granulocyte infiltrates were stained by histochemistry using chloroacetate esterase (CAE). Examination and classification for neutrophil infiltration of sections from each kidney was made by an experienced renal pathologist who was unaware of the treatment group. Briefly, depending on the degree of infiltration, 0 to 4 points were awarded: grade 0, normal renal tissue; Grade 1, slight local infiltration; Grade 2, moderate infiltration in different areas; Grade 3, strong infiltration ⁇ 50%; Grade 4, severe diffuse infiltration of more than 50% of the renal tissue. Under each condition, six animals were used and rated three representative sections of each kidney.
  • reaction buffer 50 mM potassium phosphate buffer pH 6.0, 0.68 mM O-dianisidine, 0.0005% hydrogen peroxide.
  • the change in absorbance was measured spectrophotometrically (Victor 3V, Perkin Elmer) at 450 nm for 2 minutes.
  • MPO activity was expressed as changes in optical density (OD) per minute and per milligram of protein detected by the Lowry protein assay.
  • ecto-5'-nucleotidase enzyme activity was assessed by measuring the conversion of [ 14 C] IMP into [ 14 C] inosine as described in the prior art; see. Thompson LF et al. (1979), "Ecto-5'-nucleotidase activity in T and B lymphocytes from normal subjects and patients with congenital X-linked agammaglobulinemia", /. Immunol. 123, 2475-8.
  • APCP Sigma-Aldrich
  • Ecto-5'-nucleotidase enzyme activity was calculated as a percentage of total IMP-hydrolyzing activity inhibited by APCP. The results are expressed in nmol of IMP hydrolyzed / h / mg protein.
  • RNA isolation NucleoSpin RNA II kit was isolated from cardiac or renal tissue using the total RNA isolation NucleoSpin RNA II kit according to the manufacturer's instructions (Macherey & Nagel, Düren, Germany).
  • tissue frozen in liquid nitrogen was homogenized in the presence of RAI lysis buffer (Micra D8 Homogenizer, ART-Labortechnik, Müllheim, Germany) and after filtration, the lysates were assayed for nucleo-spin RNA II. Columns loaded, followed by desalting and DNasel digestion (Macherey & Nagel, Düren, Germany). The RNA was washed and the concentration was quantitated.
  • the primer sets for the PCR reaction contained 1 ⁇ M sense strand and 1 ⁇ M antisense strand with SYBR Green I (Molecular Probes Inc.).
  • Primer sets were used for the following genes: CD73 [5'-CAA ATC CCA CAC AAC CAC TG-3 1 (SEQ ID NO: 1), 5 1 TGC TCA CTT GGT CAC AGG AC-3 1 (SEQ ID NO: 2), 123 bp]; Ai [5'-AGG GAG GGG TCA AGA ACT GT-3 1 (SEQ ID NO: 3), 5'-TCC CAG TCT CTG CCT CTG TT-3 1 (SEQ ID NO: 4), 109 bp]; A 2A [5'-GAA GAC CAT GAG GCT GTT CC-3 '(SEQ ID NO: 5), 5'-GAG TAT GGG CCA ATG GGA GT-3' (SEQ ID NO: 6), 253 bp] ; A 2B [5'-GGA AGG ACT TCG TCT CTC CA-3 '(SEQ ID NO: 7), 5'-GGG CAG CA
  • Mouse ⁇ -actin [primer sequence, 5'-ACA TTG GCA TGG CTT TGT TT-3 1 (SEQ ID NO: 11) and antisense primer sequence, 5'-GTT TGC TCC AAC CAA CTG CT-3 '(SEQ ID No. 12)] was used in identical reactions as a control for the starting template.
  • CD73-dependent adenosine formation might also play a crucial role in cardioprotection or renal protection during IP. Therefore, the transcriptional changes of renal and cardiac IP on CD73 expression and function were investigated.
  • Renal immunohistological staining and imaging by confocal laser scanning microscopy confirmed the strong induction of the CD73 protein after renal IP ( Figure 2d).
  • Control experiments using the previously described mice mutated for the cd73 gene confirmed the specificity of the anti-CD73 antibody.
  • mice were challenged by intra-arterial infusion of the specific CD73 inhibitor alpha-beta-methylene-ADP [APCP, 40 mg / kg / h (myocardium) or 2 mg / kg ip (kidney)] or vehicle control before cardiac or renal IP and / or ischemia.
  • APCP alpha-beta-methylene-ADP
  • mice were subjected to ligation of the left coronary artery (LCA) for 60 min, followed by 2 h of reperfusion with or without prior IP, consisting of 4 cycles with 5 min ischemia / 5 min reperfusion. All mice survived this experiment. Body weight and blood pressure did not differ between the APCP-treated and untreated mice.
  • LCA left coronary artery
  • APCP treatment results in a two-fold reduction in renal CD73 enzyme activity.
  • mice C57BL / 6J underwent 30-minute renal arterial occlusion with or without previous IP treatment (4 cycles, 4 min ischemia, 4 min reperfusion) followed by 24 hrs of reperfusion while animals were assayed in metabolic cages renal hemodynamics were studied. All mice survived this experiment.
  • IP increased plasma creatinine (Figure 4b), plasma potassium ( Figure 4c), creatinine clearance ( Figure 4d), urine flow rate ( Figure 4e) and excretion of sodium (Fig 4f) and potassium (Fig. 4g) via the urine.
  • mice showed markedly larger infarcts after 60 min of sole ischemia compared to controls.
  • cd73 A mice were reconstituted by intra-arterial application of soluble ecto-5'-nucleotidase (1 U 5'-nucleotidase from the venom of Crotalus atrox).
  • soluble ecto-5'-nucleotidase 1 U 5'-nucleotidase from the venom of Crotalus atrox.
  • Fig. 5f the 5'-Nucleotidase- treatment with an almost complete reconstitution of a wild-type phenotype was associated in cd73 A mice.
  • 5'-nucleotidase treatment of WT mice was associated with a significant reduction in infarct size (Figure Sf).
  • this data provides for the first time the genetic evidence for CD-dependent cardioprotection by IP. It has also been shown that treatment with soluble nucleotide phosphohydrolase or 5'-nucleotidase represents a new therapeutic option during acute myocardial ischemia.
  • this data provides for the first time genetic evidence for CD73-dependent renal protection by IP.
  • Cardiac IP is associated with selective induction of the adenosine A ⁇ receptor
  • mice After a functional contribution to cardioprotection by the IP was demonstrated, the studies were continued in A 2B "7" mice. For this purpose, the absence of adenosine A 2B receptor-mediated responses was first confirmed in these mice. In fact, PCR genotyping, measurement of adenosine A 2 B receptor mRNA expression, and A 2B receptor protein expression confirmed intermediate levels of adenosine A 2B receptors in heterozygous mice and absence of adenosine. A 2B receptors in homozygous mice ( Figure 8a-c). Furthermore, the transcript levels of the other adenosine receptors (Ai / A 2A / A 3 ) were unchanged compared to the controls in the A 2B ' 7 ' mice (data not shown).
  • mice mutated for the adenosine A2B receptor have greater infarct size and kidney damage compared to the wild type, and where no cardio or renal protection can be achieved by IP, a potential therapeutic role for a specific target group was identified A2 ⁇ agonists (BR4887 or BAY 60-6583).
  • A2 ⁇ agonists BR4887 or BAY 60-6583.
  • the chemical structure of BR4487 / BAY 60-6583 and the EC 50 values on the various adenosine receptors are shown in FIG.
  • mice were first subjected to normoxia or hypoxia (8% oxygen, 4 h) after treatment with BR4887 / BAY 60-6583 (10 ⁇ g / kg ip, 30 min before hypoxia / normoxia) or vehicle control.
  • normoxia or hypoxia 8% oxygen, 4 h
  • BR4887 / BAY 60-6583 10 ⁇ g / kg ip, 30 min before hypoxia / normoxia
  • BL6 mice were treated with a single bolus of intra-arterial BR4887 / BAY 60-6583 (10 ⁇ g / kg / body weight for 30 min prior to ischaemia) and exposed to myocardial ischemia for 60 min.
  • BR4887 / BAY 60-6583 treatment resulted in a significant reduction in infarct size ( Figure IIb, c).
  • cd73 / mice were next rendered more soluble via ip injection 5'-nucleotidase (Sigma 5'-nucleotidase from the venom of Crotalus atrox) reconstituted and treated with or without IP 30 min prior to renal ischemia.
  • BL6 mice were treated with soluble C. atrox 5'-nucleotidase and exposed to renal ischemia for 30 minutes with or without prior IP treatment. After 24 hours of reperfusion, studies of renal function were performed. As shown in Figures 14a-f, 5 'nucleotidase treatment was associated with nearly equal protection of renal function as in IP treatment.
  • mice received soluble 5'-nucleotidase i.p. or vehicle 30 min prior to renal ischemia with or without IP treatment. After 24 h of reperfusion histological sections were made.
  • this data provides for the first time the genetic evidence for CD73-dependent renal protection by IP. Furthermore, treatment with soluble 5'-nucleotidase as a potential new therapy during acute renal ischaemia was demonstrated.

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Abstract

L'invention concerne un médicament destiné à la prophylaxie, au traitement ou au diagnostic de maladies ischémiques, des procédés pour diagnostiquer chez un être vivant une prédisposition à souffrir d'une maladie ischémique ou une maladie ischémique, ainsi qu'une composition pharmaceutique ou diagnostique.
PCT/EP2007/008215 2006-09-20 2007-09-20 Médicament destiné à la prophylaxie, au traitement ou au diagnostic de maladies ischémiques Ceased WO2008034623A2 (fr)

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