WO2008034623A2 - Medicament for the prophylaxis, treatment or diagnosis of ischaemic diseases - Google Patents

Abstract

The invention relates to nucleotide-phosphohydrolase and adenosine-A2B receptor agonists used as medicaments for the prophylaxis, treatment or diagnosis of ischaemic diseases, to methods for diagnosing a predisposition to an ischaemic disease or for diagnosing the disease in a living being and to a pharmaceutical or diagnostic composition.

Description

 Medicaments for the prophylaxis or treatment or diagnosis of ischemic

Diseases

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. Furthermore, an improvement in the microcirculation by administration of vasodilators, such as. Nitric oxide donors, and antibodies to adhesion molecules and increasing the ischemic tolerance by reducing oxygen radicals by glutathione is taken.

A conditioning of organs, the so-called ischemic preconditioning (IP), has also been described as an instrument for protecting against ischaemia. It has been shown that short, controlled periods of occlusion of the arterial blood supply with subsequent reperfusion can protect against prolonged tissue ischemia. IP means that additional, controlled tissue ischemia with a consecutive reperfusion phase precedes the actual ischemia resulting from surgery. This results in organ protection by increasing ischemia tolerance.

Despite a large number of studies, it has not yet been possible to identify the molecular mechanisms underlying ischemic preconditioning in order to be able to use them therapeutically in a targeted manner. For example, Miki T. et al. (1998), "Ecto-5'-nucleotidase is not required for ischemic preconditioning in rabbit myocardium in situ", Am. J. Phyrosiol. 275, H1329-37, the role of the enzyme ecto 5'-nucleotidase during ischemic preconditioning and concluded that it does not appear to play a role here.

Van Waarde A. et al. (1989), "Protection of the kidney against ischemic injury by inhibition of 5'-nucleotidase", Am. J. Physiol. 256 (2 Pt 2): F298-305, however, contravening the findings of Miki T. et al. (supra) that renal ischemia could be improved by inhibition of 5'-nucleotidase.

Despite intensive research efforts, ischemic diseases, such as 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. 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.

Against this background, it is urgently necessary to develop new therapeutic strategies that can prevent ischemic diseases or with which such diseases can be treated and diagnosed.

It is therefore an object of the present invention to provide a medicament for the prophylaxis and / or treatment and / or diagnosis of ischemic diseases, which specifically targets the molecular causes of ischemic diseases and thereby supports the existing therapy concepts for the treatment of these diseases or diagnostics or possibly even replaced.

This object is achieved by the use of nucleotide phosphohydrolase for the manufacture of a medicament for the prophylaxis and / or treatment and / or diagnosis of ischemic diseases.

The inventors have surprisingly found in animal experiments that the administration of 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. 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.

In this case, 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. Another synonym for the 5 'nucleotidase is 5'-NT. 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.

The use according to the invention of 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.

These observations of the inventors were particularly surprising, since the statements in the prior art point in exactly the opposite direction. Thus, both Miki et al. (supra) and Van Waarde et al. (supra) that the 5'-nucleotidase is not required for protection of ischemic risk areas, or whose activity or expression should even be inhibited for this purpose. The solution according to the invention thus represents a departure from the findings of the prior art.

It is preferred according to the invention, if as 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.

In this case, it is preferred according to the invention if the nucleotide phosphohydrolase or 5'-nucleotidase is present in soluble form.

The inventors have discovered that 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.

It is further preferred according to the invention if the nucleotide phosphohydrolase is in micronized form.

Under a micronized form according to the invention an embodiment of a nucleotide phosphohydrolase understood, which is reduced in size compared to the natural or wild-type variant. Preferably, 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.

In the present invention, it is preferable that 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. Thus, 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.

According to the invention, the nucleotide phosphohydrolase can furthermore be present in the form of a coding sequence in an expression vector.

This measure has the advantage that the patient being treated can "self-procure" the required nucleotide phosphohydrolase and, if necessary, compensate for genetic defects that have led to a mutation of endogenous nucleotide phosphohydrolase. "The coding sequences of 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 These references as well as sequences are incorporated herein by reference. It is understood that the expression vector may have other portions, such as promoters, enhancers, etc., which ensure expression of the nucleotide phosphohydrolase in the patient.

According to the invention it is preferred if the drug has an activity enhancer.

As an 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.

According to the invention, the action enhancer used is preferably an adenosine A ₂ B receptor agonist, for example BR 4887.

This measure has the advantage that such an action enhancer is used, which is particularly suitable according to findings of the inventors for the further development of the drug. BR4887, also referred to as BAY 60-6583, is a specific adenosine A ₂ B receptor agonist manufactured by Bayer HealthCare. Thus, the inventors have found that even the isolated administration of BR4887 can reduce hypoxia-induced vascular leakage. A combined administration with nucleotide phosphohydrolase even leads to a significant improvement of the medicament according to the invention.

According to the invention, 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.

By this measure, the inventors take advantage of recent findings in the prior art to advantage. 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.

Here, too, the inventors take advantage of recent findings, according to which in mice that have been pretreated with dipyridamole (Boehringer Ingelheim), after the induction of hypoxia significantly decreased albumin leakage and detectable the ischemic cascade was significantly weaker in their extent; see. Eltzschig H.K. et al. (Supra).

Against this background, 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. Examples of a biological sample are tissues at risk areas (AAR), for example myocardial, renal, intestinal or liver tissue, a salivary or blood, hair sample, etc. However, 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.

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.

Upon determination that 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.

With respect to the 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.

For example, 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 (IP) 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. The inventors have now surprisingly found that treatment with nucleotide phosphohydrolase, ie with 5'-nucleotidase (CD73) or apyrase (CD39), causes almost the same protection as the IP treatment. The findings of the inventors therefore show that the administration of nucleotide phosphohydrolase, for example in soluble form, leads to an IP.

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.

The inventors have surprisingly found that administration of a specific adenosine AΣB receptor agonist after induction of hypoxia results in a markedly reduced infarct or improved renal function, respectively, than in untreated control animals. This demonstrated a cardio-renoprotective effect of adenosine A2B receptor agonists, such as BR4887 / BAY 60-6583. 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.

As such, 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.

It is understood that the features mentioned above and those yet to be explained below can be used not only in the particular combination given, but also in other combinations or in isolation, without departing from the scope of the present invention.

In the following, the invention will be explained in more detail by means of exemplary embodiments, which are of purely illustrative nature and do not limit the scope of the invention. Reference is made to the attached figures, in which the following is shown.

Figure 1 CD73 is induced by cardiac ischemic preconditioning (IP), (a) mouse model of cardiac IP. 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. (B) 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 (β-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. A representative experiment from a total of 3 experiments is shown, (d) In order to investigate the influence of IP on cardiac CD73 expression, 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). In further experiments, 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 After IP in situ, the risk area was excised at the indicated time points, snap frozen, and enzyme activity was evaluated by measuring the conversion of [ ¹⁴ C] IMP to [ ¹⁴ C] inosine For special purposes 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).

Figure 2 CD73 is induced by renal ischemic preconditioning (IP), (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 data were calculated relative to an internal housekeeping gene (β-actin) and are 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 IP. The kidneys were excised, flash frozen, lysed at the indicated time points, and the proteins were separated by SDS-PAGE and transferred to nitrocellulose. The membranes were incubated with anti-CD73 antibody. Shown is a representative experiment of 3 experiments. The same blot was assayed for β-actin expression as a control for protein loading, (d) CD73 protein is induced by IP - immunohistochemistry. Wild type and cd73 ^/ mice were subjected to IP. 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). (a) Measurement of CD? '3 activity in hearts treated with APCP. Age, sex and weight matched littermate controls were anesthetized and infused with the specific CD73 inhibitor alpha-beta-methylene-ADP (APCP) via a carotid artery catheter (40 mg / kg / h). To confirm the effective inhibition of CD73 activity, hearts were excised after 30 minutes of infusion and enzyme activity was evaluated by measuring the APCP-inhibited conversion of [ ¹⁴ C] IMP to [ ¹⁴ C] inosine. The results are expressed in nmol of IMP hydrolyzed / h / mg protein. (b) AMP-induced bradycardia is blocked by APCP infusion. To confirm the functional inhibition of CD73 by APC treatment, a bolus of 5'-adenosine monophosphate (AMP) was administered (50 μl 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. In order to investigate the influence of APCP infusion on cardioprotection by IP, preconditioning was next performed in situ with 4 cycles of IP (5 min ischemia, 5 min reperfusion, with or without APCP infusion). The mice were sacrificed after 2 hours of reperfusion and the plasma levels of troponin I (cTnI) were measured by ELISA. It should be noted that the cardioprotective effects of IP are attenuated by the APCP treatment, (d) In further experiments, infarct sizes were determined by double staining with Evan's Blue and 2,3,5-triphenyltetrazolium chloride in APCP-treated or -untreated mice (+/- APCP). In the- Farts sizes of mice treated with or without IP (- / + IP) are expressed as percent of infarct in the risk area. The results are shown as mean ± SD from 6 animals per condition, (e) Representative images of the infarcts are shown.

Fig. 4 Inhibition of CD73 by APCP treatment abolishes renal protection by ischemic preconditioning (IP). (a) 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. Experiments were performed in APCP-treated or untreated mice with or without preconditioning in situ (4 cycles of IP, 4 min ischemia, 4 min reperfusion) 30 min before ischemia. Renal function tests were performed after 24 hours of reperfusion, (b) measurement of plasma creatinine concentration. (c) Measurement of plasma potassium (plasma K ⁺ ). (d) Measurement of creatinine clearance. (e) measurement of urinary flow rate, (f) measurement of urinary sodium excretion (urinary Na ⁺ excretion), (g) measurement of urinary potassium excretion (urinary K ⁺ excretion). (h) Quantitation of PMN tissue accumulation by measurement of myeloperoxidase (MPO) tissue levels. The data are from 5 to 8 mice per condition and the results are expressed as means ± SD.

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. The results are expressed in nmol hydrolyzed IMP / h / mg protein, (b) the AMP-induced bradycardia is attenuated in CD73 ^{~ A} mice. Wiild-type cd73 ^{+ / +} or mice mutated for cd73 (cd73 ^"Λ ) were treated with a bolus of adenosine monophosphate (AMP, 50 μl of a 3 mg / ml solution of AMP) via a carotid artery catheter. Results are expressed as percent change in heart rate after AMP injection. (C) IP cardioprotection is abolished in cd73 ^~ ' ^~ mice.To examine cardiac IP in mice mutated for cd73, preconditioning was performed in situ with 4 cycles of IP (5 min ischemia, 5 min reperfusion) in wild type (cd73 ^{+ / +} ) or in those mice with targeted deletion of cd73 (cd73 ^"A ). Mice were sacrificed after 2 hours of reperfusion and infarct sizes measured by double staining with Evan's Blue and 2,3,5-triphenyltetrazolium chloride. Infarct sizes are expressed as percent of infarct in the risk area. The results are presented as mean ± SD from 6 animals per condition, (d) Troponin I (cTnl) plasma levels were measured in wild type mice (cd73 ^{+ / +} ) or cd73 mutant mice (cd73 ^Λ ) with or without IP (+/- IP) were treated. It should be noted that the cardioprotective effects of IP are attenuated in cd / '3 ^{' 1} mice (e, f) Reconstitution of cd73 ^~ - mice with adenosine. Cd73 ^{/ ~} mice (e) and age-, weight- and sex-matched littermate controls (f) received intra-arterial infusion of adenosine (200 μl / 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. Cd73 ^Λ mice (f) and age-, weight- and sex-matched littermate controls (g) received ip treatment with 1 U of soluble 5'-nucleotidase derived from C. atrox venom 60 min before IP. 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 ^Λ 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.

Figure 7 The adenosine A2β receptor is selectively induced by ischemic preconditioning (IP), (a) modulation of the level of adenosine receptor transcript by IP. 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 (β-actin) and expressed as x-fold change compared to simulated animals without IP treatment (controls, C ± SD). The 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. The animals 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. (D) preconditioning in all adenosine receptor knockout mice - except in adenosine-AzB receptor knockout mice To study cardiac IP in mice mutated for each adenosine receptor, 60 min before ischemia occurred in all adenosine receptor preconditioning in situ (4 cycles of IP, 5 min ischemia, 5 min reperfusion) or simulation-based in-age, weight or sex-matched littermate controls (torpedoed mice, Ai, A ₂ A, A _2B , A ₃ ). Operations. Mice were sacrificed 2 hours post-reperfusion and infarct sizes measured by double staining with Evan's Blue and 2,3,5-triphenyltetrazolium chloride. Infarct sizes are expressed as a percentage change from littermate controls with IP. The results are given as mean ± SD from 6 animals per condition.

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). (b) PCR for genotyping according to the instructions of the manufacturer in mice mutated for the adenosine A _2B receptor (WT: wild type, HZ: heterozygous deletion of the adenosine A2B receptor, KO: homozygous deletion of the adenosine A2β receptor). (d) Hγpoxia-induced vascular leakage increases in cardiac tissue from mice mutated for the adenosine A2β receptor. A ₂ B " ^Λ mice and age-, weight- and sex-matched controls were given intravenously to Evan's Blue solution (0.2 ml of 0.5% in PBS) and those to normobalic hypoxia (H, 8%). O ₂ , 92% N ₂ ) or room air (N) for 4 h The animals were killed and the hearts isolated The concentrations of Evan's Blue in the organ were measured after formamide extraction (55 C for 2 h) by measuring the absorbances quantified at 610 nm after subtraction of the reference absorbance at 450 nm, Evans blue in Azs mice after cardiac hypoxia exposure is expressed as mean ± SD of Evan's Blue OD / mg wet tissue and pooled from 4 to 6 Animals per condition, (e) IP is abolished in Az ^ 'mice To study cardiac IP in mice mutated for the adenosine A _2B receptor, in situ preconditioning was performed with 4 cycles of IP ( 5 min ischemia, 5 min reperfusion) in wild-type M nozzles (A ₂ B ^{+ / +,} white bars) or in Mice with targeted deletion of the adenosine A2B receptor (A ₂ B ^{7 "} , black bars) .The mice were killed after 2 hours of reperfusion and the troponin I (cTnI) plasma levels were measured with or without IP (+/- IP In addition, infarct sizes were measured by double staining with Evan's Blue and 2,3,5-triphenyltetrazolium chloride Infarct sizes are expressed as infarct percent in the risk area The results are presented as mean ± SD from 6 animals per condition Cardioprotective effects of IP are attenuated in A2B ^A mice (f) Representative images from mice deleted for the adenosine A2B receptor (A ₂ B ^{7 "} ) with or without IP 60 min before ischemia and 2 h before reperfusion with Evan's Blue / 2,3,5-triphenyltetrazolium chloride double stain. (g) order on the Kardioprotekti- to investigate the influence of pSBL 115, a water-soluble highly specific adenosine A _2A receptor antagonists by IP was preconditioning in age- weight in situ, and gender-matched littermate controls and conducted in mice mutated for the adenosine Ai receptor using 4 cycles of IP (5 min ischemia, 5 min reperfusion) 60 min before ischemia. During the experimental procedure, the mice were infused with PSB1115 (5 mg / kg / hr via a carotid artery catheter, black bars) or vehicle control (white bars). Mice were sacrificed after 2 hours of reperfusion and infarct sizes measured by double staining with Evan's Blue and 2,3,5-triphenyltetrazolium chloride. Infarct sizes treated with or without IP (- / + IP) are expressed as infarct percent in the risk area. The results are presented as mean ± SD from 6 animals per condition. It should be noted: The attenuation of infarct size limitation by IP due to PSB1115 in wild-type mice and Ai "mice.

Figure 9 Histologic signs of renal protection by IP are absent in cd73 'mice. In order to examine the histological aspects as well as the renal infiltration with polymorphonuclear neutrophils (PNM), the kidneys were treated 24 hours post ischemia with or without IP (4 IP cycles with 4 min ischemia and 4 min Reperfusion 30 min before ischemia), (a) Representative sections (x400, after H & E staining) are shown. (B) Quantification of ischemic damage using the Jablonski scale (see Materials and Methods section). Reduced renal infiltration with polymorphonuclear neutrophils (PMN) is abolished in cd73 ^~ 'mice, (c) Representative sections (chloroacetate esterase staining, x400) after ischemia (+ IP) and without IP (- IP) in cd73 ^/ mice and littermates, (d) Quantification of granulocyte infiltration. The data are from 6 to 7 mice per condition, and the results are expressed as means ± SD.

Fig. 10BR4887 / BAY 60-6583. (a) Chemical structure and (b) ECso values of BR4887 / BAY 60-6583 on the adenosine receptors AIAAR, AΣAAR and A ₂ BAR.

Fig. 11 Treatment with adenosine AΣβ receptor agonist protects against myocardial ischemia. (a) Function of vascular barrier after treatment with BR4887 / BAY 60-6583. Age-, weight- and sex-matched littermates received treatment ip with BR4887 (10 μg / kg, 30 min before hypoxia, normoxia) or with vehicle control. The mice were intravenously administered Evan's Blue (0.2 ml of 0.5% in PBS) intravenously and the mice were challenged with normobaric hypoxia (H, 8% O ₂ , 92% N ₂ ) or room air (N) h exposed. The animals were killed and the hearts were isolated. The 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. Representative images (b) or quantitative analysis (c) myocardial infarction size (60 min of ischemia, 2 h of reperfusion, double staining with Evan's Blue and 2,3,5-triphenyltetrazolium chloride) after IP treatment with BR4887 (10 μg / kg ip, 30 min before the induction of myocardial ischemia) or vehicle control in age-, weight- and sex-matched littermates. The results are displayed as mean values ± sd from 6 animals per condition, (d) Quantitative analysis of myocardial infarction size (60 min of ischemia, 2 h of reperfusion, double staining with Evan's Blue and 2,3,5-triphenyltetrazolium chloride) after IP treatment with BR4887 (10 μg / kg Lp., 30 min before the induction of myocardial ischemia) or vehicle control in mice mutated for the adenosine A ₂ B receptor. It should be noted that cardioprotection with BR4887 treatment occurs in wild-type mice, but not in A ₂ B ⁴ mice.

A treatment with adenosine A ₂ B receptor agonist protects against renal ischaemia. (a) Prevention of the kidney-protective effect of IP by the specific adenosine A ₂ B receptor antagonist PSBI15 in wild-type mice (WT). (b) Improved glomerular filtration rate by BR4887 / BAY 60-6583 after 45 min of ischemia of the kidney, (c) The effect shown in (b) is abolished in mice deficient in the adenosine A ₂ B receptor (A2B- / -). (d) The Joblonski Index, the internationally accepted measure of kidney tissue damage, shows significantly less tissue damage in WT mice administered BR4887 / BAY 60-6583. (e) Histological recordings of the kidneys of the mice treated with the vehicle or with BR4887 / BAY 60-6583.

Fig. 13 Reconstitution of cd? 3 'mice with soluble 5'-nucleotidase. After induction of anesthesia, 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. Treatment with soluble 5'-nucleotidase improves renal hemodynamics in cd73 ^{~ / ~} mice, (a) measurement of plasma creatinine concentration. (b) Measurement of potassium in plasma (plasma K ⁺ ). (c) Measurement of creatinine clearance. (d) measurement of urinary flow rate, (e) measurement of urinary sodium excretion (urinary Na ⁺ excretion), (f) measurement of urinary potassium excretion (urinary K ⁺ excretion). The data from 5 to 7 mice per condition, the results are expressed as means ± SD.

Figure 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. (d) measurement of urinary flow rate, (e) measurement of urinary sodium excretion (urinary Na ⁺ excretion), (f) measurement of urinary potassium excretion (urinary K ⁺ excretion). The data are from 5 to 7 mice per condition, the results are expressed as means ± SD.

Fig. 15 Histological signs of renal protection by treatment of wild-type mice with soluble 5'-Nudeotidase. In order to examine the histological aspects as well as the renal infiltration with polymorphonuclear neutrophils (PMN), 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). (b) Quantification of ischemic injury with the Jablonski scale (see Materials and Methods section), (c) Representative sections with chloroacetate esterase staining (x400). (d) quantification of granulocyte infiltration. The data are from 6 to 7 mice per condition, and the results are expressed as means ± SD. embodiments

L material and methods

1.1 mice

All animal testing protocols were in accordance with the German guidelines on the use of live animals and were approved by the Animal Welfare Committee of the University Hospital Tübingen and the Regional Council Tübingen. 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. Consistent with previous reports, A ^ 'mice appear to have a normal immune system and to be healthy if kept in a special pathogen-free environment. As a control littermates were used, which were matched in terms of age, gender and weight. 1.2 mouse models

(a) Cardiac IP

Anesthesia was induced with pentobarbital (70 mg / kg body weight ip) and maintained (10 mg / kg / h). The 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 tracheal tube was connected to a mechanical ventilator (Servo 900C, Siemens, Germany, with a pediatric tube system) and the animals were ventilated with a pressure controlled ventilation mode (peak inspiratory pressure 10 mbar, frequency 110 breaths / min, final positive expiratory pressure of 3 mbar Fi2 = 0.3). Blood gas analysis revealed a regular paCh (115 ± 15 mmHg) and paCO2 (38 ± 6 mmHg) level with the ventilator used. After induction of anesthesia, the animals were monitored with an ECG (Hewlett Packard, Boeblingen, Germany). The liquid replacement was carried out with normal saline solution, 0.1 ml / h ip The carotid artery was catheterized for continuous uptake of blood pressure with a Statham element (WK 280, WKK, Kaltbrunn, Switzerland). The operations were performed under an upright dissecting microscope (Olympus SZX 12). After left thoracotomy, cardiac exposure, and preparation of the pericardium, the left coronary artery (LCA) was visually identified. Subsequently, an 8.0 nylon thread (Prolele, Ethicon, Norderstedt, Germany) was placed around the vessel. LCA occlusion for ischemia and IP studies was performed using a hanging weight system; see. Eckle T. et al. (2006), "Systematic evaluation of a novel model for cardiac ischemic preconditioning in mice", Am. J. Physiol. Heart Circ. Physiol. The successful LCA occlusion was characterized by an immediate color change of the vessel from light red to dark violet, the color change of the myocardium delivered by the vessel (from bright red to white), and the presence of ST increases in the ECG approved. During reperfusion, the color changes disappeared immediately when the suspended weights were lifted and the LCA perfused again.

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). 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.

(b) Renal IP

The 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). In brief, an incision was made on the right side of the body and the right kidney was removed. After sealing the surgical wound with a continuous suture (muscle wall and skin), 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. At the end of the surgical procedure, the 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.

1.3 Heart enzyme measurement

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).

1.4 Measurement of renal function

The 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.

1.5 Histological examination

Histological examination of the kidneys was performed 24 hours after renal ischemia. The renal tissues were fixed in 4.5% buffered formalin, dehydrated and embedded in paraffin. The sections (3 μm) were stained with hematoxylin, eosin and periodic acid (PAS). The examination and classification of the entire incision of each kidney was made by an experienced kidney pathologist who had no knowledge of the experimental group. A rating scale from 0 to 4 was used as described by Jablonski et al. to perform the histopathological evaluation of proximal tubular injury through ischemia and reperfusion in kidneys with or without IP. Four animals under each condition and three representative sections from each kidney were evaluated.

1.6 Immunoblotting experiments

In some experiments, the content of CD73 and adenosine A ₂ B receptor protein was determined from the risk area. For these purposes, 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 μl, 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 μg / 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.

1.7 Immunohistochemistry

(a) myocardium

To examine the influence of IP on cardiac CD73 and the AΣBAR, 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, Munich, 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. After three washes with TBS, sections were taken for 45 minutes the secondary antibody (CD73: goat anti-rabbit Cy3, Amersham Pharmacia, Freiburg, Germany; A2B: rabbit anti-goat Cy3, Dianova Hamburg, Germany) at room temperature. The fluorescence was visualized with a confocal laser scanner microscope (Leica, Germany).

(b) kidney

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.

1.8. Myeloperoxidase (MPO activity

To quantify neutrophil tissue infiltration, the known methods of MPO measurements were modified. Samples of renal tissue were snap frozen 24 hours after the experimental procedure (renal ischemia with or without IP), homogenized in ice-cold 50 mM potassium phosphate buffer pH 7.4 and then centrifuged at 20,000 g for 15 minutes at 4 ° C. The supernatant was discarded and the pellet sonicated in 50 mM potassium phosphate buffer pH 7.4 containing 0.5% hexadecyltrimethylammonium bromide for 30 seconds. The homogenates were centrifuged at 20,000 g for 15 minutes at 4 ° C. Five μl of the supernatant was added to 195 μl of 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.

1.9 Ecto 5'-Nucleotidase Enzyme Assays

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) was used as a non-specific inhibitor of CD73. 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.

1.10 In v / vo hypoxia model

Mice mutated on the Fl line for A2BAR were prepared, validated, and characterized as described above. Control mice were matched for gender, age and weight. The vascular permeability of the entire organ was quantified by intravascular administration of Evan's Blue as described in the prior art; see. Thompson LF et al. (Supra). For the purpose of quantifying permeability, 0.2 cc of Evan's Blue 0.5% in PBS was injected intravenously. The animals were then exposed to normobaric hypoxia (8% O ₂ , 92% N ₂ ) or room temperature for 4 hours (n = 4 animals per condition). After exposure to hypoxia / normoxia, the animals were killed and hearts were isolated. The concentrations of cardiac Evan's Blue were after quantitation of formamide extraction (55 C for 2 h) by measuring the absorbances at 610 nm after subtraction of the reference absorbance at 450 nm.

1.11 Transcription analysis

To determine the influence of IP on the transcript levels of cd73, adenosine Ai, -A2A, -A2B and Aβ receptors, 4 cycles of IP (5 and 4 min ischemia respectively, 5 and 4 min., Respectively) were performed Reperfusion), the AAR was visualized by Evan's Blue staining and excised at the indicated times, followed by isolation of RNA and quantitation of transcript levels by real-time RT-PCR (iCycler, Bio-Rad Laboratories Inc.). Briefly, total RNA 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). For these purposes, 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 μM sense strand and 1 μM antisense strand with SYBR Green I (Molecular Probes Inc.). Primer sets (sense primer sequence, antisense primer sequence and transcript size) 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 CAA CTC AGA AAA CT-3' (SEQ ID NO: 8), 322 bp] ; A ₃ [5'-CAA TTC GCT CCT TCT GTT CC-3 ¹ (SEQ ID NO: 9), 5'-TCC CTG ATT ACC ACG GAC TC-3 ¹ (SEQ ID NO: 10), 334 bp] , The primer set was made using increasing cycle numbers of 94 ° C for 1 min, 58 ° C for 0.5 min, 72 ° C for 1 min, amplified. Mouse β-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.

1.12 Data analysis

The data concerning the classification of renal damage are given as median (range), all other data are given as mean ± SD from 5 to 10 animals per condition. Statistical analysis using Student's t-test (two-tailed, α <0.05) or analysis of variance was performed to identify group-specific differences. Renal damage was analyzed by the Kruskal-Wallis Placement Test.

2 results

2.1 CD73 is induced by IP

The inventors hypothesized that 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.

(a) myocardium

A prior art model of cardiac IP (Figure Ia) was used and the risk area (AAR) was isolated at the indicated times. Using a real-time reverse transcriptase Polymerase chain reaction (real-time RT-PCR), 90 min after cardiac IP, marked induction of cd73 mRNA (14.5 + 2, 7-fold, p <0.0001, Fig. Ib).

Similarly, a Western blot analysis of the risk area confirmed the induction of the CD73 protein 90 and 120 min after IP (Fig. Ic).

Immunohistological staining of the risk area and imaging by confocal laser scanning microscopy confirmed strong induction of the CD73 protein after cardiac IP. Control experiments using previously described cd73 mutant mice confirmed the specificity of the experiments (Fig. Id).

After it was shown that the cd73 mRNA and the CD73 protein are induced by cardiac IP, the functional induction of CD73 by IP was next examined, using a previously described technique for measuring ecto-5'-nucleotidase activity , As shown in Figure Ie, ecto-5'-nucleotidase activity is already induced 30 minutes after IP.

Taken together, these data provide strong evidence that CD73 is induced in the AAR by cardiac IP.

(b) kidney

In a siru model, four cycles of intermittent, renal, arterial occlusion and reperfusion (4 min ischemia, 4 min reperfusion) were performed at IP (Figure 2a). Intermittent renal arterial occlusion was achieved using a suspended weight system which did not cause any visible tissue tissue trauma during preconditioning. After IP, the kidneys were compared to renal tissue from non-preconditioned age-, weight- and sex-matched littermates. To define transcriptional effects of IP, preconditioned kidney tissue was isolated at the indicated time points after IP treatment and used for real-time RT-PCR. Significant induction of cd73 mRNA was observed (eg, 90 min after renal IP, 3.9 ± 1.4 fold, p <0.01, Figure 2b).

Similarly, renal Western blot analysis confirmed CD73 protein induction after IP (Figure 2c).

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.

After it was shown that the cd73 mRNA and the CD73 protein are induced by renal IP, the functional induction of CD 73 by IP was next examined using a previously described technique for measuring Ecto 5'-nucleotidase enzyme activity has been. As shown in Figure 2e, ecto-5'-nucleotidase activity was induced more than twice as early as 30 minutes after IP treatment.

Taken together, these data provide strong evidence that CD73 is induced by renal IP.

2.2 The inhibition of CD73 weakens the protection by IP

Having demonstrated that CD73 was induced by IP, its functional contribution to IP protection was next examined. To For this purpose 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.

(a) myocardium

As shown in Figure 3a, the above pharmacological approach results in a threefold reduction in cardiac CD73 activity upon APCP treatment.

Similarly, AMP-induced bradycardia was markedly attenuated in APCP-treated animals (Figure 3b). In fact, intravascular AMP treatment (50 μl, 8 mg / ml of AMP) resulted in a reduction in heart rate from 480 / min to 120 / min. After APCP treatment, this response was almost completely abolished (480 / min to 360 / min, p <0.0001).

After effective inhibition of cardiac CD73 activity was demonstrated, these conditions were used to examine cardioprotection by IP. For these purposes, 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.

To determine the myocardial tissue damage, the plasma levels of a previously described marker for murine myocardial ischemia [troponin I (cTnl)] were measured. Consistent with previous studies, there was an attenuation of the plasma cTnI values by IP (FIG. 3c). However, APCP treatment reversed the cardioprotective effects of IP. Likewise, measurement of infarct size by 2,3,5-triphenyltetrazolium chloride (TCC) staining confirmed inhibition of cardioprotection by IP after APCP treatment (Figure 3d, e).

In summary, these pharmacological studies provide evidence for a crucial role of CD73 in cardioprotection by IP.

(b) kidney

As shown in Figure 4a, APCP treatment results in a two-fold reduction in renal CD73 enzyme activity.

Having demonstrated effective inhibition of renal CD73 enzyme activity by APCP, these conditions were used to examine the role of CD73 in renal protection by IP. Thus, 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.

As shown in Figure 4, 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.

In contrast, APCP treatment abolished the renal protective effects of IP (Figure 4b-g). Furthermore, mice treated with IPP before IP ischemia showed an increase in myeloperoxidase activity in the renal tissue (Figure 4h). Taken together, these studies provide pharmacological evidence for a critical role of CD73 in renal protection by IP.

2.3 Protection by IP is abolished in cd73 ^y mice

Based on these pharmacological findings demonstrating that CD73 inhibition attenuated the protective effects of IP, studies in the previously described mice with targeted deletion of the cd73 gene were next performed.

(a) myocardium

Next, the absence of cardiac CD73 activity in these mice was confirmed. As shown in Figure 5a, in the cd73 ^/ mice almost no cardiac CD73 activity could be measured compared to littermate controls. Similarly, AMP-induced bradycardia in cd73 ^A mice was significantly attenuated compared to littermates.

Next, an IP was performed in the cd73 ^/ mice and littermate controls. In fact, infarct size was not attenuated at 60 min after ischemia by IP in cd73 ^/ mice (Figure 5c).

Furthermore, cd73 ^/ mice showed markedly larger infarcts after 60 min of sole ischemia compared to controls.

To confirm that IP was attenuated in cd73 ^A mice, cTnI levels in plasma samples were again measured. As shown in Figure 5d, there was no difference in cTnI plasma levels in cd73 ^/ mice after 60 minutes of ischemia with or without cardiac IP. As a "proof of principle" and to demonstrate that the absence of cardioprotection by IP in cd73 ^/ mice reflects the absence of extracellular adenosine, extracellular adenosine levels were reconstituted via intra-arterial infusion (200 μl / h, adenosine 8 mg / ml). at a dose previously shown to induce no hypotension or bradycardia (data not shown). Indeed, this treatment resulted in partial reconstitution of cardioprotection by IP in cd73 ^A mice (Figure 5e). The same treatment of wild-type mice resulted in the attenuation of infarct sizes following IP alone, suggesting additional mechanism (eg, increase in A denosin receptor-mediated signaling by IP) (Figure 5e).

In further experiments, cd73 ^A mice were reconstituted by intra-arterial application of soluble ecto-5'-nucleotidase (1 U 5'-nucleotidase from the venom of Crotalus atrox). As shown in Fig. 5f, the 5'-Nucleotidase- treatment with an almost complete reconstitution of a wild-type phenotype was associated in cd73 ^A mice. Furthermore, 5'-nucleotidase treatment of WT mice was associated with a significant reduction in infarct size (Figure Sf).

In summary, 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.

(b) kidney

First, the absence of CD73 enzyme activity in renal tissue of these mice was confirmed (Figure 6a). Next, an IP in cd73 ^{~ A} mice and littermate controls was performed. In sharp contrast to the results with the wild-type mice, renal hemodynamics by IP, including plasma creatinine (Figure 6b), plasma potassium (Figure 6c), creatinine clearance (Figure 6d), were in cd73 ^Λ mice Urine flow rate (Figure 6e), excretion of sodium (Figure 6f) and potassium (Figure 6g) via the urine, not improved. That is, the presence of a functioning CD73 activity is crucial for the tissue-protective effect of IP.

Furthermore, cd73 ^/ mice did not show a decrease in myeloperoxidase activity in renal tissue after IP treatment (Figure 6h).

In summary, this data provides for the first time genetic evidence for CD73-dependent renal protection by IP.

2.4 Cardiac IP is associated with selective induction of the adenosine A ^ receptor

After demonstrating that extracellular adenosine formation via CD73 is critical for IP, the transcriptional consequences of cardiac IP on the expression levels of all four adenosine receptors were next examined (Figure 7a-d).

In fact, these studies revealed a selective induction of the adenosine A2B receptor transcript with a maximum at 90 min after IP (14-fold induction, p <0.0001).

In contrast, the mRNA levels of the adenosine Ai and As receptors remained unchanged by the IP, while the adenosine A2A receptor was significantly suppressed. Equally confirmed immunohistochemistry using confocal laser scanning microscopy, the induction of the A _2B protein 90 min after IP (Figure 7c).

After showing that the A ₂ B mRNA and the A ₂ B protein are induced by cardiac IP, the contribution of each adenosine receptor to cardioprotection by IP was next studied. For this purpose, the previously described Ar ^Λ , A ₂ A ⁷ 'or A ₃ ^7- mice and commercially available A _2B ⁷ MaUSe [Deltagen Inc. (San Carlos, CA)] were subjected to cardiac IP. As shown in Figure 7d, all adenosine receptor knockout mice exhibited cardioprotection by IP, except for the adenosine A _2B receptor knockout mouse.

In summary, genetic evidence for a central role of the adenosine A _2B receptor in cardioprotection by IP is provided for the first time.

2.5 The IP is lifted in App ⁷ mice

(a) myocardium

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). To demonstrate functional consequences of the mutation in the adenosine A2B receptor, hypoxia-induced vascular leakage was measured. In fact, previous studies have shown an increase in Evan's Blue after hypoxia exposure with pharmacological inhibition of the adenosine A _2B receptor (MRS1754); see. Thompson LF et al. (Supra). In Ü accord with this work showed the A2 _B ^'A mice an increase in Evan's blue leakage in the cardiac tissue after hypoxia (4 h, 8% oxygen, Fig. 8d), suggesting a functional contribution of the adenosine A ₂ B signaling in vascular barrier function during hypoxia.

After functional consequences of the gene deletion of the adenosine A2B receptor in the heart were demonstrated, cardioprotection was examined by IP. In fact, the cardioprotective effects of IP as assessed by cTnI or by measuring infarct size were abolished in AΣB ^" '^" mice (Figure 8e-g).

To confirm these findings, a pharmacological approach was chosen. The highly specific, water-soluble adenosine A2B receptor antagonist PSB1115 was used (5 mg / kg / h). As shown in Figure 8, intra-arterial treatment with PSB1115 abolished the attenuation of infarct size by IP, confirming our findings in the AΣβ 'mice.

To demonstrate the specificity of PSB1115 for the adenosine A2B receptor, adenosine Ai receptor knockout mice were also treated with PSB1115. These studies revealed cardioprotection by IP in Ai ^Λ mice, which is attenuated by the specific blockade of the adenosine A _2B receptor (Figure 8g). In summary, these studies provide genetic and pharmacological evidence for a role of the adenosine A ₂ B receptor in cardioprotection by IP.

(b) kidney

As shown in Fig. 9a, 30 min ischemia resulted in wild-type or CD73 in mice ^A severe acute tubular necrosis, as is shown in the cortex and the outer stripe medullary with almost complete destruction of the proximal tubular brush border revealed by the loss of Tubuluszellkerne , In the outer medulla, a large number of rejected brush-border-bearing protuberances were observed. Both in the cortex and in the outer medulla, the formation of hyaline repellents and intraluminal necrotic cell debris was observed. In contrast, wild-type mice treated with IP before ischemia showed only mild histological signs of acute tubular necrosis as evidenced by intact tubular cell morphology. The proximal damage to the brush border was sporadic and relatively small. Furthermore, only a small number of hyaline repellents were found in the IP-treated group. Renal tissue protection by IP remained in cd73 ^{/ ~} mice. In fact, semiquantitative histological analysis in the wild-type group with IP showed a reduction of the Jablonski index of 3 (range 3 to 4) and without IP to 2 (range 1 to 3), Fig. 9b, p <0.01), while no reduction in histological grading was observed in cd73 ^A mice.

Likewise, signs of acute renal inflammation after IP were attenuated, as demonstrated by cytochemical staining of the granulocytes with chloroacetate esterase (CAE) (Figure 9c). The renal tissue showed a significant increase in granulocyte infiltration after 30 min of ischemia in wild-type and cd73 " ^A mice without IP, preferably located in peritubular areas." This granulocyte infiltration remained only in the wild-type group with IP treatment before ischemia Thus, the quantitative analysis showed a reduction in granulocyte infiltration without IP from 3.5 (range 3 to 4) to 2 (range 1 to 3, Fig. 9d, p = 0.001).

In summary, these data demonstrate histologically the absence of renal protection by IP in cd73 ^/ mice.

2.6 Adenosine A ^ B receptor agonist treatment results in lower infarct sizes during acute myocardial ischemia and less renal damage during acute renal ischemia

Having shown that 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 A2β 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.

To demonstrate the adenosine A _2B receptor-specific effects of BR4887 / BAY 60-6583, the mice were first subjected to normoxia or hypoxia (8% oxygen, 4 h) after treatment with BR4887 / BAY 60-6583 (10 μg / kg ip, 30 min before hypoxia / normoxia) or vehicle control. (a) myocardium

Consistent with studies showing increasing vascular leakage with adenosine A2B antagonist treatment, the administration of BR4887 / BAY 60-6583 resulted in a significant attenuation of hypoxia-induced vascular leakage, as measured by Evan's Blue tissue retention (Figure IIa).

In the next step, BL6 mice were treated with a single bolus of intra-arterial BR4887 / BAY 60-6583 (10 μg / kg / body weight for 30 min prior to ischaemia) and exposed to myocardial ischemia for 60 min. In fact, BR4887 / BAY 60-6583 treatment resulted in a significant reduction in infarct size (Figure IIb, c).

To confirm the specificity of BR4887 / BAY 60-6583, this experiment was repeated in adenosine-Aββ mice. As shown in Fig. 10d, no significant attenuation of the infarct size in mice A ₂ B ^Λ could be detected, confirming the specificity of the cardioprotective effects of BR4887 / BAY 60-6583 by adenosine A _2B receptor signaling.

In summary, these studies provide strong evidence for the possibility of therapeutic treatment of the adenosine A2B receptor during myocardial ischemia.

(b) kidney

Corresponding experiments were performed on the kidney. Here, as expected, it first shows that the kidney-protective effect of IP in wild-type mice (WT) is prevented by the specific adenosine A2B receptor antagonist PSBI15 (FIG. 12a). By contrast, administration of BR4887 / BAY 60-6583 after 45 minutes of kidney ischemia significantly improves the glomerular filtration rate (Figure 12b). However, this effect is abolished in mice that are deficient in the adenosine A ₂ B receptor (A2B - / -); see. Fig. 12c.

Furthermore, significantly less renal tissue damage was seen in WT mice administered BR4887 / BAY 60-6583; see. Fig. 12d and e.

In summary, these studies provide evidence of the potential for therapeutic treatment of the adenosine A2B receptor during renal ischemia.

2.7 Reconstitution of cd73 ⁷ mice with soluble 5'-nucleotidase

As a "proof of principle" and to demonstrate that the decrease in renal functional parameters in cd 73 'MAUSCn reflects the lack of extracellular 5'-nucleotidase activity, 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.

As shown in Figures 13a and 13b, the increases in serum creatinine and serum potassium are reduced after i.p. nucleotidase treatment.

Similarly, creatinine clearance (Fig. 11c), urine flow rate (Fig. 13d), excretion of sodium (Fig. 13e) and potassium (Fig. 13f) via the urine in cd73 ^/ mice with and without IP treatment decreased to.

In summary, these studies confirm the genetic studies that CD 73 seems to play a crucial role in the increase in renal resistance to ischemia after IP treatment. 2.8 Treatment of renal ischemia with soluble 5'-nucleotidase in wild-type mice

After it was shown that renal protection by IP can be successfully restored by the treatment of cdZS 'mice with soluble 5'-nucleotidase, nucleotidase treatment of renal ischemia in wild-type mice was next investigated.

For these purposes, 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.

In summary, these data demonstrate for the first time a therapeutic effect of treatment with soluble 5'-nucleotidase in renal ischemia.

2.9 Histological signs of renal protection by 5'-nucleotidase in cd73 ^{- /} mice and littermates

To confirm the findings of renal protection by 5'-nucleotidase treatment on a histological level, the renal histology and inflammation after treatment of renal ischemia with soluble 5'-nucleotidase was also examined. For these purposes, 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.

In accordance with the studies of renal function, renal histology confirmed renal protection against ischemia by ip 5'- Nucleotidase treatment as previously observed with IP treatment (Figure 15).

In summary, 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.

Claims

claims 1. Use of nucleotide phosphohydrolase for the manufacture of a medicament for the prophylaxis and / or treatment and / or diagnosis of ischemic diseases. 2. Use according to claim 1, characterized in that as nucleotide phosphohydrolase 5'-nucleotidase (CD73) is used. 3. Use according to claim 1 or 2, characterized in that as nucleotide phosphohydrolase apyrase (CD39) is used. 4. Use according to any one of claims 1 to 3, characterized in that the nucleotide phosphohydrolase is in soluble form. 5. Use according to one of claims 1 to 4, characterized in that the nucleotide phosphohydrolase is in micronized form. Use according to any one of claims 1 to 5, characterized in that the ischemic disease is selected from the group consisting of: myocardial ischemia, renal ischemia, intestinal ischemia and liver ischemia. 7. Use according to one of claims 1 to 6, characterized in that the nucleotide phosphohydrolase is present in the form of a coding sequence in an expression vector. 8. Use according to one of claims 1 to 7, characterized in that the drug has an activity enhancer. 9. Use according to claim 8, characterized in that an adenosine λ2B receptor agonist is used as the action enhancer. 10. Use according to claim 9, characterized in that as adenosine A ₂ B receptor agonist BR4887 is used. 11. Use according to one of claims 8 to 10, characterized in that an inhibitor of the nucleoside transporter is used as an effect enhancer. 12. Use according to claim 11, characterized in that the nucleoside transporter is ENT-I and / or ENT-2. 13. Use according to claim 12, characterized in that as inhibitor of ENT-I and / or ENT-2 dipyridamole is used. 14. 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) examination of the biological sample for the presence of a nucleotide phosphohydrolase, (3) if necessary Determination of the functionality and / or activity of the nucleotide phosphohydrolase, (4) Correlation of the absence of nucleotide phosphohydrolase in the biological sample or a reduced in the biological sample against the wild type operability and / or activity of the nucleotide phosphohydrolase in the biological sample with a positive diagnosis. 15. The method according to claim 14, characterized in that in step (2) a mutation screening is performed. 16. The method according to claim 14 or 15, characterized in that in step (3) the adenosine formation rate of the present in the biological sample nucleotide phosphohydrolase is determined in comparison to wild-type nucleotide phospho-phohydrolase. 17. A pharmaceutical and / or diagnostic composition comprising a nucleotide phosphohydrolase in a therapeutically effective amount and a pharmaceutically acceptable carrier and optionally an enhancer. 18. A method of diagnosing a subject in an animal for an ischemic disease or disease of an ischemic disease, comprising the steps of: (1) administering the diagnostic composition of claim 17 to a subject showing symptoms of ischemic disease; (2) determining if the symptoms are improving, and (3) correlation of improvement of symptoms with a positive diagnosis. 19. Use of nucleotide phosphohydrolase for the preparation of a medicament for ischemic preconditioning (IP). 20. Use according to claim 19, characterized in that as nucleotide phosphohydrolase apyrase (CD39) is used. 21. Use according to claim 19, characterized in that 5'-nucleotidase (CD73) is used as nucleotide phosphohydrolase. 22. Use according to any one of claims 19 to 21, characterized in that the nucleotide phosphohydrolase is in soluble form. 23. Use of an adenosine A2B receptor agonist, preferably BR4887 (BAY 60-6583), for the manufacture of a medicament for the prophylaxis and / or treatment and / or diagnosis of ischemic diseases. 24. Use according to claim 23, characterized in that the ischemic disease is myocardial ischemia. Use according to claim 23, characterized in that the ischemic disease is renal ischemia.