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WO2008104199A1 - Use of ecto-phosphatases for the treatment of (acute) myocardial infarction - Google Patents

Use of ecto-phosphatases for the treatment of (acute) myocardial infarction Download PDF

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Publication number
WO2008104199A1
WO2008104199A1 PCT/EP2007/001979 EP2007001979W WO2008104199A1 WO 2008104199 A1 WO2008104199 A1 WO 2008104199A1 EP 2007001979 W EP2007001979 W EP 2007001979W WO 2008104199 A1 WO2008104199 A1 WO 2008104199A1
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Prior art keywords
ecto
phosphatase
alkaline phosphatase
acute
myocardial infarction
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French (fr)
Inventor
Rudi Brands
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Gelato Corp NV
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Gelato Corp NV
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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)
    • 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
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial 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
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03001—Alkaline phosphatase (3.1.3.1)
    • 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

Definitions

  • the present invention relates to the use of ecto- phosphatases for the treatment or prophylaxis of (acute) myocardial infarction.
  • the present invention also relates to a method for treating a mammal, such as a human, suffering from (acute) myocardial infarction and to a pharmaceutical composition for treating (acute) myocardial infarction.
  • (Acute) myocardial infarction commonly known as a heart attack, is a disease state that occurs when the blood supply to (a part of) the heart is interrupted.
  • the resulting oxygen shortage i.e., ischemia, causes damage and potential death of heart tissue. It is a medical emergency, and the leading cause of death for both men and women all over the world.
  • Important risk factors are a previous history of vascular disease such as atherosclerotic coronary heart disease and/or angina, a previous heart attack or stroke, any previous episodes of abnormal heart rhythms or syncope, older age- especially men over 40 and women over 50, smoking, excessive alcohol consumption, the abuse of certain illicit drugs, high triglyceride levels, elevated creatine kinase or troponin levels in the blood, high LDL and low HDL, diabetes, high blood pressure, obesity, and chronically high levels of stress in certain persons.
  • myocardial infarction is derived from myocardium (the heart muscle) and infarction (tissue death due to oxygen starvation) .
  • Classical symptoms of (acute) myocardial infarction include chest pain, shortness of breath, nausea, vomiting, palpitations, sweating, and/or anxiety. Patients frequently feel suddenly ill. Women often experience different symptoms than men. The most common symptoms of myocardial infarction in women include shortness of breath, weakness, and fatigue. Approximately one third of all myocardial infarctions are silent, without chest pain or other symptoms . Immediate treatment for suspected (acute) myocardial infarction includes oxygen, aspirin, glyceryl trinitrate and pain relief.
  • Further treatment may include either medications to break down blood clots that block the blood flow to the heart, or mechanically restoring the flow by dilatation or bypass surgery of the blocked coronary artery.
  • Common treatments for a cardiogenic shock include infusing fluids comprising inotropica and administering anti-arrhythmic agents such as adenosine, verapamil, amiodarone, and beta- blockers.
  • anti-arrhythmic agents such as adenosine, verapamil, amiodarone, and beta- blockers.
  • an intra-aortic balloon pump (which reduces workload for the heart, and improves perfusion of the coronary arteries) can be considered or a left ventricular assist device (which augments the pump-function of the heart) .
  • the prophylaxis according to the present invention can be envisaged as a precautionary treatment of patients at high risk of developing (acute) myocardial infarction.
  • prophylaxis also encompasses administering the ecto-phosphatases according to the present invention to patients undergoing heart surgery, before, during or after the surgery, preferably before.
  • Phosphatases are a group of enzymes capable of dephosphorylating a substrate, i.e., the enzyme hydrolyzes phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group.
  • ecto-phosphatases denotes a subclass a phosphatases which function extracelluarly, i.e., are capable of dephosphorylating an extra-cellular substrate in the extra-cellular space. This in contrast with intracellular phosphatases dephosphorylating an intracellular substrate inside the cell, i.e., the intracellular space. These intra-cellular phosphatases are often involved in signal transduction. Such ecto-phosphatases can be in the form of integral membrane or GPI-anchored proteins displaying their catalytic domain, i.e., the domain involved in the actual dephosphorylating of a substrate, to the extra-cellular space .
  • ecto-phosphatases can be present in the extra-cellular space as secreted or soluble proteins .
  • the ecto-phosphatase according to the present invention is apyrase (ecto- nucleoside triphosphate diphosphohydrolase 1).
  • Ecto-nucleoside triphosphate diphosphohydrolase 1 or apyrase which terms are interchangeably used herein, is a nucleotide metabolizing enzyme belonging to a family of acid anhydride hydrolases. Examples of other enzymes belonging to this family are GTP phosphohydrolase, pyrophosphatase and thiamin-triphosphatase .
  • the ecto-enzyme was first identified in 1949 and in 1963 partially purified from potato.
  • the enzyme is also known under its registry number EC 3.6.1.5. or as CD39.
  • Apyrases are naturally occurring transmembrane glycoproteins that can activate intracellular pathways upon activation. Apyrases are found in a large number of microbial species such as E.coli, Aspergillus fumigatus and Kluyveromyces lactis, plants such as Arabidopsis thaliana, Glycine max and Oryza sativa, insects such as Drosophila melanogaster and mammals like Rattus norvegicus, Mus musculus and Homo sapiens .
  • An apyrase enzyme comprises three domains, an extracelluar, a transmembrane and an intracellular domain.
  • the extracellular domain comprises a conserved catalytic region responsible for the catalytic activity of the extracellular enzyme.
  • the catalytical domain catalyzes the hydrolysis of ATP to yield AMP and orthophosphate .
  • Such activity can thus be characterized as an ATP-diphosphatase or ATP diphosphohydrolase . It can also act on ADP, again yielding AMP and orthophosphate.
  • This activity can be characterized as an ADPase or ADP phosphohydrolase .
  • the enzyme can also be regarded as an ATP-ADPase.
  • Reported physiological functions of apyrases address their possible involvement in maintenance of hemostasis and inhibition of platelet aggregation through hydrolysis of extracellular ADP, which is released from activated thrombocytes upon vascular injury.
  • This ADP is known to function in recruitment and induction of platelet aggregation if not cleared by endothelial cell plasma membrane apyrase.
  • the ecto-phosphatase according to the present invention is an alkaline phosphatase such as preferably bovine intestinal alkaline phosphatase (BIAP,) or human placental phosphatase.
  • alkaline phosphatase such as preferably bovine intestinal alkaline phosphatase (BIAP,) or human placental phosphatase.
  • alkaline phosphatases are intestinal alkaline phosphatase, tissue unspecific (liver, bone and kidney) alkaline phosphatase, placental-like alkaline phosphatase and germ-cell like alkaline phosphatase.
  • Alkaline phosphatases (EC 3.1.3.1) exhibit their optimal catalytic properties, i.e. dephoshorylation, at alkaline pH' s such as between pH 8 to 11. These phosphatases are found in a large variety of organisms varying van bacteria, plants, and animals including humans. They are believed to be involved in maintaining homeostasis, barrier integrity, signal-transducing and general defense against pathogens .
  • Bovine intestinal alkaline phosphatase catalyzes the hydrolysis of many phosphomonoesters, including 5 ' -nucleotides, RNA and DNA. It is widely used for the dephoshorylation of 5 ' -phosphorylated ends of DNA or RNA for subsequent labeling with 32 P. Dephosphorylation also prevents religation of linearized plasmid DNA in cloning experiments.
  • Human placental phosphatase is assumed to be essential for normal fetal development, especially in the last trimester when placental alkaline phosphatase protein levels are high.
  • An decreased cardiac function possibly reduces bowel perfusion, leading to bowel hypoperfusion and ischemia of the intestinal mucosa. This could potentially result in an increased permeability of the of gut permeability, and the subsequent translocation of endotoxin into the circulation.
  • Lipopolysaccharide an endotoxin present in the outer cell membrane of Gram-negative bacteria, is a known potent stimulator of the innate immune response. Only minute amounts of this compound could cause a lethal septic shock.
  • LPS binds to the lipopolysaccharide-binding protein (LBP) which interacts with CD14, MD2, and Toll-like receptor 4 (TLR4) to start a signaling cascade leading to an inflammatory response possibly a cause of a cardiogenic shock.
  • LBP lipopolysaccharide-binding protein
  • TLR4 Toll-like receptor 4
  • the ecto- phosphatase according to the present invention is a recombinant protein.
  • ecto-phosphatases are readily available in the public databases such as for apyrase from various organisms including human (see fig. 1, accession number CAB40415) , placental alkaline phosphatase from various mammals including human (fig. 3, accession number AAA51709) , intestinal alkaline phosphatase (see fig. 2, accession number NP 776412, bovine) from various organisms including human (accession number NP001622), etc.
  • the recombinant ecto-phosphatase proteins according to the present invention can readily be obtained using well known cloning methods based on these already known sequences.
  • a coding sequence of an ecto-phosphatase apyrase can be identified in and isolated from a selected source, preferably human, using probes derived from known coding, genomic or amino acid sequences to screen cDNA or genomic libraries.
  • nucleic acid amplification techniques can be used for the identification, isolation and cloning of the coding sequence of an ecto-phosphatase.
  • the coding sequence can be placed under the control of appropriate regulation signals and transformed in a suitable host allowing the expression and isolation of the enzyme.
  • Appropriate regulation signals comprise a promoter, one or more enhancer sequences, a transcription and/or translation initiation site and a transcription and/or translation termination site.
  • the present ecto-phosphatase is a soluble or exogenous, most preferably recombinant, ecto-phosphatase.
  • ecto-phosphatase can be any recombinant protein under the condition that it comprises a functionally intact catalytic domain such as GDA1/CD39 catalytic domain (PSSM-Id 41214) or the alkPPC catalytic domain (PSSM-Id 58306) .
  • a soluble recombinant protein comprising the catalytic domain of an ecto-phosphatase, such as a fusion protein, in frame deletion of only the transmembrane domain, amino acid modifications such as substituting hydrophobic amino acids for hydrophilic ones, etc .
  • the present invention also relates " to a method for treating a mammal suffering from (acute) myocardial infarction comprising administering a therapeutically effective amount of an ecto-phosphatase.
  • the method comprises administering a therapeutically effective amount of an apyrase or an alkaline phosphatase, preferably bovine intestinal alkaline phosphatase or human placental alkaline phosphatase .
  • the ecto-phosphatase preferably soluble, is a recombinant protein.
  • the present invention also relates to a pharmaceutical composition for treatment of prophylaxis of (acute) myocardial infarction comprising a therapeutically effective amount of an ecto-phosphatase according to the present invention and one or more pharmaceutically acceptable excipients .
  • Preferred therapeutically effective amounts of an ecto-phosphatase according to the present invention are between 10 to 400 IU/kg body weight.
  • One IU unit is defined as the amount of apyrase capable of liberating 1 micromole inorganic phosphate from ATP or ADP per minute at pH 6.5 at 30 0 C.
  • Fig. 1 shows the amino acid sequence of the GDA1/CD39 catalytic domain (aa 86 to 540) of an ecto- phosphatase according to the present invention i.e, human apyrase (accession number CAB40415);
  • Fig. 2 shows the amino acid sequence of the alkPPc catalytic domain (aa 52-487) of an ecto- phosphatase according to the present invention, i.e., bovine intestinal alkaline phosphatase (accession number NP 776412);
  • Fig. 1 shows the amino acid sequence of the GDA1/CD39 catalytic domain (aa 86 to 540) of an ecto- phosphatase according to the present invention i.e, human apyrase (accession number CAB40415);
  • Fig. 2 shows the amino acid sequence of the alkPPc catalytic domain (aa 52-487) of an ecto- phosphatase according
  • FIG. 3 shows the amino acid sequence of the alkPPc catalytic domain (aa 50-490) of an ecto- phosphatase according to the present invention, i.e., human placental alkaline phosphatase (accession number AAA51709) ;
  • Fig. 4 shows production of the pro-inflammatory cytokine
  • FIG. 8 shows attenuation of proinflammatory MCP-I induction in a MI mouse model using the ecto- phosphatases recombinant BIAP and apyrase;
  • FIG. 9 shows the attenuation of an LPS insult in mice by recombinant intestinal alkaline phosphatase and apyrase;
  • Fig. 10 shows that recombinant soluble human apyrase (NTPase-1, CD39-1, expressed in tabacco) and recombinant human placental alkaline phosphatase
  • BIAP Bovine Intestinal Alkaline Phosphatase
  • Clinical grade bovine intestinal alkaline phosphatase was obtained from Biozyme (Blaenavon, UK) .
  • One unit is defined as that amount of BIAP able to hydrolyse 1 ⁇ mole of p-nitrophenyl phosphate per minute using a Tris-glycine buffer at pH 9.6 at 25 "C.
  • mice were anaesthetized by inhalation of a mixture of O 2 air and 4% isoflurane, endotracheally intubated, and mechanically ventilated.
  • Myocardial infarction was induced as described previously (Salto-Tellez, M., et al., Myocardial infarction in the C57BL/6J mouse: A quantifiable and highly reproducible experimental model. Cardiovascular Pathology, 2004. 13(2) : p. 91-97) .
  • LAD coronary artery was exposed via a left thoractomy and double ligated with an 8.0 prolene suture. Animals were sacrificed 4 hours after myocardial infarction after which blood was collected. Heart, lung, liver and kidneys were removed and fixed in 4% paraformaldehyde in PBS.
  • ELISA Enzyme-linked Immunosorbent Assay
  • Mouse mast cell protease-1 (mMCP-1) ELISA was from Moredun (Midlothian, Scotland, UK) and performed according to the manufacturer's instructions.
  • IL-6 One of the major pro-inflammatory cytokines produced in AMI patients is IL-6.
  • IL-6 One of the major pro-inflammatory cytokines produced in AMI patients.
  • mice Underwent permanent ligation of the LAD coronary artery to induce AMI and were sacrificed at three different time points.
  • IL-6 concentration was below detection limit ( ⁇ 4 pg/ml) (Fig. 4) .
  • AP activity was determined (Fig. 5) in serum samples by measuring hydrolysis of p-nitrophenyl phosphate by AP. All animals that received BIAP had slightly elevated serum levels of AP activity 4 hours after AMI compared to control animals
  • TNF-cx and IL-10 are observed in patients with AMI. Therefore, presence of these cytokines was determined.
  • BIAP pro-inflammatory cytokine IL-6 and IL-l ⁇
  • IL-6 levels were reduced by approximately 40% and IL- l ⁇ by approximately 30%.
  • No difference in the antiinflammatory cytokine IL-10 (Fig. 6b) production could be observed between the control group and the BIAP treated group.
  • mast cells are activated to release chymases. Activation of mast cells in mice can be measured by the release of the mouse mast cell protease-1 (mMCP-1) chymase.
  • mMCP-1 mouse mast cell protease-1
  • Serum levels of mMCP-1 were 14.7 ng/ml 4 hours after LAD coronary artery ligation.
  • BIAP treatment reduced mMCP-1 levels in serum to 8.4 ng/ml (approximately 40%), implying a significant reduction in mast cell activation (P ⁇ 0.05).
  • TNF- ⁇ concentration in serum generally believed to be an early-onset proinflammatory cytokine, was below detection limit, suggesting that the chosen time point is not relevant to detect this cytokine in Balb/c mice after LAD coronary artery ligation.
  • a reduction in pro-inflammatory cytokine production indicates a diminished systemic innate immune response, which decreases myocardial dysfunction and reduces the development of CS after AMI.
  • Chymases are abundantly produced after AMI in mammals, and are known to be involved in the cleaving of angiotensin I (Ang I) to form angiotensin II (Ang II).
  • Ang II The excessive formation of Ang II, which is observed in the acute phase after AMI, is arrhythmogenic, and several studies in different animal models have shown that decreasing Ang II formation by a specific chymase inhibitor contributes to a reduction in the mortality rate in the acute phase after AMI.
  • BIAP The influence of BIAP on the formation of the mouse chymase mMCP-1 was determined. In BIAP-treated mice, mMCP-1 production was significantly reduced by approximately 40% when compared to vehicle-treated mice. This result indicates a reduction of Ang II formation and consequently a decrease in arrhythmias, which improves cardiac function and reduce CS complication .
  • Example 2 Attenuation of proinflammatory MCP-I induction in a MI mouse model using the ecto-phosphatases recombinant BIAP and apyrase
  • Clinical grade bovine intestinal alkaline phosphatase was obtained from Biozyme (Blaenavon, UK) .
  • One unit is defined as that amount of BIAP able to hydrolyse 1 ⁇ mole of p-nitrophenyl phosphate per minute using a Tris-glycine buffer at pH 9.6 at 25 °C.
  • Clinical grade apyrase was obtained from Sigma (Sigma Aldrich, St. Louis, MO) . One unit is defined as that amount of apyrase able to liberate 1 micromole inorganic phosphate from ATP or ADP per minute at pH 6.5 at 30 0 C.
  • mice were injected with an equal volume of PBS. The study was approved by the animal ethics committee of the Faculty of Veterinary Medicine, University Utrecht.
  • mice were anaesthetized by inhalation of a mixture of O 2 air and 4% isoflurane, endotracheally intubated, and mechanically ventilated.
  • Myocardial infarction was induced as described previously (Salto-Tellez, M., et al., Myocardial infarction in the C57BL/6J mouse: A quantifiable and highly reproducible experimental model. Cardiovascular Pathology, 2004. 13(2) : p. 91-97) .
  • the LAD coronary artery was exposed via a left thoractomy and double ligated with an 8.0 prolene suture. Animals were sacrificed 4 hours after myocardial infarction after which blood was collected. Heart, lung, liver and kidneys were removed and fixed in 4% para- formaldehyde in PBS.
  • Mouse mast cell protease-1 ( ⁇ MCP-1) ELISA was from Moredun (Midlothian, Scotland, UK) and performed according to the manufacturer's instructions.
  • mast cells are activated to release chymases. Activation of mast cells in mice can be measured by the release of the mouse mast cell protease-1 (mMCP-1) chymase.
  • mMCP-1 mouse mast cell protease-1
  • Serum levels (fig. 8) of mMCP-1 were 14.7 ng/ml 4 hours after LAD coronary artery ligation.
  • BIAP treatment reduced mMCP-1 levels in serum to 8.4 ng/ml (approximately 40%), implying a significant reduction in mast cell activation (P ⁇ 0.05).
  • Example 3 Apyrase and recombinant intestinal alkaline phosphatase are capable of in vivo attenuation of LPS mediated inflammation
  • mice C57B16 were i.p. administered both CD39 (apyrase) (4.5 units) and alkaline phosphatase (rec IAP 0502, 1.5 units) and LPS (5 micrograms) .
  • mice show normalized body temperatures .
  • Example 4 Recombinant human apyrase (NTPase-1 , CD39-1) and recombinant human placental alkaline phosphatase have anti-inflammatory activity in a LPS challenge model comparable with bovine intestinal alkaline phosphatase
  • mice C57B16 were i.p. administered recombinant human apyrase (NTPase-1, CD39-1) , recombinant human placental alkaline phosphatase or bovine intestinal alkaline phosphatase and were subsequently challenged with LPS. Mice only injected with the carrier served as control group.
  • NTPase-1 recombinant human apyrase
  • recombinant human placental alkaline phosphatase or bovine intestinal alkaline phosphatase resulted in a comparable attenuation of inflammation as demonstrated by a stabilization of body temperature .
  • mice LPS administered alone, resulted in lower body temperature which recovered after 5 hours. (In mice reduced body temp is generally observed after LPS administration) .
  • recombinant human apyrase NTPase-1, CD39-1
  • recombinant human placental alkaline phosphatase or bovine intestinal alkaline phosphatase injected mice show comparable normalized body temperatures indicating an attenuation of the systemic inflammatory response due to endotoxins.

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Abstract

The present invention relates to the use of ectophosphatases such as (soluble) apyrase, human placental alkaline phosphatase or bovine intestinal alkaline phosphatase, for the treatment or prophylaxis of (acute) myocardial infarction. The present invention also relates to a method for treating a mammal, such as a human, suffering from (acute) myocardial infarction and to a pharmaceutical composition for treating (acute) myocardial infarction.

Description

USE OF ECTO- PHOS PHATASES FOR THE TREATMENT OF (ACUTE) MYOCARDIAL INFARCTION
description
The present invention relates to the use of ecto- phosphatases for the treatment or prophylaxis of (acute) myocardial infarction. The present invention also relates to a method for treating a mammal, such as a human, suffering from (acute) myocardial infarction and to a pharmaceutical composition for treating (acute) myocardial infarction.
(Acute) myocardial infarction, commonly known as a heart attack, is a disease state that occurs when the blood supply to (a part of) the heart is interrupted. The resulting oxygen shortage, i.e., ischemia, causes damage and potential death of heart tissue. It is a medical emergency, and the leading cause of death for both men and women all over the world.
Important risk factors are a previous history of vascular disease such as atherosclerotic coronary heart disease and/or angina, a previous heart attack or stroke, any previous episodes of abnormal heart rhythms or syncope, older age- especially men over 40 and women over 50, smoking, excessive alcohol consumption, the abuse of certain illicit drugs, high triglyceride levels, elevated creatine kinase or troponin levels in the blood, high LDL and low HDL, diabetes, high blood pressure, obesity, and chronically high levels of stress in certain persons.
The term myocardial infarction is derived from myocardium (the heart muscle) and infarction (tissue death due to oxygen starvation) .
Classical symptoms of (acute) myocardial infarction include chest pain, shortness of breath, nausea, vomiting, palpitations, sweating, and/or anxiety. Patients frequently feel suddenly ill. Women often experience different symptoms than men. The most common symptoms of myocardial infarction in women include shortness of breath, weakness, and fatigue. Approximately one third of all myocardial infarctions are silent, without chest pain or other symptoms . Immediate treatment for suspected (acute) myocardial infarction includes oxygen, aspirin, glyceryl trinitrate and pain relief.
Further treatment may include either medications to break down blood clots that block the blood flow to the heart, or mechanically restoring the flow by dilatation or bypass surgery of the blocked coronary artery.
The above treatments of (acute) myocardial failure all relate to restoring or influencing the blood flow to the heart thereby restoring the oxygen supply of the heart. However, there is growing evidence that (acute) myocardial infarction is also associated with an increased inflammatory (systemic) reaction. This inflammatory reaction is an important cause of a, in most cases, a fatal cardiogenic shock (CS), i.e., a primary failure of the ventricles of the heart to function effectively.
Thus, even in case the common medical treatment for (acute) myocardial infarction is able to restore or to alleviate the ischemic conditions in of the heart muscle, the also induced inflammatory (systemic) reaction of the body can be still fatal as the result of a cardiogenic shock (CS) .
Common treatments for a cardiogenic shock, depending on the type of myocardial infarction, include infusing fluids comprising inotropica and administering anti-arrhythmic agents such as adenosine, verapamil, amiodarone, and beta- blockers.
As a last option, an intra-aortic balloon pump (which reduces workload for the heart, and improves perfusion of the coronary arteries) can be considered or a left ventricular assist device (which augments the pump-function of the heart) .
However, the above treatments are directed to combat the effects of a cardiogenic shock resulting from (acute) myocardial infarction. It would be far preferred to be able to control the underlying inflammatory (systemic) reaction during (acute) myocardial infarction thereby alleviating or preventing the development of a cardiogenic shock. It is therefore an objective of the present invention to provide such treatment of (acute) myocardial infarction resulting, optionally in combination with other (common) treatments, in a higher survival rate due to alleviating or even avoiding a cardiogenic shock. This objective, amongst others, is met by the present invention by using ecto-phosphatases for the preparation of a medicament for the treatment or prophylaxis of (acute) myocardial infarction.
The prophylaxis according to the present invention can be envisaged as a precautionary treatment of patients at high risk of developing (acute) myocardial infarction.
In addition, prophylaxis also encompasses administering the ecto-phosphatases according to the present invention to patients undergoing heart surgery, before, during or after the surgery, preferably before.
For heart surgery, the heart pump function is generally reduced or stopped thereby causing comparable ischemic conditions as during (acute) myocardial infarction. Phosphatases are a group of enzymes capable of dephosphorylating a substrate, i.e., the enzyme hydrolyzes phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group.
The term "ecto-phosphatases", as used herein, denotes a subclass a phosphatases which function extracelluarly, i.e., are capable of dephosphorylating an extra-cellular substrate in the extra-cellular space. This in contrast with intracellular phosphatases dephosphorylating an intracellular substrate inside the cell, i.e., the intracellular space. These intra-cellular phosphatases are often involved in signal transduction. Such ecto-phosphatases can be in the form of integral membrane or GPI-anchored proteins displaying their catalytic domain, i.e., the domain involved in the actual dephosphorylating of a substrate, to the extra-cellular space .
As an alternative, such ecto-phosphatases can be present in the extra-cellular space as secreted or soluble proteins .
In a preferred embodiment, the ecto-phosphatase according to the present invention is apyrase (ecto- nucleoside triphosphate diphosphohydrolase 1).
Ecto-nucleoside triphosphate diphosphohydrolase 1 or apyrase, which terms are interchangeably used herein, is a nucleotide metabolizing enzyme belonging to a family of acid anhydride hydrolases. Examples of other enzymes belonging to this family are GTP phosphohydrolase, pyrophosphatase and thiamin-triphosphatase .
The ecto-enzyme was first identified in 1949 and in 1963 partially purified from potato. The enzyme is also known under its registry number EC 3.6.1.5. or as CD39.
Apyrases are naturally occurring transmembrane glycoproteins that can activate intracellular pathways upon activation. Apyrases are found in a large number of microbial species such as E.coli, Aspergillus fumigatus and Kluyveromyces lactis, plants such as Arabidopsis thaliana, Glycine max and Oryza sativa, insects such as Drosophila melanogaster and mammals like Rattus norvegicus, Mus musculus and Homo sapiens .
An apyrase enzyme comprises three domains, an extracelluar, a transmembrane and an intracellular domain. The extracellular domain comprises a conserved catalytic region responsible for the catalytic activity of the extracellular enzyme. The catalytical domain catalyzes the hydrolysis of ATP to yield AMP and orthophosphate . Such activity can thus be characterized as an ATP-diphosphatase or ATP diphosphohydrolase . It can also act on ADP, again yielding AMP and orthophosphate. This activity can be characterized as an ADPase or ADP phosphohydrolase . Based on the combined enzymatic activities of the catalytic domain, the enzyme can also be regarded as an ATP-ADPase.
Reported physiological functions of apyrases address their possible involvement in maintenance of hemostasis and inhibition of platelet aggregation through hydrolysis of extracellular ADP, which is released from activated thrombocytes upon vascular injury.
This ADP is known to function in recruitment and induction of platelet aggregation if not cleared by endothelial cell plasma membrane apyrase.
In another preferred embodiment, the ecto-phosphatase according to the present invention is an alkaline phosphatase such as preferably bovine intestinal alkaline phosphatase (BIAP,) or human placental phosphatase.
Other examples of alkaline phosphatases are intestinal alkaline phosphatase, tissue unspecific (liver, bone and kidney) alkaline phosphatase, placental-like alkaline phosphatase and germ-cell like alkaline phosphatase. Alkaline phosphatases (EC 3.1.3.1) exhibit their optimal catalytic properties, i.e. dephoshorylation, at alkaline pH' s such as between pH 8 to 11. These phosphatases are found in a large variety of organisms varying van bacteria, plants, and animals including humans. They are believed to be involved in maintaining homeostasis, barrier integrity, signal-transducing and general defense against pathogens . Bovine intestinal alkaline phosphatase (BIAP) catalyzes the hydrolysis of many phosphomonoesters, including 5 ' -nucleotides, RNA and DNA. It is widely used for the dephoshorylation of 5 ' -phosphorylated ends of DNA or RNA for subsequent labeling with 32P. Dephosphorylation also prevents religation of linearized plasmid DNA in cloning experiments.
Human placental phosphatase is assumed to be essential for normal fetal development, especially in the last trimester when placental alkaline phosphatase protein levels are high.
Without wishing to be bound to any theory, one possible explanation of the observed beneficial effects of the ecto-phosphatases according to the present invention could be the clearance of inactivation of endotoxins entering the blood circulation during (acute) myocardial infarction.
An decreased cardiac function possibly reduces bowel perfusion, leading to bowel hypoperfusion and ischemia of the intestinal mucosa. This could potentially result in an increased permeability of the of gut permeability, and the subsequent translocation of endotoxin into the circulation.
Lipopolysaccharide (LPS) , an endotoxin present in the outer cell membrane of Gram-negative bacteria, is a known potent stimulator of the innate immune response. Only minute amounts of this compound could cause a lethal septic shock. When entering the circulation, LPS binds to the lipopolysaccharide-binding protein (LBP) which interacts with CD14, MD2, and Toll-like receptor 4 (TLR4) to start a signaling cascade leading to an inflammatory response possibly a cause of a cardiogenic shock. According to a preferred embodiment, the ecto- phosphatase according to the present invention is a recombinant protein. Many sequences, both DNA and amino acid, of the ecto- phosphatases according to the present invention are readily available in the public databases such as for apyrase from various organisms including human (see fig. 1, accession number CAB40415) , placental alkaline phosphatase from various mammals including human (fig. 3, accession number AAA51709) , intestinal alkaline phosphatase (see fig. 2, accession number NP 776412, bovine) from various organisms including human (accession number NP001622), etc. The recombinant ecto-phosphatase proteins according to the present invention can readily be obtained using well known cloning methods based on these already known sequences. For example, a coding sequence of an ecto-phosphatase apyrase can be identified in and isolated from a selected source, preferably human, using probes derived from known coding, genomic or amino acid sequences to screen cDNA or genomic libraries.
As an alternative, using PCR primers based on the known ecto-phosphatase sequence of a selected source, preferably human, nucleic acid amplification techniques can be used for the identification, isolation and cloning of the coding sequence of an ecto-phosphatase.
Once isolated, the coding sequence can be placed under the control of appropriate regulation signals and transformed in a suitable host allowing the expression and isolation of the enzyme.
Appropriate regulation signals comprise a promoter, one or more enhancer sequences, a transcription and/or translation initiation site and a transcription and/or translation termination site.
In a particularly preferred embodiment of the present invention, the present ecto-phosphatase is a soluble or exogenous, most preferably recombinant, ecto-phosphatase. Such preferably recombinant ecto-phosphatase can be any recombinant protein under the condition that it comprises a functionally intact catalytic domain such as GDA1/CD39 catalytic domain (PSSM-Id 41214) or the alkPPC catalytic domain (PSSM-Id 58306) .
In most cases, this would mean a deletion of one or more transmembrane and intracellular domains while leaving at least the catalytic domain of the extracellular part of the enzyme functionally in tact, i.e., the domain providing dephoshorylation activity.
As non-limiting alternatives, other constructions can be envisaged yielding a soluble recombinant protein comprising the catalytic domain of an ecto-phosphatase, such as a fusion protein, in frame deletion of only the transmembrane domain, amino acid modifications such as substituting hydrophobic amino acids for hydrophilic ones, etc .
Considering the beneficial effects of the use of an ecto-phosphatase according to the present invention for the preparation of a medicament for the prophylaxis or treatment of (acute) myocardial infarction, the present invention also relates "to a method for treating a mammal suffering from (acute) myocardial infarction comprising administering a therapeutically effective amount of an ecto-phosphatase. In a preferred embodiment, the method comprises administering a therapeutically effective amount of an apyrase or an alkaline phosphatase, preferably bovine intestinal alkaline phosphatase or human placental alkaline phosphatase . In the method according to the present invention, the ecto-phosphatase, preferably soluble, is a recombinant protein. The present invention also relates to a pharmaceutical composition for treatment of prophylaxis of (acute) myocardial infarction comprising a therapeutically effective amount of an ecto-phosphatase according to the present invention and one or more pharmaceutically acceptable excipients .
Preferred therapeutically effective amounts of an ecto-phosphatase according to the present invention are between 10 to 400 IU/kg body weight. One IU unit is defined as the amount of apyrase capable of liberating 1 micromole inorganic phosphate from ATP or ADP per minute at pH 6.5 at 30 0C.
The present invention will further be described in the following examples which show certain preferred embodiments of the present invention. However, these examples are not intended in any way to limit the scope of the present invention which is only determined by the attached claims.
In the examples, reference is made to the following figures wherein:
Fig. 1 shows the amino acid sequence of the GDA1/CD39 catalytic domain (aa 86 to 540) of an ecto- phosphatase according to the present invention i.e, human apyrase (accession number CAB40415); Fig. 2 shows the amino acid sequence of the alkPPc catalytic domain (aa 52-487) of an ecto- phosphatase according to the present invention, i.e., bovine intestinal alkaline phosphatase (accession number NP 776412); Fig. 3 shows the amino acid sequence of the alkPPc catalytic domain (aa 50-490) of an ecto- phosphatase according to the present invention, i.e., human placental alkaline phosphatase (accession number AAA51709) ; Fig. 4 shows production of the pro-inflammatory cytokine
IL-6 after acute myocardial infarct. Mice were sacrificed at different time points (n=3 per time point) and IL-6 production was determined. Values are depicted as mean ± SEM; Fig. 5 shows alkaline phosphatase activity 4 hours after acute myocardial infarct. Values are depicted as mean ± SEM (n = 4 per treatment group) . * P <
0.05 versus control;
Fig. 6 shows the effect of BIAP on the production of the pro-inflammatory cytokines IL-6 and IL-lβ (a) and on the anti-inflammatory cytokine IL-10 (b) 4 hours after acute myocardial infarct. Values are depicted as mean ± SEM (n = 4 per treatment group) . * P < 0.05 versus control; Fig. 7 shows the effect of BIAP on the production of mMCP-1, 4 hours after acute myocardial infarct. Values are depicted as mean ± SEM (n = 4 per treatment group) . * P < 0.05 versus control; Fig. 8 shows attenuation of proinflammatory MCP-I induction in a MI mouse model using the ecto- phosphatases recombinant BIAP and apyrase; Fig. 9 shows the attenuation of an LPS insult in mice by recombinant intestinal alkaline phosphatase and apyrase;
Fig. 10 shows that recombinant soluble human apyrase (NTPase-1, CD39-1, expressed in tabacco) and recombinant human placental alkaline phosphatase
(soluble, expressed in tabacco) have antiinflammatory activity in a LPS challenge model comparable with bovine intestinal alkaline phosphatase .
EXAMPLES
Example 1 Bovine Intestinal Alkaline Phosphatase (BIAP) reduces inflammation after acute myocardial infarction (AMI) induction in mice
Materials and methods
Bovine intestinal alkaline phosphatase
Clinical grade bovine intestinal alkaline phosphatase was obtained from Biozyme (Blaenavon, UK) . One unit is defined as that amount of BIAP able to hydrolyse 1 μmole of p-nitrophenyl phosphate per minute using a Tris-glycine buffer at pH 9.6 at 25 "C.
Animal treatment Specific pathogen free female BALB/c mice (23-27 gram) were purchased from Charles River (Sulzfeld, Germany) . Mice were acclimatized for 1 week under barrier conditions in filter-topped macrolon cages with drinking water and standard food pellets ad libitum. Animals were divided into two groups: an AMI group treated with BIAP (n=4) and an AMI control group treated with vehicle alone (n=4) . BIAP was injected into the tail vein just before anaesthezation as a single intravenous dose of 5 IU in 100 μl PBS (approximately 100 times above plasma levels) . Control mice were injected with an equal volume of PBS. The study was approved by the animal ethics committee of the Faculty of Veterinary Medicine, University Utrecht. Myocardial infarction
Mice were anaesthetized by inhalation of a mixture of O2 air and 4% isoflurane, endotracheally intubated, and mechanically ventilated. Myocardial infarction was induced as described previously (Salto-Tellez, M., et al., Myocardial infarction in the C57BL/6J mouse: A quantifiable and highly reproducible experimental model. Cardiovascular Pathology, 2004. 13(2) : p. 91-97) .
Briefly, the LAD coronary artery was exposed via a left thoractomy and double ligated with an 8.0 prolene suture. Animals were sacrificed 4 hours after myocardial infarction after which blood was collected. Heart, lung, liver and kidneys were removed and fixed in 4% paraformaldehyde in PBS.
Determination of alkaline phosphatase activity
Five μl of serum was incubated for 60 minutes at 37 0C with 200 μl assay mix containing incubation buffer (0.025 M glycine/NaOH, pH 9.6), p-nitrophenyl phosphate and MgCl2 at final concentrations of 1.25 and 2 mM respectively. The end product p-nitrophenol was quantatively determined by measuring the extinction at 405 nm.
Enzyme-linked Immunosorbent Assay (ELISA) Blood samples were centrifuged and serum was collected for determination of mice IL-6, TNF-α, IL-lβ and IL-10 protein concentrations by commercially available ELISA kits according to the manufacturers' protocol (IL-6 and TNF-α from Biosource, Etten-Leur, The Netherlands; IL-lβ from R&D Systems, Abingdon, UK; and IL-10 from BD Biosciences,
Erembodegem, Belgium) . Mouse mast cell protease-1 (mMCP-1) ELISA was from Moredun (Midlothian, Scotland, UK) and performed according to the manufacturer's instructions. Statistics
All data presented are mean ± SEM. Statistical analysis was performed using Student's t-test for unpaired data (GraphPad Prism) .
Results
Determination of the IL-6 response
One of the major pro-inflammatory cytokines produced in AMI patients is IL-6. First, it had to be determined at which time point after AMI in Balb/c mice IL-6 production could be detected. Therefore, mice underwent permanent ligation of the LAD coronary artery to induce AMI and were sacrificed at three different time points. Before operation, IL-6 concentration was below detection limit (< 4 pg/ml) (Fig. 4) .
Peak IL-6 serum levels were observed 4 hours after AMI. Elevated serum levels of IL-6 could still be detected 6 and 24 hours after AMI. Based on these results, mice were sacrificed 4 hours after AMI in the BIAP treatment experiments .
Alkaline phosphatase activity
AP activity was determined (Fig. 5) in serum samples by measuring hydrolysis of p-nitrophenyl phosphate by AP. All animals that received BIAP had slightly elevated serum levels of AP activity 4 hours after AMI compared to control animals
(P < 0.05), indicating that BIAP was still present in the circulation.
Cytokine response
Systemically elevated concentrations of IL-6, IL-lβ,
TNF-cx and IL-10 are observed in patients with AMI. Therefore, presence of these cytokines was determined.
Before LAD coronary artery ligation, concentrations of the different cytokines were below detection levels. In contrast, 4 hours after AMI IL-6, IL-lβ and IL-IO were excessively produced (Fig. 6) . TNF-α production could not be determined at this time-point.
Administration of BIAP resulted in a significant reduction of the pro-inflammatory cytokines IL-6 and IL-lβ (Fig. 6a) when compared to controls. IL-6 levels were reduced by approximately 40% and IL- lβ by approximately 30%. No difference in the antiinflammatory cytokine IL-10 (Fig. 6b) production could be observed between the control group and the BIAP treated group.
Mast cell activation
After AMI in mammals, mast cells are activated to release chymases. Activation of mast cells in mice can be measured by the release of the mouse mast cell protease-1 (mMCP-1) chymase.
Serum levels of mMCP-1 were 14.7 ng/ml 4 hours after LAD coronary artery ligation. BIAP treatment reduced mMCP-1 levels in serum to 8.4 ng/ml (approximately 40%), implying a significant reduction in mast cell activation (P < 0.05).
Discussion
Four hours after AMI, a significant reduction in the concentrations of the two most prominent pro-inflammatory cytokines present in serum in the acute phase after AMI, IL-6 and IL-lβ, was observed when compared to vehicle controls.
TNF-α concentration in serum, generally believed to be an early-onset proinflammatory cytokine, was below detection limit, suggesting that the chosen time point is not relevant to detect this cytokine in Balb/c mice after LAD coronary artery ligation.
A reduction in pro-inflammatory cytokine production indicates a diminished systemic innate immune response, which decreases myocardial dysfunction and reduces the development of CS after AMI.
Chymases are abundantly produced after AMI in mammals, and are known to be involved in the cleaving of angiotensin I (Ang I) to form angiotensin II (Ang II).
The excessive formation of Ang II, which is observed in the acute phase after AMI, is arrhythmogenic, and several studies in different animal models have shown that decreasing Ang II formation by a specific chymase inhibitor contributes to a reduction in the mortality rate in the acute phase after AMI.
The influence of BIAP on the formation of the mouse chymase mMCP-1 was determined. In BIAP-treated mice, mMCP-1 production was significantly reduced by approximately 40% when compared to vehicle-treated mice. This result indicates a reduction of Ang II formation and consequently a decrease in arrhythmias, which improves cardiac function and reduce CS complication .
Example 2 Attenuation of proinflammatory MCP-I induction in a MI mouse model using the ecto-phosphatases recombinant BIAP and apyrase
Materials and methods
Bovine intestinal alkaline phosphatase and apyrase
Clinical grade bovine intestinal alkaline phosphatase was obtained from Biozyme (Blaenavon, UK) . One unit is defined as that amount of BIAP able to hydrolyse 1 μmole of p-nitrophenyl phosphate per minute using a Tris-glycine buffer at pH 9.6 at 25 °C.
Clinical grade apyrase was obtained from Sigma (Sigma Aldrich, St. Louis, MO) . One unit is defined as that amount of apyrase able to liberate 1 micromole inorganic phosphate from ATP or ADP per minute at pH 6.5 at 30 0C.
Animal treatment Specific pathogen free female BALB/c mice (23-27 gram) were purchased from Charles River (Sulzfeld, Germany) . Mice were acclimatized for 1 week under barrier conditions in filter-topped macrolon cages with drinking water and standard food pellets ad libitum. Animals were divided into three groups: an AMI group treated with BIAP (n=4), an AMI group treated with apyrase (n=4) and an AMI control group treated with vehicle alone (n=4) . BIAP and apyrase were injected into the tail vein just before anaesthezation as a single intravenous dose of 5 IU in 100 μl PBS (approximately 100 times above plasma levels).
Control mice were injected with an equal volume of PBS. The study was approved by the animal ethics committee of the Faculty of Veterinary Medicine, University Utrecht.
Myocardial infarction
Mice were anaesthetized by inhalation of a mixture of O2 air and 4% isoflurane, endotracheally intubated, and mechanically ventilated. Myocardial infarction was induced as described previously (Salto-Tellez, M., et al., Myocardial infarction in the C57BL/6J mouse: A quantifiable and highly reproducible experimental model. Cardiovascular Pathology, 2004. 13(2) : p. 91-97) .
Briefly, the LAD coronary artery was exposed via a left thoractomy and double ligated with an 8.0 prolene suture. Animals were sacrificed 4 hours after myocardial infarction after which blood was collected. Heart, lung, liver and kidneys were removed and fixed in 4% para- formaldehyde in PBS.
Enzyme-linked Immunosorbent Assay (ELISA)
Blood samples were centrifuged and serum was collected for determination of mouse mast cell protease-1 (mMCP-1) . Mouse mast cell protease-1 (πιMCP-1) ELISA was from Moredun (Midlothian, Scotland, UK) and performed according to the manufacturer's instructions.
Statistics All data presented are mean ± SEM. Statistical analysis was performed using Student's t-test for unpaired data (GraphPad Prism) .
Results
Mast cell activation
After AMI in mammals, mast cells are activated to release chymases. Activation of mast cells in mice can be measured by the release of the mouse mast cell protease-1 (mMCP-1) chymase.
Serum levels (fig. 8) of mMCP-1 were 14.7 ng/ml 4 hours after LAD coronary artery ligation. BIAP treatment reduced mMCP-1 levels in serum to 8.4 ng/ml (approximately 40%), implying a significant reduction in mast cell activation (P < 0.05).
A similar reduction in mMCP-1 levels was observed for apyrase . Example 3 Apyrase and recombinant intestinal alkaline phosphatase are capable of in vivo attenuation of LPS mediated inflammation
In an LPS challenge model, mice (C57B16) were i.p. administered both CD39 (apyrase) (4.5 units) and alkaline phosphatase (rec IAP 0502, 1.5 units) and LPS (5 micrograms) .
As shown in fig. 9, the administration of apyrase or alkaline phosphatase resulted in a comparable attenuation of inflammation as demonstrated by a stabilization of body temperature .
LPS (5 micrograms) administered i.p. alone, resulted in lower body temperature which recovered after 5 hours. (In mice reduced body temp is generally observed after LPS administration) . Both alkaline phosphatase and apyrase
(potato-apyrase) injected mice show normalized body temperatures .
The above results demonstrate that apyrase and recombinant intestinal alkaline phosphatase are capable of controlling an endotoxin associated systemic inflammatory response at comparable levels.
Example 4 Recombinant human apyrase (NTPase-1 , CD39-1) and recombinant human placental alkaline phosphatase have anti-inflammatory activity in a LPS challenge model comparable with bovine intestinal alkaline phosphatase
In an LPS challenge model, mice (C57B16) were i.p. administered recombinant human apyrase (NTPase-1, CD39-1) , recombinant human placental alkaline phosphatase or bovine intestinal alkaline phosphatase and were subsequently challenged with LPS. Mice only injected with the carrier served as control group.
As shown in fig. 10, administration of recombinant human apyrase (NTPase-1, CD39-1), recombinant human placental alkaline phosphatase or bovine intestinal alkaline phosphatase resulted in a comparable attenuation of inflammation as demonstrated by a stabilization of body temperature .
LPS administered alone, resulted in lower body temperature which recovered after 5 hours. (In mice reduced body temp is generally observed after LPS administration) . recombinant human apyrase (NTPase-1, CD39-1), recombinant human placental alkaline phosphatase or bovine intestinal alkaline phosphatase injected mice show comparable normalized body temperatures indicating an attenuation of the systemic inflammatory response due to endotoxins.

Claims

1. Use of ecto-phosphatases for the preparation of a medicament for the treatment or prophylaxis of (acute) myocardial infarction.
2. Use according to claim 1, wherein the ecto- phosphatase is apyrase or alkaline phosphatase.
3. Use according to claim 2, wherein the alkaline phosphatase is bovine intestinal alkaline phosphatase or human placental alkaline phosphatase.
4. Use according to any of the claims 1 to 3 , wherein the ecto-phosphatase is a recombinant protein.
5. Use according to any of the claims 1 to 4 , wherein the ecto-phosphatase is soluble.
6. Method for treating a mammal suffering from
(acute) myocardial infarction comprising administering a therapeutically effective amount of an ecto-phosphatase.
7. Method according to claim 6, wherein the ecto- phosphatase is apyrase or alkaline phosphatase.
8. Method according to claim 7, wherein alkaline phosphatase is bovine intestinal alkaline phosphatase or human placental alkaline phosphatase.
9. Method according to any of the claims 6 to 8 , wherein the ecto-phosphatase is a recombinant protein.
10. Method according to any of the claims 6 to 9, wherein the ecto-phosphatase is soluble.
11. Pharmaceutical composition for treatment of prophylaxis of (acute) myocardial infarction comprising a therapeutically effective amount of an ecto-phosphatase and one or more pharmaceutically acceptable excipients.
12. Pharmaceutical composition according to claim 11 comprising 10 to 400 IU/kg body weight ecto-phosphatase.
13. Pharmaceutical composition according to claim 11 or claim 12, wherein the ecto-phosphatase is apyrase or an alkaline phosphatase.
14. Pharmaceutical composition according to claim 13, wherein the alkaline phosphatase is bovine intestinal alkaline phosphatase or human placental alkaline phosphatase.
15. Pharmaceutical composition according to any of the claims 11 to 14, wherein the ecto-phosphatase is a recombinant protein.
16. Pharmaceutical composition according to any of the claims 11 to 15, wherein the ecto-phosphatase is soluble.
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