USE OF UROKINASE RECEPTOR ANTAGONISTS TO MODULATE ISCHEMIA- REPERFUSION INJURY Field of the invention The invention relates to a method of prevention and/or treatment of ischemia-reperfusion injury. In particular, the invention indicates the importance of the urokinase receptor (u- PAR) in the pathogenesis following ischemia. Consequently, the use of selective inhibitors of u-PAR are beneficial for the prevention and/or treatment of patients suffering from reperfusion injury in the heart, in organ transplantation and in the brain.

Background of the invention Peri-operative myocardial ischemia (MI) causes serious morbidity and mortality for over 5 million patients each year. Once MI occurs, the prognosis of the patient is dramatically altered: post-operative recovery times are lengthened, while non-cardiac complications are increased. Notably, 20 to 30% of those patients who experience new onset MI, will suffer fatal myocardial infarctions or dysrhythmias within 6 years. Patients who have required CPR, have a poor prognosis. Only 4-33% of the approximately 750.000 resuscitated Americans per year survive to hospital discharge. A large proportion of deaths are associated with the"post-resuscitation"syndrome, characterized by multi- organ damage. Regional MI and global ischemia share features of hypoxia-induced endothelial cell (EC) damage and ensuing reperfusion injury: Global ischemia after cardiac arrest results in diffuse EC damage, with increased plasma levels of soluble thrombomodulin and adhesion molecules, as well as enhanced expression of P-selectin and ICAM-1 in the brain. The latter is particularly relevant, since clinical outcome after global ischemia is inversely correlated with the extent of neurologic damage that is in part due to reperfusion. There is ample evidence that transient ischemia induces long-lasting changes in excitatory synaptic transmission in CA1 hippocampal neurons. Anoxic longterm synaptic potentiation is considered by some, an early marker of delayed neuronal death induced by transient ischema. After an ischemic event, blood flow is restored, and tissue repair is initiated with an inflammatory response characterized by influx of leukocytes. Murine models of MI, both transient and permanent, have recently been well characterized and provide reliable means to study myocardial wound healing and ventricular remodelling that closely reflect these processes in man. Transient ischemia induces myocyte damage and microvascular endothelial cell (EC) activation.

Upon restoration of blood flow, tissue repair is initiated by an inflammatory response characterized by influx of leukocytes and platelets. Leukocyte/platelet/EC interactions are

tightly regulated by adhesion molecules. Members of the selectin family and their ligands mediate tethering and rolling events of leukocytes and platelets on the EC surface.

Subsequently, integrins engage members of the immunoglobulin superfamily, ICAM-1 and VCAM-1, on EC. Inflammatory cells such as monocytes and polymorphonuclear cells (PMNs) are, partly through platelets, recruited to clear damaged tissue. This is followed by mesenchymal cell division, migration, matrix synthesis and eventual ventricular remodelling. Unfortunately, concomitant with this local inflammatory response, the infarct region may expand into otherwise salvageable tissue. Although blocking or targeted gene-disruption of adhesion molecules can protect mice from acute myocardial ischemia with reperfusion (MI/R), total abrogation of the inflammatory response is not desirable, as this would impair the removal of necrotic material and the formation of a mechanically strong scar. An intermediate situation would therefore be ideal-one in which MI/R- induced inflammation is modulated to limit tissue damage, yet optimised to preserve ventricular remodeling and improve ventricular function. The urokinase receptor (u-PAR) is directly involved in regulating adhesion molecule-ligand binding affinities. The function of u-PAR is to localize plasmin activity by recruiting urokinase activity to the cell surface.

Recently, however, u-PAR has been recognized to also play a pivotal role in integrin activation and cell signalling in leukocyte-EC interactions in vitro. And indeed, in vivo it has been shown that disruption of the u-PAR gene markedly reduces integrin-mediated recruitment of PMNs to inflamed peritoneum ( (May AE, Kanse SM, Lund LR, Gisler RH, Imhof BA, Preissner KT. Urokinase receptor (CD87) regulates leukocyte recruitment via beta 2 integrins in vivo J Exp Med. 1998 Sep 21; 188 (6): 1029-37). ischemia-reperfusion injury in the heart is a complex biological process with many factors involved but the present invention surprisingly shows that mice lacking a functional u-PAR gene (u-PAR-/- mice) exhibit a significantly smaller infarction after 30 minutes ischemia and 3 or 24 hours reperfusion compared to wild type mice. The data show that reducing u-PAR-mediated integrin activation is a useful therapeutic target for the treatment of myocardial ischemia and/or infarction.

Figure Figure 1: In 2-D-guided M-mode echocardiography, u-PAR mice demonstrate preserved global function after MI and better regional function of the ischemic anterior wall. The results indicate that reduced size of infarctions in u-PAR-mice due to reduced PMN- homing translates into functional benefits with respect to global and regional LV-function.

(n=5 each group, * p<0. 05).

Aims and detailed description of the invention The present invention shows that the urokinase receptor is a target for the prevention and/or the treatment of ischemia-reperfusion injury.

In a first embodiment at least one urokinase receptor inhibitor can be used for the preparation of a medicament to prevent and/or to treat ischemia-reperfusion injury. Upon the detection of an ischemic insult in the body a pharmaceutical composition comprising urokinase receptor inhibitors can be applied to prevent and/or to treat reperfusion injury.

Urokinase receptor inhibitors (or antagonists) are described in the art and comprise cyclic peptidomimetics, alkoxyalkylidenecoumarones, peptide inhibitors, oligothiophenes, porphyrins, bis (carbamoylphenyl) methanes, oligonucleotides encoding peptide inhibitors of urokinase receptor activity, sulfonic acid sulfonylamino N- (heteroaralkyl)- azaheterocyclylamide compounds, 2-alkylidene hydroxycumaranone and these inhibitors are described in WO 2001005811, EP 1026165, WO 2000001802, WO 9906393, EP 792647, WO 9846731, WO 9806386, US 6030940, US 5679782, US 5656726, WO 9428014, WO 9735969, US 6281227 and US 6200989 which are hereby incorporated by reference.

The term'ischemia-reperfusion injury'refers to injury that occurs when the flow of blood to a region of the body is temporarily halted (ischemia) and then re-established (reperfusion).

Ischemia-reperfusion injury can occur as a consequence of disease processes or surgical interventions associated with temporary interruption of blood flow. Such surgical procedures include adult and pediatric cardiac surgery procedures, peripheral vascular surgical procedures, neurosurgical procedures, i. e. brain arterial aneurysm resection and clipping, repair of certain aortic aneurysms and organ transplantation. Clinically, ischemia- reperfusion injury is manifested by such complications as for example pulmonary dysfunction, including adult respiratory distress syndrome, renal dysfunction, consumptive coagulopathies including thrombocytopenia, fibrin deposition into the microvasculature and disseminated intravascular coagulopathy. These events may lead to transient and permanent brain or spinal cord injury, cardiac arrhythmias and acute ischemic events, hepatic dysfunction including acute hepatocellular damage and necrosis, These gastrointestinal dysfunction including hemorrhage and/or infarction and multisystem organ dysfunction or acute systemic inflammatory distress syndromes. The injury may occur in the parts of the body to which the blood supply was interrupted, or it can occur in parts fully supplied with blood during the period of ischemia. Ischemia-reperfusion injury can also occur in the brain because of a stroke. For example, in the heart, myocardial ischemia occurs when myocard blood flow is interrupted for a period of more than 2 minutes. Reperfusion occurs when blood flow is restored leukocytes are recruited to the

tissue and inflammatory mediators as well as oxygen radicals are generated at an increased rate in reperfused formerly ischemic myocardium. In the heart, the end result is myocardial cell death, the extent of which is dependent on the duration of the ischemic insult and on the intensity of the subsequent reperfusion injury. The histological changes are characterized by cell swelling, calcium deposition, and contraction band formation.

In another embodiment at least one urokinase receptor inhibitor can be used for the preparation of a medicament to prevent and/or to treat ischemia-reperfusion injury that occurs in the heart and is caused by a myocard infarct.

In another embodiment at least one urokinase receptor inhibitor can be used for the preparation of a medicament to prevent and/or to treat ischemia-reperfusion injury that occurs in the brain and is caused by a stroke.

In another embodiment at least one urokinase receptor inhibitor can be used for the preparation of a medicament to prevent and/or to modulate ischemia-reperfusion injury that occurs during an organ transplant.

The term'medicament to treat'relates to a composition comprising inhibitors as described above and a pharmaceutically acceptable carrier or excipient (both terms can be used interchangeably) to treat or to prevent diseases as described herein. The administration of an inhibitor as described above or a pharmaceutically acceptable salt thereof may be by way of oral, inhaled or parenteral administration. The active compound may be administered alone or preferably formulated as a pharmaceutical composition. An amount effective to treat disorders described herein depends on the usual factors such as the nature and severity of the disorders being treated and the weight of the mammal.

However, a unit dose will normally contain 0.01 to 50 mg for example 0.01 to 10 mg, or 0.05 to 2 mg of urokinase receptor antagonist or a pharmaceutical acceptable salt thereof. Unit doses will normally be administered continuously or once or more than once a day, for example 2,3, or 4 times a day, more usually 1 to 3 times a day, such that the total daily dose is normally in the range of 0.0001 to 1 mg/kg; thus a suitable total daily dose for a 70 kg adult is 0.01 to 50 mg, for example 0.01 to 10 mg or more usually 0.05 to 10 mg. It is greatly preferred that the compound or a pharmaceutically acceptable salt thereof is administered in the form of a unit-dose composition, such as a unit dose oral, parenteral, or inhaled composition. Such compositions are prepared by admixture and are suitably adapted for oral, inhaled or parenteral administration, and as such may be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, reconstitutable powders, injectable and infusable solutions or suspensions or suppositories or aerosols. Tablets and capsules for oral administration are usually

presented in a unit dose, and contain conventional excipients such as binding agents, fillers, diluents, tabletting agents, lubricants, disintegrants, colourants, flavourings, and wetting agents. The tablets may be coated according to well known methods in the art.

Suitable fillers for use include cellulose, mannitol, lactose and other similar agents.

Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycollate. Suitable lubricants include, for example, magnesium stearate.

Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulphate.

These solid oral compositions may be prepared by conventional methods of blending, filling, tabletting or the like. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of fillers. Such operations are, of course, conventional in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non- aqueous vehicles (which may include edible oils), for example, almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol ; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents. Oral formulations also include conventional sustained release formulations, such as tablets or granules having an enteric coating. Preferably, compositions for inhalation are presented for administration to the respiratory tract as a snuff or an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case the particles of active compound suitably have diameters of less than 50 microns, preferably less than 10 microns, for example between 1 and 5 microns, such as between 2 and 5 microns. A favored inhaled dose will be in the range of 0.05 to 2 mg, for example 0.05 to 0.5 mg, 0.1 to 1 mg or 0.5 to 2 mg. For parenteral administration, fluid unit dose forms are prepared containing a compound of the present invention and a sterile vehicle. The active compound, depending on the vehicle and the concentration, can be either suspended or dissolved. Parenteral solutions are normally prepared by dissolving the compound in a vehicle and filter sterilising before filling into a suitable vial or ampoule and sealing. Advantageously, adjuvants such as a local anaesthetic, preservatives and buffering agents are also dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum.

Parenteral suspensions are prepared in substantially the same manner except that the

compound is suspended in the vehicle instead of being dissolved and sterilised by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the active compound. Where appropriate, small amounts of bronchodilators for example sympathomimetic amines such as isoprenaline, isoetharine, salbutamol, phenylephrine and ephedrine; xanthine derivatives such as theophylline and aminophylline and corticosteroids such as prednisolone and adrenal stimulants such as ACTH may be included. As is common practice, the compositions will usually be accompanied by written or printed directions for use in the medical treatment concerned.

Examples 1. Evaluation of the role of u-PAR as a target for the modulation of ischemia-reperfusion injury that is caused by a mvocardial infarction.

Twelve week old gender-matched mice (bw: 234 g) were barbiturate-anesthetized, intubated and ventilated with isoflurane in 02. Temperature and blood pressure were maintained constant. The left anterior descending coronary artery (LAD) was ligated over PE10 tubing for 30 minutes to induce myocardial ischemia with reperfusion (Mi/R).

Reperfusion times were 3 or 24 hours. Mice were NaCI-perfused and after the ligation had been re-tied coomassie blue was injected via a carotid artery catheter to planimetrically detect the area at risk (AAR). Infarcted tissue was measured by lack of TTC staining. In some mice polymorphnuclear leukocytes (PMN) from bone marrow were fluophor labeled, injected upon reperfusion and counted on 100/im sections by epifluorescent microscopy.

It was observed that left ventricular size (17. 31. 3 mm2 vs. 17. 41. 3 mm2, wt vs. u-PAR-/-, n=10) and AAR (9. 80. 9 mm2 vs. 9. 40. 8 mm2, n=10) were similar. At 3h, u-PAR-were smaller than wt infarcts (353. 5 vs. 213. 5 Infarct/AAR (%), n=5, p<0.05). After 24h, infarctions had enlarged but were significantly smaller in u-PAR-/- (483 % vs 28. 2.2 % wt vs. u-PAR, n=10, p<0.01). To test whether reperfusion injury depended on endothelial versus leukocytic u-PAR, PMN homing to ischemic myocardium was examined. 30% of u- PAR-/--compared to wt-PMN (18960 PMN/LV vs. 6416 PMN/LV, n=3/6, p<0.05) were detected. Only a trend for a reduction of PMN-homing was observed when wt-PMN were injected into wt or u-PAR~/~-mice. In contrast to the ongoing inflammation observed in the hearts of wt mice, u-PAR~/~-mice had ameliorated myocardial wound healing in preliminary studies. In echocardiography, u-PAR~/~-mice display better regional and global LV-function than wt-controls (Fig 1).

2. u-PAR-peptides are tested in large animals models of MI/R.

Large animal modelling is essential prior to extrapolating observations to human disease.

Murine peptides, however, are not suitable for these studies because 1. integrin binding

may be species-dependent, and 2. immune responses to the"foreign"peptides might confound the data. For these reasons, canine and porcine u-PAR cDNAs are traditionally cloned-hybridization of commercial canine and porcine cDNA phagemid libraries with human and/or murine probes, followed by isolation of positive clones, confirmation by DNA sequencing, subcloning and expression for functional evaluation. A model of MIIR, in chronically instrumented, awake dogs has been established in our team29. We will, for practical and ethical reasons, adapt this model to the pig. We monitor the corresponding polypeptides that are protective in mice are active in the larger animal models.

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