WO2007134813A2 - Use of desmodus salivary plasminogen activator (dspa) for treating venous thromboembolism - Google Patents

Abstract

Use of a plasminogen activator for the manufacture of a medicament for the treatment of venous thromboembolism, wherein the plasminogen activator has an at least more than 550 fold increased activity in the presence of fibrin compared to the activity without fibrin.

Description

"Method for treating venous thromboembolism"

The invention pertains to a method for treating venous thromboembolism, in particular pulmonary embolism, and to pharmaceutical compositions suitable for being used in such methods and claims the priority of the international application PCTEP2006/004771 referring to its contents.

Venous thromboembolism (VTE) is the common term for two related disorders, namely deep vein thrombosis (DVT) and pulmonary embolism (PE), both of which are serious medical conditions caused by the abnormal formation of the blood clots. The incidence of VTE varies widely in different patient populations depending on the presence or absence of risk factors. Overall, the incidence is estimated to 1 ,5-2 cases per 1000, but is substantially higher in high-risk populations, such as patients undergoing major orthopaedic surgery. Those patients have a 50% risk of developing VTE.

Given the large numbers of patients involved and the potentially fatal nature of VTE, management of the condition places a heavy burden, both financial and otherwise, on healthcare systems globally. Management of VTE can be divided into prophylaxis and acute treatment, when the former fails or is not employed.

A DVT may itself cause chronic symptoms of pain and decreased mobility, but more dangerous is its potential to cause a PE. The diagnosis of DVT heavily depends on clinical suspicion, as well as on the individual experience of the physician regarding the evaluation of lab tests and imaging studies. This uncertainty in diagnosis makes DVT an even more threatening condition. More recent technological advances have resulted in enhanced diagnostic capabilities. However, more adequate and efficient diagnostic tests are cited among the most pressing unmet needs in VTE management.

Deep venous thromboembolism (deep vein thromboembolism or deep vein thrombosis) is defined as a clinical condition, which results from a blood clot in deep venous circulation, most commonly in the lower leg or thigh. In addition to causing pain, swelling and redness, the clot may dislodge from the leg veins and travel up the circulation to the right side of the heart and the lungs, from where it may lead to a PE.

Pulmonary embolism (PE) is the sudden obstruction of the pulmonary artery, or one of its branches, most commonly the result of a blood clot. A PE is a medical emergency that can cause a irreversible damage to the lungs and death, even with an appropriate therapy. In about 50% of patients with pulmonary embolism, the embolism is massive, i.e., accompanied by at least one of several potentially compromising conditions. These compromising conditions comprise hypotension or shock, severe hypoxemic respiratory failure, acute right-sided heart dysfunction, or obstruction of the pulmonary vasculature that exceeds 50% as demonstrated by angiogram or ventilation/perfusion scan (V/Q scan). Thus, major/massive pulmonary embolism can be defined as a pulmonary embolism accompanied by haemodynamic instability. It is assumed to be the result of an interaction of embolism size and the underlying cardiopulmonary function.

The presence of compromising factors, in particular shock - and thus the occurrence of a massive PE - defines a three fold to seven fold increase in mortality, with a majority of death occurring within one hour of presentation (symptom onset). A rapid integration of historical patient information and physical findings within a readably available laboratory data in a structured physiologic approach to diagnosis and resuscitation are necessary for optimal therapeutics in this "golden hour".

Although VTE is recognized across the medical community as a serious condition with potentially fatal social consequences - pulmonary embolism continuous to be the third most common cardiovascular cause of death with a mortality rate between 20% and 30% - effective treatment remains elusive. Insufficient treatment carries with it many risks, including re-occurrence, post-thrombotic syndrome, progression from DVT to PE, and death.

Several approaches exist for treating DVT and PE. One of these is the administration of unfractionated heparin (UFH). Others concentrate on the administration of orally available anticoagulants or the administration of low molecular weight heparin (LMWHS). Another possibility is the thrombolysis of the blood clot.

However, it is important to consider, that VTE therapy has its own risks, most notably bleeding complications, and the consequences of overzealous treatment are at least as dangerous as undertreating. Thus, save and effective treatment requires skilled and experienced physicians with up-to-date knowledge of current guidelines and protocols, and access to all therapeutic alternatives.

It is known, than thrombolysis in pulmonary embolism reverses right heart failure rapidly. Alteplase, to be administered by intravenous infusion, is one of the few approved thrombolytic drugs in this indication. However, the use of thrombolytics in patients with a pulmonary embolism remains heavily debated. Those clinical studies, which have been conducted, have failed to address survival as an endpoint, instead measuring such outcomes as reduction in clot burden, V/Q scan results, improvements in capillary blood flow and other pulmonary function tests. While these studies have demonstrated superiority of thrombolytics over UFH in terms of long-term lung function, long-term survival must be assessed in order to convince the medical community of the utility of thrombolytic therapy for PE patients.

To date, on the balance lies in favour the evidence for the efficacy of thrombolysis in PE patients. However, the majority of clinical studies and registries have shown an increased incidence of major bleeding, including an increase in the incidence of catastrophic intracranial bleeds. Hence, the disfavour of the balance of the thrombolytic therapy is the bleeding risk. It is indeed the increased risk of bleeding that precludes the routine use of thrombolytics in VTE patients.

Also of significance, there remains a discrepancy between the perceived and actual treatment window for thrombolytics in PE patients. Many physicians believe, that thrombolysis is only effective if administered relatively soon after the onset of symptoms, despite the modern view among PE specialists, that the time window for effective treatment of PE is up to 14 days.

Unlike the anticoagulant therapies mentioned above, that prevent clot formation, thrombolytic agents promote the dissolution of blood clots once they have formed.

Thrombolytic agents are plasminogen activators (PA) that convert plasminogen, the inactive pro-enzyme of the fibrinolytic system in blood, to the proteolytic enzyme plasmin, which dissolves the fibrin of a blood clot into soluble degradation products. In this way, thrombolytic agents deplete stores of the protein that functions as scaffolding for a clot assembly and consolidation - and therewith increases the risk of bleeding.

Hence, the optimal profile of a thrombolytic agent is related to its fibrin selectivity, which is indicated by low systemic plasminogen and thus fibrinogen consumption. Fibrin selectivity is an established clinical parameter used in the assessment of the risk of potential bleeding complications: The higher the fibrin specificity, the lower the risk. This has led to thrombolytic agents or plasminogen activators being perceived as "double edged swords".

Because of their high cost and potential for increasing the risk of bleeding, thrombolytics are rarely routinely used to treat DVT, except in patients with massive extensive thrombosis. In addition to lysing the clot to prevent subsequent PE, thrombolytic agents may decrease pain, swelling and loss of venous valves, thus reducing the incidence of post-thrombotic syndrome. Although more research is needed, studies suggest that the use of thrombolytic therapy in patients with massive DVT of recent onset can reduce the incidence of the occurrence of pulmonary embolism. However, the known thrombolytic therapy is limited to patients with no contraindications. Contraindications include e.g. active internal bleeding or recent surgery or other vascular incidences within the last months.

Of course, the benefits of rapid reversal of right heart failure must be balanced against the risks of major bleeding, most notably intracranial haemorrhage. Although rt-PA is the only approved contemporary PE thrombolysis regime, other agents have been shown in limited trials to be effective. These agents are all administered in high concentration over a certain time period. Promising regimes that have been previously studied include two hour infusion of urokinase and one to two hours infusion of streptokinase in addition to pilot trials of pro-urokinase (recombinant single chain urokinase type plasminogen activator, rscu-PA) and staphylokinase. Furthermore reteplase was tested in a "double bolus" study, according to which the efficacy and safety is compared to the approved two hours regime of rt-PA. A summary of these regimes is given below (source: Goldhaber: American Heart Journal 1999, page 2 (editorial):

In a further study the hypothesis was evaluated, if a reduced dose of bolus rt-PA (0,6 mg/kg/15 min, maximum of 50 mg) would result in fewer bleeding complications than standard 100 mg of rt-PA administered as a continuous infusion over two hours among haemodynamically stable patients with PE. Subsidiary objectives were to compare the two rt-PA regimes with respect to

1. the rate of adverse clinical events;

2. the magnitude of change from baseline of perfusion lung scans, pulmonary angiograms or echocardiograms; and

3. the differences in coagulation parameters over time.

However, this study did not detect any significant differences between the bolus rt-PA and the two hours rt-PA with respect to bleeding complications, adverse clinical events or imaging studies. There was only less fibrinogenolysis with a bolus dosing regime. Thus, the bolus regime was not considered as being favourable (Goldhaber SZ, Agnelli G., Levine MN: Reduced Dose Bolus Alteplase vs Conventional Alteplase Infusion for Pulmonary Embolism Thrombolysis. An International Multicenter Randomized Trial. CHEST, 106, 3, September, 1994, pages 718-724).

Since even the reduced bolus did not confer protection against hemorrhagic complications, the avoidance of plasma proteolytic state may not be linked to the prevention of bleeding. It is thus thought to be more important to subject possible patients to meticulous evaluation of possible contraindications.

According to product characteristics provided by the EMEA for actilyse, contraindications for administering rt-PA to a patient with DVT are:

known haemorrhagic diathesis patients receiving oral anticoagulants, e.g. warfarin sodium manifest or recent severe or dangerous bleeding - known history of or suspected intracranial haemorrhage suspected subarachnoid haemorrhage or condition after subarachnoid haemorrhage from aneurysm any history of central nervous system damage (i.e. neoplasm, aneurysm, intracranial or spinal surgery) - haemorrhagic retinopathy, e. g. in diabetes (vision disturbances may indicate haemorrhagic retinopathy) recent (less than 10 days) traumatic external heart massage, obstetrical delivery, recent puncture of a non-compressible blood-vessel (e.g. subclavian or jugular vein puncture) - severe uncontrolled arterial hypertension bacterial endocarditis, pericarditis acute pancreatitis documented ulcerative gastrointestinal disease during the last 3 months, oesophageal varices, arterial-aneurysm, arterial/venous malformations - neoplasm with increased bleeding risk severe liver disease, including hepatic failure, cirrhosis, portal hypertension

(oesophageal varices) and active hepatitis. major surgery or significant trauma in past 3 month - any history of stroke.

For the purpose of the present invention, these contraindications are summarized as "bleeding risk factors".

Thus it is the objective of the present invention to provide improved means for a thrombolytic therapy of venous thromboembolism, in particular pulmonary embolism and deep venous thrombosis, even more particular of massive pulmonary embolism.

This objective is solved by using a plasminogen activator for the treatment of VTE, which favourably is essentially non-activatable by beta-amyloid or prion protein and which has in the presence of fibrin an enhanced activity of more than 550 fold, preferred more than

5500 fold, most preferred more than 10,000 fold, compared to the activity in the absence of fibrin. In a particular preferred embodiment the increase of activity of the PA in the presence of fibrin compared to its activity in the absence of fibrin is more than 100,000. Since the increase in activity of rt-PA is 550, the particularly preferred PA for the use according to the invention have an approximately 180-200 fold higher fibrin specificity/selectivity compared to rt-PA.

The preferred PA according to the invention are non-neurotoxic and have a half-life of more than 2.5 min, preferably more than 50 min, even more preferred more 100 min. The neurotoxicity can be assessed by methods known to the skilled person, e.g. with animal models in particular kainic acid models as described in detail in the international laid open WO03/037363.

In one preferred embodiment of the invention, DSPA alpha 1 or PA with a biological activity and pharmacological properties essentially corresponding to DSPA alpha 1 are used. DSPA alpha 1 has a half-life of 138 min and a 105,000 fold increased activity in the presence of fibrin compared to its activity in the absence of fibrin.

In a preferred embodiment DSPA alpha 1 is administered to the patients as an intravenous single bolus, i.e. without a further administration/infusion. A bolus is defined as the intravenous administration of the medicament in a short period of time, i.e. in less than 5 min, preferably not more than 2 min. In one embodiment DSPA alpha 1 is administered over 1 to 2 min.

If suitable, the administration of DSPA alpha 1 can be accompanied by the administration of anti-coagulants, e.g. heparin or aspirin. These medicaments can be given to the patient as an intravenous infusion after the bolus of DSPA alpha 1.

DSPA alpha 1 is a plasminogen activator, which originally was isolated or derived from the saliva of Desmodus rotundus {Desmodus Salivary Plasminogen Activator). Within the saliva four variants of DSPA had been isolated which, similarly to alteplase and urokinase, are composed of various conserved domains previously established in related families of proteins. The variants rDSPA alphal and rDSPA alpha2 exhibit the structural formula Finger (F), Epidermal Growth Factor (EGF) (E), Kringle (K), Protease (P), whereas rDSPA beta and rDSPA gamma are characterised by the formulas EKP and KP, respectively. Subtle sequence differences and data from southern blot hybridisation analysis indicate that the four enzymes are coded by four different genes and are not generated by differential splicing of a single primary transcript.

The variant DSPA alpha 1 has an at least 70% structural homology to alteplase; the difference being that alteplase has two kringles (FEKKP), whereas DSPA alpha 1 has only one (FEKP). DSPA alpha 1 is a serine protease with a molecular weight of 52 KD and 441 amino acids. Like other plasminogen activators (PA), DSPA alpha 1 activates plasminogen by catalysing the conversion of plasminogen into plasmin, which in turn breaks down the cross-linked fibrin present in abundance in blood clots.

DSPA alpha 1 has been found to have unique characteristics when compared to other thrombolytic agents, in particular with reference to alteplase. These include high specificity and selectivity for plasminogen-bound fibrin, low fibrinogen specificity, no neurotoxicity, and negligible activation by beta-amyloid and human cellular prion protein, in addition to a long dominant half-life of more than 2 hours. All these characteristics result in a better safety and pharmacological profile for use in clinical settings not accessible for the thrombolytic agents currently available.

DSPA alpha 1 has been extensively studied in vitro and in vivo, using various thrombosis models and preferentially with alteplase as the comparator. In these studies, DSPA alpha 1 equalled or exceeded alteplase in potency, displaying shorter lysis times and lower re- occlusion rates. Favourable pharmacokinetic properties appear to make an important contribution to its higher potency, in as much as DSPA alpha 1 has a lower total clearance and a longer terminal half-life than alteplase (see above).

This means that single bolus application over a short time period is possible. The latter is regarded as a considerable advantage in the clinical use of thrombolytic agents, especially in the emergency room or even in the ambulance.

In vivo animal studies have confirmed that the administration of DSPA alpha 1 is associated with an apparent reduction in the number of haemorrhagic events. In addition, the negligible sensitivity of DSPA alpha 1 and activation by beta-amyloid and prion protein, which has been observed in in vitro studies comparing DSPA alpha 1 to alteplase, translates into a reduced incidence of intra-parenchymal bleeding in the elderly population.

Rekombinant DSPA alpha 1 is obtained from Chinese hamster ovary cells containing a recombinant plasmid carrying the DSPA alpha 1 gene from Desmodus rotundus. Fig. 1 and fig. 2 show the structures of DSPA alpha 1 and alteplase. The sequence of DSPA alpha 1 is shown in fig. 3.

The plasminogen activator from Desmodus rotundus and its recombinant form was first disclosed in the US patents US 6,008,019. The US 5,830,849 discloses the sequence data of DSPA alpha 1. Both patents are incorporated herein by reference in terms of the structure, properties and manufacture of plasminogen activators from Desmodus rotundus, in particular DSPA alpha 1 alpha 1.

According to the present invention the term "desmoteplase" is used for any plasminogen activator with identical or essentially the same biological activity of DSPA alpha 1 regarding the activation of plasminogen and its enhanced fibrin selectivity/specificity. Preferably, the fibrin selectivity is at least 180 fold compared to rt-PA. The PA defined as desmoteplase according to the invention are preferably at least 80 or 90%, more preferred at least 95 %, most preferred at least 98% identical to the amino acid sequence according to fig. 3 (DSPA alpha 1). The plasminogen activators can include microheterogeneities, e.g. in terms if glycosylation and/or N-terminal variations, which are merely due to production systems.

According to one embodiment of the invention, the plasminogen activators are used for treating VTE patients, in particular PE or massive PE patients, who have a higher risk of bleeding compared to other VTE patients. This group of patients is defined as suffering from at least one bleeding risk factor as defined above.

In a preferred embodiment, the PA according to the invention are used for the treatment of pulmonary embolism, which is accompanied by a haemodynamic instability e.g. due to a cardiopulmonary dysfunction or a shock. Favourably, desmoteplase, even more favourable, DSPA alpha 1 can be used for such an indication.

In a preferred embodiment the PA according to the invention are administered in a single bolus. The bolus advantageously contains 100 to 300 μg/kg body weight of the pharmaceutically effective PA (preferred DSPA alpha 1 ). Particularly preferred is a bolus with 125 μg to 250μg/kg PA, e.g. 180 μg / kg body weight. The total amount can vary between 10 mg and 25 mg, preferably 12.5 mg, 18.5 or 25 mg.

Thus, this invention also pertains to a medicament (for example a vial or ampul) which is manufactured in order to enable drug administration to the patient according to the above mentioned dosages. Accordingly the invention also relates to dosage unit forms containing 10 to 30 mg of a pharmaceutically effective PA (preferred DSPA alpha 1 ), in particular a dosage unit form with 12.5, 18.5 or 25 mg drug substance. I. NON-CLINICAL STUDIES

1. Non-clinical pharmacology

1.1 Efficacy in clot lysis models in vitro

The efficacy of DSPA alpha 1 was studied in different in vitro clot lysis systems (overall clot lysis or thrombolysis including fibrinolysis) and for clot specificity (thrombolysis versus fibrinogenolysis). The studies are summarised in Table 1.

Human whole blood and plasma clots. Human whole blood and plasma clots, over one hour old, were incubated in vitro for 180 minutes with 500 μl of human plasma (at 37°C) in the presence of either DSPA alpha 1 or alteplase.

DSPA alpha 1 and alteplase were approximately equipotent on a molar basis with regard to the lysis of plasma clots. Complete visual plasma clot lysis was observed for both

DSPA alpha 1 and alteplase at concentrations of 30 nmol/l (1.56 and 1.95 mg/l, respectively; mean ± SEM, n = 9), at 39 ± 4 and 44 ± 5 minutes, respectively¹⁷. Whole blood clots were also lysed faster by DSPA alpha 1 than by alteplase. At DSPA alpha 1 concentrations of up to 100 nmol/l and over a period of 180 minutes, plasma fibrinogen levels did not drop significantly. In contrast, incubation with alteplase resulted in significant fibrinogenolysis at concentrations of 30 nmol/l and above.

In a further in vitro system, human whole blood clots (formed around a thread) were incubated in 2 ml of autologous plasma with either DSPA alpha 1 or alteplase for 6 h at 37°C. Lysis was monitored by the reduction in clot weight. DSPA alpha 1 and alteplase were approximately equipotent on a molar basis with respect to the lysis of whole blood clots. A slight, but significantly higher efficacy was obtained with DSPA alpha 1 than compared to alteplase. At the highest plasminogen activator concentration tested (50 nmol/l), fibrinogen, plasminogen and alpha₂-antiplasmin levels decreased by 26%, 17% and 77% for DSPA alpha 1 , and by 97%, 76% and 90% for alteplase, respectively (mean, n = 6).

Rat whole blood clots. Rat whole blood was diluted with acetate buffer, clotted by the addition of thrombin and then incubated with either DSPA alpha 1 or alteplase. In the control samples, the blood clots dissolved spontaneously within 75-95 minutes but in the presence of DSPA alpha 1 , at a concentration of 0.4 nmol/l (21 μg/l), the clot lysis time was reduced by approximately 50%. The activity of DSPA alpha 1 was observed to be approximately equivalent to that of alteplase.

Comparison of DSPA alpha 1 with alteplase. With respect to its clot lysis activity in vitro, DSPA alpha 1 appears to be as potent as, or slightly more potent than alteplase (Table 1 ). At higher concentrations, DSPA alpha 1 continues to maintain its clot specificity, i.e. plasmin formation at the clot surface only. This is in contrast to alteplase, which loses its clot specificity, i.e. causes plasmin formation in plasma, resulting in fibrinogen depletion and degradation of coagulation factors at higher concentrations (fig. 4; the values in fig. 4 are taken from a study in which human clots were incubated in autologous plasma containing DSPA alpha 1 or alteplase.). Thus, at plasma levels equivalent to alteplase, DSPA alpha 1 shows improved efficacy in human patients or fibrinolytic activity in human volunteers and, because of higher clot specificity, a better safety to efficacy ratio with respect to bleeding episodes.

Table 1 Overview of in vitro clot l sis studies with DSPA al ha 1

1.2 Efficacy in animal models of thrombosis

The thrombolytic potency, efficacy and clot specificity (thrombolysis versus fibrinogeno- lysis) of DSPA alpha 1 in vivo were determined in three different animal models of thrombosis, i.e. carotid artery and coronary artery thrombosis in rabbits and dogs, respectively, and a venous thrombosis model (stasis of the rat vena cava combined with thromboplastin infusion). Fibrinolytic efficacy was also studied in experimental lung embolism. The studies are summarised in Table 2. Also pertinent are studies on the effects of DSPA alpha 1 on infarct volume in rabbits and in a rat embolic stroke model.

Rat pulmonary thromboembolism. DSPA alpha 1 was compared to alteplase, in two studies, using a rat model of pulmonary thromboembolism. Radiolabeled, rat whole blood clots were embolised in the lungs of anaesthetised rats. The speed and efficacy of thrombolysis were monitored by measuring blood. In the first study, the total dose of the plasminogen activator was administered lung radioactivity intravenously as a bolus injection. In the second study, 10% of the total dose was given as a bolus injection with the remainder infused over 60 minutes.

In contrast to DSPA alpha 1 , alteplase (administered by either route) induced significant fibrinogenolysis at the highest dose of 100 nmol/kg. Fibrinolysis with alteplase was accompanied by significant plasminogen depletion, whereas alpha₂-antiplasmin levels decreased with both plasminogen activators, albeit more notably with alteplase. DSPA alpha 1 was approximately two to three times more potent on a molar basis than alteplase when administered by either route (fig. 5, 6). The time courses of blood radioactivity indicated a faster rate of lysis with DSPA alpha 1 when administered by either route. Both DSPA alpha 1 and alteplase were more potent when applied by single bolus injection: full thrombolytic efficacy was achieved at 30 nmol/kg DSPA alpha 1 bolus and at approximately 100 nmol/kg DSPA alpha 1 bolus with infusion.

Table 2 Effects of DSPA alpha 1 and alteplase in animal models of thrombosis

Rabbit carotid artery thrombosis. Thrombosis was induced by insertion of a copper coil into the common carotid artery of anaesthetised rabbits. The perfusion state of the artery was monitored by Doppler blood flow measurements. One hour after thrombotic occlusion of the artery, the animals received heparin (200 IU/kg i.v. and i.m.) and aspirin (5 mg/kg i.v.) followed by i.v. bolus injections of either 1 , 2, 4, or 20 nmol/kg DSPA alpha 1 or 2, 6, or 20 nmol/kg alteplase. DSPA alpha 1 effectively reperfused the carotid artery with about three times more potency than alteplase. Alteplase significantly decreased fibrinogen levels at doses of 6 nmol/kg and above, whereas this was not observed for DSPA alpha 1 at any dose level (Table 2). Streptokinase, at dose levels of 3 000, 10 000, and 30 000 IU/kg, was also included in this study. Thrombolysis with this agent was less efficient (2/4, 4/6, and 1/6 animals, respectively) and showed no clear dose-dependency.

Dog coronary artery thrombosis. Thrombosis was induced by insertion of a copper coil into a branch of the left coronary circumflex (LCX) artery of anaesthetised dogs. The perfusion state of the artery was monitored by repeated angiography. Two studies were performed, one with an intravenous bolus injection of the total plasminogen activator dose and a second in which a constant amount (about 10% of the total dose) was given as a bolus injection with the remainder being infused until 15 minutes after successful restoration of blood flow. Data are summarised in Table 2.

In the bolus only study, dogs were heparinised (200 IU/kg i.v. + s.c.) one hour after occlusion of the coronary artery, followed by bolus injection of DSPA alpha 1 (0.48, 0.96, or 1.92 nmol/kg) or alteplase (0.96 or 1.92 nmol/kg). In a further set of animals, 0.96 nmol/kg DSPA alpha 1 was administered without prior heparinisation. All dogs treated with heparin and DSPA alpha 1 (all doses) exhibited recanalisation of their coronary arteries, as opposed to alteplase (2/6 or 3/6 recanalisation) (Table 2). The mean time to reperfusion was shorter with all doses of DSPA alpha 1 than with high or low dose alteplase. Within the observation period of 180 minutes post dosing, the total patency time was longer with DSPA alpha 1 than with either alteplase dose. DSPA alpha 1 administered at a dose of 0.96 nmol/kg without heparin was not efficacious. There was no significant change in the plasma level of alpha₂-antiplasmin in any experimental group.

In the bolus plus infusion study all dogs received heparin (200 IU/kg i.v. and s.c.) one hour after occlusion of the coronary artery, followed by equal doses of DSPA alpha 1 or alteplase (0.77 or 2.31 nmol/kg complemented by 1.54 or 0.46 nmol/kg. min, respectively).

All dogs treated with either dose of DSPA alpha 1 showed reperfusion of their coronary arteries. In one dog receiving low dose alteplase the coronary artery was not reopened.

With low dose DSPA alpha 1 , 83% of the coronary arteries remained patent until the end of the observation period (180 minutes after starting treatment), compared to 40% with low dose alteplase. To summarise, the two fibrinolytics showed marked differences at the lower dose schedule, a persisting reperfusion being obtained in 2/6 and 5/6 animals treated with alteplase and DSPA alpha 1 , respectively. Neither fibrinogen nor plasminogen nor alpha₂-antiplasmin decreased with either dose of DSPA alpha 1 , while high dose alteplase led to a substantial drop in alpha₂-antiplasmin levels (not significant because of small number of animals). DSPA alpha 1 (measured as antigen using a specific enzyme- linked immunosorbent assay, ELISA) had a longer plasma half-life and a lower clearance with respect to alteplase.

Rat venous thrombosis. Venous thrombosis was induced in anaesthetised rats by a short thromboplastin infusion combined with a ligation of the vena cava. One hour after ligation, the vena cava was opened and the thrombus was removed to determine its wet weight. DSPA alpha 1 and alteplase were administered at 1 , 10, or 100 nmol/kg i.v. bolus five minutes prior to vena cava ligation. Both thrombolytic agents significantly reduced thrombus wet weight at 10 nmol/kg and above. DSPA alpha 1 was more potent than alteplase. Due to systemic fibrin formation (which stimulates DSPA alpha 1 and alteplase) in this model, both plasminogen activators induced a similar degree of fibrinogenolysis (Table 2).

Comparison of DSPA alpha 1 with alteplase. DSPA alpha 1 is more potent and, under certain conditions, produces more efficient and rapid lysis than alteplase in rat, rabbit and dog thrombi in vivo (Table 2). At higher doses, DSPA alpha 1 appears to maintain its clot specificity, whereas alteplase evokes plasminogen depletion and degradation of coagulation factors. The in vivo studies confirm the higher clot specificity of DSPA alpha 1 already described in vitro. With regard to potency, DSPA alpha 1 was found to be superior to alteplase, a property most probably related to its prolonged half-life.

1.3 Efficacy in animal models of embolic stroke

The efficacy of DSPA alpha 1 in acute cerebral ischaemia was investigated in rabbit and rat models of embolic stroke.

Rabbit embolic stroke. Cerebral ischaemia was induced by embolization with an autologous whole blood clot (length approximately 1.5 mm) injected into the internal carotid artery of anaesthetised rabbits. Two studies were performed; in the first, the animals received either DSPA alpha 1 (38 nmol/1 ml/kg) or 1 ml/kg of phosphate-buffered saline as an intravenous bolus injection 2 h after embolization (Table 3). In the other study, the rabbits were given either DSPA alpha 1 or phosphate-buffered saline as an intravenous bolus injection 3 h after embolization (Table 4). 26 h after embolization, the surviving animals were sacrificed. The brains were removed, stained with 2,3,5- triphenyltetrazolium chloride (TTC), then fixed and cut in 2 mm thick sections. Infarct volume was evaluated morphometrically.

A single intravenous bolus of DSPA alpha 1 , given 2 h after the induction of thromboembolic stroke, significantly reduced infarct volume as measured 24 h later. Qualitatively similar results were observed when DSPA alpha 1 was given after a delay of 3 h but did not reach statistical significance. DSPA alpha 1 was not associated with significantly increased oedema or increased evidence of intracerebral bleeding in surviving animals. Almost all brains showed what appeared to be haemorrhagic foci external to the infarction areas. The reason for this is unclear at present.

Mortality was higher in the DSPA alpha 1 treated group (38% compared to 9%), although this difference was not statistically significant.

Table 3 Effects of DSPA alpha 1 on infarct volume in rabbits (given 2 h after thromboembolic stroke

Median (Q1 , Q3)* P<0.05 compared to vehicle control (Mann-Whitney Rank Sum Test)

Table 4 Effects of DSPA alpha 1 on infarct volume in rabbits (administered 3 h after thromboembolic stroke

Data were obtained from 10 surviving animals in the vehicle group and 11 rabbits in the DSPA alpha 1 -treated group (the 10 rabbits that survived the entire experimental protocol plus 1 rabbit that died); Median (Q1 , Q3)

Rat embolic stroke. In a rat embolic stroke model, DSPA alpha 1 (96 nmol/kg = 5 mg/kg) given 45 minutes after stroke onset, as a 10% bolus plus 90% infusion over a period of 60 minutes, produced a significant reduction in the ischaemic lesion size in vivo. Post mortem data also indicated a reduction in infarct size. A second independent series of experiments in a rat embolic stroke model was designed to compare the therapeutic efficacy and safety of DSPA alpha 1 , reteplase, and alteplase when administered either 1 or 3 h after embolic middle cerebral artery occlusion in rats.

Male Wistar rats were divided into eight experimental groups and one control group, with a total of 9-15 animals per group. Middle cerebral artery (MCA) occlusion was achieved in male rats by introduction of an autologous blood clot at the base of the right MCA by means of a catheter extended up the right internal carotid artery and entered via the external carotid artery. Drugs were administered 1 or 3 h post occlusion as detailed in Table 5.

MCA₁ middle cerebral artery

Rats were sacrificed 48 h post occlusion. The percentages of right hemisphere infarcts, mortality rates, neurological deficit scores (used for verification of occlusional damage), and the incidence of fatal haemorrhagic transformation were analysed.

In the control group, as determined one hour after embolisation, the mean infarct size was significantly larger than that in each of the drug-treated groups. There were no significant differences between the various drug-treated groups with respect to infarct size (fig. 7).

Rats that survived 48 h were sacrificed and analysed;

Control, control group; rt-PA, alteplase (154 nmol/kg); rtp, reteplase (25.6 nmol/kg); rDSPAalphal , DSPA alpha 1 ; low rDSPAalphal , 10 nmol/kg; med ^• rDSPAalphal , 30 nmol/kg; high rDSPAalphal ,

90 nmol/kg;

* significantly different from control; mean values ± SEM

Rats treated after 3 h had higher mortality rates and a greater incidence of haemorrhagic transformation than controls. Because of the low number of remaining animals, infarct sizes were not analyzed. The rise in mortality was largely due to an increase in fatal haemorrhagic transformation. In the 1 h treatment groups, haemorrhagic events accounted for 0-2 fatalities per group (alteplase, 2/11 ; reteplase, 1/12; low rDSPAalphal , 0/13; medium rDSPAalphal , 0/11 ; high rDSPAalphal , 1/15), whereas the respective numbers were 3/9 (tr-PA), 3/9 (rtp), and 4/9 (DSPA) on treatment 3 h after embolisation. No such changes were seen in control animals (0/12).

Conclusion. The rabbit and rat embolic stroke studies confirm both the efficacy of DSPA alpha 1 and the contention that higher fibrin specificity and selectivity lead to a lower risk of haemorrhagic transformation, which is of particular importance with regard to a reduction of intracranial haemorrhage as catastrophic side effect in the thrombolytic therapy of pulmonary embolism. The latter is supported by the observation that rats receiving reteplase or alteplase 1 h post embolisation experienced occasional haemorrhagic transformation (3/25 rats in the two groups), whereas only 1/42 rats in the three DSPA alpha 1 groups was affected and this was in the highest dose group (90 nmol/kg).

Ischaemia-induced damage to the vasculature results in increased permeability and contributes to the haemorrhagic transformation that is associated with delayed thrombolytic therapy. Alteplase has been shown to degrade the blood brain barrier, thus instigating a further increase in the risk of intracerebral haemorrhage. This has not been studied in the case of either reteplase or DSPA alpha 1. However, in contrast to DSPA alpha 1 , alteplase is susceptible to activation by a variety of molecules including prion protein, beta-amyloid and fibrinogen. Thus, it is feasible that alteplase can exacerbate early ischaemia-induced injury to the blood brain barrier whereas DSPA alpha 1 activity remains more clot-specific. It is noteworthy that the experimental use of thrombin- enriched, as opposed to spontaneously formed clots, may per se promote blood-brain barrier perturbation. While this does not account for the apparent difference between the thrombolytic agents, this observation suggests that the use of thrombolytic agents may be safer than would be predicted from such animal studies.

The rise in mortality after delayed treatment, which was seen with each of the thrombolytic agents studied, can be attributed to haemorrhagic transformation. There was a higher incidence of haemorrhagic transformation in the 3 h post embolisation groups than in the 1 h or control groups. It is important to note that alteplase and reteplase were administered at the doses found to be effective 1 h after occlusion. DSPA alpha 1 was given at a dose of 90 nmol/kg, which would appear to be higher than that actually required. Thus, a 9-fold lower dose of 10 nmol/kg, that was shown to be equally effective in the 1 h post occlusion setting, might be better tolerated in the delayed treatment setting. 1.4 Specificity of activation

There is consensus that high fibrin specificity is an essential factor in tolerability of plasminogen activators. In addition, unfolding evidence indicates that activation by non- fibrin molecules is also an issue not only with fibrinogen as a possible stimulant, but also with molecules such as beta-amyloid and prion protein.

1.4.1 Fibrin specificity

In vitro studies on the kinetics of plasminogen activation showed that DSPA alpha 1 , without a stimulating cofactor, was virtually devoid of plasminogen-activating activity, whereas rt-PA activated plasminogen to substantial extents, being 260-fold more active than DSPA alpha 1. In the presence of fibrin, DSPA alpha 1 activity was 105 000 times higher than without, while rt-PA, owing to its intrinsic, fibrin-independent activity, only displayed a 550-fold increase.

For a further demonstration of DSPA alpha 1 's fibrin specificity, fibrin and casein zymographic experiments were performed. These experiments involved electrophoresing increasing concentrations of rt-PA or DSPA on SDS-PAGE and overlaying the gels onto either a fibrin or a casein matrix containing plasminogen. As shown in fig. 8, rt-PA activity was readily detected on both fibrin and casein substrates (fig. 8, panels A and B). DSPA alpha 1 was clearly detectable on a fibrin matrix (fig. 8, panel A, lanes 6-9), but was only seen on a casein matrix at the highest concentration used (100 nmol/l; fig. 8, panel B, lane 9). The discrepant fibrin specifies of the two thrombolytics are also evident from the study covered under 4.1.4.2 (see fig. 8).

1.4.2 Fibrinogen and fibrin degradation products

In vitro clot lysis studies were performed on whole human blood clots. Clots were generated in vitro, aged for 1 h, and placed in autologous plasma in the presence of varying concentrations of either rt-PA or DSPA alpha 1. The thrombolytic potential of the two plasminogen activators was determined after a 6 h incubation. While the thrombolytic profiles were very similar, fibrinogen degradation was negligible with DSPA alpha 1 , but substantial with rt-PA. Sensitivity to fibrinogen activation may be defined as fibrin selectivity and expressed as the ratio of activity in the presence of fibrin over activity in the presence of fibrinogen. The resultant values for DSPA alpha 1 and rt-PA are 12 900 and 72, respectively. The differences in fibrin selectivity are consistent with the study covered under 4.1.4.3 (see fig. 9). 1.4.3 beta-amyloid

Alteplase has been characterised as a multiligand receptor; it can be activated by a number of non-fibrin molecules including fibrinogen, prion protein, and amyloid peptides. For a further characterisation of DSPA alpha 1 , the efficacies of fibrin, fibrinogen and amyloid beta (1-42) as cofactors for the activation of alteplase and DSPA alpha 1 were determined in vitro by the absorbance change produced by plasminogen activation in the presence of a chromogenic plasmin substrate.

There was no difference in activity when fibrin was added as a cofactor. Moreover, as illustrated in fig. 8, the advantage of DSPA alpha 1 in terms of fibrin specificity (activity without any cofactor) and selectivity (activity in the presence of fibrinogen) was confirmed. Most interestingly, DSPA alpha 1 was 30-fold less efficient than alteplase in the presence of beta-amyloid. This difference may be clinically relevant: in an analysis of 23 patients with intracerebral haemorrhage in association with alteplase/heparin treatment for acute myocardial infarction, pathology findings were available for five patients; of these, three patients displayed cerebral amyloid angiopathy.

Normalised against value in DSPA alpha 1 control experiments (no cofactors)

1.4.4 Prion protein

Comparative efficacies of alteplase and DSPA alpha 1 in the presence of prion protein are available from two studies. In the first study, 200 nM PrP23-110 stimulated alteplase and DSPA alpha 1 , both at a concentration of 1.8 nmol/l, by factors of 43 and 1.2, respectively. Similar results were obtained in the second study, in which two different prion protein preparations were used. Stimulation of alteplase activity was 89- and 250-fold, in contrast to DSPA alpha 1 , which showed a negligible increase (factor 10) or even a nominal decrease (factor 0.6). In summary, there is no or only negligible stimulation of DSPA alpha 1 by prion protein, whereas alteplase activity is substantially enhanced.

1.4.5 Conclusions

Although DSPA alpha 1 and rt-PA share a 70% structural homology, their proteolytic activities differ in clinically relevant aspects.

In practical terms, this means that the action of DSPA alpha 1 is fibrin sensitive and its activity is not increased by the presence of fibrinogen or fibrin degradation products. Thus, the key pharmacological characteristics of DSPA alpha 1 are its high fibrin specificity and selectivity. As a result, DSPA alpha 1 mainly initiates local fibrinolysis without the systemic activation that would lead to fibrinogen consumption and thus, a higher risk of bleeding episodes. In this context, it may be pertinent to note that tenecteplase exceeds rt-PA in fibrin specificity by a factor of ~ 10.

II. Clinical studies

DSPA alpha 1 was investigated in a clinical phase Il study in the indication of massive pulmonary embolism. The safety, tolerability, and efficacy of single intravenous administration of DSPA alpha 1 at doses of 125, 180, and 250 microg/kg compared with alteplase 100 mg in subjects with acute massive pulmonary embolism was assessed.

The main efficacy parameter was reperfusion measured by reduction in total pulmonary resistance (TPR), mean pulmonary artery pressure (mPAP), and Miller index. The reductions in TPR, mPAP, and Miller index were assessed by the relative change from baseline (percentage change) defined as (actual value - baseline) / baseline x 100.

The main safety parameters were proportions of patients with: Major bleedings including all ICH events, ICH events and serious adverse events.

Summary:

Efficacy endpoints included TPR, mPAP, and Miller Index. The efficacy endpoints showed improvements with each DSPA alpha 1 treatment group starting as early as 2 h after drug administration and reaching full effect after 6 h. This does not apply for alteplase, since its effects continued to improve with maximum effects observed after 24 h. With regard to

DSPA alpha 1 , these effects were dose related and changes in the DSPA alpha 1

180 μg/kg and 250 μg/kg dose groups. Mostly, they were greater than those observed with alteplase in most analyses.

The overall differences in the qualitative assessments of the Miller Index between any of the treatment groups were unremarkable, irrespective of local or central assessment. An additional approach using "overall response criteria" (composite of decrease in TPR, mPAP by at least 40% from baseline with or without normalization and decrease in Miller Index by at least 40% from baseline) did not yield different overall results.

In subjects with acute pulmonary embolism, administration of single-dose, 1- to 2-min intravenous injections of DSPA alpha 1 at doses of 125, 180, and 250 μg/kg BW showed dose-related improvements in invasively monitored hemodynamic parameters, especially with regard to mPAP. With regard to most analyses, effects observed with DSPA alpha 1 180 μg/kg and 250 μg/kg were similar or greater than those observed with alteplase. The effects of DSPA alpha 1 established more rapidly than those of alteplase.

The overall incidence rate of treatment-emergent adverse events ranged between 66.7% (DSPA alpha 1 180 μg/kg) and 100% (alteplase).

TPR

Table Fehler! Kein Text mit angegebener Formatvorlage im Dokument.-1 : Total pulmonary resistance - relative changes from baseline after 2, 6, 12, and 24 h (LOCF; mean ± SD; ITT population; n=34)

Desmote- Desmote- Desmote- Alteplase plase plase plase 100 mg

125 μg/kg 180 μg/kg 250 μg/kg (n=12)

BW BW BW

(n=7) (n=9) (n=6)

Change from baseline at 2 h -6.5 + 27.2% -33.0 ± 19.9% -32.9 ± 26. ,5% -33 .8 + 22.0%

Change from baseline at 6 h -24.9 ± 26.0% -23.9 + 23.3% -42.2 ± 29. .1% -33 .3 + 39.3%

Change from baseline at 12 h -30.3 + 27.1 % -30.3 ± 27.5% -43.2 ± 33. ,1% -33.9 ± 41.4%

Change from baseline at 24 h -38.5 ± 21.4% -48.0 + 22.4% -56.0 ± 29.4% -50. .4 + 16.3%

mPAP

Table Fehler! Kein Text mit angegebener Formatvorlage im Dokument.-2: Mean pulmonary artery pressure - relative changes from baseline after 2, 6, 12, and 24 h (LOCF; mean ± SD; ITT population; n=34)

Desmote- Desmote- Desmote- Alteplase plase plase plase 100 mg

125 μg/kg 180 μg/kg 250 μg/kg (n=12)

BW BW BW

(n=7) (n=9) (n=6)

Change from baseline at 2 h -5.6 ± 21 .9% -27.9 ± 14.3% -30.4 ± 20 .5% -29.6 + 21. .8%

Change from baseline at 6 h -16.8 ± 14 .9% -28.7 + 23.0% -40.1 + 18.0% -27.1 ± 38.1%

Change from baseline at 12 h -19.8 + 12.6% -37.0 ± 14.6% -42.8 ± 19 .3% -34.5 ± 35 .2%

Change from baseline at 24 h -17.7 ± 12 .6% -43.9 + 16.8% -47.1 ± 20 .5% -42.3 ± 13 .1 % Miller Index

Table Fehler! Kein Text mit angegebener Formatvorlage im Dokument.-3: Miller Index - absolute and relative mean changes from baseline between 2 and 12 h (mean ± SD; ITT population; n=34)

Desmote- Desmote- Desmote- Alteplase plase plase plase 100 mg

125 μg/kg 180 μg/kg 250 μg/kg (n=12)

BW BW BW

(n=7) (n=9) (n=6)

Local assessment

Baseline 22.0 + 3.0 22.9 ± 2.8 20.5 ± 2.5 22.9 ± 3.7

Absolute change from -3.0 + 2.8 -5.4 ± 3.8 -7.2 ± 4.7 -8.9 ± 5.5 baseline between 2 to 12 h

Relative change from -14.1 ± 13.9% -23.6 ± 15.6% -35.0 ± 21.7% -41.6 ± 27.2% baseline between 2 to 12 h

Central assessment

Baseline 21.3 + 6.8 14.2 ± 6.8 15.2 ± 6.8 19.8 ± 8.1

Absolute change from -2.8 + 4.0 -3.0 ± 4.8 -7.0 ± 9.8 -6.6 ± 6.0 baseline between 2 to 12 h

Relative change from -10.8 ± 19.8% -12.3 ± 23.8% -28.1 ± 56.9% -30.5 ± 32.0% baseline between 2 to 12 h

Brief description of the legend

Fig. 1 Shows the structure of DSPA alpha 1

Fig. 2 Shows the structure of alteplase

Fig. 3 Represents the amino acid sequence of DSPA alpha 1.

Fig. 4 Fibrinogen levels after 180 minutes incubation with either DSPA alpha 1 or alteplase

Fig. 5 Thrombolytic effects of DSPA alpha 1 and alteplase administered as a bolus in pulmonary thromboembolism in the rat

Fig. 6 Thrombolytic effects of DSPA alpha 1 and alteplase administered as an infusion in pulmonary thromboembolism in the rat

Fig. 7 Effect of thrombolytic agents on right hemisphere percentage occupied by infarct

Fig. 8 Discrepant fibrin specificities of DSPA alpha 1 and rt-PA in zymographic experiments

Fig. 9 Plasminogen activation by alteplase and DSPA alpha 1 in the presence of various cofactors

Claims

Claims

1. Use of a plasminogen activator for the manufacture of a medicament for the treatment of venous thromboembolism, wherein the plasminogen activator has an at least more than 550 fold increased activity in the presence of fibrin compared to the activity without fibrin and is administered to the patient with a dosis of 100 to 300 μg/kg body weight, in particular 125, 180 or 250 μg/kg.

2. Use of a plasminogen activator of claim 1 , wherein a total amount of 10 to 30 mg plasminogen activator, preferably 12.5, 18.0 or 25.0 mg, is administered to the patient.

3. Use of a plasminogen activator according to claims 1 and 2, wherein the plasminogen activator i. is essentially non-activatable by beta-amyloid and/or prion protein and/or ii. is non-neurotoxic and/or iii. has a half-life of at least more than 2.5 min.

4. Use of a plasminogen activator according to one of the above claims, wherein the plasminogen activator has an at least more than 5500 fold increased activity in the presence of fibrin compared to the activity without fibrin and has a half-life of at least more than 50 min.

5. Use of a plasminogen activator according to one of the above claims, wherein the plasminogen activator is desmoteplase.

6. Use of a plasminogen activator according to one of the above claims, wherein the plasminogen activator i. has an amino acid sequence according to fig. 3 or microheterogeneous forms thereof or ii. is at least 90 %, more preferred 95 %, even more preferred 98 % identical to the amino acid sequence of fig. 3.

7. Use of a plasminogen activator according to one of the above claims, wherein the venous thromboembolism is deep venous thrombosis or pulmonary embolism, in particular massive pulmonary embolism.

8. Use of a plasminogen activator according to one of the above claims, wherein the patients have an increased risk of bleeding.

9. Use of a plasminogen activator according to one of the above claims, wherein the patients have at least one of the risk factors as follows: known haemorrhagic diathesis, receipt of oral anticoagulants, manifest or recent severe or dangerous bleeding, known history of or suspected intracranial haemorrhage, suspected subarachnoid haemorrhage or condition after subarachnoid haemorrhage from aneurysm, any history of central nervous system damage (i.e. neoplasm, aneurysm, intracranial or spinal surgery), haemorrhagic retinopathy, e. g. in diabetes (vision disturbances may indicate haemorrhagic retinopathy), recent (less than 10 days) traumatic external heart massage, obstetrical delivery, recent puncture of a non-compressible blood-vessel (e.g. subclavian or jugular vein puncture), severe uncontrolled arterial hypertension, bacterial endocarditis, pericarditis, acute pancreatitis, documented ulcerative gastrointestinal disease during the last 3 months, oesophageal varices, arterial-aneurysm, arterial/venous malformations, neoplasm with increased bleeding risk, severe liver disease, including hepatic failure, cirrhosis, portal hypertension (oesophageal varices) and active hepatitis, major surgery or significant trauma in past 3 months, any history of stroke.

10. Use of a plasminogen activator according to one of the above claims, wherein the medicament is administered as a single bolus.