DE202006015207U1 - Use of plasminogen activator for the preparation of medicament to treat vein thromboembolism, where the plasminogen activator in presence of fibrin exhibits an increased activity in comparison with the activity without fibrin - Google Patents

Abstract

Use of plasminogen activator for the preparation of medicament to treat vein thromboembolism, where the plasminogen activator in presence of fibrin exhibits an increased activity of at least around 550 folds in comparison with the activity without fibrin. ACTIVITY : Thrombolytic. MECHANISM OF ACTION : None given.

Description

The Invention relates to a method for the treatment of venous thromboembolism, in particular pulmonary embolism, and pharmaceutical compositions for use in these methods.

venous thromboembolism (VTE) is the common one Designation for two related diseases, namely deep vein thrombosis (DVT) and pulmonary embolism (PE), both of which are serious medical conditions is the result of abnormal blood clots. The Incidence of VTE varies in different patient populations strong depending on the presence or absence of risk factors. All in all is the incidence estimated 1.5 to 2 cases per 1000 patients, but in populations high risk, e.g. Patients undergoing a major orthopedic Have to undergo surgery, it is much higher. These patients are at 50% risk of developing from VTE.

in view of the big Number of affected patients and potentially fatal Nature of VTE makes the treatment of this condition both financially as well as otherwise put a huge strain on health systems The treatment of VTE may be in prophylaxis and acute treatment when prophylactic failed or not become.

A Deep vein thrombosis itself can cause chronic symptoms with pain and reduced mobility cause, but more dangerous is their potential to cause a pulmonary embolism. The diagnosis the deep vein thrombosis depends strongly of clinical suspicion and the individual experience of the Physician with regard to the evaluation of laboratory results and imaging Examinations. This uncertainty in the diagnosis makes the deep vein thrombosis to an even more threatening condition. newer Technological advances have improved diagnostic options guided. In the most urgent yet unmet needs of the Treatment of deep vein thrombosis will be more appropriate and more efficient diagnostic tests called.

depth Venous thromboembolism (deep vein thromboembolism or deep vein thrombosis) is defined as a clinical condition resulting from a blood clot in the deep venous circulation, most often in the calf or in the Thighs, results. The clot does not just cause pain, Swelling and redness, It can also be released from the leg veins and through the bloodstream to the right Side of the heart and lungs where there is a pulmonary embolism trigger can.

A Pulmonary embolism (PE) is the sudden Closure of the pulmonary artery or one of its branches, the most common resulting from a blood clot. A pulmonary embolism is a medical one Emergency, irreversible damage in the lungs and can lead to death, even if one appropriate therapy takes place.

at About 50% of patients with pulmonary embolism, the embolism is massive, i.e. she goes with at least one of several possible ones other states associated. These include Hypotension or shock, severe hypoxemic respiratory failure, acute Right ventricular dysfunction or obstruction of the pulmonary vessels over 50%, demonstrated by angiogram or ventilation / perfusion recording (V / Q scan). A strong / massive pulmonary embolism can thus be defined as pulmonary embolism with haemodynamic instability goes hand in hand. It is assumed that this instability is the result an interaction between size of the embolism and the underlying cardiopulmonary function.

The Presence of compromising factors, in particular shock - and thus the occurrence of a massive pulmonary embolism - defines a threefold increased up to seven times Mortality, with most deaths within an hour of presentation (onset of symptoms) enter. A quick integration of historical information of the patient and the physical Findings in easily available Laboratory data in a structured physiological approach for the Diagnosis and revival are optimal in this "golden hour" Therapies necessary.

Even though deep vein thrombosis in medical circles as serious Condition with potentially fatal social consequences is known - is pulmonary embolism still the third most common cardiovascular Cause of death with a mortality rate between 20% and 30% - one effective treatment is not yet available. An inadequate Treatment involves many risks, including renewal Onset, post-thrombotic syndrome, progression from deeper Vein thrombosis to pulmonary embolism and death.

It There are several possibilities for the treatment of deep vein thrombosis and pulmonary embolism. A is the administration of unfractionated heparin (UFH). Other focus on the administration of orally available anticoagulants or the administration of low molecular weight heparin (LMWHS). A different possibility is the thrombolysis of the blood clot.

It But it is important to remember that the therapy of venous thromboembolism own risks are most notable Bleeding complications, and the consequences of too ambitious Treatments are at least as dangerous as those of undersupply. A safe and effective treatment thus requires competent and experienced doctors, the most up-to-date knowledge of current guidelines and protocols are and access to all therapeutic alternatives to have.

It It is known that thrombolysis in pulmonary embolism quickly reverses of right heart failure leads. Alteplase by intravenous Infusion is one of the few approved thrombolytic agents in this indication area. The use of thrombolytics at Patients with pulmonary embolism remains very controversial. The clinical Studies done were not concerned with survival as an endpoint, but results such as reduction of blood clot load, V / Q scan results, Improvements in capillary blood flow and other pulmonary function tests measured. Although these studies have the superiority of thrombolytics over UFH shown in terms of long-term lung function, but that Long-term survival must be judged to medical circles from the benefits of thrombolytic Therapy for Convince patients with pulmonary embolism.

To Today, the evidence lies in favor of the efficacy of thrombolysis in patients with pulmonary embolism on the scales. Most clinical However, studies and registries have an increased incidence of heavy bleeding shown, including an increase in the incidence of catastrophic intracranial hemorrhage. Therefore, the risk of bleeding is to the disadvantage of thrombolytic Therapy. Indeed is it the heightened Bleeding risk that is routine use of thrombolytic agents in patients with pulmonary embolism.

significantly is also that there is still a discrepancy between the perceived and actual Treatment window for Thrombolytics in patients with pulmonary embolism. Many doctors believe that Thrombolysis is only effective if it is relatively soon after Onset of symptoms is administered, although specialists on The area of pulmonary embolism of modern view are that Time window for effective treatment of pulmonary embolism is up to 14 days.

in the Contrary to the ones mentioned above anticoagulant therapies that reduce the formation of blood clots prevent, promote Thrombolytics the dissolution the blood clot after their formation. Thrombolytics are plasminogen activators (PA), the plasminogen, the inactive proenzyme of fibrinolytic Systems in the blood, convert to the proteolytic enzyme plasmin, the dissolves the fibrin of a blood clot into soluble degradation products. On exhaust that way Thrombolytics are the stores of protein that serve as scaffolding for the arrangement and consolidation of a clot and thus the risk of bleeding elevated.

The optimal profile of a thrombolytic agent is therefore related to its Fibrinselektivität, the due to low systemic plasminogen and thus fibrinogen consumption is shown. The fibrin selectivity is an established clinical one Parameters in the assessment of the risk for potential bleeding complications: The higher the fibrin specificity, the lower the risk. This has meant that thrombolytics or Plasminogen activators as "double-tailed Sword "viewed become.

by virtue of their high cost and potential for increasing the risk of bleeding Thrombolytics are rarely routinely used to treat deep Used for venous thrombosis, except in patients with massive extensive thrombosis. In addition to the dissolution of the clot to prevent a later one Pulmonary embolism can Thrombolytics relieve the pain and swelling and the loss reduce the venous valves, thereby reducing the incidence of postthrombotic Syndrome is reduced. Although more research is needed Studies indicate that the application of thrombolytic therapy in patients with fresh massive deep vein thrombosis the Incidence of occurrence of pulmonary embolism may decrease.

The However, known thrombolytic therapy is on patients without contraindications limited. Contraindications are e.g. active internal bleeding or a recent one Surgery or other vascular incidents in the last few months.

The benefits of rapid reversal of right heart failure must, of course, be weighed against the risks of heavy bleeding, and most intracranial bleeding. Although rt-PA is the only approved modern thrombolytic regimen for pulmonary embolism, other agents have been shown to be effective in limited studies. These agents are all administered in high concentration over a period of time. Promising treatments that have been studied include the two-hour infusion of urokinase and the one to two hour infusion of streptokinase in addition to pilot studies with pro-urokinase (recombinant single-chain urokinase-type plasminogen activator, rscu-PA) and staphylokinase. Furthermore, reteplase was also studied in a "double bolus" study comparing efficacy and safety with the approved two-turn scheme of rt-PA, a summary of these regimens is given below (Source: Goldhaber: American Heart Journal 1999, page 2 (Editorial):

• 1. die Rate unerwünschter klinischer Ereignisse;
• 2. den Umfang von Veränderungen gegenüber der Ausgangswerterhebung in Perfusionslungen-Scans, Lungenangiogrammen oder Echokardio-grammen; und
• 3. die Unterschiede in den Gerinnungsparametern im Zeitverlauf.

Another study assessed the hypothesis that a reduced bolus dose of rt-PA (0.6 mg / kg / 15 min, maximum 50 mg) would result in fewer bleeding complications than the standard dose of 100 mg rt-PA when administered as a continuous infusion over two hours in haemodynamically stable patients with pulmonary embolism. Secondary objectives were the comparison of the two rt-PA schemes with regard to

• 1. the rate of adverse clinical events;
• 2. the extent of changes from baseline in perfusion lung scans, lung angiograms or echocardiograms; and
• 3. the differences in the coagulation parameters over time.

These However, study showed no significant differences between bolus rt-PA and two-hour rt-PA with regard to bleeding complications, undesired clinical Events or imaging studies. It was only found less fibrinogenolysis under a bolus dosing regimen. The bolus regime was therefore not considered advantageous (Goldhaber SZ, Agnelli G., Levine MN: Reduced Dose Bolus Alteplase Vs. Conventional alteplase Infusion for Pulmonary Embolism Thrombolysis. To International Multicenter Randomized trial. CHEST, 106, September 3, 1994, pages 718-724).

There not even the decreased bolus protection against hemorrhagic complications was able to confer a prevention of a proteolytic plasma state perhaps not related to the prevention of bleeding. It is therefore for important, possible Patients to a close examination for possible contraindications too undergo.

• – Bekannte hämorrhagische Diathese
• – Patienten unter oralen Antikoagulanzien, z.B. Warfarin-Natrium
• – Bestehende oder frische schwere oder gefährliche Blutungen
• – Bekannte Vorgeschichte oder Verdacht auf intrakranielle Blutungen
• – Verdacht auf subarachnoidale Blutung oder Zustand nach subarachnoidaler Blutung aus einem Aneurysma
• – Jede zentralnervöse Schädigung in der Vorgeschichte (d.h. Neoplasien, Aneurysmen, intrakranielle oder spinale Operation)
• – Hämorrhagische Retinopathie, z.B. bei Diabetes (Sehstörungen können auf hämorrhagische Retinopathie hinweisen)
• – Neuere (weniger als 10 Tage alte) traumatische externe Herzmassage, Entbindung, neuere Punktion eines nichtkomprimierbaren Blutgefäßes (z.B. V. subclavia oder jugularis)
• – Starker unkontrollierter arterieller Bluthochdruck
• – Bakterielle Endokarditis, Perikarditis
• – Akute Pankreatitis
• – Dokumentierte ulzerative gastrointestinale Erkrankung in den letzten 3 Monaten, Ösophagusvarizen, Arterienaneurysma, arterielle/venöse Fehlbildungen
• – Neoplasie mit erhöhtem Blutungsrisiko
• – Schwere Lebererkrankung, einschließlich Leberinsuffizienz, Zirrhose, Portalhypertonie Ösophagusvarizen) und aktive Hepatitis
• – Große Operation oder signifikantes Trauma in den letzten 3 Monaten
• – Jeder Schlaganfall in der Vorgeschichte.

According to the product characteristics provided by EMEA for Aktilyse, the following contraindications to the administration of rt-PA in patients with deep vein thrombosis are:

• - Known hemorrhagic diathesis
• - Patients on oral anticoagulants, eg warfarin sodium
• - Existing or fresh severe or dangerous bleeding
• - Known history or suspicion of intracranial hemorrhage
• - Suspected subarachnoid hemorrhage or condition after subarachnoid hemorrhage from an aneurysm
• Any history of central nervous system damage (ie neoplasia, aneurysm, intracranial or spinal surgery)
• - Hemorrhagic retinopathy, eg in diabetes (blurred vision may indicate haemorrhagic retinopathy)
• - Newer (less than 10 days old) traumatic external cardiac massage, childbirth, newer puncture of an incompressible blood vessel (eg V. subclavia or jugularis)
• - Severe uncontrolled arterial hypertension
• - Bacterial endocarditis, pericarditis
• - Acute pancreatitis
• - Documented ulcerative gastrointestinal disease in the last 3 months, esophageal varices, Ar retinal aneurysm, arterial / venous malformations
• - neoplasia with increased risk of bleeding
• - Severe liver disease, including hepatic insufficiency, cirrhosis, portal hypertension, esophageal varices) and active hepatitis
• - Major surgery or significant trauma in the last 3 months
• - Every stroke in the history.

For the purpose the present invention, these contraindications are under the term "bleeding risk factors".

It is therefore an object of the present invention an improved Funds for a thrombolytic therapy of venous thromboembolism, in particular Pulmonary embolism and deep vein thrombosis and especially massive To provide pulmonary embolism.

These The object is achieved by using a plasminogen activator for treatment solved by venous thromboembolism, which is not substantially beneficial by beta-amyloid or prion protein is activatable and in the presence of fibrin improved activity by more than 550 times, preferably more than 5500 times and completely more preferably more than 10,000 times as compared with the activity in the absence of fibrin. In a particularly preferred embodiment is the increase in activity of PA in the presence of fibrin in comparison with the activity in the absence of fibrin more than 100,000. As the increase in the activity of rt-PA 550, have the most preferred PA for use according to the invention about 180 to 200 times higher Fibrin specificity / selectivity as rt-PA.

The preferred PA according to the invention are non-neurotoxic and have a half-life of over 2.5 Min, preferably over 50 min, especially over 100 min. Neurotoxicity can be assessed by methods known to those skilled in the art, e.g. with animal models in particular kainsic acid models as detailed in International Publication WO 03/037363.

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

In a preferred embodiment DSPA alpha 1 is administered to the patient as an intravenous single bolus, i.e. without further administration / infusion. A bolus is defined as intravenous Administration of the drug in a short period, i. in less than 5 minutes, preferably at most 2 minutes. In one embodiment becomes DSPA alpha 1 over Administered for 1 to 2 min.

If suitable is the administration of DSPA alpha 1 from the administration of anticoagulants, e.g. Heparin or acetylsalicylic acid, accompanied be. These medications can to the patient as intravenous Infusion after the bolus of DSPA alpha 1 can be administered.

DSPA Alpha 1 is a plasminogen activator, originally derived from the saliva of Desmodus rotundus was isolated (Desmodus salivary plasminogen activator). In saliva, four variants of DSPA were isolated, similar to Alteplase and urokinase consist of different conserved domains, previously established in related families of proteins. The variants rDSPA alpha 1 and rDSPA alpha 2 show the structural formula Finger (F), epidermal growth factor (EGF) (E), kringle (K), Protease (P) while rDSPA beta and rDSPA gamma are identified by the formulas EKP and KP, respectively are.

light Sequence differences and data from Southern blot hybridization analysis point out that the four enzymes of four different genes and not by different splicing of a single one primary Transcripts are generated.

The Variant DSPA alpha 1 has at least 70% structural Homology to alteplase; the difference is that alteplase owns two Kringles (FEKKP) while DSPA alpha 1 only has one (FEKP). DSPA alpha 1 is a serine protease with a molecular weight of 52 KD and 441 amino acids. As other plasminogen activators (PA) activate DSPA alpha 1 plasminogen by catalyzing the conversion of plasminogen to plasmin, thereby in turn, the cross-linked abundant fibrin in blood clots is reduced.

DSPA Alpha 1 has, in comparison with other thrombolytic agents, in particular with alteplase, unique features. These include the high specificity and selectivity for plasminogen bound Fibrin, low fibrinogen specificity, no neurotoxicity and negligible Activation by beta-amyloid and human cellular prion protein, additionally to a long dominant half-life of more than 2 hours. All these properties give a better safety and pharmacological Profile for use in the clinical setting, for the thrombolytic agents currently not yet accessible are.

DSPA alpha 1 has been extensively studied in vitro and in vivo, with different thrombosis models and preferably alteplase as reference substance were used. In these studies, the effect of DSPA was alpha 1 alteplase equal or better, and it showed shorter lysis times and lower Reokklusionsraten. Cheap pharmacokinetic properties seem to be an important contributor to the higher effect because DSPA alpha 1 has a lower overall clearance and a longer one terminal half-life has as alteplase (see above).

The means that the administration of a single bolus over a short period possible is. This is considered a significant advantage in clinical use of thrombolytics, especially in the emergency room or even in the Ambulance.

animal studies in vivo have confirmed that the administration of DSPA alpha 1 with an apparent reduction the number of hemorrhagic Events go hand in hand. About that translated the negligible sensitivity DSPA alpha 1 and activation by beta-amyloid and prion protein, those in study in vitro with comparison of DSPA alpha 1 and alteplase was observed in a decreased incidence of intraparenchymal Bleeding in the elderly Patients.

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

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

In accordance with the invention, the term "desmoteplase" is used for any plasminogen activator having identical or substantially the same biological activity as DSPA alpha 1 with respect to the activation of plasminogen and its better fibrin selectivity / specificity Preferably, the fibrin selectivity is at least 180 compared to rt-PA The PAs defined as desmoteplase according to the invention are preferably at least 80 or 90%, in particular at least 95% and very particularly preferably at least 98% identical to the amino acid sequence in FIG 3 (DSPA alpha 1). The plasminogen activators may contain microheterogeneities, for example with respect to glycosylation and / or N-terminal variations, which are solely due to production systems.

According to one embodiment The invention relates to the plasminogen activators for the treatment of patients with venous thromboembolism, especially pulmonary embolism or massive Pulmonary embolism is used in which a higher risk compared with other patients with venous thromboembolism. This patient group by definition suffers at least a bleeding risk factor as defined above.

In a preferred embodiment are the PA of the invention for the treatment used by pulmonary embolism associated with hemodynamic instability, e.g. due to cardiopulmonary dysfunction or shock. Advantageous can Desmoteplase and more favorably DSPA alpha 1 for this indication be used.

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

I. NON-CLINICAL STUDIES

1. Non-clinical pharmacology

1.1 Efficacy in clot lysis models in vitro

The Efficacy of DSPA alpha 1 has been demonstrated in various clot lysis systems in vitro (Total clot lysis or thrombolysis with fibrinolysis) and clot specificity (thrombolysis versus fibrinogenolysis). The investigations are in table 1 summarized.

Human Whole blood and plasma clot. Human thoroughbred and plasma clot, the above were one hour old, were in vitro for 180 minutes with 500 .mu.l of human Plasma (at 37 ° C) in the presence of either DSPA alpha 1 or alteplase.

DSPA alpha 1 and alteplase were on a molar basis with respect to lysis of plasma clots about equally effective. A complete visual plasma clot lysis was reported for both DSPA alpha 1 and alteplase at concentrations of 30 nmol / l (1.56 and 1.95 mg / l, mean ± SEM, n = 9) after 39 ± 4 or 44 ± 5 Minutes observed. Whole blood clots were identified by DSPA alpha 1 also resolved faster as by alteplase. At concentrations of DSPA alpha 1 from to to 100 nmol / l over for a period of 180 minutes, plasma fibrinogen levels fell not significant. In contrast, led Incubation with alteplase to a significant fibrinogenolysis at concentrations of 30 nmol / l and above.

In another in vitro system, human whole blood clots (which had formed around a suture) 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 reduction in clot weight. DSPA alpha 1 and alteplase were approximately equally effective on a molar basis with respect to lysis of whole blood clots. With DSPA alpha 1, a slight but significantly higher efficacy was found in comparison with alteplase. At the highest concentration tested of the plasminogen activator (50 nmol / l) increased the fibrinogen, plasminogen and alpha _{2-antiplasmin} levels by 26%, 17% and 77% for DSPA alpha 1 and by 97%, 76% and 90% off Alteplase (mean, n = 6).

full blood clots of rats. Whole blood from rats was diluted with acetate buffer, by Add from Thrombin coagulated and then with DSPA alpha 1 or alteplase incubated. In the control samples, the blood clots dissolved spontaneously within 75-95 Minutes, but in the presence of DSPA alpha 1 at one concentration of 0.4 nmol / l (21 μg / l) the clot lysis time was reduced by about 50%. The activity of DSPA alpha 1 was approximately equivalent to alteplase.

Comparison of DSPA alpha 1 with alteplase. With respect to clot lysis activity in vitro, DSPA alpha 1 appears to be as effective as or somewhat more effective than alteplase (Table 1). At higher concentrations, DSPA alpha 1 retains its clot specificity, ie, plasmin formation occurs only at the surface of the clot. This differs from alteplase, which loses its clot specificity, ie, causes plasma plasmin formation, resulting in fibrinogen depletion and degradation of coagulation factors at higher concentrations ( 4 ; the values in 4 come from a study in which human clots in autologous plasma were incubated with DSPA alpha 1 or alteplase). Thus, at plasma levels similar to alteplase, DSPA alpha 1 shows improved efficacy in human subjects or fibrinolytic activity in subjects and, due to the higher clot specificity, a better safety / efficacy ratio for bleeding episodes. Table 1 Overview of clot lysis studies in vitro with DSPA alpha 1

1.2 Efficacy in animal models with thrombosis

The thrombolytic effect, efficacy and clot specificity (thrombolysis versus fibrinogenolysis) of DSPA alpha 1 in vivo were evaluated in three different animal models with thrombosis, i. thrombosis the carotid and coronary artery in rabbits and dogs and a venous thrombosis model (Stasis of rat vena cava in combination with thromboplastin infusion). In addition, the fibrinolytic activity became more experimental Pulmonary embolism examined. The investigations are summarized in Table 2. Also useful are studies on the effects of DSPA alpha 1 on the infarct volume of rabbits and in an embolic stroke model in rats.

pulmonary thromboembolism in rats. DSPA alpha 1 was used in two studies with a Rat model of pulmonary thromboembolism compared to rats. radioactive labeled whole blood clots of rats were in the lungs of Anesthetized rats are embolized. The speed and effectiveness Thrombolysis was monitored by blood measurement. In the first investigation the total dose of plasminogen activator was given intravenously as a bolus injection administered to the lungs. In the second study, 10% the total dose administered as a bolus injection during the Rest over Was infused for 60 minutes.

Tabelle 2 Wirkungen von DSPA alpha 1 und Alteplase in Tiermodellen mit Thrombose In contrast to DSPA alpha 1, alteplase (administered by both routes) elicited significant fibrinogenolysis at the highest dose of 100 nmol / kg. Fibrinolysis with alteplase was associated with significant plasminogen depletion, while alpha ₂ -antiplasmin levels decreased under both plasminogen activators but more severely under alteplase. DSPA alpha 1 was approximately two to three times as effective on a molar basis as alteplase when administered by either route ( 5 . 6 ). The blood radioactivity time courses indicated a faster lysis rate of DSPA alpha 1 when administered by either route. Both DSPA alpha 1 and alteplase were more effective when administered as a single bolus injection: complete thrombolytic efficacy was achieved at 30 nmol / kg DSPA alpha 1 bolus and approximately 100 nmol / kg DSPA alpha 1 bolus with infusion.

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

thrombosis the carotid of rabbits. Thrombosis was induced by introduction of a Copper coil into the common carotid artery of anesthetized rabbit triggered. The perfusion status of the artery was monitored by Doppler blood flow measurements. One hour after the thrombotic occlusion of the artery the animals heparin (200 IU / kg i.v. and i.m.) and acetylsalicylic acid (5 mg / kg i.v.) and then iv Bolus injections of 1, 2, 4 or 20 nmol / kg DSPA alpha 1 or 2, 6 or 20 nmol / kg of alteplase. DSPA alpha 1 resulted in effective reperfusion the carotid, the effect was about three times as high as the from alteplase. Alteplase led to a significant decrease in fibrinogen levels at doses of 6 nmol / kg and above, while this for DSPA alpha 1 was not observed at any dose (Table 2). streptokinase was in this study at doses of 3000, 10000 and 30000 IU / kg contain. Thrombolysis with this agent was less efficient (2/4, 4/6 and 1/6 animals) and showed no clear dose-dependency.

Coronary Artery Thrombosis in dogs. Thrombosis was induced by introduction of a copper coil in a branch of the left circumflex coronary artery (LCX) of anesthetized Raised dogs. The perfusion status of the artery was monitored by repeated angiography. Two examinations were performed, one with intravenous bolus injection the total dose of plasminogen activator and a second, in the one constant amount (about 10% of the total dose) administered as a bolus injection with the rest until 15 minutes after successful recovery the blood flow was infused. The data are summarized in Table 2.

In the study in which only one bolus was administered, the dogs were heparinized one hour after occlusion of the coronary artery (200 IU / kg iv + sc), 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 another series of Tie 0.96 nmol / kg DSPA alpha 1 was administered without prior heparinization. All dogs treated with heparin and DSPA alpha 1 (all doses) showed recanalization of the coronary arteries, which was unlike alteplase (2/6 or 3/6 with recanalization) (Table 2). The mean time to reperfusion was shorter for all doses of DSPA alpha 1 than for the high or low dose of alteplase. Within the observation period of 180 minutes after administration, the total transit time under DSPA alpha 1 was longer than at both alteplase doses. DSPA alpha 1 at a dose of 0.96 nmol / kg without heparin was not effective. There was no significant change in the plasma levels of alpha ₂ -antiplasmin in any experimental group.

In the study with bolus administration plus infusion, all dogs received heparin (200 IU / kg iv and sc) one hour after occlusion of the coronary artery, following which equal doses of DSPA alpha 1 or alteplase were administered (0.77 or 2.31 nmol / kg supplemented at 1.54 and 0.46 nmol / kg.min, respectively). All dogs treated with both doses of DSPA alpha 1 showed reperfusion of the coronary arteries. In a dog receiving the low dose alteplase, the coronary artery did not reopen. At the low dose of DSPA alpha 1, 83% of the coronary arteries remained consistent until the end of the observation period (180 minutes after initiation of treatment), whereas only 40% of the low dose of alteplase did. In summary, the two fibrinolytic agents showed marked differences at the lower dose, with sustained reperfusion being achieved in 2 out of 6 and 5 out of 6 old and DSPA alpha 1 animals, respectively. Neither fibrinogen nor plasminogen or alpha ₂ -antiplasmin decreased below either dose of DSPA alpha 1, whereas the high dose of alteplase resulted in a significant decrease in alpha ₂ -antiplasmin levels (which was insignificant due to the small number of animals). , DSPA alpha 1 (measured as antigen using a specific ELISA) had a longer half life and a lower clearance than alteplase.

venous thrombosis in rats. Venous thrombosis was performed in anesthetized rats a short thromboplastin infusion in combination with a ligature the vena cava is triggered. One hour after ligature, the vena cava was opened and the thrombus was removed to determine its wet weight. DSPA alpha 1 and alteplase were reported as i.v. Bolus in a dose of 1, 10 or 100 nmol / kg five Administered minutes before ligature of the vena cava. Both thrombolytic Funds passed to a significant reduction in the wet weight of the thrombus at 10 nmol / kg and above. DSPA alpha 1 was more effective than alteplase. Due to systemic Fibrin formation (which stimulates DSPA alpha 1 and alteplase) in this Model solved both plasminogen activators fibrinolysis to a similar extent (Table 2).

comparison from DSPA alpha 1 with alteplase. DSPA alpha 1 is more effective and leads under certain conditions to more efficient and faster lysis as alteplase in rat, rabbit and dog thrombi in vivo (Table 2). At higher Doses, DSPA alpha 1 appears to maintain its clot specificity, while Alteplase plasminogen exhaustion and degradation of coagulation factors. The studies in vivo to confirm the higher one clot specificity of DSPA alpha 1, which has already been described in vitro. In terms of DSPA alpha 1 was superior to Alteplase. This attribute depends on most likely with the longer one Half-life together.

1.3 Efficacy in animal models with embolic stroke

The Effectiveness of DSPA alpha 1 in acute cerebral ischemia investigated in rabbit and rat models with embolic stroke.

embolic Stroke in rats. Cerebral ischemia was caused by embolization with an autologous whole blood clot (length approx. 1.5 mm), the injected into the internal carotid artery of anesthetized rabbits has been. Two studies were carried out; received in the first the animals either DSPA alpha 1 (38 nmol / 1 ml / kg) or 1 ml / kg phosphate buffered Saline as intravenous Bolus injection 2 h after embolization (Table 3). In the other In the study, the rabbits were given either DSPA alpha 1 or phosphate buffered Saline as intravenous Bolus injection 3 h after embolization (Table 4). 26 h after the embolization became the survivors Animals killed. The brains were removed with 2,3,5-triphenyltetrazolium chloride (TTC) stained and then fixed and cut into 2 mm thick preparations. The infarct volume was assessed morphometrically.

One single intravenous Bolus of DSPA alpha 1 2 h after initiation of thromboembolic stroke led to a significant reduction in infarct volume measured 24 h later.

Qualitatively similar results were observed when DSPA alpha 1 was released after a delay of 3 h was administered, but statistical significance was not achieved. DSPA alpha 1 was not associated with a significant increase in edema or evidence of intracerebral haemorrhage in the surviving animals. Almost all brains apparently had hemorrhagic foci outside the infarcted area. The reason for this is still unclear at the moment.

• Median (Q1, Q3)* p < 0,005 im Vergleich zur Vehikelkontrolle (Mann-Whitney-Rangsummentest)

Tabelle 4 Wirkungen von DSPA alpha 1 auf das Infarktvolumen von Kaninchen (Verabreichung 3 h nach thromboembolischem Schlaganfall)

Mortality was higher in the DSPA alpha 1-treated group (38% vs. 9%), but this difference was not statistically significant. TABLE 3 Effects of DSPA alpha 1 on the infarct volume of rabbits (administration 2 h after thromboembolic stroke)

• Median (Q1, Q3) * p <0.005 versus vehicle control (Mann-Whitney rank sum test)

TABLE 4 Effects of DSPA alpha 1 on the infarct volume of rabbits (administration 3 hours after thromboembolic stroke)

The Data was surviving by 10 Animals in the vehicle group and 11 rabbits in the DSPA alpha 1 treated group (the 10 rabbits receiving the whole Experimental period survived and the 1 died rabbit); Median (Q1, Q3).

Embolic stroke in rats. In an embolic stroke model in rats resulted DSPA alpha 1 (96 nmol / kg = 5 mg / kg) administered 45 minutes after the onset of stroke as a 10% bolus plus 90% infusion over a period of 60 minutes significantly reduced the size of the ischemic lesion in vivo. The post-mortem data also indicates a reduction in infarct size.

A second independent Series of experiments in an embolic stroke model on rats designed so that the therapeutic efficacy and safety of DSPA alpha 1, reteplase and alteplase when administered 1 or 3 h after embolic occlusion of the middle cerebral artery of Rats could be compared.

Male Wistar rats were divided into eight experimental groups and one control group, with a total of 9-15 animals in each group. Central cerebral artery occlusion (MCA) was achieved in male rats by introducing an autologous blood clot at the base of the right MCA through a catheter that was advanced into the right internal carotid artery and inserted through the external carotid artery. The drugs were administered 1 or 3 hours after occlusion as shown in Table 5. Effects of thrombolytics on the infarct volume of rats with embolic stroke: experimental groups

MCA, middle cerebral artery

The Rats were sacrificed 48 h after closure. The percentages of right hemispheric infarcts, Mortality rates, neurological deficit scores (for verification of closure damage) and the incidence is more deadly haemorrhagic Transformation were analyzed.

In the control group, the mean infarct size determined one hour after embolization was significantly greater than that in the drug-treated groups. There were no significant differences between the various drug-treated groups in terms of infarct size ( 7 ).

Rats still alive after 48 hours were killed and examined;
Control, control group; rt-PA, alteplase (154 nmol / kg); rtp, reteplase (25.6 nmol / kg); rDSPAalpha1, DSPA alpha 1;
Low dose rDSPAalpha1, 10 nmol / kg; median dose rDSPAalpha1, 30 nmol / kg; high dose rDSPAalpha1, 90 nmol / kg;
* significant difference from the control; Mean values ± SEM

To 3 h treated rats had higher mortality rates and a higher one Incidence of hemorrhagic Transformation as the controls. Due to the small number remaining animals, infarct sizes were not evaluated. Of the Increase in mortality was mainly on an increase deadlier haemorrhagic Attributed transformation. In The groups treated after 1 h were haemorrhagic Events for 0-2 deaths per Group responsible (alteplase 2/11; reteplase 1/12; low dose rDSPAalpha1 0/13; median dose rDSPAalpha1 0/11; high dose rDSPAalpha 1 1/15) while the corresponding figures at treatment 3 h after embolization 3/9 (tr-PA), 3/9 (rtp) and 4/9 (DSPA). In the control animals there were no corresponding changes (0/12).

Conclusion. The studies of embolic stroke in rabbits and rats both confirm the efficacy of DSPA alpha 1 and the assumption that higher fibrin specificity and selectivity Risk for hemorrhagic transformation is reduced, which is particularly important in reducing intracranial hemorrhage as a catastrophic side effect of thrombolytic therapy of pulmonary embolism. This is supported by the observation that rats receiving reteplase or alteplase 1 h after embolization occasionally showed haemorrhagic transformation (3/25 rats in the two groups), while only 1/42 rats in the three DSPA alpha 1 groups thereof were affected, and then only in the highest dose group (90 nmol / kg).

By ischemia triggered damage the vessels lead to increased permeability and contribute to the hemorrhagic Transformation at the time of delayed thrombolytic therapy is related. Alteplase demonstrably builds the blood / brain barrier off and triggers such a further increase in the risk of intracerebral hemorrhage out. This has not been investigated in the case of reteplase or DSPA alpha 1. Unlike DSPA alpha 1, alteplase is for activation susceptible to a variety of molecules, including Prion protein, beta-amyloid and fibrinogen. Thus, it is conceivable that alteplase early ischemic damage strengthen the blood / brain barrier can, while the activity of DSPA alpha 1 remains more clot specific. It is remarkable that the experimental use of thrombin-enriched blood clots, as opposed to spontaneously formed blood clots, per se the disruption of the blood / brain barrier promote can. This explains While not the apparent difference between the thrombolytic Means, but this observation indicates that the use of thrombolytics may be safer than from such animal experiments Studies are predicted.

Of the Increase in mortality after delayed Treatment observed with each of the studied thrombolytic agents was, is on the hemorrhagic Attributed transformation. It gave a higher one Incidence of hemorrhagic Transformation in groups treated 3 h after embolization than in the control groups treated after 1 h. It must be noticed that alteplase and reteplase were given in doses, which are effective 1 h after closure. DSPA alpha 1 was in a Dose of 90 nmol / kg administered, which seems to be higher than actually necessary. Thus could a 9-fold lower dose, 10 nmol / kg, in the study 1 h after closure similar was effective at delayed Treatment better tolerated be.

1.4 Specificity of activation

It There is a unanimous opinion that high fibrin specificity is an important factor Factor for the compatibility of Represents plasminogen activators. In addition, new hints show that activation by non-fibrin molecules is not only for Fibrinogen as possible Stimulant but also for molecules such as. Beta-amyloid and prion protein play a role.

1.4.1 Fibrin specificity

In vitro studies on the kinetics of plasminogen activation showed that DSPA alpha 1 without stimulating cofactor practically no plasminogen activating activity showed while rt-PA plasminogen significantly activated and 260 times more effective was as DSPA alpha 1. In the presence of fibrin was the activity of DSPA alpha 1 105,000 times higher as without, while rt-PA due to its own fibrin-independent activity only one Showed a 550-fold increase.

To further demonstrate the fibrin specificity of DSPA alpha 1 zymographic experiments were performed with fibrin and casein. In these experiments, increasing concentrations of rt-PA or DSPA on SDS-PAGE were electrophoresed and the gels were placed on a plasminogen-containing fibrin or casein matrix. As in 8th can be seen, the activity of rt-PA was easily detected on both the fibrin and casein substrates ( 8th , Panels A and B). DSPA alpha 1 was clearly detectable on a fibrin matrix ( 8th , Panel A, lines 6-9), but was seen on the casein matrix only at the highest concentration used (100 nmol / l; 8th , Panel B, line 9). The differing fibrin specificities of the two thrombolytic agents are also evident from the study discussed in 4.1.4.2 (see 8th ).

1.4.2 Fibrinogen and fibrin degradation products

In vitro clot lysis studies were performed on human whole blood clots. Clots were generated in vitro, aged for 1 h and placed in autologous plasma in the presence of various concentrations of rt-PA or DSPA alpha 1. The thrombolytic potential of the two plasminogen activators was determined after 6 hours of incubation. While the thrombolytic profiles were very similar, the Fib Rinogen degradation under DSPA alpha 1 negligible, but considerably under rt-PA. The sensitivity to fibrinogen activation can be defined as fibrin selectivity and expressed as the ratio of activity in the presence of fibrin to activity in the presence of fibrinogen. The resulting values for DSPA alpha 1 and rt-PA are 12900 and 72, respectively. The differences in fibrin selectivity correspond to the study discussed in 4.1.4.3 (see 9 ).

1.4.3 Beta Amyloid

alteplase was characterized as a multi-ligand receptor; it can by a Number of non-fibrin molecules including Fibrinogen, prion protein and amyloid peptides are activated. For further characterization of DSPA alpha 1, the efficacies of fibrin, fibrinogen and amyloidbeta (1-42) as cofactors for activation of alteplase and DSPA alpha 1 in vitro by plasminogen activation caused change absorption in the presence of a chromogenic plasmin substrate determined in vitro.

There was no difference in activity when adding fibrin as a cofactor. As in 8th In addition, the advantage of DSPA alpha 1 in terms of fibrin specificity (activity without cofactor) and selectivity (activity in the presence of fibrinogen) was confirmed. Most interestingly, DSPA alpha 1 was 30 times less potent than alteplase in the presence of beta-amyloid. This difference may be clinically relevant: in an analysis of 23 patients with intracerebral haemorrhage associated with Alteplase / heparin treatment for acute myocardial infarction, pathologic findings were present in five patients; Of these, three showed cerebral amyloid angiopathy.

Control experiments (without Cofactors) with normalization against the value with DSPA alpha 1

1.4.4 prion protein

comparative Efficiencies of alteplase and DSPA alpha 1 in the presence of Prion protein are available from two studies. In the first study stimulated 200 nM PrP23-110 alteplase and DSPA alpha 1, both in a concentration of 1.8 nmol / l, factors of 43 and 1.2, respectively. Similar Results were obtained in the second study, in which two different Prion protein preparations were used. The stimulation of alteplase activity was 89- increased to 250 times unlike DSPA alpha 1, which has a negligible increase (factor 10) or even a nominal decrease (factor 0.6). In summary can be said that DSPA alpha 1 not at all or only negligible prion protein is stimulated, while the alteplase activity considerably is improved.

1.4.5 Conclusions

Even though DSPA alpha 1 and rt-PA are 70% structurally homogeneous their proteolytic activities in clinically relevant Aspects.

Practically This means that the effect of DSPA alpha 1 is fibrin-sensitive and that his activity is not increased by the presence of fibrinogen or fibrin degradation products. The main pharmacological Properties of DSPA alpha 1 are thus its high fibrin specificity and selectivity.

Thereby Solves DSPA alpha 1 mainly local fibrinolysis without systemic activation resulting in fibrinogen consumption and thus to a higher one Risk for Bleeding episodes lead would. In this regard, it may be important to note that Tenecteplase exceeds rt-PA by a factor of about 10 in terms of fibrin specificity.

II. Clinical Studies

DSPA Alpha 1 was in a Phase II clinical study with massive pulmonary embolism examined as an indication. Safety, tolerability and effectiveness of a single intravenous Administration of DSPA alpha 1 at a dose of 125, 180 or 250 μg / kg compared with alteplase 100 mg in subjects with acute massive Pulmonary embolism examined.

The most important efficacy parameter was reperfusion as measured by a reduction in total pulmonary resistance (TPR), mean pulmonary artery pressure (mPAP) and the Miller index. The reductions in TPR, mPAP and Miller Index were due to the relative change from the Output value (percentage change) defined as (actual value - output value) / output value × 100 evaluated.

The most important safety parameters were the proportions of patients with: severe bleeding, including all ICH events, ICH events, and serious unwanted ones Events.

Summary:

The Efficacy endpoints included TPR, mPAP and Miller Index. The Efficacy endpoints showed as early as 2 h after administration of the drug Improvements with each DSPA alpha 1 treatment group, and the full effect was achieved after 6 h. This does not apply to alteplase because its effects continued to improve and reached after 24 h its maximum. With regard to DSPA alpha 1 these were Effects dose-dependent changes in the groups with DSPA alpha 1 in doses of 180 μg / kg and 250 μg / kg. In most cases they were bigger than the effects observed with alteplasia in most analyzes.

The Overall differences in qualitative assessments of the Miller index between the treatment groups were independent of the local or central Investigation not remarkable. An additional approach with "overall response criteria" (composed from decrease of TPR, mPAP by at least 40% compared to Initial value with or without normalization and decrease of the Miller index at least 40% over the baseline) did not bring any other results.

at Subjects with acute pulmonary embolism showed the administration of Single doses in the form of intravenous 1 to 2 minute doses of DSPA alpha 1 injections of 125, 180 or 250 μg / kg body weight dose-dependent Improvements of invasively monitored hemodynamic Parameters, in particular with regard to mPAP. For most analyzes were those with 180 μg / kg and 250 μg / kg DSPA alpha 1 observed effects similar to or greater than the effects observed under alteplase. The effects of DSPA alpha 1 came in faster than Alteplase's.

mPAP Tabelle 2: Mittlerer pulmonaler Arteriendruck – relative Veränderungen gegenüber dem Ausgangswert nach 2, 6, 12 und 24 h (LOCF; Mittelwert ± SD; ITT-Kollektiv; n = 34)

Miller-Index Tabelle 3: Miller-Index – absolute und relative mittlere Veränderungen gegenüber dem Ausgangswert zwischen 2 und 12 h (LOCF; Mittelwert ± SD; ITT-Kollektiv; n = 34)

The overall incidence rate of treatment-related adverse events varied between 66.7% (DSPA alpha 1 180 μg / kg) and 100% (alteplase). TPR Table 1: total lung resistance - relative changes from baseline at 2, 6, 12 and 24 h (LOCF, mean ± SD, ITT collective, n = 34)

mPAP Table 2: Mean pulmonary arterial pressure - relative changes from baseline at 2, 6, 12 and 24 h (LOCF, mean ± SD, ITT collective, n = 34)

Miller Index Table 3: Miller Index - absolute and relative mean changes from baseline between 2 and 12 h (LOCF, mean ± SD, ITT collective, n = 34)

Short description of Legend:

1 shows the structure of DSPA alpha 1

2 shows the structure of alteplase

3 represents the amino acid sequence of DSPA alpha 1

4 Fibrinogen level after 180 minutes incubation with DSPA alpha 1 or alteplase

5 Thrombolytic effects of DSPA alpha 1 and alteplase when administered as a bolus in pulmonary thromboembolism of rats

6 Thrombolytic effects of DSPA alpha 1 and alteplase when administered as an infusion in pulmonary thromboembolism of rats

7 Effect of thrombolytic agents on the infarcted portion of the right hemisphere

8th Deviating fibrin specificities of DSPA alpha 1 and rt-PA in zymographic experiments

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

Claims (11)

Use of a plasminogen activator for the preparation a medicament for the treatment of venous thromboembolism, wherein the plasminogen activator in comparison with the activity without fibrin at least 550-fold increased activity in the presence of fibrin having. Use of the plasminogen activator according to claim 1, wherein the plasminogen activator i. essentially not is activatable by beta-amyloid and / or prion protein, and / or ii. is not neurotoxic and / or iii. a half-life of at least more than 2.5 minutes. Use of a plasminogen activator after a the above claims, wherein the plasminogen activator in comparison with the activity without fibrin an at least 5500-fold increased activity in the presence of fibrin and has a half-life of at least more than 50 minutes. Use of a plasminogen activator after a the above claims, wherein the plasminogen activator is Desmoteplase. Use of a plasminogen activator according to any one of the preceding claims, wherein the plasminogen activator i. an amino acid sequence after 3 or microheterogeneous forms thereof or ii. at least 90%, in particular 95% and most preferably 98% with the amino acid sequence of 3 is identical. Use of a plasminogen activator after a the above claims, where venous thromboembolism is deep vein thrombosis or pulmonary embolism is, especially massive pulmonary embolism. Use of a plasminogen activator after a the above claims, wherein the patients have an elevated Bleeding risk. Use of a plasminogen activator according to claim 7, wherein the patients have at least one of the following risk factors have: known hemorrhagic Diathesis, intake of oral anticoagulants, manifest or fresh serious or dangerous Bleeding, known history of or suspected intracranial Bleeding, suspected subarachnoid hemorrhage or condition after a subarachnoid hemorrhage by an aneurysm, history of damage of the central nervous system (i.e., neoplasia, aneurysm, intracranial or spinal surgery), hemorrhagic Retinopathy, e.g. in diabetes (blurred vision may indicate haemorrhagic retinopathy), newer (less than 10 days old) traumatic external cardiac massage, Delivery, recent puncture of an incompressible blood vessel (e.g. V. subclavia or jugularis), strong uncontrolled arterial Hypertension, bacterial endocarditis, pericarditis, acute pancreatitis, documented ulcerative gastrointestinal disease in recent years 3 months, esophageal varices, Arterial aneurysm, arterial / venous malformations, neoplasia with elevated Bleeding risk, severe liver disease, including hepatic insufficiency, Cirrhosis, portal hypertension (esophageal varices) and active hepatitis, great Surgery or significant trauma in the last 3 months, everyone Stroke in prehistory. Use of a plasminogen activator according to any one of the preceding claims, wherein the plasminogen activator is administered to the patient at a dose of 100 to 300 μg / kg of body weight, in particular 125, 180 or 250 μg / kg is administered. Use of a plasminogen activator after a the above claims, wherein a total amount of 10 to 30 g plasminogen activator, preferably 12.5, 18.0 or 25.0 mg is administered to the patient. Use of a plasminogen activator after a the above claims, wherein the drug is administered as a single bolus.