No abstract

Recent clinical studies using hirudin as anticoagulant have demonstrated that an efficient method to determine the current blood level of hirudin is imperative for exact dose finding and adjustment. Only the exact determination of the hirudin content in blood, performed within a few minutes, prevents overdosage involving side effects or, otherwise, a subtherapeutic dose regimen. Therefore, a method for rapid, sensitive, and reproducible measurement of hirudin in blood, plasma, and other body fluids has been developed. The method, which is based on coagulation measurement, is called ecarin clotting time (ECT). In this test, ecarin, a purified enzyme of the Echis carinatus snake venom, acts as a prothrombin activator. In contrast to the "solid phase" prothrombin activation by prothrombinase, the ecarin-induced prothrombin activation proceeds in an alternative way, i.e., without the need of cofactors, resulting in intermediates such as meizothrombin. Compared to thrombin, meizothrombin has a lower procoagulant activity, but it still binds hirudin, which leads to the inhibition of meizothrombin. Depending on the sample's concentration of hirudin, ecarin forms a residual, nonhirudin-bound amount of intermediates of the prothrombin-thrombin conversion that are able to concentration-dependently convert fibrinogen to fibrin. There is an excellent linear correlation between ECT prolongation and the hirudin content of the sample in a range from 50 to 5,000 ng/mL blood or plasma. This allows immediate measurement not only of the therapeutic blood level of hirudin, but also of its concentration in blood following under- or overdosage. The ECT method is nearly independent of variations in the sample's content of fibrinogen (from 60% to 100%) and prothrombin (from 20% to 100%.) Heparin is not able to catalyze the very low antithrombin inhibition of meizothrombin. Therefore, it is also possible to determine hirudin in blood containing varying amounts of heparin. Another advantage of the method is that it can be applied to different mechanical measuring systems used in coagulation diagnostics.

The ecarin clotting time (ECT) is a meizothrombin generation test that allows for precise quantification of direct thrombin inhibitors. The ECT has demonstrated its usefulness for more than 10 years in biochemical-pharmacological investigations as well as in clinical research and in the clinical routine. It has proved valuable especially as a drug-monitoring method in r-hirudin therapy. This test has been adjusted to clinical requirements by numerous modifications. Following the description of the biochemical background and the measuring principle of the ECT, this article gives a short survey of several modifications of the ECT for both preclinical and clinical use, e.g., for biochemical investigations, as a point-of-care method and for cardiac surgery. Advantages and disadvantages of these methods are discussed.

SummaryThe pharmacokinetics and the effects on the haemostatic system of hirudin were assessed in six healthy subjects after single intravenous or subcutaneous dose (1000 AT-U/kg). When hirudin was given intravenously first-order elimination kinetics followed the initial distribution phase. The decline in plasma hirudin concentration was most adequately expressed by a biexponential equation describing a two compartment model. A mean elimination half-life of 0.84 hr and a mean volume of distribution of 12.9 1 were calculated. After subcutaneous injection a low hirudin level (∼0.5 AT-U/ml) was maintained for a prolonged period of time.In the 24 hour-urine up to 50 per cent of the administered amount of hirudin was excreted in active form.Thrombin time, partial thromboplastin time and prothrombin time measured in plasma samples ex vivo were prolonged dependent on the hirudin plasma level. Platelet counts, fibrinogen level and the fibrinolytic system were unchanged. Bleeding time was prolonged twice at maximum.Subcutaneous or intravenous administration of pure hirudin was tolerated without side-effects.

SummaryThe pharmacodynamics and pharmacokinetics of hirudin were studied in dogs, rabbits and rats. Hirudin proved to be a well tolerated substance with low toxicity. After intravenous injection it was eliminated with a half time of 50 to 60 min. It was nearly completely excreted through the kidneys in biologically active form. The efficacy of hirudin in preventing venous thrombosis, vascular shunt occlusion and disseminated intravascular coagulation in rats was demonstrated.