Goetz Nowak

R-hirudin as anticoagulant in regular hemodialysis therapy: finding of therapeutic R-hirudin blood/plasma concentrations and respective dosages.

Recently heparin-induced thrombocytopenia type II has been diagnosed more frequently and does not exclude hemodialysis patients. Up to now, recombinant hirudin is the only available anticoagulant showing no immunologic cross reactions with heparin. However, the use of r-hirudin in hemo dialysis patients with different degrees of residual renal func tions is impossible using standard dosages because elimination of r-hirudin varies depending on the degree of residual renal function. Therefore the first study was carried out using con secutive r-hirudin anticoagulated hemodialyses to determine the appropriate dose of r-hirudin. Ten hemodialysis patients with creatinine clearance values ranging between 0 and 13 mL/min/1.73m2 were anticoagulated with r-hirudin. An initial bolus of 0.1 mg/kg bwt before the first hemodialysis, resulted in an average r-hirudin blood concentration of 305 ng/mL at the end of treatment. The dose for each of the following four he modialyses was adjusted individually to reach the minimum therapeutic r-hirudin blood concentration. At the end of these treatments the mean blood r-hirudin concentration was 422 ng/mL. The necessary mean doses ranged between 0.008 and 0.125 mg/kg bwt correlating to the creatinine clearance values of the patients. All hemodialyses of the study were effective and safe. Bleeding times determined during r-hirudin antico agulation were significantly lower than control values mea sured 2 days after a heparin administration. The study proved that r-hirudin may be an efficient and safe heparin alternative as a hemodialysis anticoagulant when the individuals residual renal function is noted for dosage and dose adjustment and is controlled by drug monitoring using the ecarin clotting time.

Biochemical and pharmacokinetic characterisation of two PEGylated variants of dipetarudin.

Dipetarudin was coupled to polyethylene glycol (PEG)-5000 residues in order to improve its pharmacokinetic profile and to enhance its anticoagulant efficacy. The resulting compounds, mono- and di-PEGylated dipetarudin were purified by gel filtration. Mono-PEGylated dipetarudin exhibited similar activity like its non-conjugated equivalent both in vitro and in vivo. However, di-PEGylated dipetarudin showed longer distribution and elimination half-lives and higher area under the time-concentration curve in comparison with the unmodified inhibitor which may be attributed to decreased renal clearance. Futhermore, ratio k(12)/k(21) decreased when the number of PEG chains coupled to dipetarudin increased. It means that the inter-compartment transfer of dipetarudin, characterised by a fast distribution and a high retention in the peripheral compartment, is reverted by coupling to PEG. Thus, the transfer of mono-PEGylated dipetarudin between these compartments is similar in both senses and the transfer of di-PEGylated dipetarudin is slower from vascular to extravascular compartment than vice versa. Our results show that di-PEGylated dipetarudin produces a better and longer anticoagulant effect than unmodified dipetarudin which is a desirable attribute for future therapeutic application.

Successful renal transplantation in a patient with heterozygous prothrombin gene, factor V Leiden mutation and heparin‐induced thrombocytopenia using r‐hirudin as anticoagulant

Abstract:  Vascular complications remain the most common cause of early renal allograft loss in patients with end‐stage renal failure. Underlying thrombophilic disorders increase the risk of early graft thrombosis. A male adolescent with high‐risk thrombophilia because of combined heterozygous factor V Leiden (G1691A) and prothrombin gene (G20210A) mutation developed HIT II. Hemodialysis and subsequent renal transplantation were undertaken using recombinant hirudin, a direct and selective thrombin inhibitor, as an anticoagulant. Primary function in the transplanted kidney was excellent. No thrombotic or hemorrhagic events have occurred and follow‐up showed excellent long‐term graft survival. Patients on HD have an increased risk for the development of HIT, and therefore, they need repetitive screening for the development of acquired thrombotic risk factors (e.g. HIT II or lupus anticoagulant). R‐hirudin is efficacious and safe on both HD and following renal transplantation.

Special pharmacokinetics of dipetarudin suggests a potential antitumor activity of this thrombin inhibitor.

Thrombin is a potent mitogen for many tumor cells, suggesting that this enzyme may be involved in tumor genesis and metastasis. Inhibition of thrombin expressed on the surface of tumor cells may improve outcomes in some tumor cases. For this reason, a thrombin inhibitor to be applied in antitumor therapy must have favorable pharmacokinetic attributes to exert its action as long as possible in the extravascular compartment of the extracellular space, with a short action intravascularly, avoiding bleeding and/or other undesirable side-effects. None of the thrombin inhibitors in clinical use has these properties. Here, we report for first time a direct thrombin inhibitor, named dipetarudin that could be very useful in antitumor therapy because of its pharmacokinetic behavior characterized by a rapid distribution in the extravascular space with a slow elimination from this compartment.

Reversible Crystallization of Argatroban after Subcutaneous Application in Pigs

Argatroban is a thrombin inhibitor used as anticoagulant in patients with heparin-induced thrombocytopenia. It is usually administered as an intravenous bolus followed by infusion. Nevertheless, its pharmacokinetics after subcutaneous administration is unknown. The aim of this study was to assess the pharmacokinetics of two different formulations of argatroban in pigs after subcutaneous administration. Antithrombotic activity in plasma was determined by ecarin chromogenic assay. To visualize the formation of crystals, argatroban was administered to rats into the subcutaneous tissue exposed after removing the skin, and the injection site was photographed at different times. After subcutaneous administration of a sorbitol/ethanol formulation of argatroban in pigs was observed a slow absorption phase was followed by long-lasting levels of this inhibitor. C max and AUC(0−24) showed dose-dependent increases, while elimination half-life and t max value did not change significantly with dose. In contrast, saline-dissolved argatroban showed a faster absorption phase followed by a shorter elimination half-life. Argatroban dissolved in sorbitol/ethanol leads to long-lasting plasma levels due to the formation and permanent dissolution of a crystalline depot at the injection place. This represents a simple way to deliver argatroban continuously over an extended period which can be beneficial for prophylaxis or treatment of chronic coagulations disorders.

Improvement of the specificity of dipetarudin by site directed mutagenesis

Protease specificity is crucial to the design of thrombin inhibitors as inhibition of other physiologically relevant serine-proteases can compromise their clinical use. Dipetarudin, a potent thrombin inhibitor, also inhibits trypsin and plasmin. Due to the specificity of an inhibitor being influenced by the amino acid residue at the P1 position, we replaced the Arg10 at P1 position of dipetarudin by a histidine, which is the P1 residue of rhodniin, a very specific thrombin inhibitor. The amino acid replacement was carried out by site directed mutagenesis. The mutant, dipetarudinR10H, showed a loss of plasmin and trypsin inhibitory activities present in its wild-type counterpart and a 3-fold higher dissociation constant for thrombin than dipetarudin. However, compared to dipetarudin and r-hirudin, dipetarudinR10H showed similar activity in coagulation screening assays such as activated partial thromboplastin time (aPTT), prothrombin time (PT), ecarin clotting time (ECT) and ecarin chromogenic assay (ECA).