ALBERT

Description: Background: The pathogenesis of thrombosis involves both cellular and humoral processes. Most antithrombotic drugs exhibit either anti-protease or anti-platelet effects. A combination of anti-protease and anti-platelet drugs provides better efficacy in the management of thrombotic disorders. A series of synthetic low molecular weight serine protease inhibitors with varying anti-platelet effects (Medicure Inc.) are being assessed for antithrombotic properties. Materials and Methods: This investigation reports on a compound with low antithrombin/high anti-platelet activity MC 45301 (A) and a compound with high antithrombin/low anti-platelet activity MC 45308 (B) activity in in-vitro and in-vivo settings used to profile antithrombotic drugs. Results: A exhibited strong anti-platelet actions as measured using ADP as an agonist (IC50=1.1 g/ml), whereas B had a higher IC50 (9.4 g/ml). In the antithrombin titration assay A (〉100 μg/ml) showed a relatively higher IC50 than B (45 μg/ml). In the global anticoagulant assays, A exhibited somewhat weaker effects than B. In the Xa generation assay, both compounds exhibited similar effects. However, in the thrombin generation assays B exhibited stronger effects. In whole blood assays both compounds produced anticoagulant and anti-platelet effects. Intravenous administration of these compounds to rabbits over a dose range of 50–500 g/kg produced strong dose dependent antithrombotic actions. In comparison to direct antithrombin agents such as argatroban, at a comparable dose, B produced identical antithrombotic actions, which were disproportional to the systemic anticoagulant effects. A produced modest antithrombotic actions with minimal ex vivo clotting effects. This data is highly suggestive that compounds with dual targets are able to produce stronger antithrombotic actions relative to monotherapeutic agents. Additional studies in arterial thrombosis may provide newer insights into the antithrombotic actions of compounds with dual sites of action. Moreover, these agents may be more effective in thrombotic conditions where both platelets and the coagulation system are involved.

Description: Background: Because of the relative insensitivity of the global clot based assays such as the activated partial thromboplastin time (APTT), the low molecular weight heparins (LMWHs) are potency evaluated/optimized in the anti-Xa (AXa) and heptest. A new clot based assay namely, prothrombin induced clotting time (PiCT) is sensitive to the anticoagulant effects of LMWHs and related drugs. As the LMWHs are standardized using the anti-Xa methods, using the International Standards, this study was designed to cross validate the 2nd International Standard for LMWHs(NIBSC 01/608) in various assay methods. Methods: Commercially available LMWHs, Dalteparin (D), Enoxaparin (E), Tinzaparin (T) and the 1st International Standard (85/600) were crossed referenced against the 2nd International Standard (NIBSC 01/608) using an amidolytic AXa method. Each of these LMWHs were compared in the AXa, adjusted concentration range of 0–1.0 U/ml using the Heptest, AXa, AIIa and PiCT. In addition plasma samples from patients receiving a LMWH, E for therapeutic and interventional purposes were measured using various tests. Results: The AXa potency adjusted LMWHs (D, E, and T) and 1st International Standard provided superimposable concentration curves in the amidolytic AXa assays. However marked differences in the heptest and PiCT were noted. Major differences were noted in the AIIA levels, even between the two International standards. When patients samples (n=75) from a therapeutic trial (1.0 mg/kg BID/SC) were evaluated, assay based differences were further amplified. The amidolytic AXa assay consistently measured higher AXa levels. When the two standards were cross-referenced with one another in different assays, major differences were noted in the clot-based assays. Even in the AXa assay at equivalent AXa levels, differences were obvious. Conclusions: These results suggest that both of the International Standards of LMWH are valid for only the cross standardization of the AXa activities of LMWHs. If any of the other methods were used, significantly different results were obtained with each of the individual LMWHs. Thus, the 2nd International Standard should only be used for amidolytic AXa assay for potency referencing purposes. Moreover, the stated potency of the 2nd Internation Standard may need to be readjusted against the 1st Standard to obtain valid results. These standards are of limited value in the clinical monitoring of LMWHs. It is therefore proposed that each of the LMWHs should be cross referenced by its own standard and the clinical monitoring of these drugs should only be carried out utilizing the specific drug used in a given patient. The PiCT test offers a global test which is capable of monitoring the effects of all components of heparins regardless of their affinity to serpines. Moreover, the effect of TFPI released on clotting processes is also measured. Thus, the PiCT test provides a physiologically relevant anticoagulant index.

Description: Low Molecular Weight Heparins (LMWHs) are currently developed for anticoagulation in various intravenous indications such as DVT treatment and percutaneous interventions (PCI). Traditionally, these drugs are administered in anti-Xa units. The purpose of this study was to compare the relative levels of anticoagulation produced by different LMWHs in whole blood ACT (Hemochron), an amidolytic anti-Xa method and a plasma-based global anticoagulant assay, the PiCT test (Pentapharm, Basel, Switzerland). Various LMWHs such as dalteparin, enoxaparin, reviparin and a synthetic pentasaccharide were supplemented to freshly drawn native blood samples to simulate patient samples containing 1 U/ml anti-Xa levels. Three generic versions of enoxaparin were also included in this study. In addition to the ACT measurement in whole blood, citrated plasma samples were also tested for APTT, Heptest, PiCT, amidolytic anti-Xa and IIa activities, and protease generation assays. All agents produced assay-based effects on different tests, which were not proportional to the adjusted anti-Xa activity. Correlation of the ACT data resulted in a good relationship with PiCT for the commercially available branded products(r2=0.97). ACT and Heptest (r2=0.51), ACT and anti-Xa (r2