Abstract
Introduction: Heparin cleavage under alkaline conditions results in low molecular weight heparin (LMWH) chains, a significant proportion of which contain 1,6-anhydromannosamine and/or 1,6-anhydroglucosamine at the reducing end. Despite the widespread use of the LMWHs for the prophylaxis and treatment of thrombosis, it remains unclear whether such structural modifications impact the pharmacologic activity of the drug. This study examined the in vitro anticoagulant and in vivo pharmacokinetic/pharmacodynamic (PK/PD) behavior of LMWHs containing varying levels of 1,6-anhydrosugar content.
Materials and Methods: By altering the temperature and pH of the depolymerization reaction, LMWHs containing 0, 5, 10, 20 and 40% 1,6-anhydrosugar were produced. These compounds were supplemented to normal human plasma and normal primate plasma and assayed for anticoagulant (APTT and Heptest) and antiprotease (anti-IIa and anti-Xa) activity. The effect of 1,6-anhydrosugar on the PK/PD profile of LMWHs was assessed by administering the 40% 1,6-anhydro LMWH or enoxaparin (~20% 1,6-anhydrosugar) intravenously to groups of non-human primates (n=4–6) at a dose of 1 mg/kg. Blood samples were collected at baseline and at various time points up to 24 hours post-administration for determination of Heptest clotting times, anti-IIa and anti-Xa activity. The biologic activities were converted to equivalent LMWH concentrations using calibration curves prepared in normal primate plasma.
Results: The molecular weight profiles of these LMWHs were comparable. No effect on anticoagulant or antiprotease activity was observed when the 1,6-anhydro content varied between 0 and 10%. When the 1,6-anhydro content was increased to 20 and 40%, a content-dependent reduction in anticoagulant activity was observed such that the prolongation of the APTT and Heptest by the 40% 1,6-anhydro LMWH was 58 and 23% less, respectively, than that produced by the LMWH lacking the 1,6-anhydro group when tested in the linear range of the concentration-response curve. This effect appears to be related primarily to an interference with antithrombin activity. Inhibition of thrombin activity in an amidolytic assay was 35% lower with the 40%-anhydro LMWH compared to the 0% anhydro compound (10 mg/ml), whereas anti-Xa activity was only 7% lower. Assay dependent variations were observed in the PK/PD profiles of the 40% anhydro LMWH and enoxaparin. As expected, the half-life of antithrombin activity was considerably shorter than that of the anti-Xa activity. The pharmacokinetic behavior of the 40% 1,6-anhydro LMWH and enoxaparin in terms of half-life, area under the curve, systemic clearance and volume of distribution was not significantly different when calculated using plasma concentrations determined by anti-IIa or anti-Xa assay. When concentrations determined by Heptest were used, the AUC determined for enoxaparin was approximately 2-fold higher than that determined with the 40% anhydro LMWH.
Conclusions: Microchemical changes in the structure of low molecular weight heparin oligosaccharides can induce measurable changes in the biologic activity of LMWHs. While the pharmacokinetic profile does not appear to be altered by an enhanced 1,6-anhydro content, the effect of 1,6-anhydro content on the clinical efficacy and safety of LMWHs is unknown. Such findings may have particular impact on the development of generic LMWHs.
Carbohydrate PEGylation, an approach to improve pharmacological potency