Search

Hydrogen peroxide (H2O2) and Cu(OAc)2 or FeSO4 (Fenton type reagents) perform heparin (Hep) depolymerisation to low-molecular-weight heparin (LMWH) following a radical chain mechanism. Hydroxyl (OH) radicals which are initially generated from H2O2 reduction by transition metal ions abstract hydrogen atoms on the heparin chain providing carbon centred radicals whose decay leads to the depolymerisation process. The main depolymerisation mechanism involves Hep radical intermediates that cleave the glycosidic linkage at unsulphated uronic acids followed by a 6-O-nonsulphated glucosamine, thus largely preserving the pentasaccharide sequence responsible for the binding to antithrombin III (AT). Both the transition metal ions influence the overall efficiency of the radical chain processes: Fe2+ acting as a catalyst, while Cu2+ acts as a reagent. LMWHs, especially those afforded by Cu2+, are somewhat unstable to the usual basic workup. However, this lack of stability can be eliminated by a previous NaBH4 reduction. Furthermore, with Cu2+, the process is much more reproducible than with Fe2+. Therefore, for the process of Fenton type depolymerisation of heparin, the use of Cu(OAc)2 is clearly preferable to the more "classical" FeSO4. The resulting activities and characteristics of these LMWHs are peculiar to these oxidative radical processes. In addition, LMWH provided by H2O2/Cu(OAc)2 in optimised conditions was found to posses anti-Xa and anti-IIa activities comparable to those of LMWHs currently in clinical use.

Low molecular weight (LMW) heparins have prolonged circulating half-lives relative to unfractionated heparin, but the rates of plasma clearance differ between different LMW preparations. To determine the impact of method of production on their pharmacokinetic and ex vivo biological properties, two LMW heparins of similar molecular weight distribution, Logiparin and Fragmin, were radiolabelled with 125I, administered intravenously with 4 mg/kg of carrier drug into rabbits, and the circulating radiolabelled material and anti-Xa activity were analysed by size exclusion chromatography and affinity for antithrombin and Polybrene. Following administration of Logiparin, the anti-Xa amidolytic activity was eliminated with the same half-life as the anti-thrombin-binding radiolabel and was not neutralised by antibody against tissue factor pathway inhibitor (TFPI). Larger molecules were cleared preferentially and were no longer detectable 8 h post injection. These findings resemble those we have previously described for Enoxaparin. After Fragmin administration the antithrombin binding radiolabel was cleared more rapidly than the anti-Xa activity, and at late times after injection a significant amount of this activity was neutralised by antibody against TFPI. Sulphated radiolabel was eliminated with a similar half-life to the anti-Xa activity and sulphated molecules > 6000 Da remained in the circulation 8 h after administration. Fragmin, unlike Logiparin and Enoxaparin, has no negatively charged sulphamino group at the reducing end of the molecule. We suggest that this minimises cellular interaction and protects the larger molecules from elimination. They remain in the circulation, contributing to anti-Xa activity by binding TFPI. Thus the method of production of LMW heparins may significantly influence their pharmacokinetic properties and circulating anticoagulant activities.