Characterisation of Persistent Anti-Xa Activity Following Administration of the Low Molecular Weight Heparin Enoxaparin Sodium (Clexane)

Summary

It is widely reported that persistent anti-Xa activity follows administration of low molecular weight heparins. To identify the effectors of this activity we have injected ¹²⁵I-labelled Enoxaparin sodium into rabbits and subsequently analysed the circulating radiolabelled material and anti-Xa activity by affinity and size exclusion chromatography. Antithrombin III-binding material derived from the injected drug was responsible for all the anti-Xa amidolytic activity. At early times after injection additional anticoagulant activity which was largely attributable to tissue factor pathway inhibitor was measured by the Heptest clotting assay after removal of glycosaminoglycans from plasma samples. Small radiolabelled fragments, including penta/hexasaccharide with affinity for antithrombin III, were detectable in the circulation 1 week later, and sulphated oligosaccharides persisted for 3-4 weeks. Significant quantities of radiolabel remained in the liver and kidney several weeks post-injection; these organs may sequester some of the injected drug and give rise to circulating biologically active material by degradation and secretion of catabolic products into the plasma.

• References

• 1 Barrowcliffe TW, Johnson EA, Thomas DP. Low molecular weight Heparin. John Wiley and Sons. Chichester, New York, Brisbane, Toronto, Singapore 1992: 101-123
• 2 Dawes J, Bara L, Billaud E, Samama M. Relationship between biological activity and concentration of a low-molecular-weight heparin (PK 10169) and unfractionated heparin after intravenous and subcutaneous administration. Haemostasis 1986; 16: 116-122
• 3 Briant L, Caranobe C, Saivin S, Sie P, Bayrou B, Houin G, Boneu B. Unfractionated heparin and CY 216: pharmacokinetics and bioavailabilities of the antifactor Xa and Ha effects after intravenous and subcutaneous injection in the rabbit. Thromb Haemost 1989; 61: 348-353
• 4 Kassis J, Hirsh J, Ofosu F. Evidence for the prolonged clearance of antithrombin activity of the low molecular weight heparin Enoxaparine. Thromb Haemost 1991; 65: 1298
• 5 Agnelli G, Iorio A, Alatri A, Boschetti E, Mazzolai L, Nenci GG, Ofosu F, Hirsh J. Plasma half-life of the anti-IIa activity of low molecular weight heparin assessed with a sensitive test (PTNA). Thromb Haemost 1993; 69: 768
• 6 Dawes J, Pepper DS. Catabolism of low dose heparin in man. Thromb Res 1979; 14: 845-860
• 7 Boneu B, Dol F, Caranobe C, Sie P, Houin G. Pharmacokinetics of heparin and related polysaccharides. Ann NY Acad Sci USA 1989; 556: 282-291
• 8 Thomas DP, Barrowcliffe TW, Merton RE, Stocks J, Dawes J, Pepper DS. In vivo release of anti-Xa clotting activity by a heparin analogue. Thromb Res 1980; 17: 831-840
• 9 Sandset M, Abildgaard U, Larsen ML. Heparin induces release of extrinsic coagulation pathway inhibitor (EPI). Thromb Res 1988; 50: 803-813
• 10 Dawes J. The pharmacokinetics of LMW dermatan sulphate: long-term persistence of intact material. Thromb Haemost 1993; 69: 339-313
• 11 Dawes J, Prowse CV, Pepper DS. The measurement of heparin and other therapeutic sulphated polysaccharides in plasma, serum and urine. Thromb Haemost 1985; 54: 630-634
• 12 Lam L, Silbert J, Rosenberg R. The separation of active and inactive forms of heparin. Biochem Biophys Res Commun 1976; 69: 570-577
• 13 Greenwood F, Hunter W, Glover J. The preparation of 131I-labelled human growth hormone of high specific radioactivity. Biochem J 1963; 14: 114-123
• 14 Andersson L, Barrowcliffe T, Holmer E, Johnson E, Sims G. Anticoagulant properties of heparin fractionated by affinity chromatography on matrix-bound antithrombin III and by gel filtration. Thromb Res 1976; 9: 575-583
• 15 Hodson B, Pepper D, Dawes J. Novel affinity chromatographic system for the single-step purification of glycosaminoglycans from complex systems using volatile buffers J Chromatog 1991; 565: 416-423
• 16 Teien A, Lie M, Abildgaard U. Assay of heparin in plasma using a chromo-genic substrate for activated factor X. Thromb Res 1976; 8: 413-416
• 17 Yin ET, Wessler S. Heparin-accelerated inhibition of activated Factor X by its plasma inhibitor. Biochim Biophys Acta 1970; 201: 387-390
• 18 Frydman AM, Bara L, Le Roux Y, Woler M, Chauliac F, Samama MM. The antithrombotic activity and pharmacokinetics of enoxaparine, a low molecular weight heparin, in humans given single subcutaneous doses of 20 to 80 mg. J Clin Pharmacol 1988; 28: 609-618
• 19 Lindahl AK, Abildgaard U, Larsen ML, Aamodt LM, Nordfang O, Beck TC. Extrinsic pathway inhibitor (EPI) and the post-heparin anticoagulant effect in tissue thromboplastin induced coagulation. Thromb Res 1990; XIV 39-48
• 20 Dawes J, Pepper DS. A competitive binding assay for heparin, heparan sulphates and other sulphated polymers. In: Radioimmunoassay in Basic and Clinical Pharmacology. Handbook of Experimental Pharmacology Vol. 82. Patrono C, Peskar BA, eds. Springer-Verlag, Berlin, Heidelberg, New York, London, Paris, Tokyo 1987: 543-558
• 21 Kristensen HI, Ostergaard PB, Nordfang O, Abildgaard U, Lindahl AK. Effect of tissue factor pathway inhibitor (TFPI) in the Heptest assay and in an amidolytic anti factor Xa assay for LMW heparin. Thromb Haemost 1992; 68: 310-314
• 22 Warn-Cramer B, Maki S, Rapaport S. Heparin-releasable and platelet pools of tissue factor pathway inhibitor in rabbits. Thromb Haemost 1993; 69: 221-226
• 23 Larsen AK, Hetekidis S, Langer R. Disposition and anticoagulant activity of biologically active heparin fragments in the rat. J Pharmacol Exp Ther 1984; 231: 373-378
• 24 van Amsterdam RGN, Vogel GMT, Visser A, Kop W, Buiting M, Meule-man DG. Plasma disappearance of synthetic pentasaccharides derived from heparin explained by antithrombin III. Thromb Haemost 1993; 69: 893
• 25 Freeman C, Hopwood J. Lysosomal degradation of heparin and heparan sulphate. In: Heparin and Related Polysaccharides. Lane DA, Lindahl U, eds. Plenum Press, New York 1992: 121-134
• 26 Thunberg L, Backstrom G, Wasteson A, Robinson HC, Ogren S, Lindahl U. Enzymatic depolymerisation of heparin-related polysaccharides: substrate specificities of mouse mastocytoma and human platelet endo-B-D-glucuro-nidases. J Biol Chem 1982; 257: 10278-10282