Quantification of Bovine and Porcine Heparins Utilizing the Heparin Red Assay, Applications in the Study of Pharmacokinetics and Pharmacodynamics

Abstract

Introduction Currently there is a major interest in developing bovine intestinal heparin for anticoagulant purposes. It is well known that bovine intestinal heparins exhibit a relatively lower potency than porcine intestinal heparins. The pharmacokinetic behavior of heparins has traditionally been based on antithrombin-dependent anti-Xa and anti-IIa activities. Heparin Red is a polycationic substance whose intrinsic fluorescence is quenched upon binding to heparin. As such, assays utilizing Heparin Red detect all heparin oligosaccharides in a sample: those that bind to AT and produce anti-Xa and anti-IIa activities, and those that do not. These non-AT binding heparin oligosaccharides can still impact coagulation through their interaction with heparin cofactor II, TFPI and platelets. Thus, the pharmacokinetic behavior of heparin (half-life, etc.) as determined using a Heparin Red assay may more closely reflect the clinical behavior of heparin (antithrombotic, bleeding) than pharmacodynamic measures where heparin levels are determined based only on AT-dependent activities (anti-Xa or anti-IIa). Methods Primates were administered PMH (Medefil, Glendale Heights, IL) or BMH (KinMaster, Passo Fundo, Brazil) at a dose of 100 anti-Xa U/kg IV. Blood samples were collected prior to and at 15, 30, 60 and 120 minutes post-heparin administration. Blood samples were centrifuged to produce platelet poor plasma which was aliquoted and stored frozen until analysis. Heparin levels were assessed using a chromogenic anti-Xa assay and a Heparin Red assay relative to product-specific calibration curves. Pharmacokinetic parameters were assessed using a non-compartmental model. Results Circulating functional drug levels based on anti-Xa activity were the same in PMH and BMH-treated primates. Peak levels of 1.45 ± 0.11 and 1.48 ± 0.08 U/ml were observed in PMH and BMH-treated primates, respectively. Using drug levels determined by anti-Xa assay, AUCs for bovine and porcine heparin treated animals were calculated to be 111.5 ± 11.0 and 108.8 ± 26.7 U*min*ml-1, respectively. By Heparin Red assay, peak circulating heparin levels were higher following BMH administration (10.7 ± 0.5 vs. 9.0 ± 0.2 mg/ml; t-test p<0.001) and the AUC for BMH-treated primates was approximately 22% larger than for PMH-treated primates (728.2 ± 35.3 vs. 594.9 ± 5.4 mg*min*ml-1; Mann-Whitney test p=0.029). Conclusions BMH, when administered at equivalent anti-Xa unit doses, produces comparable pharmacodynamic effects as PMH despite the presence of higher circulating GAG levels as measured by the Heparin Red assay. Measurement of BMH levels using the Heparin Red assay may be useful for studying its absolute pharmacokinetics and optimization of protamine sulfate dosage for its neutralization.