USP Potency Adjusted Bovine Mucosal Heparins (BMH) Are Comparable to Porcine Mucosal Heparin (PMH) at Equivalent Levels

Introduction: Unfractionated heparin (UFH) remains to be the only parenteral anticoagulant used in the management of various thrombotic disorders such as deep vein thrombosis (DVT), pulmonary embolism (PE), and cardiovascular interventions. Most of the heparins used clinically are derived from porcine intestinal mucosa. There is likelihood of supply shortage of this important anticoagulant which is crucial for hemodialysis, cardiopulmonary bypass surgery and other vascular interventions. BMH are currently being developed for re-introduction for both medical and surgical indications. In contrast to the PMH, the active pharmaceutical ingredient (API) of BMH exhibit a somewhat weaker USP potency as cross-referenced against PMH. We hypothesized that at equivalent potencies as adjusted by using the USP reference, the BMH may exhibit comparable in vitro and in vivo effects. Therefore, in vitro and in vivo studies were used to compare the APIs of the bovine (140 U/mg) and the PMH (190 U/mg) to demonstrate their bioequivalence.

Materials and Methods: API versions of PMH (190 U/mg) were obtained from Celsus Laboratories (Franklin, OH). API versions of BMH (140 U/mg) were obtained from KinMaster (Paso Fundo, Brazil). Each of these heparins was assayed for their molecular weight profile, AT affinity, USP potency, protamine and platelet factor 4 neutralization and anticoagulant/antiprotease profiles using standard laboratory methods. In the primate studies, potencies of each heparin were determined by amidolytic anti-Xa assay in relation to the USP heparin standard. Individual groups of primates (n=4) were administered 100 anti-Xa U/kg doses of bovine or porcine heparin via intravenous route. Blood samples were collected prior to dosing and at 15-, 30-, 60- and 120-minutes post-administration. Anti-Xa and anti-IIa activities were measured to determine circulating heparin concentrations using commercially available USP compliant kits (Aniara Diagnostica, West Chester, OH). These drug concentrations were used to determine pharmacokinetic parameters such as area under the curve (AUC), half-life (t1/2), clearance (Cl) and volume of distribution (Vd) using the PKSolver add-in for Excel.

Results: BMH exhibited higher molecular weight profiles compared to PMH as determined by size exclusion chromatography (BMH (Mw) 18.6 ± 0.5 kDa and PMH 15.4 ± 0.4 kDa). BMH exhibited a potency of 140 U/mg and PMH had a potency of 195 U/mg. In the anticoagulant and antiprotease assays, the BMH exhibited lower functionality which was proportional to USP potency. In vitro, when the BMH was compared at a potency adjusted concentration with PMH, it showed identical calibration curves in the aPTT and anti-protease assays. However, in the protamine neutralization and platelet factor 4 studies, BMH required slightly higher amounts of the agents in contrast to PMH. The concentration vs. time curves for both heparins were almost superimposable. Peak drug levels of approximately 1.5 and 1.4 U/mL were measured using anti-Xa and anti-IIa assays, respectively. After 2 hours, circulating drug levels were decreased to approximately 0.4 U/mL for all heparins. Pharmacokinetic parameters calculated from plasma concentration-time curves indicated that both heparins behaved similarly. Mean half-life based on anti-Xa activity ranged from 54 ± 11 min for porcine heparin to 71 ± 18 min for bovine heparin. Slightly longer half-lives were observed using plasma concentrations determined using anti-IIa activity. Mean AUC values based on anti-Xa or anti-IIa activities were comparable for both heparins. Mean Vd (~60 ml/kg) and Cl (~0.75 ml/kg/min) were also comparable for both heparins.

Conclusion: In vitro, BMH at adjusted biologic potency is comparable to PMH, however, it requires proportionally higher amount of protamine and platelet factor 4 due to the increased mass for adjusting to higher potency. In the non-human primates, USP cross-referenced anti-Xa potency adjusted based dosing results in comparable pharmacokinetic profiles for bovine and porcine heparins. Therefore, such dosing may provide uniform levels of anticoagulation for the parenteral indications for heparins. These observations warrant clinical validations in the specific indications.