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

Objective Primate studies have shown that bovine and porcine heparins dosed at equivalent anti-Xa units produce equivalent anti-IIa and anti-Xa responses in vivo. Heparin’s pharmacokinetic behavior is typically based on antithrombin (AT)-dependent anti-Xa and anti-IIa activities. Heparin Red is a polycationic substance whose intrinsic fluorescence is quenched upon binding to heparin. Assays utilizing Heparin Red detect heparin oligosaccharides that bind to AT and those that do not. Non-AT binding heparin oligosaccharides can impact coagulation through their interaction with heparin cofactor II, TFPI and platelets. Thus, the pharmacokinetic behavior of heparin as determined using a Heparin Red assay may more closely reflect the clinical behavior of heparin than pharmacodynamic measures where heparin levels are determined based only on AT-dependent activities.

Material and 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. 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 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, respectively. By Heparin Red assay, peak heparin levels were higher following BMH administration (10.7 ± 0.5 vs. 9.0 ± 0.2 ug/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 ug*min/ml; Mann-Whitney test p=0.029).

Conclusion At equivalent anti-Xa unit doses, BMH 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 identifying the appropriate dose of protamine to completely neutralize BMH.