Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmacology and Experimental Therapeutics

Abstract

Heparin and heparin related drugs have remained the anticoagulant of choice for medical and surgical indications. These drugs are comprised of poly-component sulfated chains of varying length and biophysical structure with direct and indirect pharmacological effects. Heparins produce their therapeutic effects by multiple mechanisms involving both the cellular and humoral targets. These drugs represent natural products which are isolated from mammalian tissue rich in mast cells. Currently used heparin and LMWHs are obtained from porcine mucosal tissue. Other animal sources including bovine and ovine tissues also provide additional sources for the manufacturing of heparins. While there may be microchemical and biophysical differences among these agents, heparins from different origins produce anticoagulant effects, which can be standardized using various assay methods which include the whole blood clotting, global anticoagulant studies and Serpin mediated inhibition of such enzymes as Thrombin and factor Xa. The primary purpose of this dissertation was to compare the structural and functional characteristics of BMH, OMH and PMH and their depolymerized derivatives (LMWHs). Furthermore, to test the hypothesis that "Despite molecular components distribution and minor structural differences, potency referenced heparins of different animal origins and their depolymerized derivatives may exhibit similar pharmacological activities". For this investigation, one batch of each heparin and LMWH source were utilized. Their activities in biochemical and biological assays were compared in order to elucidate how their molecular composition and structural characteristics affect their pharmacological actions. To compare the molecular and structural features of UFHs and LMWHs, such analytical methods as GPC, NMR and LC-MS were utilized. Other biophysical methods including light scattering analysis (Photon correlation spectroscopy and Z-potential measurements) were also used. A comprehensive profiling of the anticoagulant and antiprotease actions of these agents with refence to Serpin mediated modulation of the hemostatic system along with cellular effects of these agents were investigated in whole blood and defined biochemical systems. The in vivo actions of UFHs and LMWHs have been investigated in terms of multiple PK and PD parameters in simulated settings and by utilizing a validated non-human primate (Macaca Mulatta) model. These investigations have demonstrated that BMH, OMH and PMH exhibit similar molecular and structural profiles with minor differences. BMH showed weaker anticoagulant and antiprotease activities compared to OMH and PMH and this difference was not observed when compared potency adjusted-based BMH. The molecular and structural profiles of all LMWHs were comparable and each produced similar anticoagulant and antiprotease activities. The in vitro and in vivo neutralization profiles of all heparins were comparable and both the PS and PF4 effectively neutralized these agents. However, the LMWHs were only partially neutralized in a comparable and an assay dependent fashion. Studies in platelet-based systems showed that AIPA profile and the HIT antibody interactions among all heparins and LMWHs from different species were comparable. In vivo studies in animal model showed that, the anti-Xa and anti-IIa effects of OMH and PMH were significantly stronger compared to BMH after IV administration. However, BMH showed relatively longer half-life (t1/2) compared to PMH and OMH with no major variations in AUC, Vd and Cl parameters between each agent. In regard to LMWHs, all agents demonstrated similar anti-Xa and anti-IIa effects and comparable PK/PD parameters with no significant differences. Additional functional PD parameters including endogenous TFPI release and thrombin generation potential assays showing comparable effects with potency adjusted UFHs and LMWHs. The absolute PK parameters as measured by quantifying GAG contents utilizing a fluorescence quenching method were consistent to the results obtained in the functional PK/PD studies. In conclusion, this dissertation has validated the hypothesis that potency adjusted UFHs and LMWHs of different origins are comparable in mediating their biologic and pharmacologic actions. Thus, the cross-referencing of these agents with established pharmacopeial standards provide heparins with comparable anticoagulant actions which may be interchangeable warranting clinical validation.

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