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MODULATING PLASMIN ACTIVITY USING REVERSIBLE MULTIVALENT INHIBITORS FOR DRUG DELIVERY APPLICATIONS

thesis
posted on 2022-12-07, 19:10 authored by Tanmaye Nallan ChakravarthulaTanmaye Nallan Chakravarthula

Deep vein thrombosis (DVT) and Pulmonary embolism (PE) are responsible for over 900,000 cases and 100,000 deaths each year in the US. Direct fibrinolytic agents such as plasmin are being investigated for their treatment. However, plasmin administration is not widely studied as low plasmin concentrations are rapidly inactivated by antiplasmin in vivo, whereas high plasmin doses would deplete endogenous antiplasmin and impose bleeding risks. Thus, a plasmin delivery system that can achieve efficient clot lysis while minimizing inactivation by antiplasmin and has reduced bleeding risks is needed. To address this, we propose using reversible inhibitors of plasmin that can sequester plasmin from antiplasmin and release it on the surface of a fibrin clot to achieve clot lysis. The inhibition must be tuned such that it is strong enough to protect plasmin from antiplasmin and weak enough to release plasmin at the clot for lysis. To achieve this, we utilize principles of multivalency to synthesize three classes of inhibitors with varying potencies and mechanisms of inhibition: (i) Multivalent benzamidines (ii) Multivalent tranexamic acids (TXA), and (iii) Hetero-multivalent inhibitors having both benzamidine and TXA. Benzamidine is a competitive inhibitor of plasmin’s active site. TXA, on the other hand, is an FDA-approved weak active site inhibitor that is primarily used to disrupt plasmin(ogen) from binding to fibrin on the clot by inhibiting plasmin’s kringle domains. Multivalent inhibitors were synthesized using amine-reactive chemistry, purified using RP-HPLC and confirmed with Mass Spectrometry. Inhibition assays were performed to assess inhibition potency by determining Ki values (inhibition constants). Lower Ki values indicate stronger inhibition. With multivalent benzamidine derivatives, it was observed that changing valency and linker length substantially impacted inhibition and resulted in Ki values ranging from 2.1 to 1,395 μM. Inhibitors of higher valencies and shorter linker lengths exhibited stronger inhibition. Multivalent TXAs of valencies 1 to 16 were also tested and they exhibited Ki values varying from 2.5 to 21,370 μM indicating up to 8,548-fold improvement in inhibition due to valency. It was found that monovalent TXA, primarily a kringle inhibitor, can be converted into a stronger active site inhibitor by multivalency. With hetero-bivalent TXA-dPEG36-AMB, simultaneous binding of benzamidine to the active site and TXA to the kringle domains was achieved to attain improved inhibition. These results indicate that multivalency can significantly alter the potency of inhibitors and can modulate plasmin inhibition for drug delivery.

History

Degree Type

  • Doctor of Philosophy

Department

  • Biomedical Engineering

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

Nathan J. Alves

Advisor/Supervisor/Committee co-chair

Sherry L. Harbin

Additional Committee Member 2

David H. Thompson

Additional Committee Member 3

Luis Solorio