Novel Antagonists for Low Molecular Weight Heparin and Heparin-Like Drugs.

Abstract 2092

Poster Board II-69

Unfractionated heparin and other heparin derivatives such as low molecular weight heparins (LMWHs) are used for treatment of coagulation diseases and in surgical applications. Currently neutralization is achieved using protamine sulfate, whose cationic properties allow charge-based binding to heparin. However, protamine is unable to completely reverse LMWH's anticoagulant effects and has potential for side-effects. Our objective was to characterize the ability of a series of salicylamide derivatives (PolyMedix Corp, Radnor, PA) to neutralize the activities of a LMWH (enoxaparin) and a heparin-derived drug containing the antithrombin-binding sequence (fondaparinux) to better understand the structural features required for effective neutralization. Human plasma (n=4) was supplemented with enoxaparin (0.9 – 15 μg/ml) or fondaparinux (0.6 – 10 μg/ml) to develop concentration-response curves. Each of 18 salicylamide derivatives (i.e. PMX 60056, etc.) or protamine sulfate was added to the enoxaparin- and fondaparinux-supplemented plasma samples at concentrations of 2.5 or 5.0 μg/ml. Anticoagulant activity was measured by coagulation (activated partial thromboplastin time and Heptest) and anti-protease (anti-factor Xa and anti-thrombin) assays using an automated coagulation analyzer. Data was analyzed in terms of percent neutralization of therapeutic (7.5 and 1.25 μg/mL) and prophylactic concentrations (3.75 and 0.625 μg/mL) of enoxaparin and fondaparinux, respectively. For the LMWH, the ratios of neutralizing agent to enoxaparin ranged from 0.33 to 1.33. As the ratio increased, more effective neutralization was observed. Consistent with previous results, protamine was only capable of partially neutralizing the in vitro activities of LMWH. While approximately 60% of the anti-thrombin activity was neutralized, much less neutralization was observed in terms of anti-Xa and anticoagulant activity. The prototype salicylamide derivative (PMX 60056) more effectively neutralized the anti-thrombin and anti-Xa activities of enoxaparin. Of the 17 new derivatives, approximately half were as effective as PMX 60056 at neutralizing anti-thrombin activity, and several were notably better at neutralizing anti-Xa activity. It was also noted that several compounds were not effective at neutralizing anticoagulant or anti-protease activities. The ratios of neutralizing agent to fondaparinux ranged from 2.0 to 8.0. At any of the ratios tested, protamine was ineffective at neutralizing anti-Xa or Heptest activity. Although all salicylamide derivatives exhibited a reduced capacity to neutralize fondaparinux in comparison to enoxaparin, at higher ratios, several of the derivatives were more potent than PMX 60056. This study shows that salicylamide derivatives can neutralize the anticoagulant and anti-protease actions of LMWH and fondaparinux. Manipulation of chemical structure may allow for the identification of agents that are more effective than currently available antagonists.