In Vitro Characterization of the Neutralization of Unfractionated Heparin and Low Molecular Weight Heparin by Novel Salicylamide Derivatives.

Introduction: Unfractionated heparin and the low molecular weight heparins (LMWHs) are commonly used in the treatment of acute coronary syndromes, as prophylaxis against deep vein thrombosis and pulmonary embolism and to prevent clotting during interventional and surgical procedures. The neutralization of unfractionated heparin is critical following the completion of coronary bypass surgery to avoid excessive blood loss. Unfractionated heparin can be neutralized by protamine sulfate, a highly cationic peptide that binds to heparin in a charge-dependent manner. However, the use of protamine can be associated with serious side-effects such as hypotension, bronchoconstriction, or pulmonary hypertension, possibly due to the release of histamine. Additionally, large doses of protamine can produce an anticoagulant effect. This study characterizes the ability of a series of low molecular weight, homogeneous, synthetic, polycationic salicylamide derivatives (PolyMedix, Radnor, PA) to neutralize the anticoagulant actions of unfractionated heparin and enoxaparin.

Methods: Human plasma was supplemented with unfractionated heparin or enoxaparin (Sanofi-Aventis, Paris, France) at a concentration of 10 μg/ml. Protamine sulfate or one of six of the structurally distinct salicylamide derivatives was added to aliquots of heparinized plasma to achieve final concentrations of 50, 25 and 12.5 μg/ml. The supplemented plasmas were immediately analyzed using clotting (aPTT, Heptest, thrombin time) and amidolytic (anti-Xa, anti-IIa) assays.

Results: Using the in vitro assays, protamine sulfate was shown to concentration-dependently neutralize the actions of unfractionated heparin in all of the assays. Two of the salicylamide derivatives tested produced an effect comparable to protamine, while three derivatives exhibited a relatively stronger neutralization of unfractionated heparin. The extent of neutralization measured by anti-Xa and anti-IIa assays was also greater with the derivatives. While residual anti-Xa and anti-IIa activities (20% and 10%, respectively) were observed even with a 5-fold gravimetric excess of protamine, complete neutralization was observed with the salicylamide derivatives. Protamine is known to be less effective at neutralizing LMWHs. In this study, the anticoagulant activity of enoxaparin as measured by aPTT and Heptest was neutralized approximately 50% by protamine even at a 5:1 protamine to enoxaparin ratio. The derivatives were able to completely neutralize the anticoagulant effects of enoxaparin. A similar pattern was observed with the amidolytic assays. While protamine was unable to neutralize the anti-Xa activity of enoxaparin, 5 of the 6 salicylamide derivatives concentration-dependently inhibited the anti-Xa activity.

Discussion: These studies demonstrate that the PolyMedix series of salicylamide derivatives can effectively neutralize the anticoagulant and anti-protease actions of unfractionated heparin and LMWHs such as enoxaparin. Initial results suggest that such agents are more effective than protamine at neutralizing other LMWHs. Future studies are designed to characterize the compounds’ PK/PD profiles. These results warrant further studies on the neutralization profile of PolyMedix series of salicylamide derivatives in animal models of bleeding and thrombosis.