In Vivo Neutralization of Unfractionated Heparin and Low Molecular Weight Heparin by a Novel Salicylamide Derivative.
Authors: Kuziej Jacqueline , Litinas Evangelos , Hoppensteadt Debra , Liu Dahui , Walenga Jeanine M. , Fareed Jawed , Jeske Walter 
Published: April 2020

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. Although unfractionated heparin can be neutralized by protamine sulfate, its use can be associated with serious side-effects such as hypotension, bronchoconstriction, or pulmonary hypertension. Additionally, large doses of protamine can produce an anticoagulant effect. We had previously reported on the ability of salicylamide derivatives to neutralize anticoagulant activity following supplementation to heparinized plasma. This study evaluates the ability of one such salicylamide derivative (PMX 60056; PolyMedix, Radnor, PA) to neutralize the hemorrhagic and antithrombotic actions of unfractionated heparin and enoxaparin in defined animal models.

Methods: Male, Sprague-Dawley rats were anesthetized and then treated with an IV bolus of heparin or enoxaparin. Five minutes later, rats were treated with either saline or one of three doses of protamine sulfate or PMX 60056. Five minutes later, the distal 2 mm of the rat tail was transsected and the time for bleeding to stop was measured. Upon completion of the bleeding model, the jugular veins were isolated and residual antithrombotic activity was measured using a jugular vein clamping model (Raake et al, Thromb. Res53(1):73–7, 1989). A blood sample was collected prior to euthanasia to evaluate levels of circulating anticoagulant activity.

Results: At a dose of 2 mg/kg, both UFH and enoxaparin significantly prolonged the bleeding time compared to vehicle-treated control (UFH: 29.1±5.1 min vs. 7.3±1.4 min, p&lt;0.001; enoxaparin: 16.7±3.6 min vs. 7.3±1.4 min; p&lt;0.001). Over a concentration range of 0.5 to 2.0 mg/kg, both protamine and PMX 60056 dose-dependently reduced the heparin-induced increase in bleeding time; at equigravimetric doses, both protamine and PMX 60056 produced near complete neutralization of the heparin-induced bleeding. Both protamine and PMX 60056 were also able to neutralize the hemorrhagic effects of enoxaparin, and PMX 60056 appeared to be somewhat more effective than protamine (7.4±1.2 vs. 8.7±1.5 min) at equigravimetric concentrations of enoxaparin and neutralizing agent. Residual antithrombotic activity was comparable in the heparin and enoxaparin-treated rats (7.2±1.1 and 7.0±0.6 clampings, respectively vs. 3.4±0.5 for vehicle treated rats). Comparable neutralization of antithrombotic activity by both protamine and PMX 60056 was observed. Blood samples collected prior to euthanasia were analyzed for anticoagulant (aPTT and Heptest) and anti-protease (anti-Xa and anti-IIa) activities. While variable degrees of neutralization could be observed in these assays, overall there was a poor correlation between residual anticoagulant/anti-protease activity and neutralization of bleeding time.

Discussion: These studies demonstrate that novel salicylamide derivatives can effectively neutralize the antithrombotic and hemorrhagic actions of unfractionated heparin and LMWHs such as enoxaparin. Initial results suggest that PMX 60056 is equally effective as protamine at neutralizing UFH and may be slightly better at neutralizing enoxaparin. Further studies to characterize the PK/PD profiles of the salicylamide derivative are warranted.