Arylamides: Discovery of a Novel Class of Fully-Synthetic Small Molecule Heparin and LMWH Antagonists.

Abstract 1096

Structural studies of the binding interaction between the negatively charged heparin pentasaccharide binding site and antithrombin III suggested that an amphiphilic structure resembling features designed into compounds prepared for antimicrobial activity might also provide an efficient design basis for the development of heparin antagonists. A key design element of our antimicrobial approach at PolyMedix involves compounds exhibiting polycationic arrays which are appended to a diverse set of rigid backbones. A broad screening of the antimicrobial compound library in an enoxaparin (ENX) anti-factor Xa assay, coupled with subsequent secondary testing, identified compounds with potent and broad neutralizing activity. A lead compound (PMX-60056) was identified in one structural series, the salicylamides, and this compound has successfully completed Phase 1 human clinical studies. Recently, a new series of antagonists identified in the initial screen, the arylamides, was investigated for heparin neutralizing activities. Compounds were first tested for their ability to neutralize the anticoagulant activity of ENX, tinzaparin (TZP) or fondaparinux (FPX) in an in vitro amidolytic assay for factor Xa activity. Activated partial thromboplastin time (aPTT) assays demonstrated that the compounds were also active against unfractionated heparin (UFH) in a plasma-based clotting assay. The lead compound PMX150 was comparable to or better than PMX-60056 versus all anticoagulants tested (EC50s: UFH 0.6 uM, ENX 0.1 uM, TZP 0.1 uM, FPX 0.7 uM). Early compounds have shown an interesting structure activity relationship (SAR) which has identified specific regions of the molecule that are important for activity. Additionally, rotation thromboelastometry (ROTEM) shows that compounds are able to neutralize heparin and ENX in human whole blood, restoring normal coagulation profiles. As an initial test for safety, compounds were evaluated in hemolysis and cytotoxicity assays using isolated human erythrocytes, a transformed human liver cell line (HepG2 cells) and a mouse fibroblast cell line (NIH3T3). PMX150 was not cytotoxic (or hemolytic) at concentrations 180 fold greater than the EC50 concentrations in the anti-coagulation assays, indicating a high selectivity index between toxicity and efficacy. Furthermore, the SAR has identified synthetic strategies that should further improve this safety margin. The in vivo efficacy of PMX150 was evaluated in a rat coagulation model using ENX (2 mg/kg) administered by IV injection followed 3 minutes later by saline, protamine or PMX150. Blood was collected before dosing with ENX, and at 1, 3, 10, and 60 min after dosing, for aPTT and factor Xa analysis. PMX150 was more efficacious than protamine; neutralizing 89–100% of the ENX anti-factor Xa activity over the entire 60 minute time course. The in vivo assays concur with the in vitro studies, showing that compounds have been identified with high efficacy versus low molecular weight heparin (LMWH). Protamine, the heparin antagonist currently used in the clinic, routinely causes a transient decrease in blood pressure upon dosing. Hemodynamic effects with PMX-60056 have also been observed in human subjects in the absence of heparin. As PMX150 represents a new chemical series, investigations into potential adverse hemodynamic effects were performed. Blood pressure and heart rate were measured via arterial catheters in rats administered protamine or PMX150 by a 10 minute IV infusion. As expected, protamine displayed transient and prolonged blood pressure reductions at 8 and 16 mg/kg dosages, respectively. However, PMX150 had little to no effect on blood pressure at 8 mg/kg and only a minor transient reduction at 16 mg/kg. In conclusion, we have identified a new series of fully synthetic small molecule antagonists that display potent in vitro activity against UFH, the LMWHs, and fondaparinux. The lead compound in this series, PMX150 is able to neutralize ENX in vivo with more efficacy than protamine and, furthermore, has an improved hemodynamic profile with respect to protamine.