Design, synthesis, and SAR of a series of activated protein C (APC) inhibitors with selectivity against thrombin for the treatment of haemophilia.

A design strategy was used to identify inhibitors of activated protein C with selectivity over thrombin featured by a basic and/or aromatic functionality for binding to the S2 pocket. Our strongest inhibitor showed an IC50-material value and selectivity for APC vs thrombin similar to a compound previously reported in the literature. However, in contrast to the reference compound, our compound showed a retained coagulant effect of thrombin with increasing substrate concentration in a modified Calibrated Automated Thrombogram (CAT) method. This was likely related to our compound being inactive against FVIIa, while the reference compound showed an IC50 of 8.9 µM. Thus, the higher selectivity of our compound against all relevant coagulation factors likely explained its higher therapeutic potential in comparison to the reference compound. The data indicate that at least a 100-fold selectivity over other serine proteases in the coagulation cascade will be required for an effective APC inhibitor.

Activated protein C inhibitor for correction of thrombin generation in hemophilia A blood and plasma1.

BACKGROUND: Replacement therapy for hemophilic patient treatment is costly, because of the high price of pharmacologic products, and is not affordable for the majority of patients in developing countries. OBJECTIVE: To generate and evaluate low molecular weight agents that could be useful for hemophilia treatment. METHODS: Potential agents were generated by synthesizing specific inhibitors [6-(Lys-Lys-Thr-[homo]Arg)amino-2-(Lys[carbobenzoxy]-Lys[carbobenzoxy]-O-benzyl)naphthalenesulfonamide] (PNASN-1)] for activated protein C (APC) and tested in plasma and fresh blood from hemophilia A patients. RESULTS: In the activated partial thromboplastin time-based APC resistance assay, PNASN-1 partially neutralized the effect of APC. In calibrated automated thrombography, PNASN-1 neutralized the effect of APC on thrombin generation in normal and congenital factor VIII-deficient plasma (FVIII:C < 1%). The addition of PNASN-1 to tissue factor-triggered (5 pm) contact pathway-inhibited fresh blood from 15 hemophilia A patients with various degrees of FVIII deficiency (FVIII:C < 1-51%) increased the maximum level of thrombin generated from 78 to 162 nm, which approached that observed in blood from a healthy individual (201 nm). PNASN-1 also caused a 47% increase in clot weight in hemophilia A blood. CONCLUSIONS: Specific APC inhibitors compensate to a significant extent for FVIII deficiency, and could be used for hemophilia treatment.