Evaluation of Potential Safety Biomarkers for Monitoring Thrombogenic Potential of FXa^I16L

FXa^I16L is a variant of activated coagulation factor Xa in which the conformational transition from zymogen to active protease is impaired. Binding to FVa facilitates its transition to the active conformation, rescues procoagulant activity and is hypothesized to localize FXa^I16L hemostatic effect to sites of hemorrhage.

In nonclinical toxicology studies, thrombi/emboli were observed in animals administered doses exceeding the projected efficacious dose. To identify a possible biomarker predictive of FXa^I16L -related thrombi/emboli, effects of FXa^I16L on platelets (PLAT), fibrinogen (FBGN), activated partial thromboplastin time (aPTT), prothrombin time (PT), factor V activity (FV), protein C (PROT C) activity, tissue factor pathway inhibitor (TFPI), and antithrombin III (AT-III), were evaluated in in male cynomolgus monkeys (3/group) given FXa^I16L for 5 days at IV bolus dosages of 0, 0.1, or 1 mg/kg/day (administered as 0, 0.05, 0.5 mg/kg/dose; BID, 8 h apart). Changes in these parameters were evaluated 5 and 30 min after each dose on Days 1, 3 and 5, and histological evaluation for thrombi/emboli occurred approximately 45 min after first daily dose on Day 5. A single thrombus was observed in the heart of a single animal given 0.1 mg/kg/day, multiple thrombi/emboli were observed in the heart, lung and/or kidney of all animals given 1 mg/kg/day. Compared with pretest values, decreased (range) PLAT (24%-45%), FBGN (11%-86%), aPTT (11%-30%), FV (11%-76%), PROT C (60%-100%), ATIII (2%-26%), and prolonged PT (5%-524%) were observed at ³0.1 mg/kg/day, and prolonged aPTT (17%-367%) and increased TFPI (10%-49%) occurred at 1 mg/kg/day. PROT C activity was below detectable levels 5 min after dosing 1 mg/kg/day, a dose that caused multiple thrombi/emboli. Thus, PROT C activity (as measured by StaClot activity assay) may have utility as a safety biomarker for monitoring treatment with FXa^I16L.

Depletion of PROT C activity in the 5-day biomarker study in monkeys may reflect actual depletion of PROT C, or may reflect pharmacologic activity of FXa^I16L that interferes with the clot based endpoint of this assay since the compound may inherently decrease clotting times. To distinguish between these possibilities, in vitro experiments were performed wherein varying concentrations of FXa^I16L were added to normal human plasma and specimens were assayed by the StaClot® (clot based)PROT C activity assay, the StaChrome® (chromogenic) PROT C activity assay and by the Asserachrom® (ELISA) PROT C assay. In a similar manner, effect of FXa^I16L on protein S levels was assessed using the StaClot® (clot based) protein S activity assay and an antigenic assay for free protein S. Following in vitro incubation of human plasma with increasing concentrations of FXa^I16L, clot-based methods for PROT C and Protein S showed dose-dependent decreases in activity. In contrast, chromogenic and ELISA-based methods for PROT C or antigenic assay for Protein S remained unchanged from baseline levels, confirming that FXa^I16L interferes with the StaClot® assay and does not deplete Protein S or PROT C.

Data presented as Mean ± S.D. n = 5 donor plasmas/drug concentration.

^aStaClot PROT C or Protein S – clot-based endpoint.

*Statistically different from baseline (0 ng/mL). P<0.05.

Conclusion: Pharmacologic activity of FXa^I16L interferes with the clot-based PROT C and protein S assays. However, because marked decreases in the clot-based PROT C assay correlated with the observation of thrombi/emboli in monkeys, this assay remains a useful safety biomarker for monitoring treatment with FXa^I16L. Regardless, the impact of drug pharmacology on the respective assay mechanism should be considered when interpreting results.