Dose Selection for Ex Vivo Experiments

In designing ex vivo experiments to evaluate potential biomarkers, selection of the appropriate drug concentration is critical. Furthermore, when experiments are conducted in multiple species, selection of the same drug concentration for all species is typically not ideal, due to species-specific drug sensitivity. Factor X concentrations vary by species, and the level of FXa inhibition is also variable. Therefore, the concentrations of FXa inhibitor utilized in this particular ex vivo evaluation were selected to achieve a range of FXa inhibition that was modest to nearly complete in all species examined. Pharmacology studies predicted that this FXa inhibitor would result in species-specific factor Xa sensitivity in the order human > dog > rat. Interestingly, this species specificity was not identical to that observed with Otamixaban [10], demonstrating that extrapolating biomarker data even between compounds in the same class may be misleading. For this developmental FXa inhibitor, human plasma was spiked to obtain final drug concentrations of 0, 0.2, 0.6, 1.2, and 6.0 |g/mL. Drug concentrations of 0, 0.4, 2.0, 8.0, and 15.0 |g/mL were selected for dog assessments and 0, 1.0, 4.0, 12.0, and 24.0 |g/mL were used for ex vivo assessments in rats to achieve a comparable range of FXa inhibition compared to that observed in human samples.

Thromboplastin is the reagent that induces clot formation in the PT assay. There is ample documentation that the type and sensitivity of thromboplastin is a critical factor in the effective and safe monitoring of coumadin administration [13-17]. To minimize this variability in PT assays a calibration system was adopted by the World Health Organization (WHO) in 1982. This system converts the PT ratio observed with any thromboplastin into an international normalized ratio (INR). This value was calculated as follows: INR = observed PT ratioc, where the PT ratio is subject PT/control PT and c is the power value representing the International Sensitivity Index (ISI) of the particular throm-boplastin [18]. This system has proven to be an effective means of monitoring human oral anticoagulant therapy with coumadin and has been implemented almost universally. It allows individuals to be monitored at multiple clinics using varying reagents and instrumentation, while still achieving an accurate assessment of true anticoagulation. However, there is little or no information regarding selection of thromboplastin reagents or use of the INR for monitoring of FXa inhibitors.

Typically, the higher the ISI value, the less sensitive the reagent, and the longer the PT time produced. The most commonly used thromboplastin reagents for PT evaluation are either rabbit brain thromboplastin (of variable ISI values, depending on manufacturer and product) or human recombinant thromboplastin, typically with an ISI of approximately 1.0. Use of the INR is accepted as a more relevant biomarker of anticoagulant efficacy than are absolute increases in PT alone, at least for coumadin therapy [13] . To more fully evaluate the effect of this FXa inhibitor on INR, PT was evaluated using rabbit brain thromboplastin, with ISI values of 1.24, 1.55, and 2.21 and a human recombinant thromboplastin (0.98 ISI). Although either human recombinant thromboplastin or rabbit thromboplastin are considered acceptable reagents for the conduct of PT testing, it was unclear whether these reagents would produce similar results in the presence of an FXa inhibitor or whether the sensitivity of the thromboplastin itself would affect results.

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Project Management Made Easy

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