Pursuing Biomarkers Beyond Pt Inr And aPTT

Values obtained with aPTT under ex vivo conditions were less sensitive than PT to FXa inhibitor-i nduced elevations and often underestimated drug concentration (data not shown). Beyond PT, INR, and aPTT, the most commonly used assay to evaluate FXa inhibitors is probably the anti-factor Xa assay (anti- FXa). It seems logical that a parameter named anti- Factor Xa assay should be the ideal biomarker for a FXa inhibitor. Additionally, this assay is used routinely in clinical settings to monitor the safety of heparin, a substance that also inhibits FXa production [20]. However, this assay is little more than a surrogate marker for drug concentration. A standard curve is prepared using the administered heparin (or other FXa inhibitor), and the chromagenic assay allows determination of the drug concentration in the plasma samples via production of FXa [ 21] - For heparin, the anti - FXa assay appears relevant. Years of use has allowed the development of a strong correlation between the number of international units of heparin determined via the assay and clinical safety. Reference ranges have been defined for the assay and provide a rapid estimation of under, over, or therapeutic levels of heparin administration [22]. Still variability in the anti-FXa assay has been reported and is attributable to a number of factors, including instrumentation, assay technique, specificity of the commercially available kits, heparin preparations used in generating the standard curve, and approaches to data fitting [21]. In contrast, this experience does not exist for anti-Xa values obtained during FXa inhibitor administration. Just as the PT and INR may not be as beneficial for predicting FXa inhibitor effects as they are for coumadin, it should not be assumed that the anti-FXa assays have equivalent predictivity for heparin and other FXa inhibitors. For the Pfizer developmental FXa inhibitor, the anti-FXa assay offered little more than the PT as a monitor of drug concentration.

An additional assay called the factor X clotting (FX:C) assay was also evaluated. This assay is conducted using genetically engineered factor-deficient plasma spiked with serial dilutions of purified human factor X [23,24]. Concentrations of factor X in plasma are then determined by extrapolation from the standard curve. Since factor X must be converted to factor Xa for clot formation to occur, a functional clotting assay for factor X can also be used to assess the effects of factor Xa inhibitors. The FX:C assay provides several unique features that may make it a valuable biomarker for monitoring factor Xa inhibitor therapy: (1) the assay provides a rapid, reliable assessment of drug concentration and the percent inhibition of FXa achieved during drug inhibitor administration; (2) the assay can be performed on a high-throughput automated platform that is available in most hospital-based coagulation laboratories; and (3) individual factor X concentrations range from 60 to 150% between subjects [25]. This fairly high level of baseline intersubject variability suggests that a standard dose of drug may have a substantially different impact on total factor X inhibition. The FX:C assay defines baseline factor X activity and thereby allows continued dosing to achieve a targeted factor X concentration [4]. Literature is available concerning factor X concentrations and bleeding history in patients with either inherited or acquired factor X deficiency, so minimally there is some understanding that correlates the impact of reductions in FX:C evaluations and bleeding potential [26-28]. By determining the actual concentration of functional factor X remaining, physicians may have increased confidence in the administration of factor Xa inhibitors.

As with all the other coagulation biomarkers used for monitoring FXa inhibition, it was not immediately clear whether the FX:C assay was applicable in multiple species. Ex vivo experiments allowed this evaluation. To provide effective anticoagulant activity a 30% reduction in FX:C activity was predicted to be the minimal requirement for this compound. The FXa inhibitor concentrations in the ex vivo experiments were selected to bracket a range of factor X inhibition predicted to range from approximately 30% to 100%. Table 6 shows the intended concentrations of this FXa inhibitor in each species, the resulting FX:C activity, and the percent inhibition achieved. Assessment of these drug concentrations induced factor Xa inhibition of approximately 20% to >90%, showing that the targeted range could be predicted and achieved in

TABLE 6 Factor X Activity and Percent Inhibition in Plasma Samples Containing Increasing Concentrations of a Factor Xa Inhibitor"
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