Darexaban has high sensitivity in the prothrombin time clotting test

Most synthetic factor Xa (FXa) and thrombin inhibitors examined in pharmacokinetic studies and phase II/III clinical trials have had predictable pharmacokinetics and have been found to be effective and safe without the need for laboratory monitoring [1]. However, assessment of the degree of antithrombotic effect may be beneficial in certain patients, such as those with renal or liver impairment, or in emergency situations, although precisely how such assessments should be carried out is an open issue. Current pharmacodynamic markers for these agents include well-established clotting assays that measure prothrombin time (PT) and activated partial thromboplastin time (APTT), more sensitive methods that measure diluted PT (DPT), and specific enzyme assays for FXa inhibition and thrombin generation [1]. Changes in these markers correlate with the plasma concentration of the investigated inhibitor; however, their accuracy in predicting systemic exposure to inhibitors is unsatisfactory, owing to poor sensitivity or non-standardized procedures. Darexaban (YM150) is a novel, potent, oral direct FXa inhibitor that prevents the prothrombin activation induced by free and clot-associated FXa, prothrombinase, and whole blood clots [2]. After oral administration, darexaban is rapidly and almost entirely metabolized to darexaban glucuronide. Although darexaban and darexaban glucuronide are equipotent, darexaban glucuronide is the main active moiety in humans in vivo [3]. Consistent with this, a high correlation between darexaban glucuronide plasma concentration and PT prolongation has been observed, and relatively low doses of darexaban (20 mg twice-daily) result in a four-fold PT prolongation, indicating that PT is highly responsive to changes in darexaban glucuronide plasma concentrations [3]. Here, we compared the effects of darexaban on PT with those of other direct FXa inhibitors, using several thromboplastin reagents. We also examined the relationship between PT prolongation and FXa inhibition for each inhibitor. Human plasma was spiked with a 1/100 volume of vehicle, darexaban, darexaban glucuronide, rivaroxaban or apixaban to achieve different final concentrations, selected on the basis of reported clinical exposure concentrations during the treatment of venous thrombosis: approximately 0.3–3 lmol L (trough to peak concentrations) for darexaban glucuronide, and 0.03– 0.3 lmol L for rivaroxaban and apixaban [3–5]. The chromogenic assay for FXa activity in plasma was performed as described previously, with minor modifications [6]. PT was measured with three commercially available thromboplastin reagents, according to each manufacturer s protocol. The PT ratio (PTR) was calculated as the PT for the inhibitor-spiked plasma divided by the PT for the vehicle-spiked plasma. The local international sensitivity index values were determined to be 1.12, 0.91 and 0.84 for HemosIL PT-fibrinogen HS PLUS, PT-fibrinogen recombinant, andRecombiPlasTin, respectively. For theDPT test, thromboplastin reagents were diluted 16-fold as compared with conventional PT test quantities with 50 mmol L calcium chloride solution. All sample handling and FXa inhibitor-spiked plasma to vehicle-spiked plasma ratio calculations were otherwise similar to those applied in the conventional PT tests. Data are expressed as the mean ± standard error of the mean of four independent assays. ED50 values were calculated with a sigmoid-Emax model for the inhibition of FXa activity. Darexaban, darexaban glucuronide, rivaroxaban and apixaban inhibited FXa activity in human plasma in a concentration-dependent fashion, with typical Hill-shaped concentration–FXa curves, but different potencies (EC50 values and 95% confidence intervals: 3.4 [3.1–3.7], 1.1 [1.0–1.2], 0.12 [0.11–0.13], and 0.15 [0.14–0.15] lmol L, respectively). At clinical exposure concentrations, all tested FXa inhibitors prolonged the PT obtained when mixed with commercial thromboplastins (Fig. 1A). Furthermore, darexaban and its human metabolite darexaban glucuronide produced higher PTR values than rivaroxaban or apixaban, regardless of the thromboplastin reagent used. The relationship between PTR and FXa inhibition is shown in Fig. 1B. Interestingly, darexCorrespondence: Toshiyuki Funatsu, Applied Pharmacology Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki, Japan. Tel. : +81 3 5916 5329; fax: +81 3 5916 5606. E-mail: toshiyuki.funatsu@astellas.com