Dose-effect relationship for several coagulation markers during administration of the direct thrombin inhibitor S 18326 in healthy subjects

Abstract

Aims: We conducted a phase I placebo-controlled trial with two i.v. doses (0.5 mg h-1 and 3 mg h-1) of S 18326, a selective thrombin inhibitor that interacts with the catalytic site of thrombin, with the aim to study the relationships between increasing plasma levels of S 18326 and changes in coagulation tests and thrombin generation markers.

Methods: Thirty-six healthy male volunteers were divided into three groups. In each group, 10 volunteers were randomly assigned to receive S 18326 and two to receive a placebo. Following a bolus of 4.5 mg, doses were 0.5 mg h-1 in the first group and 3 mg h-1 in the two other groups, administered as an i.v. infusion for 24 h. Blood was drawn repeatedly up to 36 h after the bolus, and tested for the activated clotting time (ACT) and activated partial thromboplastin time (APTT). The APTT reagent was chosen among five commercial reagents to yield a linear increase in the clotting time among possible therapeutic S 18326 concentrations in vitro. To accurately measure the thrombin-inhibiting effects of low doses of S 18326 (< 0.5 microm), we developed a specific chromogenic assay. We also measured F1 + 2 prothrombin fragment levels to assess the effect of S 18326 on thrombin generation in vivo.

Results: A two-compartment pharmacokinetic model was fitted to the S 18326 plasma concentration vs time data by using population pharmacokinetic methods. Results of the pharmacodynamic-pharmacokinetic relationships showed that both the ACT and APTT methods yielded a linear increase according to the S 18326 concentration measured using a highly sensitive analytical method. At the end of infusion, ACT was prolonged 1.20 and 1.95-fold in the 0.5 mg h-1 and the 3 mg h-1 groups, respectively, and APTT was prolonged 1.27 and 2.75-fold. Thrombin inhibition plateaued above 0.5 microm of S 18326 according to an Emax model, confirming that the test was highly sensitive. F1 + 2 levels fell significantly after the 24 h S 18326 infusion (0.83 nm to 0.6 nm and 0.80 nm to 0.44 nm in the 0.5 mg h-1 and the 3 mg h-1 groups, respectively), but remained stable after the placebo infusion.

Conclusions: Our results support specific monitoring of the thrombin inhibitor S 18326 with ACT and APTT to establish the safety range of the drug in further studies. Moreover, the fall in F1 + 2 prothrombin fragments suggests that S 18326 effectively reduces the retroactivation of factors V and VIII by thrombin.

Figure 1

APTT response to plasma spiking with 0–7.5 µm S 18326. The reagents used were: Pathromtin® (•), CK Prest® (○), Silimat® (□), aAPTT® (▴) and PTTA®(▵). Plasma (50 µl) was incubated with 50 µl of the reagent for 3 min at 37 ° C, and the reaction was started by adding 50 µl of 0.025 m calcium chloride (Diagnostica Stago). The clotting time was recorded on a ST4 coagulometer. Mean of three experiments, each performed in duplicate.

Figure 2

Determination of plasma dilutions required to measure S 18326 thrombin inhibition in a chromogenic assay. A pool of normal plasma containing 1 µm S 18326 was diluted 1 : 25–1 : 400 (40–2.5 nm) and 50 µl of each dilution was incubated with the same volume of 13 nm thrombin solution for 5 min at room temperature. The reaction was started by adding 100 µl of 0.2 mm S 2238 and absorbance (OD) was read at 405 nm for 3 min on a BCT analyser. Results are given as milli ΔOD405 min−1.

Figure 3

Mean plasma concentration-time profiles. Mean (s.d.) plasma concentration (ng ml−1) of S 18326 after an intravenous bolus of 4.5 mg followed by infusion of the low dose (♦) or the high dose (▴ and □) over 24 h. The lines represent the mean plasma concentration of S 18326 predicted by the population PK model for each dose. The insert presents the plot of individual prediction of plasma concentration (ng ml−1) of S 18326 vs the observed concentrations.

Figure 4

Individual ACT-ratios (a), APTT-ratios (b), and anti IIa activity (c) vs predicted plasma concentrations of S 18326PK/PD analysis was performed after PK modelling, and linear regressions was used to fit ACT and APTT ratios to a function of the S 18326 plasma concentration; an Emax model best described the relationship between anti IIa activity and S 18326 plasma concentrations. 95% confidence limits around the mean predicted relationships of the three biological markers are represented in dotted lines.