EVT201 is a partial GABAA receptor agonist, which inhibits nervous system to treat
insomnia.
EVT201 can form a variety of metabolites in vivo including Ro46-1927, Ro18-5528, Ro40-9970, Ro66-9196 and Ro66-5448. This study developed a simple method to realize the simultaneous determination of
EVT201 and its five metabolites by HPLC-MS/MS with an electrospray ion source (ESI). The
deuterium substitute of
EVT201 was chosen as the internal standard and the multiple reaction monitoring (MRM) was used for the quantification. The separation of the six compounds was accomplished with an ACE Excel 3 AQ column (50 × 2.1 mm, 3 µm, ACE). The process of
protein precipitating-transferring-
nitrogen blowing-reconstituting was adopted for the sample pretreatment. This method was successfully validated according to the FDA guidance. Calibration curves were linear over the concentration range of 0.100-100 ng/mL for
EVT201, 0.0300-30.0 ng/mL for Ro46-1927, 0.0600-6.00 ng/mL for Ro18-5528, 0.0200-4.00 ng/mL for Ro40-9970, 0.100-20.0 ng/mL for Ro66-9196 and 0.100-20.0 ng/mL for Ro66-5448. The intra-run and inter-run precisions and accuracies were all within 14.5%. This fully validated method was successfully applied to a clinical pharmacokinetic study of
EVT201 and its five metabolites in Chinese healthy subjects after the single (2.5 mg and 5 mg, N = 12) and multiple dose (2.5 mg, N = 13) administration of
EVT201 capsules. The test results of 2.5 mg dose group showed that for
EVT201, Ro46-1927, Ro18-5528, Ro40-9970, Ro66-9196 and Ro66-5448, the Cmax values (ng/mL) were 39.2 ± 9.2, 10.3 ± 1.4, 0.218 ± 0.044, 0.128 ± 0.051, 7.01 ± 1.51, 8.73 ± 3.69, respectively; the AUC0-t values (h·ng/mL) were 231 ± 79, 143 ± 72, 10.9 ± 2.1, 1.84 ± 0.78, 55.9 ± 18.7, 135 ± 40 respectively. For
EVT201, Ro46-1927, Ro66-5528, Ro66-9196 and Ro40-5448, the results of Cmax and AUC0-t proved that the five compounds showed linear pharmacokinetic profile over the dose ranges of 2.5 mg to 5 mg. Meanwhile, it is the first report to evaluate the pharmacokinetic characteristics of Ro40-9970, Ro66-9196 and Ro66-5448 in human plasma. It provided meaningful parameters for the safety and tolerability evaluation of
EVT201 capsules in human.