Insight into stereoselective disposition of enantiomers of a potent antithrombotic agent, S002-333 following administration of the racemic compound to mice.

S002-333 [2-(4-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-b-carboxylic acid amide], a potent antithrombotic agent developed by CSIR-CDRI, is a racemic mixture of two enantiomers (S004-1032 (R)-isomer and S007-1558 (S)-isomer). Despite extensive research, little is known about the pharmacokinetics of S002-333 enantiomers. Given that mouse is an established model for anti-platelet/antithrombotic activity and interspecies differences exists in the direction of stereoselectivity in pharmacokinetic processes, we investigated the pharmacokinetic disposition of S002-333 enantiomers in mice. Whereas the pharmacokinetics of S002-333 was non-stereoselective after intravenous (i.v.) administration, substantial stereoselectivity was observed after oral administration of the racemate. The oral AUC0-∞ of (R)-isomer (1228.21±97.55h∗ng/mL) was higher than that of (S)-isomer (861.55±182.07h∗ng/mL) whereas it was comparable after i.v. administration. The absolute oral bioavailability of (R)-isomer was ~1.7 times higher than that of its antipode. On incubating the racemic mixture or individual isomers with mice liver microsomes, (S)-isomer depleted significantly faster than (R)-isomer. Thus, low absolute oral bioavailability of (S)-isomer in comparison to (R)-isomer could be associated to stereoselective hepatic metabolism of (S)-isomer. Furthermore, no metabolic interaction between the enantiomers was observed. Tissue distribution analysis revealed that the highest amount of the enantiomers was localized in small intestine and liver which could be due to first pass metabolism in these organs. Stereoselectivity in the distribution of S002-333 was observed in liver, kidney, spleen and brain; however no significant differences between the plasma protein binding of the enantiomers were observed. The information revealed in the present work might prove valuable in deciding the development of S002-333 as racemic mixture and/or single enantiomer.

Pre-clinical investigation of plasma pharmacokinetics and biodistribution of a novel antithrombotic agent S002-333 in mice using LC-MS/MS.

S002-333 [2-(4-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-b-carboxylic acid amide] is a novel and potent antithrombotic agent developed by CSIR-CDRI, India. The present study was aimed to develop a sensitive LC-MS/MS method for the quantification of S002-333 in mice plasma and tissues. The extraction of S002-333 from relatively small amount of mouse biomatrices (50µL) was accomplished using protein precipitation followed by liquid-liquid extraction and the separation of analytes was achieved on C18 reversed phase column using acetonitrile and triple distilled water (75:25, v/v) as mobile phase at a flow rate of 0.6mL/min. The instrument was operated in the multiple reaction monitoring (MRM) mode using electrospray ionization (ESI) in the positive scan mode. For all the biomatrices, linear relationship was attained over the concentration range of 0.39-200ng/mL with correlation coefficients ≥0.992. The lower limit of quantification for mouse plasma and tissue homogenates was 0.39ng/mL. The bioanalytical method was reproducible and reliable for all the matrices with inter-day and intra-day variability in precision being less than 15% and accuracy within ±15%. The assay was successfully applied to pharmacokinetics and tissue distribution of S002-333 in mice. The pharmacokinetic study revealed adequate gastrointestinal absorption of S002-333 into the systemic circulation of mice with absolute oral bioavailability of 45.8%. Tissue distribution data showed rapid and wide distribution of S002-333 in the following order: small intestine>liver>kidney≈lungs>heart>spleen>brain. The present findings may provide meaningful basis for further clinical development of this new chemical entity.