Metabolic profiling of a novel antithrombotic compound, S002- 333 and enantiomers: metabolic stability, species comparison and in vitro–in vivo extrapolation Amrita Saxena a , Guru R. Valicherla a , Girish K. Jain a , Rabi S. Bhatta a , Anil K. Saxena b , and Jiaur R. Gayen a, * a Pharmacokinetics and Metabolism Division, CSIR – Central Drug Research Institute, Lucknow 226031, India b Medicinal and Processing Chemistry Division, CSIR – Central Drug Research Institute, Lucknow 226031, India ABSTRACT: Objective. The aim of this research work was to characterize the metabolism of S002-333, (2-(4’-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-pyrido (3,4-b) indole-3- carboxylic acid amide) and its enantiomers, S004-1032 (R-form) and S007-1558 (S-form) in pooled human liver microsomes (PHLM) and pooled liver microsomes (LM) of rat (RLM), rabbit (RABLM), dog (DLM) and monkey (MLM). Another objective of this study was to identify suitable surrogate species to humans for further development of lead candidates. Method. In vitro metabolic stability and metabolite identification of S002-333 and enantio- mers were carried out in PHLM and LM of various species. The prediction of surrogate spe- cies and in vitro in vivo extrapolation were performed based upon the calculated in vitro intrinsic clearance (CL int ). Results/Conclusion. The in vitro CL int values for S002-333, S004-1032 and S007-1558 were 0.027 ± 0.005, 0.025 ± 0.004 and 0.036 ± 0.005 ml/min/mg, re- spectively, in PHLM, indicating that S007-1558 was the most metabolically unstable of the three. The LM of other species showed similar results. A common surrogate species to humans for S002-333 and enantiomers was predicted as rabbit where the extrapolated hepatic clearance (CL H ) did not show a significant difference to the in vivo CL H values. However, none of the species closely mimic humans with respect to the proportion of major metabolites (M-1–M-4) formed in vitro. Likewise, the CL H values were also predicted in humans for S002-333 and enantiomers using various mathematical models. During analysis, there was no chiral inversion evident among the individual isomers throughout in vitro and in vivo experiments. In conclusion, the in vitro results indicate a prominent role of phase I metabolism in the degradation of S002-333 and enantiomers and predict rabbit as an alternative species to conduct further safety and efficacy studies. Copyright © 2015 John Wiley & Sons, Ltd. Key words: metabolic stability; in vitro–in vivo extrapolation; intrinsic clearance; protein binding; chiral inversion Introduction Cardiovascular disorders associated with intravas- cular thrombosis and pulmonary embolism occur with an incidence of approximately 1 per 1000 annually in adult populations [1]. The latest under- standing of collagen-induced platelet adhesion and aggregation led to the discovery of two prominent platelet collagen receptors, glycoprotein Ia/IIa (or integrin α2β1) and glycoprotein VI (GPVI) [2–5]. These important new targets are involved in a sig- naling cascade for the initiation of platelet adhesion *Correspondence to: Pharmacokinetics and Metabolism Divi- sion, CSIR-Central Drug Research Institute, B. S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow-226031, India. E-mail: jr.gayen@cdri.res.in BIOPHARMACEUTICS & DRUG DISPOSITION Biopharm. Drug Dispos. (2015) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/bdd.1995 Received 18 May 2015 Revised 1 October 2015 Accepted 12 October 2015 Copyright © 2015 John Wiley & Sons, Ltd.