Human hepatic metabolism of a novel 2-carboxyindole glycine antagonist for stroke: in vitro-in vivo correlations.

1. The hepatic metabolism of 3-[-2(phenylcarbamoyl) ethenyl]-4,6-dichloroindole-2-carboxylic acid (GV150526), a novel glycine antagonist for stroke, was investigated. 2. After a single intravenous administration of 800 mg GV150526 to healthy volunteers, six metabolites were observed. The major metabolites detected in human plasma have been shown by mass spectrometry to be glucoronides and one sulphate conjugate. 3. After incubation of GV150526 for 6 and 24 h with human liver slices, three glucuronide metabolites were observed. After incubation of GV150526 with pooled human liver microsomes, only one metabolite was observed, with the same molecular weight and HPLC retention time as the synthetic standard GV217053 (GV150526 hydroxylated on the para-position of the phenyl ring). 4. GV150526 hydroxylase enzyme kinetics--a step before sulphation--was determined using pooled human microsomes and was shown to be catalysed by cytochrome P4502C9. Glucuronidation kinetics towards GV150526 using microsomal preparations were also determined. Glucuronidation of GV150526 was observed with UGT1A1 cDNA-expressed protein, but not with UGT1A6. 5. The above enzyme kinetic data were used to calculate intrinsic clearance after scaling-up and hepatic clearance were calculated. Since GV150526 has a high plasma protein binding capacity, the effect of GV150526 binding to microsomal protein was determined. Thus, enzyme kinetic data were corrected, plotting the free (unbound) concentration of GV150526 versus enzymatic velocities: apparent Vmax did not alter significantly but apparent Km was approximately 10-fold lower. Correlation of these corrected enzyme kinetic data to predict clearance with in vivo clearance of GV150526 was good when both fu(plasma) and fu(microsomes) were included in the clearance calculations.