Conjugation of the Enantiomers of Ketotifen to Four Isomeric Quaternary Ammonium Glucuronides in Humans In Vivo and in Liver Microsomes

The antiallergic drug ketotifen is chiral due to a nonplanar seven-membered ring containing a keto group. Earlier studies have revealed glucuronidation at the tertiary amino group as a major metabolic pathway in humans. Chemical synthesis of glucuronides from racemic ketotifen now led to four isomers separable by HPLC of which two each could be ascribed to (R) -(+)- and (S) -(−)-ketotifen by synthesis from the enantiomers. According to 1 H NMR analysis of the (S) -ketotifen N -glucuronides, the conformation of the piperidylidene ring differs between the two isomers. Enzymatic hydrolysis with Escherichia coli β-glucuronidase proceeded at a lower rate with the slower eluting (S) -ketotifen glucuronide than with the three other isomers. On incubation of the ketotifen enantiomers (0.5–200 μM) with human liver microsomes in the presence of UDP-glucuronic acid and Triton X-100, the N -glucuronides of (R) -ketotifen were produced with an apparent K M 15 μM and V max 470 pmol/min/mg protein. The two (S) -ketotifen glucuronides were formed by two-enzyme kinetics with K M1 1.3 μM and K M2 92 μM and V max values of 60 and 440 pmol/min/mg protein. After ingestion of 1 mg of racemic ketotifen, 10 healthy subjects excreted in urine 17 ± 5% of the dose in the form of N -glucuronides. The (R) -ketotifen glucuronide isomers contributed one-sixth only, whereas the remainder consisted primarily of the (S) -ketotifen glucuronide isomer, which eluted last. Differential hydrolysis or membrane transport may be responsible for the discrepancy between N -glucuronide isomer ratios in vitro and in vivo.