Enantioselective drug metabolism catalysed by the polymorphic CYP2D6 and CYP2C19 : a methodological investigation focused on mianserin, mephenytoin and omeprazole

Abstract

The aim of this thesis was to develop enantioselective analytical methods and apply them to studies of oxidative metabolism of chiral drugs catalysed by the polymorphic cytochrome P450 CYP2D6 and CYP2C 19 enzymes. The work has been focused on mianserin, mephenytoin and omeprazole. The kinetics of mianserin were studied in 15 Swedish healthy volunteers after a single oral dose of 20 mg mianserin. A significant correlation was found between the metabolic ratio (MR) of debrisoquine and the area under the plasma concentration time curve (AUC~,2 h) for both mianserin and the main metabolite, desmethylmianserin. Further, chiral HPLC analysis showed that the elimination of S(+)-mianserin but not of R(-)-mianserin is catalysed by CYP2D6. The plasma concentrations of the enamtiomers of both mianserin and desmethylmianserin were studied in 66 Japanese depressed patients during mianserin treatment.

A pronounced variation in plasma concentrations of the enantiomers was seen in patients treated with the same dose. In the majority of the samples, the plasma concentration of the more active S(+)-mianserin was higher than that of R(-)-mianserin. For desmethylmianserin, the R-enantiomer was the major form while the S-enantiomer was not detectable in most of the samples. The stereoselective metabolism of S,R-mephenytoin is widely used as a phenotyping test for CYP2CI9. An S-mephenytom conjugate is present in urine of extensive metabolisers (EM), but not of poor metabolisers (PM) of S-mephenytoin. This conjugate is easily hydrolysed back to S-mephenytoin, resulting in an increased urinary S/R ratio of mephenytoin. Earlier, two urine collections, (0-8 hour and 24-32 hour) were used, but this study shows that the two phenotypic groups can be separated in one single urine sample by estimating the S/R ratio before and after acid hydrolysis.

The S-mephenytoin conjugate was isolated in urine from one EM and its structure was tentatively determined. The compound was isolated using several HPLC purifications. Gas chromatography, mass spectrometry and amino acid analysis showed that it is a cysteine conjugate of S-mephenytoin. The absolute structure is, however, unknown, but an S-N bond between cysteine and S-mephenytoin formed via an oxidative radical mechanism catalysed by CYP2C 19 is suggested. The use of omeprazole as a probe drug for CYP2C 19 was evaluated in 160 healthy Swedish volunteers. The MR expressed as the concentration ratio of omeprazole and hydroxyomeprazole in a plasma sample drawn 3 hours after intake of 20 mg omeprazole correlated with the urinary S/R ratio of mephenytoin. Furthermore, the phenotype determined with omeprazole agreed with the CYP2CI9 genotype with respect to the defect CYP2C19m~ and CYP2CI9m2 alleles.

Enantioselective analysis of omeprazole and hydroxyomeprazole showed that the concentration of (+)-omeprazole was higher and it was eliminated at a lower rate than (-)-omeprazole in PMs while the opposite was seen jn EMs of S-mephenytoin. For hydroxyomeprazole, the (+)-enantiomer was present in much higher concentrations than the (-)-enamtiomer in EMs but not in PMs. This study shows that the CYP2CI9 catalysed hydroxylation of omeprazole is markedly stereoselective for the (+)-enantiomer. In conclusion, the studies presented in this thesis show that the variation in enzymatic activity as well as the marked enantioselectivity contribute to the pronounced variation in the rate of metabolism which is associated with pharmacogenetic polymorphisms.