Individual differences in in vitro and in vivo metabolic clearances of the antipsychotic drug olanzapine from non-smoking and smoking Japanese subjects genotyped for cytochrome P4502D6 and flavincontaining monooxygenase 3.

OBJECTIVE: The antipsychotic olanzapine is reportedly metabolized by inducible human cytochrome P450 (CYP) 1A2 and variable copy-number CYP2D6 and polymorphic flavin-containing monooxygenase 3 (FMO3) in different pathways. We investigated individual differences in the metabolite formation and clearance of olanzapine in vitro and in vivo. METHODS: Human liver microsomal olanzapine oxidation activities were evaluated, and plasma concentrations of olanzapine were determined in 21 Japanese patients (mean age: 50 years, range: 32-69 years, 14 male and 7 female, including 6 smokers) genotyped for CYP2D6 (*1, *5, and *10) and FMO3 (E158K, C197fsX, R205C, V257M, E308G, and R500X). RESULTS: Furafylline (a CYP1A2 inhibitor), quinidine (a CYP2D6 inhibitor), and heat treatment (inactivates FMO3) suppressed liver microsomal metabolic clearance of olanzapine by approximately 30%. Olanzapine N-demethylation and N-oxygenation were found to be catalyzed by CYP1A2 and CYP2D6 and by CYP2D6 and FMO3, respectively, in experiments using liver microsomes and recombinant enzymes. Plasma concentrations and clearance of olanzapine were not affected by CYP2D6 or FMO3 genotypes or smoking behavior. CONCLUSIONS: Olanzapine clearance was not affected by CYP2D6 or FMO3 genotypes or smoking behavior as a single factor under the present conditions because olanzapine clearance is mediated by multiple enzymes involved in two major and one minor pathways.

Individual differences in in vitro and in vivo metabolic clearances of antipsychotic risperidone from Japanese subjects genotyped for cytochrome P450 2D6 and 3A5.

OBJECTIVE: There are conflicting reports regarding the effects of cytochrome P450 (P450, CYP) genotypes on the plasma concentrations of risperidone and its pharmacologically active metabolite, 9-hydroxyrisperidone (paliperidone), in clinical patients. The aim of this study was to investigate individual differences in the metabolic clearance of risperidone in vitro and in vivo. METHODS: In vitro liver microsomal risperidone 9-hydroxylation activities and in vivo plasma concentrations of risperidone and paliperidone were investigated in 15 male and 12 female Japanese subjects (mean age 52 years, range: 24-75 years) genotyped for CYP2D6 and CYP3A5. RESULTS: CYP2D6 intermediate and poor metabolizers showed significantly lower liver microsomal risperidone 9-hydroxylation activities than extensive metabolizers did at 5 µM of risperidone; this difference was not evident at 50 µM of risperidone. The recombinant CYP3A5 Vmax/Km value for risperidone 9-hydroxylation was 30% that of CYP3A4, and liver microsomes from CYP3A5 expressers had similar risperidone 9-hydroxylation activities to those of CYP3A5 poor expressers. The plasma concentration/dose ratios for risperidone and paliperidone in 27 Japanese patients were not significantly influenced by the CYP2D6 or CYP3A5 genotypes. CONCLUSIONS: Individual differences in metabolic clearance of risperidone under the present conditions were not significantly influenced by the genotypes of CYP2D6 or CYP3A5.

Effects of cytochrome P450 2D6 and 3A5 genotypes and possible coadministered medicines on the metabolic clearance of antidepressant mirtazapine in Japanese patients.

A personalized treatment approach should be considered with the second-generation psychiatric drug mirtazapine because of high frequencies of side effects, including characteristic drowsiness. Plasma concentrations of mirtazapine in patients are influenced by many factors, including polymorphic cytochrome P450 enzymes contributing to its transformation to 8-hydroxymirtazapine and N-demethylmirtazapine. The aim of this study was to investigate the determinant factors for individual variations of metabolic clearance of mirtazapine using in vitro and in vivo methods. In vitro analyses using liver microsomes from individual humans in correlation assays and recombinantly expressed P450 enzymes revealed that CYP2D6 was the major contributor to mirtazapine 8-hydroxylation with high affinity, and that CYP3A5 catalyzed N-demethylation in a similar high-capacity manner to that of CYP3A4. CYP1A2 was a minor contributor to mirtazapine 8-hydroxylation. Metabolic clearance of mirtazapine determined in substrate depletion assays and mirtazapine 8-hydroxylation activities in individual liver microsomes were significantly lower in CYP2D6 intermediate metabolizers (IM) and poor metabolizers (PM) than in extensive metabolizers (EM) (p<0.05). Trough plasma concentration/dose ratios of mirtazapine from 14 patients were significantly higher in the CYP2D6 IM/PM group than in the EM group and were also higher in the CYP3A5 poor-expressors group than in the expressors group (p<0.05). Mirtazapine clearance in pooled human liver microsomes was inhibited by quinidine (a CYP2D6 inhibitor), ketoconazole (a CYP3A inhibitor), and in combination with risperidone and duloxetine, possible coadministered medicines. These results suggested that mirtazapine metabolic clearance could be variously influenced by the CYP2D6 and CYP3A5 genotypes and coadministered drugs in clinical patients.

CYP3A4 intron 6 C>T polymorphism (CYP3A4*22) is associated with reduced CYP3A4 protein level and function in human liver microsomes.

Effects of the CYP3A4 intron 6 C>T (CYP3A4*22) polymorphism, which has recently been reported to have a critical role in vivo, were investigated by measuring CYP3A4 protein expression levels and CYP3A4-dependent drug oxidation activities in individual human liver microsomes in vitro. Prior to protein analysis, analysis of DNA samples indicated that 36 Caucasian subjects were genotyped as CYP3A4*1/*1 and five subjects were CYP3A4*1/*22, with a CYP3A4*22 allelic frequency of 6.1%. No CYP3A4*22 alleles were found in the Japanese samples (106 alleles). Individual differences in CYP2D6-dependent dextromethorphan O-demethylation activities in liver microsomes from Caucasians were not affected by either the CYP3A4*1/*22 or CYP3A5*1/*3 genotype. Liver microsomes genotyped as CYP3A4*1/*22 (n = 4) showed significantly lower CYP3A-dependent dextromethorphan N-demethylation, midazolam 1'-hydroxylation, and testosterone 6ß-hydroxylation activities, as well as lower expression levels of CYP3A protein (28% of control), compared with those of the CYP3A4*1/*1 group (n = 19). The other polymorphism, CYP3A5*1/*3, did not show these differences (n = 4). The CYP3A4*22 polymorphism was associated with reduced CYP3A4 protein expression levels and resulted in decreased CYP3A4-dependent activities in human livers. The present results suggest an important role of low expression of CYP3A4 protein associated with the CYP3A4*22 allele in the individual differences in drug clearance.

A rapid multiplex PCR assay that can reliably discriminate the cytochrome P450 2D6 whole-gene deletion allele from 2D6*10 alleles.

BACKGROUND: Genetic polymorphisms of the human CYP2D6 gene can affect the metabolism of many drugs in clinical use. As a first step toward identifying poor drug metabolizers in the clinical setting, we developed a new multiplex PCR-based genotyping method to detect CYP2D6 whole-gene deletion. METHODS: We validated the new method by analyzing 500 genomic DNA samples from a Japanese population with the conventional long-PCR method and the new multiplex PCR method. The long-PCR system used a forward primer for CYP2D7P (a pseudogene closely related to CYP2D6) and a common reverse primer for the untranslated region. The multiplex PCR system used the same two primers as the long PCR and an additional forward primer for CYP2D6. RESULTS: With the long-PCR system, DNA samples identified as containing CYP2D6*5 (whole-gene deletion) formed 3.5-kb PCR products. With the multiplex PCR system, many samples yielded 4.7-kb PCR products (implying the existence of normal CYP2D6) and some DNA samples yielded 6.2-kb PCR products (probably indicating CYP2D6*10D). The long-PCR assay detected 64 CYP2D6*5 alleles among 1000 Japanese alleles; however, the new multiplex PCR system identified 5 of these 64 alleles as CYP2D6*10D. CONCLUSIONS: The new multiplex PCR method is useful for detecting CYP2D6*5. This system could reliably discriminate CYP2D6*5 from homologous pseudogene CYP2D7P and functional CYP2D6*10D.