Aims The present study was designed to define the kinetic behaviour of sertraline N-demethylation in human liver microsomes and to identify the isoforms of cytochrome P450 involved in this metabolic pathway. Methods The kinetics of the formation of N-demethylsertraline were determined in human liver microsomes from six genotyped CYP2C19 extensive (EM) and three poor metabolisers (PM). Selective inhibitors of and specific monoclonal antibodies to various cytochrome P450 isoforms were also employed. Results The kinetics of N-demethylsertraline formation in all EM liver microsomes were fitted by a two-enzyme Michaelis-Menten equation, whereas the kinetics in all PM liver microsomes were best described by a single-enzyme Michaelis-Menten equation similar to the low-aYnity component found in EM microsomes. Mean apparent Km values for the high-and low-aYnity components were 1.9 and 88mm and V max values were 33 and 554 pmol min−1mg 1 protein, respectively, in the EM liver microsomes. Omeprazole (a CYP2C19 substrate) at high concentrations and sulphaphenazole (a selective inhibitor of CYP2C9) substantially inhibited Ndemethylsertraline formation. Of five monoclonal antibodies to various cytochrome P450 forms tested, only anti-CYP2C8/9/19 had any inhibitory eVect on this reaction. The inhibition of sertraline N-demethylation by anti CYP2C8/9/19 was greater in EM livers than in PM livers at both low and high substrate concentrations. However, anti-CYP2C8/9/19 did not abolish the formation of N-demethylsertraline in the microsomes from any of the livers. Conclusions The polymorphic enzyme CYP2C19 catalyses the high-aYnity Ndemethylation of sertraline, while CYP2C9 is one of the low-aYnity componentsof this metabolic pathway.

Aims: To investigate the incidence of the CYP2C19 polymorphism in the Chinese Dai population. Methods: One hundred and ninety-three healthy Chinese Dai volunteers were identified with respect to CYP2C19 by genotype and phenotype analyses. A polymerase chain reaction-restriction fragment length polymorphism method was performed for genotyping procedures. The 4-hydroxymephenytoin (4-OH-MP) and S/R-mephenytoin (S/R-MP) excreted in the urine were determined by high-performance liquid chromatographyand gas chromatography, respectively. Results: Eighteen subjects were identified as poor metabolisers (PMs). The frequency of PMs in the Chinese Dai subjects was 9.3% (95% confidence interval 5.2, 13.4), which is lowerthan that in the Chinese Han population (P<0.05). Chinese Dai subjects had a higher frequency of the mutant CYP2C19*2 allele (0.303) and a lowerfr equency of the mutant CYP2C19*3 allele (0.034). These two mutant alleles could explain all defi-ciencies of CYP2C19 activity in the Chinese Dai subjects. The frequency of the CYP2C193 allele is significantly lowerthan that in the Chinese Han population (P<0.05). The mean S/R ratio was lower in the homozygous extensive metabolisers (EMs) compared with that in heterozygous EMs (P<0.01), and the latter was lowerthan that in the PMs (P<0.01). Furthermore, the mean S/R ratio in CYP2C193/CYP2C192 heterozygous PMs was possibly lower than that in the CYP2C192/CYP2C19*2 homozygous PMs (P<0.05). Conclusion: The frequencies of PMs and CYP2C19*3 allele in the Chinese Dai population are significantly lowerthan those in the Han population. The CYP2C19genotype analysis is largely consistent with the mephenytoin phenotype analysis. The variability of S/R ratios in EMs and PMs shows a gene-dosage e ect.

Aims To investigate the distribution of CYP3A activity in the Chinese population, and to test for gender-related differences in CYP3A activity. Methods Using midazolam as a probe drug, CYP3A activity in 202 Chinese healthy subjects (104 men) was measured by plasma 1-hydroxymidazolam: midazolam (1-OH-MDZ: MDZ) ratio at 1 h after oral administration of 7.5mg midazolam. The different phases of the menstrual cycle including preovulatory, ovulatory and luteal phases of 66 women phenotyped with midazolam were recorded. The concentrations of 1-OH-MDZ and MDZ in plasma were measured by HPLC Results A 13-fold variation of CYP3A activity (log1-OH-MDZ: MDZ: range-0.949-0.203) was shown. The CYP3A activity was normally distributed as indicated by the frequency distribution histogram, the probit plot and the Kolmogorov-Smirnov test (P>0.05). The CYP3A activity of women was higher than that of men (median: -0.36vs-0.43, P<0.05; 95% CI for difference: -0.127,-0.012). There was a significant difference in CYP3A activity between the three phases of the menstrual cycle. The activity was highest in the preovulatory phase and decreased sequentially in the ovulatory and luteal phases (P<0.05). Conclusions A normal distribution of CYP3A activity was observed in the Chinese population. The CYP3A activity is higher in female subjects than in males. CYP3A activity differed across the phases of the menstrual cycle.

This work evaluated the kinetic behavior of fluoxetine O-dealkylation in human liver microsomes from different CYP2C19 genotypes and identified the isoenzymes of cytochrome P450 involved in this metabolic pathway. The kinetics of the -trifluoromethylphenol (TFMP) formation from fluoxetine was determined in human liver microsomes from three homozygous (wt/wt) and three heterozygous (wt/m1) extensive metabolizers (EMs) and three poor metabolizers (PMs) with m1 mutation (m1/m1) with respect to CYP2C19. The formation rate of TFMP was determined by gas chromatograph with electron-capture detection. The kinetics of TFMP formation was best described by the two-enzyme and single-enzyme Michaelis-Menten equation for liver microsomes from CYP2C19 EMs and PMs, respectively. The mean intrinsic clearance (Vmax/Km) for the high- and low-affinity component was 25.2 l/min/nmol and 3.8 l/min/nmol of cytochrome P450 in the homozygous EMs microsomes and 12.8 l/min/nmol and 2.9 l/min/nmol of cytochromecytochrome P450 in the heterozygous EMs microsomes, respectively. Omeprazole (a CYP2C19 substrate) at a high concentration and triacetyloleandomycin (a selective inhibitor of CYP3A4) substantially inhibited O-dealkylation of fluoxetine. Furthermore, fluoxetine O-dealkylation was correlated significantly with S-mephenytoin 4-hydroxylation at a low substrate concentration and midazolam 1 -hydroxylation at a high substrate concentration in liver microsomes of 11 Chinese individuals, respectively. Moreover, there were obvious differences in the O-dealkylation of fluoxetine in liver microsomes from different CYP2C19 genotypes and in microsomal fractions of different human-expressed lymphoblast P450s. The results demonstrated that polymorphic CYP2C19 and CYP3A4 enzymes were the major cytochrome P450 isoforms responsible for fluoxetine O-dealkylation, whereas CYP2C19 catalyzed the high-affinity O-dealkylation of fluoxetine, and its contribution to this metabolic reaction was gene dose-dependent.

目的：建立同时测定体外肝微粒体中氟西汀及其代谓十产物去甲氟西汀的反相高效液相色谱紫外检测法。方法：含微粒体蛋白和NADPH发生系统及氟西汀的孵育液加入冰乙腈酸终止反应后，再加入去甲替林作为内标并以，z.正己烷和乙腈的混合液进行萃取，然后以反相ODS柱分离，在226nlTl处以紫外检测器进行检测。结果：孵育体系中没有明显的干扰峰出现，氟西汀和去甲氟西汀洗脱快，分离好，线性范围均为10-800ug/L，最低检测限均为5ug/L，相对回收率为94%-104%，变异系数少于9.1%。结论：本法快速，灵敏，回收率高，且萃取过程简单，可用于体外氟西汀的代谢研究。