Screening of drug metabolizing enzymes for fusidic acid and its interactions with isoform-selective substrates in vitro.

1. Fusidic acid (FA) is widely used for the treatment of infections of sensitive osteomyelitis or skin and soft tissue caused by bacteria. However, the role of cytochrome P450s (CYPs) in the metabolism of FA is unclear. In the present study, we screened the main CYPs for the metabolism of FA and studied its interactions with isoform-selective substrates in vitro. 2. The main CYP450s were screened according to the inhibitory effect of specific inhibitors on the metabolism of FA in human liver microsomes (HLMs) or recombinant CYP isoforms. Enzyme kinetic parameters including Ki, Ki', Vmax, and IC50 were calculated to determine the potential of FA to affect CYP-mediated metabolism of isoform-selective substrates. 3. FA metabolism rate was inhibited by 49.8% and 83.1% under CYP2D6, CYP3A4 selective inhibitors in HLMs. In recombinant experiment, the inhibitory effects on FA metabolism were 83.3% for CYP2D6 and 58.9% for CYP3A4, respectively. FA showed inhibition on CYP2D6 and CYP3A4 with Kis of 13.9 and 38.6 µM, respectively. Other CYP isoforms including CYP1A2, CYP2A6, CYP2C9, CYP2E1, and CYP2C19 showed minimal or no effect on the metabolism of FA. 4. FA was primarily metabolized by CYP2D6 and CYP3A4 and showed a noncompetitive inhibition on CYP2D6 and a mixed competitive inhibition on CYP3A4. Drug-drug interactions between FA and other chemicals, especially with substrates of CYP2D6 and CYP3A4, are phenomena that clinicians need to be aware of and cautious about.

Sodium tanshinone IIA sulfonate and its interactions with human CYP450s.

1.Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of tanshinone IIA, a famous Chinese medicine used for many years to treat cardiovascular disorders. However, the role of cytochrome P450 (CYP) enzymes in the metabolism of STS was unclear. In this study, we screened the main CYPs for the metabolism of STS and studied their interactions in vitro. 2.Seven CYPs were screened for the metabolism of STS by human liver microsomes (HLMs) or recombinant CYP isoforms. To determine the potential of STS to affect CYP-mediated phase I metabolism in humans, phenacetin (CYP1A2), coumarin (CYP2A6), tolbutamide (CYP2C9), metoprolol (CYP2D6), chlorzoxazone (CYP2E1), S-Mephenytoin (CYP2C19), and midazolam (CYP3A4) were used as the respective probe substrates. Enzyme kinetic studies were performed to investigate the mode of inhibition of the enzyme-substrate interactions. 3.STS inhibited the activity of CYP3A4 in a dose-dependent manner in the HLMs and CYP3A4 isoform. Other CYP isoforms, including CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on the metabolism of STS. 4.The results suggested that STS primarily inhibits the activities of CYP3A4 in vitro, and STS has the potential to perpetrate drug-drug interactions with other CYP3A4 substrates.

The influence of ABCB1 polymorphism C3435T on the pharmacokinetics of silibinin.

WHAT IS KNOWN AND OBJECTIVE: Silibinin (Silybin), a major constituent of the milk thistle, is commonly used to treat chronic liver disease in some countries. It has been reported to inhibit the transport activity of ABCB1. This study was carried out to determine whether ABCB1 C3435T polymorphism influenced the pharmacokinetics of silibinin contained in silymarin capsules. METHODS: Twenty-three healthy volunteers (10 ABCB1 CC, 8 CT and 5 TT genotypes) were enrolled in this clinical trial. Each volunteer was given a single dose of 140 mg Silymarin Capsule. Blood samples were then collected up to 12 h. HPLC-MS/MS was used to detect serial blood concentration of silybin. RESULTS AND DISCUSSION: The peak plasma concentration (Cmax) in subjects of CC (144·8 ± 60·1 ng/mL) and CT (129·3 ± 50·3 ng/mL) genotypes were 2-fold higher than in subjects of TT genotype (60·1 ± 18·3 ng/mL) (with P = 0·0007 and P = 0·0115 respectively). The area under the concentration-time curve (AUC) extrapolated to infinity [AUC(0-∞)] of CC carriers (347·1 ± 133·8 ng/mL h) was significantly higher than that of TT carriers (228·3 ± 52·9 ng/mL h) (P = 0·0115). WHAT IS NEW AND CONCLUSION: The pharmacokinetics of silibinin was significantly influenced by ABCB1 C3435T polymorphism. Dosage adjustment may be necessary for subjects of different genotypes to ensure comparative exposures. A dose-ranging clinical trial should be undertaken to determine whether the observed differences are clinically significant.

The effects of sodium ferulate on the pharmacokinetics of bupropion and its active metabolite in healthy men.

AIMS: Sodium ferulate (SF) is widely prescribed in clinic for the treatments of cardiovascular and cerebrovascular diseases. In our study, we investigated the effect of SF on the pharmacokinetics of bupropion and its active metabolite 4-hydroxybupropion in healthy Chinese men. METHODS: In a two-phase, randomized, crossover study with a 2-week wash-out period between phases, nineteen healthy male volunteers were given with or without pretreatment with SF 150 mg three times daily for fourteen days, and a single dose of bupropion 150 mg was administered. Pharmacokinetics profiles for bupropion and 4-hydroxybupropion were determined. RESULTS: After SF treatment relative to bupropion alone, the geometric mean ratios and 90% confidence interval of 4-hydroxybupropion were 1.25 (1.17, 1.34) for AUC  0-infinity, 1.10 (1.05, 1.15) for Cmax, and 1.14 (1.09, 1.20) for t1/2 respectively. The corresponding values for bupropion were 0.907 (0.811, 1.014), 0.974 (0.910, 1.045) and 1.13 (1.06, 1.21) respectively. The corresponding values for AUC  0-infinity ratio of 4-hydroxybupropion over bupropion were 1.28 (0.811, 1.014).CONCLUSIONS, Bupropion hydroxylation was slightly induced by the 14 days of SF pre-treatment. Whether co-administration with SF require a dosage adjustment of bupropion needs further exploration.

Effects of curcumin on the pharmacokinetics of talinolol in human with ABCB1 polymorphism.

This study was to investigate the effect of concomitantly administered curcumin on the pharmacokinetics of talinolol and association with ABCB1 C3435T genetic polymorphism. A two-phase, randomized, single-blind, crossover study was carried out in 18 healthy male volunteers with different genotypes of ABCB1, including C3435C (CC, n = 6), C3435T (CT, n = 6) and T3435T (TT, n = 6). The pharmacokinetics of talinolol were measured after co-administration of placebo or 1000 mg curcumin capsules once daily for 14 days. The AUC(0-48 h) and AUC(0-∞) of talinolol were increased by 67.0% (95% CI: 1.09~2.25; p = 0.002) and 80.8% (95% CI: 0.92~2.69; p = 0.005) respectively with curcumin co-administration. The C(max) of talinolol was significantly higher after curcumin administration as compared with placebo (p = 0.029).The CL/F of talinolol was decreased by 25.9% (p = 0.005) during the curcumin-treated phase. No significant change in t(max) and t(1/2) of talinolol were observed between the placebo- and curcumin-treated phases. AUC(0-48), AUC(0-∞), C(max) of talinolol were extensively increased and CL(oral)/F decreased in TT subjects. Co-administration of curcumin significantly increased the plasma concentration of talinolol in healthy volunteers. The effect of curcumin on talinolol was associated with ABCB1 genotypes (C3435T).

Effect of a traditional Chinese medicine Liu Wei Di Huang Wan on the activities of CYP2C19, CYP2D6 and CYP3A4 in healthy volunteers.

Liu Wei Di Huang Wan (LDW), a well-known traditional Chinese medicine, is widely used for the treatment of various diseases in China. This study was designed to investigate the potential herb-drug interactions of LDW in healthy volunteers and attempted to ascertain whether the interaction might be affected by genotypes. We assessed the effect of LDW on the activities of CYP2C19, CYP2D6 and CYP3A4 in 12 Chinese healthy subjects in a single-center, controlled, non-blinded, two-way crossover clinical trial. The subject pool consisted of six extensive metabolizers with CYP2C19*1/*1 and six poor metabolizers with CYP2C19*2/*2. Placebo or 4.8 g LDW (12 pills, 0.2 g/pill, twice daily) was given to each participant for 14 continuous days with a wash-out period of 2 weeks after an oral administration of 30 mg omeprazole, 30 mg dextromethorphan hydrobromide and 7.5 mg midazolam. The activities of CYP2C19, CYP2D6 and CYP3A4 were ascertained by their respective plasma or urinary metabolic ratios on day 14 post-treatment. There is no difference in the activities of the three tested enzymes before or after a 14-day administration of LDW. LDW had no effect on the pharmacokinetic parameters of the substrates and their metabolites. A 14-day administration of LDW did not affect the activities of CYP2C19, CYP2D6 and CYP3A4. LDW is unlikely to cause pharmacokinetic interaction when it is combined with other medications predominantly metabolized by these enzymes.

Effect of genistein on the activities of cytochrome P450 3A and P-glycoprotein in Chinese healthy participants.

To determine the effect of genistein on cytochrome P450 3A (CYP3A) and P-glycoprotein (P-gp) function using the probe substrates midazolam and talinolol, respectively. Eighteen healthy adult male participants were enrolled in a two-phase randomized crossover design. In each phase, the participants received placebo or genistein for 14 days. On the 15th day, midazolam and talinolol were administered and blood samples were obtained. Midazolam and talinolol pharmacokinetic parameter values were calculated and compared before and after genistein administration. Co-administration of genistein decreased the area under the concentration-time curve from 0 to 36 h (AUC 0-36) (143.65 ± 55.40 ng h/mL versus 126.10 ± 40.14 ng h/mL, p < 0.05), and the area under the concentration-time curve from zero to infinity (AUC 0-∞) (209.18 ± 56.61 ng h/mL versus 180.59 ± 43.03 ng h/mL, p < 0.05), and also maximum concentration (Cmax) of midazolam (48.86 ± 20.21 ng/mL versus 36.25 ± 14.35 ng/mL p < 0.05). Similarly, AUC 0-36 (2490.282 ± 668.79 ng h/mL versus 2114.46 ± 861.11 ng h/mL, p < 0.05), AUC 0-∞ (2980.45 ± 921.09 ng h/mL versus 2626.92 ± 1003.78 ng h/mL, p < 0.05) and Cmax of talinolol (326.58 ± 197.67 ng/mL versus 293.42 ± 127.19 ng/mL, p < 0.05) were reduced by genistein co-administration. The oral clearance of midazolam (1.68 ± 0.85 h-1 versus 3.98 ± 0.59 h-1, p < 0.05) and talinolol (3.34 ± 1.24 h-1 versus 3.79 ± 1.55 h-1, p<0.05) were increased by genistien significantly. Administration of genistein can result in a modest induction of CYP3A and possibly P-gp activity in healthy volunteers.

Effect of glycyrrhizin on CYP2C19 and CYP3A4 activity in healthy volunteers with different CYP2C19 genotypes.

The objective of this study was to investigate the interaction between glycyrrhizin and omeprazole and observe the effects of glycyrrhizin on CYP2C19 and CYP3A4 activities in healthy Chinese male volunteers with different CYP2C19 genotypes. Eighteen healthy subjects (six CYP2C19*1/*1, five CYP2C19*1/*2, one CYP2C19*1/*3, five CYP2C19*2/*2 and one CYP2C19*2/*3) were enrolled in a two-phase randomized crossover trial. In each phase, all subjects received placebo or glycyrrhizin salt tablet 150 mg twice daily for 14 consecutive days. The pharmacokinetics of omeprazole (20 mg orally on day 15) was determined for up to 12 h following administration by high-performance liquid chromatography. After 14-day treatment of glycyrrhizin, plasma omeprazole significantly decreased, and those of omeprazole sulfone significantly increased. However, plasma concenetrations of 5-hydroxyomeprazole did not significantly change. The ratio of AUC(0-infinity) of omeprazole to omeprazole sulfone decreased by 43.93% + or - 13.56% (p = 0.009) in CYP2C19*1/*1, 44.85% + or - 14.84% (p = 0.002) in CYP2C19*1/*2 or *3 and 36.16% + or - 7.52% (p < 0.001) in CYP2C19*2/*2 or *3 while those of omeprazole to 5-hydroxyomeprazole did not change significantly in all three genotypes. No significant differences in glycyrrhizin response were found among CYP2C19 genotypes. Glycyrrhizin induces CYP3A4-catalyzed sulfoxidation of omeprazole and leads to decreased omeprazole plasma concentrations, but has no significant impact on CYP2C19-dependent hydroxylation of omeprazole.

Effects of the CYP2C9*13 allele on the pharmacokinetics of losartan in healthy male subjects.

The aim of the study was to determine the pharmacokinetics of losartan in relation to the CYP2C9*13 allele. A single oral dose of 50 mg losartan was administrated to each of the 16 healthy male volunteers with a different genotype (CYP2C9*1/*1, n = 6; CYP2C9*1/*13, n = 4; and CYP2C9*1/*3, n = 6). Blood samples were collected from pre-dose up to 24 h after the drug administration. Plasma losartan and E3174 (an active metabolite of losartan) were assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). All the subjects finished the study without adverse drug effects. In the present study, the frequencies of CYP2C9*13 and *13 alleles were 0.6% and 2.6% in Chinese healthy volunteers, respectively, and both alleles were in Hardy-Weinberg equilibrium. Compared with the subjects in the CYP2C9*1/*1 group, individuals carrying the CYP2C9*1/*13 genotype showed significantly a longer t(1/2) of losartan and E3174 and markedly increased the area under the curve (AUC) of losartan. Meanwhile, the CYP2C9*1/*3 genotype group had significant differences in t(1/2) and Cmax of E3174 compared with the CYP2C9*1/*1 group. The ratio of AUC(E3174)/AUC(losartan) after losartan administration in the CYP2C9*1/*13 and CYP2C9*1/*3 groups was also statistically different from that in the CYP2C9*1/*1 group. The data indicate that the presence of the CYP2C9*13 allele results in poor metabolism of losartan after a single oral dose.

Effect of continuous silymarin administration on oral talinolol pharmacokinetics in healthy volunteers.

1. The objective of this study was to investigate the effect of concomitantly administered silymarin on the pharmacokinetics of talinolol, a typical substrate for P-glycoprotein (P-gp), in healthy Chinese volunteers and its association with a multidrug resistance 1 (MDR1) C3435T genetic polymorphism. 2. Eighteen healthy adult men (six MDR1 3435CC homozygotes, six MDR1 3435CT heterozygotes and six MDR1 3435TT homozygotes) were recruited in a two-phase, randomized, single-blind, crossover design. The pharmacokinetics of talinolol were measured after co-administration of placebo or 140 mg silymarin capsules three times daily for 14 days. Concentrations of talinolol in plasma were measured for up to 36 h after drug administration by liquid chromatography-mass spectrometry (HPLC-MS). 3. The peak plasma concentration (C(max)) of talinolol was significantly higher after silymarin administration as compared with placebo (p = 0.007). The area under the plasma concentration-time curve from zero to 36 h (AUC(0-36)) and AUC(0-infinity) of talinolol was increased by 36.2% +/- 33.2% and 36.5% +/- 37.9%, respectively, by silymarin co-administration. The oral clearance (CL/F) of talinolol was decreased by 23.1% +/- 16.6% (p < 0.001) during the silymarin-treated phase. No change in the time to peak concentration (t(max)) and the blood elimination half-life (t(1/2)) of talinolol was observed between the placebo- and silymarin-treated phases. 4. Co-administration of silymarin significantly increased the plasma concentration of talinolol in healthy volunteers.

Effect of sodium tanshinone II A sulfonate on the activity of CYP1A2 in healthy volunteers.

Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of tanshinone IIA, a famous Chinese medicine which has been used in the treatment of cardiovascular disorders for many years. Using caffeine as a probe drug, this project was designed to investigate the effect of STS on the activity of CYP1A2 in humans. Sixteen unrelated healthy volunteers were recruited for this two-phase, randomized and crossover study. The volunteers received either placebo or 60 mg day(-1) of STS injections through vein for 13 days. Pharmacokinetics of caffeine and the metabolite paraxanthine was determined by high-performance liquid chromatography. CYP1A2 activity was monitored by the ratio of paraxanthine to caffeine at 6 h in plasma. Enzyme activity analysis showed that STS significantly increased the activity of CYP1A2 by 41.1% [90% confidence interval (CI), 17.4-64.8%] (p = 0.036). The area under the curve [AUC((0-24h))] of caffeine significantly decreased by 13.3% [90% CI = 7.0-19.6%] (p = 0.005) with 13 days of treatment of STS. AUC((0-24h)) of paraxanthine significantly increased by 17.4% [90% CI = 4.3-30.5%] (p = 0.035). No significant difference was found for other parameters of caffeine and paraxanthine between two phases. STS has significantly induced the activity of CYP1A2 in vivo. Simultaneously, AUC((0-24h)) of caffeine and paraxanthine were significantly affected by STS. The findings have provided some useful information for safe and effective usage of STS in clinic.

Clopidogrel inhibits CYP2C19-dependent hydroxylation of omeprazole related to CYP2C19 genetic polymorphisms.

This study explores the impact of clopidogrel on the pharmacokinetics of omeprazole related to CYP2C19 genetic polymorphisms. Twelve healthy volunteers (6 CYP2C19*1/*1, 5 CYP2C19*2/*2, and 1 CYP2C19*2/*3) are enrolled in a 2-phase randomized crossover trial. In each phase, the volunteers are administered a single oral dose of omeprazole 40 mg after pretreatment of either placebo or clopidogrel (300 mg on the first day and then 75 mg once daily for 3 consecutive days). Plasma concentrations of omeprazole and its metabolites are quantified by high-performance liquid chromatography with UV detection. After clopidogrel treatment, the AUC(0-infinity) of omeprazole increases by 30.02% +/- 18.03% (P = .004) and that of 5-hydroxyomeprazole decreases by 24.30% +/- 11.66% (P = .032) in CYP2C19*1/*1. The AUC(0-infinity) ratios of omeprazole to 5-hydroxyomeprazole increase by 74.98% +/- 35.48% (P = .001) and those of omeprazole to omeprazole sulfone do not change significantly (P = .832) in CYP2C19*1/*1. No significant alteration is observed in CYP2C19*2/*2 or *3. Clopidogrel inhibits CYP2C19-dependent hydroxylation of omeprazole in CYP2C19*1/*1 and has no impact on CYP3A4-catalyzed sulfoxidation of omeprazole.

The effect of herbal medicine baicalin on pharmacokinetics of rosuvastatin, substrate of organic anion-transporting polypeptide 1B1.

The aim of this study was to explore potential herb-drug interaction between baicalin and rosuvastatin, a typical substrate for organic anion-transporting polypeptide 1B1 (OATP1B1) related to different OATP1B1 haplotype groups. Eighteen unrelated healthy volunteers who were CYP2C9*1/*1 with different OATP1B1 haplotypes (six OATP1B1*1b/*1b, six OATP1B1*1b/*15, and six OATP1B1*15/*15) were selected to participate in this study. Rosuvastatin (20 mg orally) pharmacokinetics after coadministration of placebo and 50-mg baicalin tablets (three times daily orally for 14 days) were measured for up to 72 h by liquid chromatography-mass spectrometry in a two-phase randomized crossover study. After baicalin treatment, the area under the plasma concentration-time curve (AUC)(0-72) and AUC(0-infinity) of rosuvastatin decreased by 47.0+/-11.0% (P=0.001) and 41.9+/-7.19% (P=0.001) in OATP1B1*1b/*1b, 21.0+/-20.6% (P=0.035) and 23.9+/-8.66% (P=0.004) in OATP1B1*1b/*15, and 9.20+/-11.6% (P=0.077) and 1.76+/-4.89% (P=0.36) in OATP1B1*15/*15, respectively. Moreover, decreases of both AUC(0-72) and AUC(0-infinity) of rosuvastatin among different haplotype groups were significantly different (P=0.002 and <0.001). Baicalin reduces plasma concentrations of rosuvastatin in an OATP1B1 haplotype-dependent manner.

A preliminary report of a new design of cast metal fixed twin-block appliance.

The present paper describes a cast metal fixed twin-block appliance utilized to correct a Class II malocclusion, which is designed for full-time wear. The object of the present paper is to achieve rapid functional correction of Class II malocclusions by transmitting favourable occlusal forces to inclined planes which are cemented to the posterior teeth. In the meantime, pre-adjusted fixed edgewise appliances can be placed on the anterior teeth to correct their malpositions. This new functional appliance design may shorten the total treatment duration and reduce the need for patient compliance.

Association of CYP3A4*18B polymorphisms with the pharmacokinetics of cyclosporine in healthy subjects.

The aim of this study is to evaluate the association of the CYP3A4*18B genotype with the cyclosporine metabolism in healthy subjects. We employed PCR-RFLP assays for analysis of the CYP3A4*18B genotype. Each of 26 subjects, comprising 12 CYP3A4*1/*1, 12 CYP3A4*1/*18B and 2 CYP3A4*18B/*18B, was given a single oral dose of cyclosporine (4 mgkg(-1)). The plasma concentrations of cyclosporine were measured for up to 24 h post dose by high-performance liquid chromatography-electrospray mass spectrometry. We found that the mean Cmax (95% confidence intervals) of cyclosporine were 2237 (2905, 1859) (*1/*1), 2247 (2916, 1869) (*1/*18B), and 905 (1192, 506) ng ml(-1) (*18B/*18B)(p = 0.037) and the mean AUCO-4 were 5026 (6181, 4372) (*1/*1), 4434 (5481, 3841) (*1/*18B) and 2561 (3155, 1736) ng ml(-1) h (*18B/*18B) (p=0.021). The CL in the *18B/*18B group was significantly higher than in the *1/*1 group. However, Tmax exhibited no difference among the three genotypes. *18B/*18B group showed 50% reduction in concentration at 2 h post dose compared with *1/*18B (p = 0.062) or *1/*1 (p = 0.047), but no statistical significance was detected between*1/*1 and *1/*18B groups (p > 0.05). The data suggest that the CYP3A4*18B genotype affects cyclosporine pharmacokinetics probably resulting from a higher enzymatic activity of this mutation in healthy subjects.

Sensitive and selective liquid chromatography-mass spectrometry method for the quantification of rosiglitazone in human plasma.

A sensitive and selective high-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (HPLC-ESI-MS-MS) method for determination of rosiglitazone in human plasma has been developed. After the addition of the internal standard, plasma samples were precipitated by acetonitrile. The compounds were separated on a proC18 column using a mixture of ammonium acetate buffer (0.02 M, pH 6.5) and acetonitrile (in the ratio of 47:53, v/v) as mobile phase. A Finnigan LCQdeca plus ion trap mass spectrometer connected to a Finnigan Surveyor HPLC was used to develop and validate the method. Linearity was established for the range of concentrations 1-1000 ng/ml with a coefficient of determination (r(2)) of 0.999. The intra-day accuracy for rosiglitazone ranged from 110.0 to 99.2% at low, medium and high levels. The inter-day accuracy was less than 15%. The lower limit of quantitation (LLOQ) was identified reproducible at 1.0 ng/ml with a precision of 5.7%. After validation, the method was used to study the pharmacokinetic profile of rosiglitazone in five healthy volunteers after administration of a single oral dose (4.0mg). The proposed method enabled the unambiguous evaluation and quantitation of rosiglitazone for pharmacokinetic, bioavailability or drug-drug interaction studies. A possible chromatography peak (m/z 121, its parent ion m/z 344) of N-demethyl rosiglitazone was observed at 3.49 min during determining rosiglitazone. This may be also a potential method for simultaneous determination of rosiglitazone and its metabolite N-demethyl rosiglitazone concentrations in plasma.

Single nucleotide polymorphisms and haplotypes of histamine N-methyltransferase in patients with gastric ulcer.

INTRODUCTION: Histamine plays a crucial role in the regulation of gastric acid secretion, which is involved in the pathogenesis of peptic ulcer. Histamine N-methyltransferase (HNMT) is the major metabolizing enzyme for histamine inactivation in human stomach. OBJECTIVE: This study aims to determine whether there exists a relationship between HNMT gene polymorphisms and the risk for gastric ulcer (GU). METHODS: 118 GU patients and 154 ethnically matched control subjects were enrolled and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays were developed to genotype all these subjects for the T-1637C, C-411T, C314T and A1097T point mutations in HNMT gene. Haplotypes were reconstructed from the genotype data. RESULTS: Frequencies of the variant alleles in cases and controls were 0.398 vs 0.396 for T-1637C, 0.144 vs 0.110 for C-411T, 0.034 vs 0.042 for C314T, and 0.242 vs 0.273 for A1097T, respectively, with no significant difference for any locus between the two groups (all P > 0.05). Also the frequencies of genotypes, haplotypes and haplotype pairs based on these polymorphisms did not differ significantly between cases and controls. CONCLUSION: This study provided no evidence for the involvement of HNMT polymorphisms in the susceptibility to GU.

Determination of estradiol metabolites in human liver microsome by high performance liquid chromatography-electrochemistry detector.

AIM: To constitute a method to determine the estradiol metabolites in human liver microsome in low concentration of estradiol. METHODS: Use high performance liquid chromatography after solvent extraction, evaporation, and reconstitution to separate the metabolites and use a electrochemistry detector to detect the metabolites. RESULTS: With a mobile phase of acetic acid buffer-acetonitrile (50:50, v/v, pH 4.5) at flow rate of 1.0 mL/min and a potential of +0.7 V vs Ag/AgCl, all six composition were well separated and satisfactorily detected. There are E3, 16alpha-OHE1, 2-OHE2, E1, and two unidentified composition. The minimum detectable amount is about 100 p g on column. This method is sensitive enough to detect E1 in a substrate concentration of 1 micromol/L. CONCLUSION: The method can be used to study the metabolism mechanism of estradiol in liver microsome.

Assessment of cytochrome P450 activity by a five-drug cocktail approach.

OBJECTIVES: Our goal was to establish and validate a modified cocktail approach including probe drugs caffeine, chlorzoxazone, mephenytoin, metoprolol, and midazolam for simultaneous phenotyping of CYP1A2, CYP2E1, CYP2C19, CYP2D6, and CYP3A. METHODS: The study was conducted in 14 healthy, nonsmoking male volunteers with a cocktail of 5 drugs consisting of 100 mg caffeine, 200 mg chlorzoxazone, 100 mg mephenytoin, 100 mg metoprolol, and 7.5 mg midazolam in a randomized manner with a 7 x 7 Latin square design. Plasma was obtained at 1, 4, and 6 hours, and urine was collected from 0 to 8 hours after oral drug administration. RESULTS: The phenotypic indexes determined for caffeine, chlorzoxazone, mephenytoin, metoprolol, and midazolam were not significantly different when the drugs were given in different combinations. There were no metabolic interactions or analytic interference of these probe drugs. CONCLUSIONS: This cocktail approach can simultaneously provide independent in vivo phenotypic measures for the cytochrome P450 (CYP) enzymes CYP1A2, CYP2E1, CYP2C19, CYP2D6, and CYP3A.

Plasma caffeine metabolite ratio (17X/137X) in vivo associated with G-2964A and C734A polymorphisms of human CYP1A2.

Either G-2964 or A734 in the human CYP1A2 gene was confirmed to be associated with high inducible enzyme activity in smokers, but not in nonsmokers. In this study, for the first time, we observed an association between phenotypes and genotypes of CYP1A2 with respect to the two genetic polymorphisms in 163 healthy Chinese volunteers living in Qidong. The ratio of plasma 17X/137X at 6 h after oral administration of 300 mg caffeine was employed in CYP1A2 phenotyping analysis, while genotyping analysis was carried out by polymerase chain reaction-restriction fragment length polymorphism. The allele frequencies of A at -2964 and A at 734 in 139 non-smoking subjects were 0.25 and 0.67, respectively. The A/A-2964C/C734, G/A-2964C/C734 or A/A-2964C/A734 genotype that was thought to have lower inducibility/activity of CYP1A2 than the other genotypes did not exist in the tested Chinese subjects. The ratio of 17X/137X was 0.46 +/- 0.26 in G/G-2964A/A734 genotypes (n = 22) and 0.36 +/- 0.19 in non-G/G-2964A/A734 (n = 117). In addition, there was significant difference between them (P = 0.036). A similar result was also achieved in 24 smokers. Since Qidong is a special region with particularly high incidence of hepatocellular carcinoma in China, the association of phenotypes with genotypes of CYP1A2 in the Qidong population might result from some inducible environmental factors such as those of cigarettes in smokers.