Prenatal pharmacogenomics: a promising area for research.

Clinical applications of prenatal genetic screening currently focus on detection of aneuploidy and other genetic diseases in the developing fetus. Growing evidence suggests that the fetal genome may also be informative about fetal exposures through contributions to placental transport as well as placental and fetal metabolism. Possible clinical applications of prenatal pharmacogenomic screening include prospective optimization of medication selection and dosage, as well as retrospective assessment of whether a fetus was previously exposed to significant risk. Newly available noninvasive methods of prenatal genetic screening mean that relevant fetal genotypes could be made available to obstetricians for use in management of a current pregnancy. This promising area for research merits more attention than it has thus far received.The Pharmacogenomics Journal advance online publication, 10 May 2016; doi:10.1038/tpj.2016.33.

Interindividual variability of CYP2C19-catalyzed drug metabolism due to differences in gene diplotypes and cytochrome P450 oxidoreductase content.

Large interindividual variability has been observed in the metabolism of CYP2C19 substrates in vivo. The study aimed to evaluate sources of this variability in CYP2C19 activity, focusing on CYP2C19 diplotypes and the cytochrome P450 oxidoreductase (POR). CYP2C19 gene analysis was carried out on 347 human liver samples. CYP2C19 activity assayed using human liver microsomes confirmed a significant a priori predicted rank order for (S)-mephenytoin hydroxylase activity of CYP2C19*17/*17 > *1B/*17 > *1B/*1B > *2A/*17 > *1B/*2A > *2A/*2A diplotypes. In a multivariate analysis, the CYP2C19*2A allele and POR protein content were associated with CYP2C19 activity. Further analysis indicated a strong effect of the CYP2C19*2A, but not the *17, allele on both metabolic steps in the conversion of clopidogrel to its active metabolite. The present study demonstrates that interindividual variability in CYP2C19 activity is due to differences in both CYP2C19 protein content associated with gene diplotypes and the POR concentration.The Pharmacogenomics Journal advance online publication, 1 September 2015; doi:10.1038/tpj.2015.58.

Associations between diet and cardiometabolic risk among Yup'ik Alaska Native people using food frequency questionnaire dietary patterns.

BACKGROUND AND AIMS: In previous analyses, we identified three dietary patterns from food frequency questionnaire data among a sample of Yup'ik Alaska Native people living in Southwest Alaska: a "subsistence foods" dietary pattern and two market-based dietary patterns "processed foods" and "fruits and vegetables". In this analysis, we aimed to characterize the association between the dietary patterns and cardiometabolic (CM) risk factors (lipids, blood pressure, glucose, adiposity). METHODS AND RESULTS: We used multilevel linear regression to estimate the mean of each CM risk factor, comparing participants in the 4th to the 1st quartile of each dietary pattern (n = 637). Models were adjusted for age, sex, past smoking, current smoking, and physical activity. Mean log triglyceride levels were significantly higher among participants in the 4th compared to the 1st quartile of the processed foods dietary pattern (ß = 0.11). Mean HbA1c percent was significantly lower (ß = -0.08) and mean diastolic blood pressure (DBP) mm Hg was significantly higher (ß = 2.87) among participants in the 4th compared to the 1st quartile of the fruits and vegetables dietary pattern. Finally, mean log triglyceride levels and mean DBP mm Hg were significantly lower among participants in the 4th compared to the 1st quartile of the subsistence foods dietary pattern (ß = -0.10 and ß = -3.99 respectively). CONCLUSIONS: We found increased CM risk, as reflected by increased triglycerides, associated with eating a greater frequency of processed foods, and reduced CM risk, as reflected by lower triglycerides and DBP, associated with eating a greater frequency of subsistence foods.

Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression.

Variation in the CYP3A enzymes, which act in drug metabolism, influences circulating steroid levels and responses to half of all oxidatively metabolized drugs. CYP3A activity is the sum activity of the family of CYP3A genes, including CYP3A5, which is polymorphically expressed at high levels in a minority of Americans of European descent and Europeans (hereafter collectively referred to as 'Caucasians'). Only people with at least one CYP3A5*1 allele express large amounts of CYP3A5. Our findings show that single-nucleotide polymorphisms (SNPs) in CYP3A5*3 and CYP3A5*6 that cause alternative splicing and protein truncation result in the absence of CYP3A5 from tissues of some people. CYP3A5 was more frequently expressed in livers of African Americans (60%) than in those of Caucasians (33%). Because CYP3A5 represents at least 50% of the total hepatic CYP3A content in people polymorphically expressing CYP3A5, CYP3A5 may be the most important genetic contributor to interindividual and interracial differences in CYP3A-dependent drug clearance and in responses to many medicines.

Influence of CYP3A5 genotype on the pharmacokinetics and pharmacodynamics of the cytochrome P4503A probes alfentanil and midazolam.

The hepatic and first-pass cytochrome P4503A (CYP3A) probe alfentanil (ALF) is also metabolized in vitro by CYP3A5. Human hepatic microsomal ALF metabolism is higher in livers with at least one CYP3A5*1 allele and higher CYP3A5 protein content, compared with CYP3A5*3 homozygotes with little CYP3A5. The influence of CYP3A5 genotype on ALF pharmacokinetics and pharmacodynamics was studied, and compared to midazolam (MDZ), another CYP3A probe. Healthy volunteers (58 men, 41 women) were genotyped for CYP3A5 *1, *3, *6, and *7 alleles. They received intravenous MDZ then ALF, and oral MDZ and ALF the next day. Plasma MDZ and ALF concentrations were determined by mass spectrometry. Dark-adapted pupil diameters were determined coincident with blood sampling. In CYP3A5(*)3/(*)3 (n=62), (*)1/(*)3 (n=28), and (*)1/(*)1 (n=8) genotypes, systemic clearances of ALF were 4.6+/-1.8, 4.8+/-1.7, and 3.9+/-1.7 ml/kg/min and those of MDZ were 7.8+/-2.3, 7.7+/-2.3, and 6.0+/-1.4 ml/kg/min, respectively (not significant), and apparent oral clearances were 11.8+/-7.2, 13.3+/-6.1, and 12.6+/-8.2 ml/kg/min for ALF and 35.2+/-19.0, 36.4+/-15.7, and 29.4+/-9.3 ml/kg/min for MDZ (not significant). Clearances were not different between African Americans (n=25) and Whites (n=68), or between CYP3A5 genotypes within African Americans. ALF pharmacodynamics was not different between CYP3A5 genotypes. There was consistent concordance between ALF and MDZ, in clearances and extraction ratios. Thus, in a relatively large cohort of healthy subjects with constitutive CYP3A activity, CYP3A5 genotype had no effect on the systemic or apparent oral clearances, or pharmacodynamics, of the CYP3A probes ALF and MDZ, despite affecting their hepatic microsomal metabolism.

Purification of macaque liver flavin-containing monooxygenase: a form of the enzyme related immunochemically to an isozyme expressed selectively in adult human liver.

A microsomal flavin-containing monooxygenase (FMO) was purified 77-fold from macacque liver microsomes on the basis of its methyl p-tolyl sulfoxidase activity. Sequential chromatography on anion- and cation-exchangers, lauryl-Sepharose and 2',5'-ADP-Sepharose provided a purified preparation which exhibited an apparent molecular mass of 59 kDa and a pI of 8.3. N-terminal amino-acid sequencing revealed the characteristic Gly-X-Gly-X-X-Gly consensus sequence for the putative FAD-binding domain of microsomal FMO. In marked contrast to the well-characterized hepatic and pulmonary forms present in experimental animals, the macacque liver enzyme displayed stereoselectivity for sulfoxidation of p-tolyl methyl sulfide on the pro-S rather than the pro-R face of the substrate. Polyclonal antibodies raised against the macacque liver form exhibited little or no cross-reactivity with major purified forms of the enzyme isolated from rabbit liver, guinea-pig liver or rabbit lung. Anti-macacque liver FMO did not cross-react with human fetal liver or adult kidney microsomes, but did recognize a 59 kDa constituent of human adult liver microsomes. The intensity of this immunoreactive 59 kDa band correlated well with human liver microsomal N,N-dimethylaniline N-oxygenase activity. We conclude that human adult liver selectively expresses a microsomal FMO which is functionally and immunochemically distinct from the FMO form(s) present in human fetal liver and adult kidney, and from the major hepatic and pulmonary forms present in common laboratory animals.

Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines for CYP3A5 Genotype and Tacrolimus Dosing.

Tacrolimus is the mainstay immunosuppressant drug used after solid organ and hematopoietic stem cell transplantation. Individuals who express CYP3A5 (extensive and intermediate metabolizers) generally have decreased dose-adjusted trough concentrations of tacrolimus as compared with those who are CYP3A5 nonexpressers (poor metabolizers), possibly delaying achievement of target blood concentrations. We summarize evidence from the published literature supporting this association and provide dosing recommendations for tacrolimus based on CYP3A5 genotype when known (updates at www.pharmgkb.org).

Selective expression of CYP3A5 and not CYP3A4 in human blood.

There is a marked variation between people in the activity of CYP3A4 in liver and intestine. We reasoned that if CYP3A4 was expressed in peripheral blood cells, a simple blood based test of CYP3A4 phenotype might be feasible. We prepared peripheral blood smears from healthy volunteers and performed immunostaining with a rabbit polyclonal antibody that selectively reacts with enzymes within the CYP3A subfamily. Staining was observed only within the cytoplasm of neutrophils (PMNs). cDNA prepared from isolated PMNs and mononuclear cells was subjected the polymerase chain reaction using as primers synthetic oligonucleotides that selectively amplify fragments of each known CYP3A cDNAs (CYP3A3, CYP3A4, CYP3A5, and CYP3A7). Amplification was only obtained with the CYP3A5 specific oligonucleotides, predominantly in PMNs, and the identity of the amplified fragment was confirmed by sequencing. Next, whole white cell homogenate prepared from human blood was reacted on immunoblots with a monoclonal antibody that recognizes all CYP3A proteins or an absorbed polyclonal antibody that recognizes only CYP3A5. Both antibodies recognized a protein in the white cells that comigrated with purified CYP3A5. However, metabolism of midazolam, a substrate of CYP3A, could not be detected in homogenates of isolated granulocytes, in homogenates of the whole WBC fractions, or in incubations with unlysed WBC preparations. We conclude that CYP3A4 is not expressed in peripheral blood and hence a blood phenotyping test for this enzyme will not be feasible. Our discovery that CYP3A5 is present may be important since this enzyme is also present in the liver intestine and kidney of many people.

Tissue distribution and interindividual variation in human UDP-glucuronosyltransferase activity: relationship between UGT1A1 promoter genotype and variability in a liver bank.

The variability in a liver bank and tissue distribution of three probe UDP-glucuronosyltransferase (UGT) activities were determined as a means to predict interindividual differences in expression and the contribution of extrahepatic metabolism to presystemic and systemic clearance. Formation rates of acetaminophen-O-glucuronide (APAPG), morphine-3-glucuronide (M3G), and oestradiol-3-glucuronide (E3G) as probes for UGT1A6, 2B7, and 1A1, respectively, were determined in human kidney, liver, and lung microsomes, and in microsomes from intestinal mucosa corresponding to duodenum, jejunum and ileum. While formation of E3G and APAPG were detectable in human kidney microsomes, M3G formation rates from kidney microsomes approached the levels seen in liver, indicating significant expression of UGT2B7. Interestingly, rates of E3G formation in human intestine exceeded the hepatic rates by several fold, while APAPG and M3G formation rates were low. The intestinal apparent Km value for E3G formation was essentially identical to that seen in liver, consistent with intestinal UGT1A1 expression. No UGT activities were observed in lung. Variability in APAPG and M3G activity across a bank of 20 human livers was modest (< or = 7-fold), compared to E3G formation, which varied approximately 30-fold. The E3G formation rates were found to segregate by UGT1A1 promoter genotype, with wild-type (TA)6 rates significantly greater than homozygous mutant (TA)7 individuals. Kinetic analyses were performed to demonstrate that the promoter mutation altered apparent Vmax without significantly affecting apparent Km. These results suggest that glucuronidation, and specifically UGT1A1 activity, can profoundly contribute to intestinal first pass metabolism and interindividual variability due to the expression of common allelic variants.

In-vivo phenotyping for CYP3A by a single-point determination of midazolam plasma concentration.

We investigated whether a single plasma midazolam concentration could serve as an accurate predictor of total midazolam clearance, an established in-vivo probe measure of cytochrome P450 3A (CYP3A) activity. In a retrospective analysis of data from 224 healthy volunteers, non-compartmental pharmacokinetic parameters were estimated from plasma concentration-time curves following intravenous (IV) and/or oral administration. Based on statistical moment theory, the concentration at the mean residence time (MRT) should be the best predictor of the total area under the curve (AUC). Following IV or oral midazolam administration, the average MRT was found to be approximately 3.5 h, suggesting that the optimal single sampling time to predict AUC was between 3 and 4 h. Since a 4-h data point was common to all studies incorporated into this analysis, we selected this time point for further investigation. The concentrations of midazolam measured 4 h after an IV or oral dose explained 80 and 91% of the constitutive interindividual variability in midazolam AUC, respectively. The 4-h midazolam measurement was also an excellent predictor of drug-drug interactions involving CYP3A induction and inhibition. Compared with baseline values, the direction and magnitude of change in midazolam AUC and the 4-h concentration were completely concordant for all study subjects. We conclude that a single 4-h midazolam concentration following IV or oral administration represents an accurate marker of CYP3A phenotype under constitutive and modified states. Moreover, the single-point approach offers an efficient means to phenotype and identify individuals with important genetic polymorphisms that affect CYP3A activity.

The human pregnane X receptor: genomic structure and identification and functional characterization of natural allelic variants.

The pregnane X receptor (PXR)/steroid and xenobiotic receptor (SXR) transcriptionally activates cytochrome P4503A4 (CYP3A4) when ligand activated by endobiotics and xenobiotics. We cloned the human PXR gene and analysed the sequence in DNAs of individuals whose CYP3A phenotype was known. The PXR gene spans 35 kb, contains nine exons, and mapped to chromosome 13q11-13. Thirty-eight single nucleotide polymorphisms (SNPs) were identified including six SNPs in the coding region. Three of the coding SNPs are non-synonymous creating new PXR alleles [PXR*2, P27S (79C to T); PXR*3, G36R (106G to A); and PXR*4, R122Q (4321G to A)]. The frequency of PXR*2 was 0.20 in African Americans and was never found in Caucasians. Hepatic expression of CYP3A4 protein was not significantly different between African Americans homozygous for PXR*1 compared to those with one PXR*2 allele. PXR*4 was a rare variant found in only one Caucasian person. Homology modelling suggested that R122Q, (PXR*4) is a direct DNA contact site variation in the third alpha-helix in the DNA binding domain. Compared with PXR*1, and variants PXR*2 and PXR*3, only the variant PXR*4 protein had significantly decreased affinity for the PXR binding sequence in electromobility shift assays and attenuated ligand activation of the CYP3A4 reporter plasmids in transient transfection assays. However, the person heterozygous for PXR*4 is normal for CYP3A4 metabolism phenotype. The relevance of each of the 38 PXR SNPs identified in DNA of individuals whose CYP3A basal and rifampin-inducible CYP3A4 expression was determined in vivo and/or in vitro was demonstrated by univariate statistical analysis. Because ligand activation of PXR and upregulation of a system of drug detoxification genes are major determinants of drug interactions, it will now be useful to extend this work to determine the association of these common PXR SNPs to human variation in induction of other drug detoxification gene targets.

Single-dose disulfiram inhibition of chlorzoxazone metabolism: a clinical probe for P450 2E1.

Disulfiram and its reduced metabolite diethyldithiocarbamate have been identified previously as selective mechanism-based inhibitors of human liver microsomal cytochrome P450 2E1 in vitro. In animals, a single oral dose of disulfiram has been shown to produce a rapid and selective inactivation of hepatic P450 2E1 content and catalytic activity in vivo. This investigation explored the efficacy of single dose disulfiram as an inhibitor of human P450 2E1 activity in vivo. Clinical P450 2E1 activity was assessed by the 6-hydroxylation of chlorzoxazone, a metabolic pathway catalyzed selectively by P450 2E1. Six healthy volunteers received 750 mg oral chlorzoxazone on two occasions in a crossover design, 10 hours after 500 mg oral disulfiram, or after no pretreatment (control subjects). Disulfiram pretreatment markedly decreased chlorzoxazone elimination clearance to 15% of control values (from 3.28 +/- 1.40 to 0.49 +/- 0.07 ml/kg/min, p < 0.005), prolonged the elimination half-life (from 0.92 +/- 0.32 to 5.1 +/- 0.9 hours, p < 0.001), and caused a twofold increase in peak plasma chlorzoxazone concentrations (20.6 +/- 9.9 versus 38.7 +/- 10.3 micrograms/ml, p < 0.001). Disulfiram also profoundly decreased the formation clearance of 6-hydroxychlorzoxazone, from 2.30 +/- 0.93 to 0.17 +/- 0.05 ml/kg/min (p < 0.005). These findings show that a single dose of disulfiram significantly diminishes the activity of human P450 2E1 in vivo. The efficacy of single-dose disulfiram as an inhibitor of human P450 2E1 suggests that this modality for manipulating clinical P450 2E1 activity may provide a useful probe for delineating P450 2E1 participation in human drug biotransformation or for the treatment of poisoning by P450 2E1-activated toxins.

Clinical enflurane metabolism by cytochrome P450 2E1.

BACKGROUND: Fluorinated ether anesthetic hepatotoxicity and nephrotoxicity are mediated by cytochrome P450-catalyzed oxidative metabolism. Metabolism of the volatile anesthetic enflurane to inorganic fluoride ion by human liver microsomes in vitro is catalyzed predominantly by the cytochrome P450 isoform CYP2E1. This investigation tested the hypothesis that P450 2E1 is also the isoform responsible for human enflurane metabolism in vivo. Disulfiram, which is converted in vivo to a selective inhibitor of P450 2E1, was used as a metabolic probe for P450 2E1. METHODS: Twenty patients undergoing elective surgery were randomized to receive disulfiram (500 mg orally; n = 10) or nothing (control subjects; n = 10) the evening before surgery. All patients received a standard anesthetic of enflurane (2.2% end-tidal) in oxygen for 3 hours. Blood enflurane concentrations were measured by gas chromatography. Plasma and urine fluoride concentrations were quantitated by ion-selective electrode. RESULTS: Patient groups were similar with respect to age, weight, gender, duration of surgery, and blood loss. Total enflurane dose, measured by cumulative end-tidal enflurane concentrations (3.9 to 4.1 MAC-hr) and by blood enflurane concentrations, was similar in both groups. Plasma fluoride concentrations increased from 3.6 +/- 1.5 mumol/L (baseline) to 24.3 +/- 3.8 mumol/L (peak) in untreated patients (mean +/- SE). Disulfiram treatment completely abolished the rise in plasma fluoride concentration. Urine fluoride excretion was similarly significantly diminished in disulfiram-treated patients. Fluoride excretion in disulfiram-treated patients was 62 +/- 10 and 61 +/- 12 mumol on days 1 and 2, respectively, compared with 1090 +/- 180 and 1200 +/- 220 mumol in control subjects (p < 0.05 on each day). CONCLUSIONS: Disulfiram prevented fluoride ion production after enflurane anesthesia. These results suggest that P450 2E1 is the predominant P450 isoform responsible for human clinical enflurane metabolism in vivo.

Single-dose disulfiram does not inhibit CYP2A6 activity.

BACKGROUND: Disulfiram and its primary metabolite diethyldithiocarbamate are effective mechanism-based inhibitors of human liver cytochrome P450 2E1 (CYP2E1) in vitro. A single dose of disulfiram, which significantly diminishes human P450 2E1 activity in vivo, has been used to investigate the role of CYP2E1 in human drug metabolism and to prevent CYP2E1-mediated biotransformation. Nevertheless, the specificity of single-dose disulfiram toward human CYP2E1 in vivo is unknown. Because diethyldithiocarbamate also inhibits human liver CYP2A6 in vitro, this investigation explored the effect of single-dose disulfiram on human CYP2A6 activity in vivo. METHODS: CYP2A6 activity was assessed by the 7-hydroxylation of coumarin, which is catalyzed selectively by CYP2A6. Ten healthy volunteers received 50 mg oral coumarin on two occasions in a randomized crossover design, approximately 10 hours after 500 mg oral disulfiram was administered or after no pretreatment (control group). Plasma and urine 7-hydroxycoumarin and plasma coumarin concentrations were determined by HPLC. RESULTS: The area under the plasma 7-hydroxycoumarin versus time curve (2.69 +/- 0.90 micrograms.hr/ml) was not decreased after disulfiram pretreatment (3.33 +/- 0.93 micrograms.hr/ml). Furthermore, maximum plasma concentration (Cmax) of 7-hydroxycoumarin (1.4 +/- 0.5 versus 1.8 +/- 0.6 micrograms/ml) and time to reach Cmax (1.0 +/- 0.2 and 1.0 +/- 0.4 hour) were unchanged by disulfiram pretreatment. Urinary 7-hydroxycoumarin excretion over a 24-hour period (38.9 +/- 10.8 mg) was also undiminished by disulfiram pretreatment (45.2 +/- 6.6 mg). CONCLUSIONS: Single-dose disulfiram does not inhibit human CYP2A6 activity in vivo. When single-dose disulfiram is used as an in vivo probe for P450, inhibition of drug metabolism suggests involvement of CYP2E1 but not CYP2A6.

The erythromycin breath test predicts the clearance of midazolam.

Midazolam, a commonly used sedative and amnestic medication, has recently been shown to be largely metabolized in the liver by a cytochrome P450, termed CYP3A4. There is at least a tenfold intersubject variability in the liver content and catalytic activity of CYP3A4, which may in part account for the known interpatient differences in the kinetics of midazolam. To test this hypothesis, we determined the intravenous midazolam kinetics of 20 medically stable, hospitalized patients, whose hepatic CYP3A4 activities were determined with use of the [14C-N-methyl]erythromycin breath test. During the kinetic study, we also performed psychometric testing designed to quantitate the level of sedation and amnesia. We found a significant positive correlation between the erythromycin breath test results and weight adjusted clearance (in milliliters per minute per kilogram) of both total midazolam (r = 0.52; p = 0.03) and unbound midazolam (r = 0.61; p < 0.01). The relatively low dose of midazolam used (0.0145 mg/kg) produced significant but transient sedation and memory impairment in some of the patients. We conclude that interpatient differences in liver CYP3A4 activity in part account for the variations in midazolam kinetics. Our observations account for reported drug interactions involving midazolam and suggest that patients with low CYP3A4 activity may be most susceptible to prolonged amnestic effects occasionally produced by this short-acting benzodiazepine.

Inhibition and induction of cytochrome P4502E1-catalyzed oxidation by isoniazid in humans.

We studied the effect of isoniazid administration on the cytochrome P4502E1-catalyzed elimination of chlorzoxazone and acetaminophen. Isoniazid, 300 mg daily, was administered for 7 days to a group of 10 volunteer slow acetylators. Acetaminophen, 500 mg, and chlorzoxazone, 750 mg, were administered on separate occasions before isoniazid, during the period of isoniazid administration, and after the discontinuation of isoniazid. Isoniazid inhibited the clearance of chlorzoxazone by 58%, as assessed from plasma data, and inhibited the formation of acetaminophen thioether metabolites (a measure of the formation of the hepatotoxin N-acetyl-p-benzoquinone imine and catechol oxidative metabolites of acetaminophen, as determined from their recovery in urine, by 63% and 49%, respectively. Two days after the discontinuation of isoniazid, the clearance of chlorzoxazone was increased over the value before isoniazid by 56%. Acetaminophen thioether but not catechol metabolites were increased by 56% 1 day after the discontinuation of isoniazid and had returned to the pre-isoniazid value 3 days after the discontinuation of isoniazid. We conclude that the time course of the interaction with regard to chlorzoxazone elimination and formation is compatible with an inhibition-induction effect of isoniazid on cytochrome P4502E1. The mechanism of this biphasic effect is probably induction by protein stabilization, which results in inhibition of catalytic activity while isoniazid is present.

CYP3A activity in European American and Japanese men using midazolam as an in vivo probe.

OBJECTIVE: To investigate putative differences in CYP3A activity between European American and Japanese subjects using midazolam as an in vivo probe. METHODS: Midazolam was administered orally (2 mg) to 22 young healthy Japanese men and, on a separate occasion, to 19 of these by the intravenous route (1 mg). The disposition of the drug and its 1'-hydroxy metabolite were determined and compared with data collected in a similar fashion in 20 young healthy European American men. RESULTS: Plasma concentrations of midazolam, especially those attained soon after drug administration, were higher after intravenous injection in Japanese subjects than those in European American men. This observation was associated with smaller initial (2.5-fold) and steady-state (1.8-fold) volumes of distribution for the drug; normalization for body weight only modestly reduced these differences. The systemic clearance value of midazolam was 25% lower (P < .03) in Japanese subjects, but this difference was not apparent after accounting for the smaller body weights of that group. No statistical differences were noted in the elimination half-life (t 1/2) of midazolam between European American and Japanese subjects. Much greater interindividual variability was observed after oral administration compared with intravenous administration, but significant differences were not found between the 2 groups with respect to the maximum midazolam plasma level or its oral clearance. Absolute oral bioavailability and its associated gastrointestinal and hepatic extraction ratios also showed no statistically significant interracial differences. CONCLUSIONS: On average, hepatic CYP3A, as measured by the metabolism of midazolam, is lower in young healthy Japanese men compared with similar European Americans. However, there is considerable interindividual variability, and body size appears to be an important determinant. After oral administration, even greater variability in the plasma level-time profile of midazolam is present, and no statistically significant or clinically important interracial/ethnic difference is present. Possibly because of smaller body mass and differences in body composition, midazolam has a smaller distribution volume(s) in Japanese men than in European American men that might be an important factor when drugs are administered intravenously.

Ethanol and production of the hepatotoxic metabolite of acetaminophen in healthy adults.

BACKGROUND: Recent case reports suggest that consumption of ethanol may increase the risk of liver injury induced by acetaminophen (INN, paracetamol). However, this possibility is at odds with previous clinical studies that showed that acute ethanol ingestion could protect against hepatotoxicity by inhibiting CYP-mediated acetaminophen oxidation. We tested the hypothesis that ethanol ingestion can increase susceptibility to acetaminophen toxicity if acetaminophen ingestion occurs shortly after ethanol is cleared from the body. METHODS: Ten healthy volunteers each received a 6-hour intravenous infusion of ethanol (to achieve a blood concentration of 100 mg/dL ethanol) or 5% dextrose in water, administered in random order. Acetaminophen (500 mg) was ingested 8 hours after the end of the infusion. Blood and urine were collected for assessment of formation of N-acetyl-p-benzoquinone imine (NAPQI), the hepatotoxic metabolite of acetaminophen. RESULTS: Mean NAPQI formation was enhanced by 22% (range, 2% to 38%; P < .03) when the acetaminophen dose was given after an ethanol infusion, compared with after 5% dextrose in water infusion. This mean increase was similar in magnitude to that predicted by a mathematical model describing the induction of CYP2E1, the main enzyme catalyzing NAPQI formation, by a mechanism of enzyme stabilization. CONCLUSIONS: Consumption of up to one 750-mL bottle of wine, six 12-ounce cans of beer, or 9 ounces of 80-proof liquor over the course of a single evening modestly increases the fraction of an acetaminophen dose converted to its toxic metabolite, NAPQI, when acetaminophen is ingested soon after ethanol has been cleared from the body. This change in acetaminophen metabolism may present an incremental increase in the risk of acetaminophen hepatotoxicity.

Metabolism of dapsone to its hydroxylamine by CYP2E1 in vitro and in vivo.

Dapsone toxicity is putatively initiated by formation of a hydroxylamine metabolite by cytochromes P450. In human liver microsomes, the kinetics of P450-catalyzed N-oxidation of dapsone were biphasic, with the Michaelis-Menten constants of 0.14 +/- 0.05 and 0.004 +/- 0.003 mmol/L and the respective maximum velocities of 1.3 +/- 0.1 and 0.13 +/- 0.04 nmol/mg protein/min (mean +/- SEM). Troleandomycin (40 mumol/L) inhibited hydroxylamine formation at 100 mumol/L dapsone by 50%; diethyldithiocarbamate (150 mumol/L) and tolbutamide (400 mumol/L) inhibited at 5 mumol/L dapsone by 50% and 20%, respectively, suggesting that the low-affinity isozyme is CYP3A4 and the high-affinity isozymes are 2E1 and 2C. Disulfiram, 500 mg, 18 hours before a 100 mg oral dose of dapsone in healthy volunteers, diminished area under the hydroxylamine plasma concentration-time curve by 65%, apparent formation clearance of the hydroxylamine by 71%, and clearance of dapsone by 26%. Disulfiram produced a 78% lower concentration of methemoglobin 8 hours after dapsone.

Influence of polymorphic N-acetyltransferase phenotype on the inhibition and induction of acetaminophen bioactivation with long-term isoniazid.

OBJECTIVE: To determine in patients receiving isoniazid prophylaxis whether an increase in the CYP2E1 dependent formation clearance of acetaminophen (paracetamol) to N-acetyl-p-benzoquinone imine (NAPQI) occurs during a normal 24-hour isoniazid dose interval and whether the interaction is dependent on acetylation status. METHODS: Acetaminophen elimination kinetics were determined on four different occasions. Ten subjects were assigned to receive acetaminophen either simultaneously with the 8 am dose of isoniazid or 12 hours after the isoniazid dose. One week later, on the last day of isoniazid therapy, subjects received acetaminophen at the alternate time of day. The control phase acetaminophen administrations were repeated 1 and 2 weeks later, following the initial randomization. Isoniazid acetylation (NAT2) genotype was determined by analysis of genomic DNA obtained from peripheral blood leukocytes. RESULTS: The mean NAPQI formation clearance was inhibited 57% when acetaminophen and isoniazid were coadministered but was unchanged compared with time-matched control when acetaminophen was given 12 hours after the isoniazid dose. However, when data from subjects was segregated according to isoniazid (INH) acetylation phenotype, the mean ratio of NAPQI formation clearances (+INH/-INH) with 8 PM acetaminophen was significantly higher for fast acetylators compared with slow acetylators (1.36 versus 0.68; p = 0.006). CONCLUSIONS: Fast metabolizers of isoniazid appeared to clear the inducer or inhibitor from the active site of CYP2E1 more rapidly, which resulted in an increased formation of NAPQI 12 hours after the isoniazid dose. In contrast, formation of NAPQI for slow isoniazid metabolizers remained inhibited.