Population pharmacokinetic analysis of circadian rhythms in hepatic CYP3A activity using midazolam.

Diurnal changes in the activity of drug metabolizing enzymes may contribute to the variability in drug disposition and drug effects. The aim of this study was to quantify the circadian rhythmicity exhibited by hepatic CYP3A. A 10 µg/kg intravenous bolus dose, followed by a 30-hour 4 µg/kg/h intravenous infusion of midazolam, used as a probe substrate for hepatic CYP3A activity, was administered to 16 healthy volunteers (8 males and 8 females). Blood samples were drawn hourly for 24 hours after achieving steady state, and plasma concentrations of midazolam and its main metabolite 1-OH midazolam were determined. Population pharmacokinetic analysis was performed using nonlinear mixed effects modeling. One-compartment pharmacokinetic models best described midazolam and 1-OH midazolam pharmacokinetic disposition. An unequivocal but minor diurnal pattern was identified in the midazolam plasma concentration profiles, which was described using a cosine function with a 24-hours period. The fluctuation in the relative CYP3A activity ranged between 10% above average around 15:00, and 10% below average around 03:00. None of the covariates tested had a significant impact on the parameters estimated. Although a diurnal pattern in hepatic CYP3A activity was identified, its magnitude suggests that it is small and without clinical significance for drug therapy.

A novel toxicokinetic modeling of cypermethrin and permethrin and their metabolites in humans for dose reconstruction from biomarker data.

To assess exposure to pyrethroids in the general population, one of most widely used method nowadays consists of measuring urinary metabolites. Unfortunately, interpretation of data is limited by the unspecified relation between dose and levels in biological tissues and excreta. The objective of this study was to develop a common multi-compartment toxicokinetic model to predict the time courses of two mainly used pyrethroid pesticides, permethrin and cypermethrin, and their metabolites (cis-DCCA, trans-DCCA and 3-PBA) in the human body and in accessible biological matrices following different exposure scenarios. Toxicokinetics was described mathematically by systems of differential equations to yield the time courses of these pyrethroids and their metabolites in the different compartments. Unknown transfer rate values between compartments were determined from best fits to available human data on the urinary excretion time courses of metabolites following an oral and dermal exposure to cypermethrin in volunteers. Since values for these coefficients have not yet been determined, a mathematical routine was programmed in MathCad to establish the possible range of values on the basis of physiological and mathematical considerations. The best combination of parameter values was then selected using a statistic measure (reliability factor) along with a statistically acceptable range of values for each parameter. With this approach, simulations provided a close approximation to published time course data. This model allows to predict urinary time courses of trans-DCCA, cis-DCCA and 3-PBA, whatever the exposure route. It can also serve to reconstruct absorbed doses of permethrin or cypermethrin in the population using measured biomarker data.

Quantifying the effect of covariates on concentrations and effects of steady-state phenprocoumon using a population pharmacokinetic/pharmacodynamic model.

BACKGROUND AND OBJECTIVES: The oral anticoagulant phenprocoumon, similar to other vitamin K antagonists, is characterized by pronounced interindividual variability in the doses needed to achieve the desired therapeutic effect. Previous studies assessed the effect of genetic and demographic covariates on empirical dose requirements of phenprocoumon to enable individualized dose prediction. The aim of the present study was to quantify major sources of interindividual variability separately on the pharmacokinetics and pharmacodynamics of phenprocoumon using a population pharmacokinetic-pharmacodynamic model. METHODS: A single steady-state blood sample was collected from 278 patients and assayed by liquid chromatography-tandem mass spectrometry for phenprocoumon and its metabolites. Genotyping was performed for variants of the cytochrome P450 (CYP) 2C9 (CYP2C9) and vitamin K epoxide reductase complex, subunit 1 (VKORC1) genes. Effects were quantified by international normalized ratio (INR). Data were analyzed simultaneously using NONMEM VII. RESULTS: The model confirmed CYP2C9 and VKORC1 variants as the major predictors of variability in phenprocoumon concentrations and effects, together with body weight, age, comedication with CYP3A modifiers (i.e. inhibitors or inducers) and presence of atrial fibrillation. These covariates explained 50.0 % of the observed variability in the model parameters. Phenprocoumon clearance fractions mediated per CYP2C9 allele were 13.4, 9.5 and 5.7 mL/h for the 1, 2 and 3 variants, respectively. An additional clearance fraction of 5.3 mL/h was independent of CYP2C9 activity. Homozygous VKORC1 wild-type carriers were estimated to have a 2.13-fold higher phenprocoumon exposure requirement than homozygous 1173 C>T carriers to achieve the same effect on INR. CONCLUSIONS: The model provides a deeper insight in the separate pharmacokinetic and pharmacodynamic parts of phenprocoumon action. Thus, it provides important information for individualized dose prediction, with the option to include further covariates not studied here with known effects on individual pharmacokinetic or pharmacodynamic processes.

The role of human cytochrome P450 enzymes in metabolism of acrylamide in vitro.

OBJECTIVE: Acrylamide (AA), a probable human carcinogen, is present in fried and baked starch-rich food. In vivo, the substance is partly biotransformed to glycidamide (GA), which may account for carcinogenic effects. Existing data suggest an important but not exclusive contribution of CYP2E1 to GA formation. The aim of this project was to derive respective enzyme kinetic parameters for CYP2E1 and to assess a possible role of other important human CYPs for this reaction in vitro. METHODS: AA (0.2-20 mM) was incubated with human liver microsomes (HLM) and human cytochrome P450 enzymes (supersomes™). GA was quantified by a specific LC-MS/MS method. Enzyme kinetic parameters were estimated assuming a single binding site. Furthermore, inhibition experiments were performed with diethyldithiocarbamate (DDC), a potent inhibitor of CYP2E1. RESULTS: The mean ± SD maximum formation rate (Vmax) and Michaelis-Menten constant (Km) for GA formation in HLM were 199 ± 36 pmol GA/mg protein/min and 3.3 ± 0.5 mM, respectively. In AA incubations with supersomes™, only for CYP2E1 measurable GA formation was detected in all tested AA concentrations (Vmax and Km were 5.4 nmol GA/nmol CYP2E1/min and 1.3 mM, respectively). Inhibition constant (IC50) of DDC was 3.1 ± 0.5 µM for HLM and 1.2 ± 0.2 µM for CYP2E1 supersomes™. Therefore, relevant participation of CYPs other than CYP2E1 in the metabolism of AA to GA in humans does not seem likely. CONCLUSION: Our results confirm the major role of CYP2E1 in GA formation from AA, albeit with low affinity and low capacity. Further studies are needed to identify other pathways of GA formation.

Profiling of mercapturic acids of acrolein and acrylamide in human urine after consumption of potato crisps.

SCOPE: Acrolein (AC) and acrylamide (AA) are food contaminants generated by heat treatment. We studied human exposure after consumption of potato crisps by monitoring excretion of mercapturic acids (MAs) in urine. METHODS AND RESULTS: MA excretion was monitored in human urine collected up to 72 h after ingestion of a test meal of experimental (study 1: 1 mg AA/150 g) or commercially available (study 2: 44 µg AA plus 4.6 µg AC/175 g) potato crisps. MA contents were analysed after purification via SPE using HPLC-ESI-MS/MS. On the basis of the area under the curve values of MAs excreted in urine, the total excretion of AC-related MAs exceeded that of AA-related MAs up to 12 times in study 1 and up to four times in study 2. Remarkably, AC content of potato crisps of study 2 was found to be only about 1/10 the AA content, as determined by isotope dilution headspace GC/MS. CONCLUSION: Our results indicate substantially higher exposure to AC from potato crisps than to AA. Total AC in such foods may encompass bioavailable AC forms not detected by headspace GC/MS. Both findings may also apply to other heat processed foods.

Pharmacokinetics, pharmacodynamics, and comparative bioavailability of single, oral 2-mg doses of dexamethasone liquid and tablet formulations: a randomized, controlled, crossover study in healthy adult volunteers.

BACKGROUND: Dexamethasone is a glucocorticoid used widely worldwide for immunosuppressive treatment, allergies, bronchiolitis, and croup, among others. For children, liquid formulations are especially suitable because, compared with other dosage forms, both exact dosing and proper intake are facilitated. OBJECTIVE: The objective was to evaluate the pharmacokinetics, pharmacodynamics, and comparative bioavailability of a commercial liquid oral dexamethasone formulation intended for pediatric use relative to those of a tablet. METHODS: In a randomized, controlled, crossover study in 24 healthy adult volunteers, we administered single doses of the liquid and tablet formulation, containing 2 mg of dexamethasone each. Blood samples were taken up to 24 hours postdose. Quantification was carried out using a validated specific and sensitive high-pressure liquid chromatography with UV detector method. Noncompartmental pharmacokinetic parameters were compared between treatments according to European Medicines Agency (EMA) bioequivalence guidelines. For AUC(0-t) and C(max), the 90% CI for the ratio of the test and reference products should be contained within the predetermined acceptance interval of 80% to 125%. As a pharmacodynamic variable, we measured suppression of endogenous cortisol (predose and postdose). RESULTS: Both preparations showed similar pharmacokinetic and pharmacodynamic profiles but high between-subject variability of pharmacokinetic key parameters and endogenous cortisol concentrations (>30%). Mean AUC(0-t), AUC(0-∞), and C(max) were 37.8 ng/mL/h, 46.0 ng/mL/h, and 9.35 ng/mL, respectively, for the liquid and 41.3 ng/mL/h, 48.1 ng/mL/h, and 9.17 ng/mL, respectively, for the tablet formulation. T(max) was 0.89 hour (liquid) and 0.97 hour (tablet). The point estimates and 90% CIs for AUC(0-t), AUC(0-∞), and C(max) ratios (liquid vs tablet) were 91.42% (82.05%-101.86%), 95.72% (84.46%-108.5%), and 102.04% (86.94%-119.76%), respectively. Thus, point estimates and 90% CIs were within the bioequivalence range of 80% to 125% for all relevant parameters, including the pharmacodynamic parameter AUEC (area under the effect curve). CONCLUSIONS: This single-dose study suggests that the test and reference products met the EMA regulatory criteria to assume bioequivalence in these fasting healthy male and female volunteers. German Register of Clinical Trials registration number: DRKS00000785.

Effect of the CYP2C8 genotype on the pharmacokinetics and pharmacodynamics of repaglinide.

The pharmacokinetics of repaglinide shows pronounced interindividual variability, for which several reasons have been considered, including interactions with drugs inhibiting CYP2C8 and CYP2C8 genetic polymorphism. However, existing data on the role of genetic polymorphisms in repaglinide disposition are not fully consistent. We studied the effect of CYP2C8*3 on the pharmacokinetics and pharmacodynamics of repaglinide in 29 healthy whites carrying CYP2C8*3/*3 (n = 4), CYP2C8*1/*3 (n = 13), or CYP2C8*1/*1 (n = 12). After administration of a single dose of 2 mg of repaglinide, blood was drawn for assessment of repaglinide pharmacokinetics and pharmacodynamics, and urine was collected to quantify the main repaglinide metabolites M1 and M4 up to 24 h postdose. Repaglinide and the metabolites were quantified by liquid chromatography-tandem mass spectrometry. Considering only the effect of CYP2C8*3, the mean (95% confidence interval) area under the time-concentration curve (AUC) from zero to infinity of repaglinide was 72.4 (6.7-138.0), 97.2 (59.2-135.2), and 105.9 (52.4-159.3) ng · ml(-1) · h and the maximal concentration (C(max)) was 38.5 (3.8-73.2), 50.3 (37.5-63.0), and 60.3 (31.5-89.1) ng · ml(-1), respectively, in carriers of CYP2C8*3/*3, CYP2C8*1/*3, and CYP2C8*1/*1 [p > 0.05, one-way analysis of variance (ANOVA)]. In addition, for urinary metabolite excretion and pharmacodynamic parameters, i.e., mean and maximal changes in insulin and glucose concentration, no significant differences between CYP2C8 genotypes were observed. Likewise, no significant effects on the pharmacokinetics or pharmacodynamics were observed when AUC and C(max) of repaglinide were corrected for reported effects of the SLCO1B1 521T>C polymorphism or when both polymorphisms were tested in a two-way ANOVA. In conclusion, CYP2C8*3 does not seem to play an important role in the pharmacokinetics and pharmacodynamics of repaglinide given in a therapeutic dose.

Impaired hepatic drug and steroid metabolism in congenital adrenal hyperplasia due to P450 oxidoreductase deficiency.

OBJECTIVE: Patients with congenital adrenal hyperplasia due to P450 oxidoreductase (POR) deficiency (ORD) present with disordered sex development and glucocorticoid deficiency. This is due to disruption of electron transfer from mutant POR to microsomal cytochrome P450 (CYP) enzymes that play a key role in glucocorticoid and sex steroid synthesis. POR also transfers electrons to all major drug-metabolizing CYP enzymes, including CYP3A4 that inactivates glucocorticoid and oestrogens. However, whether ORD results in impairment of in vivo drug metabolism has never been studied. DESIGN: We studied an adult patient with ORD due to homozygous POR A287P, the most frequent POR mutation in Caucasians, and her clinically unaffected, heterozygous mother. The patient had received standard dose oestrogen replacement from 17 until 37 years of age when it was stopped after she developed breast cancer. METHODS: Both subjects underwent in vivo cocktail phenotyping comprising the oral administration of caffeine, tolbutamide, omeprazole, dextromethorphan hydrobromide and midazolam to assess the five major drug-metabolizing CYP enzymes. We also performed genotyping for variant CYP alleles known to affect drug metabolism. RESULTS: Though CYP enzyme genotyping predicted normal or high enzymatic activities in both subjects, in vivo assessment showed subnormal activities of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in the patient and of CYP1A2 and CYP2C9 in her mother. CONCLUSIONS: Our results provide in vivo evidence for an important role of POR in regulating drug metabolism and detoxification. In patients with ORD, in vivo assessment of drug-metabolizing activities with subsequent tailoring of drug therapy and steroid replacement should be considered.

Absorption, pharmacokinetics, and safety of triclosan after dermal administration.

We evaluated the pharmacokinetics and safety of the antimicrobial agent triclosan after dermal application of a 2% triclosan-containing cream to six volunteers. Percutaneous absorption calculated from urinary excretion was 5.9% +/- 2.1% of the dose (mean +/- standard deviation). The amount absorbed suggests that daily application of a standard adult dose would result in a systemic exposure 890 times lower than the relevant no-observed-adverse-effect level. Triclosan can be considered safe for use in hydrophobic creams.

In vivo role of cytochrome P450 2E1 and glutathione-S-transferase activity for acrylamide toxicokinetics in humans.

Acrylamide, a potential food carcinogen in humans, is biotransformed to the epoxide glycidamide in vivo. Both acrylamide and glycidamide are conjugated with glutathione, possibly via glutathione-S-transferases (GST), and bind covalently to proteins and nucleic acids. We investigated acrylamide toxicokinetics in 16 healthy volunteers in a four-period change-over trial and evaluated the respective role of cytochrome P450 2E1 (CYP2E1) and GSTs. Participants ingested self-prepared potato chips containing acrylamide (1 mg) without comedication, after CYP2E1 inhibition (500 mg disulfiram, single dose) or induction (48 g/d ethanol for 1 week), and were phenotyped for CYP2E1 with chlorzoxazone (250 mg, single dose). Unchanged acrylamide and the mercapturic acids N-acetyl-S-(2-carbamoylethyl)-cysteine (AAMA) and N-acetyl-S-(2-hydroxy-2-carbamoylethyl)-cysteine (GAMA) accounted for urinary excretion [geometric mean (percent coefficient of variation)] of 2.9% (42), 65% (23), and 1.7% (65) of the acrylamide dose in the reference period. Hemoglobin adducts increased clearly following the acrylamide test-meal. The cumulative amounts of acrylamide, AAMA, and GAMA excreted and increases in AA adducts changed significantly during CYP2E1 blockade [point estimate (90% confidence interval)] to the 1.34-fold (1.14-1.58), 1.18-fold (1.02-1.36), 0.44-fold (0.31-0.61), and 1.08-fold (1.02-1.15) of the reference period, respectively, but were not changed significantly during moderate CYP2E1 induction. Individual baseline CYP2E1 activity, CYP2E1*6, GSTP1 313A>G and 341T>C single nucleotide polymorphisms, and GSTM1-and GSTT1-null genotypes had no major effect on acrylamide disposition. The changes in acrylamide toxicokinetics upon CYP2E1 blockade provide evidence that CYP2E1 is a major but not the only enzyme mediating acrylamide epoxidation in vivo to glycidamide in humans. No obvious genetic risks or protective factors in xenobiotic-metabolizing enzymes could be determined for exposed subjects.

Phenotyping of N-acetyltransferase type 2 and xanthine oxidase with caffeine: when should urine samples be collected?

OBJECTIVES: Individual activities of N-acetyltransferase 2 (NAT2) and of xanthine oxidase (XO) can be assessed using ratios of urinary caffeine metabolites. We investigated how ratios changed over time and which urine collection interval would be the best for NAT2 and XO activity assessments. METHODS: On two occasions separated by 14 days, 16 healthy male Caucasians collected urine before and 0-2, 2-4, 4-6, 6-8, 8-12, 12-16 and 16-24 h after a dose of 150 mg caffeine given in the framework of a phenotyping cocktail study. The metabolites 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 5-acetylamino-6-amino-3-methyluracil (AAMU), 1-methylxanthine (1X), and 1-methylurate (1U) were quantified with LC-MS/MS. The molar ratio (AFMU + AAMU)/(1X + 1U + AFMU + AAMU) was used as a NAT2 metric, while the ratio 1U/(1X + 1U) served as XO metric. RESULTS: The NAT2 ratios were stable in the intervals 4-24 h after caffeine dosing. Mean intra-individual coefficients of variation were 11-23% starting 4 h post-dose, while inter-individual variability reached 37-75%. The XO ratios increased gradually by 14% from the 2-4 to the 16-24 h interval. The mean intra- and inter-individual coefficients of variation of XO activity were 3-18 and 7-10% respectively. No significant differences between study occasions were observed. CONCLUSIONS: Any sampling interval at least 4 h after caffeine dosing is suitable for NAT2 and XO activity assessments. XO activities can only be compared between volunteers and studies if the same urine collection schedule has been respected. The low intraindividual variability allows for sample sizes of 16 and 6 participants in crossover interaction studies of NAT2 and XO activity respectively.

Assessment of urinary mephenytoin metrics to phenotype for CYP2C19 and CYP2B6 activity.

OBJECTIVES: (S)-Mephenytoin is selectively metabolised to (S)-4'-hydroxymephenytoin by CYP2C19. The urinary excretion of 4'-hydroxymephenytoin reflects the activity of individual enzymes. We evaluated fractioned urinary collection and beta-glucuronidase pre-treatment in order to determine the optimal CYP2C19 metrics. We also assessed whether urinary excretion of N-desmethylmephenytoin (nirvanol) might be a useful CYP2B6 metric in in vivo studies. METHODS: A 50-mg dose of mephenytoin was administered to 52 volunteers as a component of phenotyping cocktails in four separate studies. Urine was collected up to 166 h post-dose. Urinary excretion of 4'-hydroxymephenytoin and nirvanol was quantified by liquid chromatography-tandem mass spectrometry, and common CYP2C19 and CYP2B6 genotypes were determined. RESULTS: Cumulative excretion of 4'-hydroxymephenytoin in urine with beta-glucuronidase treatment collected from before mephenytoin administration up to 12-16 h thereafter showed the greatest difference between CYP2C19 genotypes and the lowest intra-individual variability (7%). Renal elimination of nirvanol was highest for a *4/*4 individual and lowest for individuals carrying the *5/*5 and *1/*7 genotype, but lasted for several weeks, thus making its use in cross-over studies difficult. CONCLUSION: Cumulative urinary excretion of 4'-hydroxymephenytoin 0-12 h post-administration is a sensitive and reproducible metric of CYP2C19 activity, enabling the effect of a drug on CYP2C19 to be assessed in a small sample size of n=6 volunteers. While nirvanol excretion may reflect CYP2B6 activity in vivo, it is not useful for CYP2B6 phenotyping.

Pharmacokinetics and pharmacodynamics of rosiglitazone in relation to CYP2C8 genotype.

OBJECTIVES: Rosiglitazone is metabolically inactivated predominantly via the cytochrome P450 (CYP) enzyme CYP2C8. The functional impact of the CYP2C8*3 allele coding for the Arg139Lys and Lys399Arg amino acid substitutions is controversial. The purpose of this was to clarify the role of this polymorphism with regard to the pharmacokinetics and clinical effects of rosiglitazone. METHODS: From a large sample of healthy volunteers, 14 carriers of the CYP2C8*1/*1 allele, 13 carriers of the *1/*3 allele, and 4 carriers the *3/*3 allele were selected for a clinical study. Rosiglitazone (8 mg) single-dose and multiple-dose pharmacokinetics and its effects on glucose level and body weight were monitored. Plasma and urine concentrations of rosiglitazone and desmethylrosiglitazone were measured, and kinetics was analyzed by noncompartmental and population-kinetic compartmental methods. RESULTS: Mean total clearance values were 0.033 L x h(-1) x kg(-1) (95% confidence interval [CI], 0.030-0.037 L x h(-1) x kg(-1)), 0.038 L x h(-1) x kg(-1) (95% CI, 0.033-0.044 L x h(-1) x kg(-1)), and 0.046 L x h(-1) x kg(-1) (95% CI, 0.033-0.058 L x h(-1) x kg(-1)) in carriers of CYP2C8 genotypes *1/*1, *1/*3, and *3/*3, respectively, on day 1 (P = .02, ANOVA [F test]). Rosiglitazone kinetics could be adequately described by a 1-compartmental model with first-order absorption. Besides CYP2C8 genotype, body weight was a significant covariate (P < .001, log-likelihood ratio test). Elimination half-lives were 4.3, 3.5, and 2.9 hours in CYP2C8*1/*1, *1/*3, and *3/*3 carriers, respectively. Clearance of desmethylrosiglitazone was also higher in CYP2C8*3 allele carriers, with mean values of 1.96 L/h (95% CI, 1.42-2.69 L/h), 2.22 L/h (95% CI, 1.61-3.04 L/h), and 2.47 L/h (95% CI, 1.80-3.39 L/h), respectively (P = .03). The plasma glucose area under the concentration curve was significantly lower after 14 days of taking rosiglitazone compared with day 1 (P = .01, paired t test), but no relationship of the glucose-lowering effect of rosiglitazone with CYP2C8 genotype was observed. CONCLUSIONS: This study showed that the CYP2C8*3 allele confers higher in vivo metabolic capacity than the wild-type CYP2C8*1 allele but the pharmacokinetic differences resulting from CYP2C8*3 were quantitatively moderate.

Toxicokinetics of acrylamide in humans after ingestion of a defined dose in a test meal to improve risk assessment for acrylamide carcinogenicity.

High amounts of acrylamide in some foods result in an estimated daily mean intake of 50 microg for a western style diet. Animal studies have shown the carcinogenicity of acrylamide upon oral exposure. However, only sparse human toxicokinetic data is available for acrylamide, which is needed for the extrapolation of human cancer risk from animal data. We evaluated the toxicokinetics of acrylamide in six young healthy volunteers after the consumption of a meal containing 0.94 mg of acrylamide. Urine was collected up to 72 hours thereafter. Unchanged acrylamide, its mercapturic acid metabolite N-acetyl-S-(2-carbamoylethyl)cysteine (AAMA), its epoxy derivative glycidamide, and the respective metabolite of glycidamide, N-acetyl-S-(2-hydroxy-2-carbamoylethyl)cysteine (GAMA), were quantified in the urine by liquid chromatography-mass spectrometry. Toxicokinetic variables were obtained by noncompartmental methods. Overall, 60.3 +/- 11.2% of the dose was recovered in the urine. Although no glycidamide was found, unchanged acrylamide, AAMA, and GAMA accounted for urinary excretion of (mean +/- SD) 4.4 +/- 1.5%, 50.0 +/- 9.4%, and 5.9 +/- 1.2% of the dose, respectively. Apparent terminal elimination half-lives for the substances were 2.4 +/- 0.4, 17.4 +/- 3.9, and 25.1 +/- 6.4 hours. The ratio of GAMA/AAMA amounts excreted was 0.12 +/- 0.02. In conclusion, most of the acrylamide ingested with food is absorbed in humans. Conjugation with glutathione exceeds the formation of the reactive metabolite glycidamide. The data suggests an at least 2-fold and 4-fold lower relative internal exposure for glycidamide from dietary acrylamide in humans compared with rats or mice, respectively. This should be considered for quantitative cancer risk assessment.

Should we use N-acetyltransferase type 2 genotyping to personalize isoniazid doses?

Isoniazid is metabolized by the genetically polymorphic arylamine N-acetyltransferase type 2 (NAT2). A greater number of high-activity alleles are related to increased acetylation capacity and in some reports to low efficacy and toxicity of isoniazid. The objective of this study was to assess individual isoniazid exposure based on NAT2 genotype to predict a personalized therapeutic dose. Isoniazid was administered to 18 healthy Caucasians (age 30 +/- 6 years, body weight 74 +/- 10 kg, five women) in random order as a 200-mg infusion, a 100-mg oral, and a 300-mg oral single dose. For the assessment of NAT2 genotype, common single nucleotide polymorphisms identifying 99.9% of variant alleles were characterized. Noncompartmental pharmacokinetics and compartmental population pharmacokinetics were estimated from isoniazid plasma concentrations until 24 h postdose by high-pressure liquid chromatography. The influence of NAT2 genotype, drug formulation, body weight, and sex on dose-normalized isoniazid pharmacokinetics was assessed by analysis of variance from noncompartmental data and confirmed by population pharmacokinetics. Eight high-activity NAT2*4 alleles were identified. Sex had no effect; the other factors explained 93% of the variability in apparent isoniazid clearance (analysis of variance). NAT2 genotype alone accounted for 88% of variability. Individual isoniazid clearance could be predicted as clearance (liters/hour) = 10 + 9 x (number of NAT2*4 alleles). To achieve similar isoniazid exposure, current standard doses presumably appropriate for patients with one high-activity NAT2 allele may be decreased or increased by approximately 50% for patients with no or two such alleles, respectively. Prospective clinical trials are required to assess the merits of this approach.

Cytochrome P450 2C9 phenotyping using low-dose tolbutamide.

OBJECTIVES: The hypoglycaemic drug tolbutamide is used for assessment of CYP2C9 activity in vivo. However, therapeutically active doses of 500 mg bear the risk of hypoglycaemia, and a tolbutamide-derived parameter based on a single plasma or urine concentration reflecting CYP2C9 activity accurately is lacking. METHODS: We examined tolbutamide and its metabolites 4'-hydroxy-tolbutamide and carboxytolbutamide in plasma and urine of 26 healthy, male volunteers up to 24 h after intake of 125 mg tolbutamide using liquid chromatography-tandem mass spectrometry. CYP2C9 genotypes were determined by sequencing of exons 3 and 7. Raw plasma and urine data were compared with pharmacokinetic parameters, CYP2C9 genotypes, and data from a study in 23 volunteers with all six CYP2C9*1-*3 combinations who received 500 mg tolbutamide. RESULTS: Plasma clearance and tolbutamide plasma concentrations 24 h after drug intake reflected the genotypes: 0.85 l/h and 1.70 microg/ml (95% confidence interval, CI, 0.80-0.89 l/h and 1.50-1.90 microg/ml) for CYP2C9*1 homozygotes (n=15), 0.77 l/h and 2.14 microg/ml (95%CI, 0.67-0.88 l/h and 1.64-2.63 microg/ml) for *1/*2 genotypes (n=7), 0.60 l/h and 3.13 microg/ml (95%CI, 0.58-0.62 l/h and 2.68-3.58 microg/ml) for *1/*3 genotypes (n=3), and 0.57 l/h and 3.27 microg/ml in the single *2/*2 carrier. Natural logarithms of tolbutamide plasma concentrations 24 h after intake correlated to plasma clearance (r(2)=0.84, P<0.0000001). This correlation was confirmed in the comparison data set (r(2)=0.97, P<0.0000001). CONCLUSIONS: A low dose of 125 mg tolbutamide can safely and accurately be used for CYP2C9 phenotyping. As a simple metric for CYP2C9 activity, we propose to determine tolbutamide in plasma 24 h after drug intake.