The generation, detection, and effects of reactive drug metabolites.

The decline in approval of new drugs during the past decade has led to a close analysis of the drug discovery process. One of the main reasons for attrition is preclinical toxicity, frequently attributed to the generation of protein-reactive drug metabolites. In this review, we present a critique of such reactive metabolites and evaluate the evidence linking them to observed toxic effects. Methodology for the characterization of reactive metabolites has advanced greatly in recent years, and is summarized first. Next, we consider the inhibition of key metabolic enzymes by electrophilic metabolites, as well as unfavorable drug-drug interactions that may ensue. One important class of protein-reactive metabolites, not linked conclusively to a toxic event, is acyl glucuronides. Their properties are discussed in light of the safety characteristics of carboxylic acid containing drugs. Many adverse drug reactions (ADRs) are known collectively as idiosyncratic events, that is, not predictable from knowledge of the pharmacology and pharmacokinetics of the parent compound. Observed ADRs may take various forms. Specific organ injury, particularly of the liver, is the most direct: we examine this in some detail. Moving to the cellular level, we also consider the upregulation of induced cellular processes. The related, but distinct, issue of hypersensitivity or allergic reactions to drugs and their metabolites, possibly via the immune system, is considered next. Finally, we discuss the impact of such data on the drug discovery process, both through early detection of reactive metabolites and informed synthetic design, which eliminates unfavorable functionality from drug candidates.

Pharmacokinetic-pharmacodynamic modeling of mood and withdrawal symptoms in relation to plasma concentrations of methadone in patients undergoing methadone maintenance treatment.

The aims of the present study were to characterize the relationship between plasma racemic methadone and its enantiomers' concentrations with respect to their pharmacodynamic effects and to investigate the influence of potential covariates on the pharmacodynamic parameters in patients on methadone maintenance treatment (MMT). Eighty-eight regular subjects at the Sheffield Care Trust Substance Misuse Services were studied. Samples of blood and urine were collected before the daily dose of methadone. Blood samples were taken up to 5 hours after dose. Total plasma concentrations of (RS)-methadone and total and unbound plasma concentrations of both enantiomers were measured by liquid chromatography-mass spectrometry. The Total Mood Disturbance Score (TMDS), the Objective Opioid Withdrawal Scale (OOWS), and the Subjective Opioid Withdrawal Scale (SOWS) were used as measures of mood and withdrawal. Population pharmacokinetic/pharmacodynamic analysis and subsequent multiple regression analysis were used to determine the factors influencing the pharmacodynamic effects of methadone. Significant decreases (P ≤ 0.04) were observed in the scores for the TMDS, SOWS, and OOWS for 5 hours after methadone dosage. The TMDS had returned to baseline by 10 hours after dose (P = 0.98), at which time the SOWS remained significantly below baseline (P = 0.001). Multiple regression analysis revealed that 33% of the overall variation in unbound (R)-methadone EC50 was explained by 3 variables, namely CYP3A activity (9%), age (16%), and sex (8%). Age also accounted for 8% and 9% of the variation in total (rac)- and (R)-methadone EC50. The present study has confirmed that the duration of mood change in the present study was shorter than the effect of methadone in stabilizing withdrawal symptoms. Thus, it is likely that a once-daily dose of methadone, albeit effective for preventing withdrawal, may not be sufficient to improve mood in some patients. Finally, it was established that CYP3A activity, years of dependent use, sex, and age are major determinants of methadone EC50 with respect to TMDS.

Contribution of the activities of CYP3A, CYP2D6, CYP1A2 and other potential covariates to the disposition of methadone in patients undergoing methadone maintenance treatment.

AIMS: To investigate the influence of different cytochrome P450 (CYP) activities and other potential covariates on the disposition of methadone in patients on methadone maintenance therapy (MMT). METHODS: Eighty-eight patients (58 male; 21-55 years; 84 White) on MMT were studied. CYP2D6 activity [3 h plasma metabolic ratio of dextromethorphan (DEX) to dextrorphan (DOR)] was determined in 44 patients (29 male; 24-55 years), CYP1A2 activity (salivary caffeine elimination half-life) in 44 patients (21 male; 24-55 years) and CYP3A activity (oral clearance of midazolam) in 49 patients (33 male; 23-55 years). Data on all three CYPs were obtained from 32 subjects. Total plasma concentrations of (RS)-methadone and total and unbound plasma concentrations of both enantiomers were measured by LC/MS. Population pharmacokinetics and subsequent multiple regression analysis were used to calculate methadone oral clearance and to identify its covariates. RESULTS: Between 61 and 68% of the overall variation in total plasma trough concentrations of (RS)-, (R)- and (S)-methadone was explained by methadone dose, duration of addiction before starting MMT, CYP3A activity and illicit morphine use. CYP3A activity explained 22, 16, 15 and 23% of the variation in unbound (R)-, unbound (S)-, total (RS)- and total (S)-methadone clearances, respectively. Neither CYP2D6 nor CYP1A2 activity was related to methadone disposition. CONCLUSIONS: CYP3A activity has a modest influence on methadone disposition. Inhibitors and inducers of this enzyme should be monitored in patients taking methadone.

Single-dose pharmacokinetic study of clomiphene citrate isomers in anovular patients with polycystic ovary disease.

The pharmacokinetics of the zuclomiphene (Zu) and enclomiphene (En) isomers of clomiphene citrate following a single oral dose (50 mg) were characterized for the first time in patients receiving the drug (ie, infertile women with polycystic ovary syndrome). Plasma concentrations of Zu and En were measured in 9 patients from the second day of their menstrual cycle (day 1 of dosing) up to 21 days. The mean (+/- coefficient of variation) of C(max), t(max), and AUC of Zu was 15 +/- 41 ng/mL, 7 +/- 87 h, and 1289 +/- 34 ng/mL.h (AUC(0-456 h)), and that of En was 15 +/- 18 ng/mL, 3 +/- 68 h, and 65 +/- 35 ng/ml.h (AUC(0-72h)), respectively. These parameters appeared to be different for Zu from those reported previously in healthy participants, except for t(max). The pharmacokinetic parameters of En in patients with polycystic ovary syndrome were not generally different from the healthy subjects. The effect of obesity on Zu kinetics was stronger than that on En. The conventional model-dependent pharmacokinetics of clomiphene citrate isomers could not be determined due to a very flat terminal half-life and the long-tailed residence time, signifying the lipophilic nature and potentially extensive distribution of the compound.

Evaluation of the relationship between plasma concentrations of en- and zuclomiphene and induction of ovulation in anovulatory women being treated with clomiphene citrate.

OBJECTIVE: To investigate the relationship between the plasma concentrations of clomiphene citrate (CC) isomers zu- (Zu) and enclomiphene (En), and ovulation outcome. DESIGN: Prospective, cohort study. SETTING: Reproductive medicine and fertility center in a university teaching hospital, United Kingdom. PATIENT(S): Forty-two women with World Health Organization type 2 infertility. INTERVENTION(S): The clinical and biochemical features of patients who were about to start CC for induction of ovulation were recorded. Plasma concentration of Zu and En were monitored at three points (days 2, 8, and 21) throughout the treatment cycle(s). MAIN OUTCOME MEASURE(S): Ovulation. RESULT(S): Thirty-nine patients completed the study. Both En and Zu accumulated throughout treatment. Among the 36 responders, there was no statistically significant relationship between the clinical and biochemical characteristics of the patients, En or Zu concentrations, and the dose required to induce ovulation. Moreover, the Zu and En concentrations were not different in the three patients who failed to respond. CONCLUSION: The concentrations of En and Zu in plasma, on their own or in combination with other covariates (e.g., weight, body mass index, free androgen index), are not a predictor of the ovulation response to CC or of the dose requirement. Further studies are needed to explore the role of additional covariates, including the presence of active metabolites, and the balance of the effects of En and Zu.

CYP2D6 is primarily responsible for the metabolism of clomiphene.

Clomiphene is a first line treatment for anovulation, a common cause of infertility. Response to clomiphene is variable and unpredictable. Tamoxifen is structurally related to clomiphene, and also shows considerable variation in response. CYP2D6 and CYP3A4 are major contributors to the metabolism of tamoxifen. The aim of the present work was to define the role of CYP2D6 and CYP3A4 in the in vitro metabolism of enclomiphene, regarded by some as the more active isomer of clomiphene. Enclomiphene (25 microM) was incubated with human liver microsomes (from 4 extensive (EM) and 1 poor metaboliser with respect to CYP2D6) and with microsomes from lymphoblastoid cells expressing CYP2D6. Microsomes from all the EM livers and recombinant CYP2D6 metabolised enclomiphene (the disappearance of drug ranged from 40-60%). No metabolism was detected in microsomes from the PM liver. Quinidine (1 microM) completely inhibited the metabolism of enclomiphene by all the EM livers and by recombinant CYP2D6 (p<0.001, one way ANOVA). Ketoconazole (2 microM) had no significant effect on enclomiphene metabolism in 3 out of the 4 EM livers. The extent of enclomiphene metabolism was correlated with the amount of CYP2D6 present (p<0.001, Pearson correlation test). The findings indicate that CYP2D6 is primarily responsible for the metabolism of enclomiphene.

Inactivation of CYP2D6 by methylenedioxymethamphetamine in different recombinant expression systems.

Recombinantly expressed CYP450 systems (rCYPs) are often used to screen for irreversible/quasi-irreversible enzyme inhibitors during drug development. The concentration- and time-dependent inactivation of CYP2D6 by methylenedioxymethamphetamine (MDMA) was compared in three different rCYP2D6 systems (yeast microsomes, Supersomestrade mark and Bactosomestrade mark) under the conditions of the most commonly used protocols in assessing mechanism-based inactivation (MBI). MDMA (2-20microM) was pre-incubated with enzyme for 0, 2.5 and 5min followed by a five-fold dilution and further incubation with dextromethorpan (DEX) (50microM). The formation of dextrorphan (DOR) from DEX was used as a specific marker of CYP2D6 activity. Concentration- and time-dependent inactivation of CYP2D6 by MDMA was observed with each rCYP system. However, the apparent kinetic parameters for MBI (k(inact), the maximum inactivation rate constant and K(I), the inhibitor concentration associated with half maximal rate of inactivation) were significantly greater (p<0.05) for Bactosomestrade mark (0.95+/-0.33min(-1), 42.9+/-20.1microM) than those found using yeast microsomes (0.28+/-0.04min(-1), 2.86+/-1.18microM) and Supersomestrade mark (0.38+/-0.05min(-1), 3.66+/-0.10microM). After correction for depletion of MDMA during pre-incubation, k(inact) and K(I) values determined using Bactosomestrade mark decreased significantly but remained higher than for the other rCYP systems (p<0.05). Substantial metabolism of DOR after its formation from DEX was also observed using Supersomestrade mark and Bactosomestrade mark. Sub-optimal study design when investigating MBI may compromise the quantitative characterization of inhibitory characteristics using some rCYP systems.

PRN prescribing in psychiatric inpatients: potential for pharmacokinetic drug interactions.

Medications are commonly prescribed to psychiatric inpatients on a PRN (pro re nata/as required) basis, allowing drugs to be administered on patient request or at nurses' discretion for psychiatric symptoms, treatment side effects or physical complaints. However, there has been no formal study of the pharmacokinetic implications of PRN prescribing. The objective of the study was to determine the prevalence of PRN drug prescription and administration, and to assess the potential for interactions involving CYP2D6 and CYP3A4 between drugs prescribed and administered to inpatients on psychiatry wards.A cross-sectional survey of prescriptions on general adult and functional elderly psychiatric wards in one city was carried out. Data were recorded from prescription charts of 323 inpatients (236 on general adult and 87 on functional elderly wards). Of 2089 prescriptions, 997 (48%) of prescriptions were on a PRN basis (most commonly benzodiazepines and other hypnotic agents, antipsychotics, analgesics and anticholinergic agents), but only 143 (14%) of these had been administered in the previous 24 hours. One fifth of patients were prescribed drug combinations interacting with CYP2D6 or CYP3A4 of potential clinical importance which included one or more drugs prescribed on a PRN basis.PRN prescribing is common among inpatients in psychiatry, and may lead to cytochrome P450 mediated interactions. Prescribers should be aware of the potential for unpredictability in plasma concentrations, side effects and efficacy which PRN prescribing may cause through these interactions, particularly in old age psychiatry and in treatment of acute psychosis.

The use of mechanistic DM-PK-PD modelling to assess the power of pharmacogenetic studies -CYP2C9 and warfarin as an example.

AIM: To assess the power of in vivo studies needed to discern the effect of genotype on pharmacokinetics (PK) and pharmacodynamics (PD) using CYP2C9 and (S)-warfarin as an example. METHODS: Information on the in vitro metabolism of (S)-warfarin and genetic variation in CYP2C9 was incorporated into a mechanistic population-based PK-PD model. The influence of study design on the ability to detect significant differences in PK (AUC(0-12 h)) and PD (AUEC(0-12 h) INR) between CYP2C9 genotypes was investigated. RESULTS: A study size of 90 (based on the natural abundance of genotypes and uniform dosage) was required to achieve 80% power to discriminate the PK of (S)-warfarin between wild type (*1/*1) and the combination of all other genotypes. About 250 subjects were needed to detect a difference in anticoagulant response. The power to detect differences between specific genotypes was much lower. Analysis of experimental comparisons of the PK or PD between wild-type and other individual genotypes indicated that only 21% of cases (20 of 95 comparisons within 11 PD and four PK-PD studies) reported statistically significant differences. This was similar to the percentage expected from our simulations (20%, chi(2) test, P = 0.80). Simulations of studies enriched with specific genotypes indicated that only three and five subjects were required to detect differences in PK and PD between wild type and the *3/*3 genotype, respectively. CONCLUSION: The utilization of prior information (including in vivo enzymology) in clinical trial simulations can guide the design of subsequent in vivo studies of the impact of genetic polymorphisms, and may help to avoid costly, inconclusive outcomes.

Incorporating in vitro information on drug metabolism into clinical trial simulations to assess the effect of CYP2D6 polymorphism on pharmacokinetics and pharmacodynamics: dextromethorphan as a model application.

In vitro-in vivo extrapolation of clearance, embedded in a clinical trial simulation, was used to investigate differences in the pharmacokinetics and pharmacodynamics of dextromethorphan between CYP2D6 poor and extensive metabolizer phenotypes. Information on the genetic variation of CYP2D6, as well as the in vitro metabolism and pharmacodynamics of dextromethorphan and its active metabolite dextrorphan, was integrated to assess the power of studies to detect differences between phenotypes. Whereas 6 subjects of each phenotype were adequate to achieve 80% power in showing pharmacokinetic differences, the power required to detect a difference in antitussive response was less than 80% with 500 subjects in each study arm. Combining in vitro-in vivo extrapolation with a clinical trial simulation is useful in assessing different elements of study design and could be used a priori to avoid inconclusive pharmacogenetic studies.

Evaluation of an existing nomogram for predicting the response to clomiphene citrate.

OBJECTIVE: To evaluate the ability of an existing nomogram to predict response to clomiphene citrate (CC) in infertile couples with World Health Organization group II ovulatory disorders, using the free androgen index, body mass index, and menstrual-cycle history. DESIGN: Retrospective case-notes study. SETTING: Reproductive medicine and fertility center at a university teaching hospital in the United Kingdom. PATIENT(S): One hundred four anovulatory women. INTERVENTION(S): One hundred four anovulatory women who had been treated with CC were studied retrospectively. Age, body mass index, free androgen index, and cycle history were used to assign a likelihood of response for each patient on the basis of a published nomogram. Predicted and observed responses were compared. MAIN OUTCOME MEASURE(S): Ovulation rate. RESULT(S): The diagnostic characteristics of the model on the basis of the optimal cutoff points were as follows: sensitivity, 96% (95% confidence interval [CI]: 90%-99%); specificity, 33% (95% CI: 18%-49%); positive predictive value, 73% (95% CI: 63%-82%); negative predictive value, 80% (95% CI: 60%-99%); likelihood ratio for ovulation, 1.34 (95% CI: 1.1-1.8); likelihood ratio for resistance, 0.13 (95% CI: 0.04-0.43); kappa, 0. 26 (95% CI: 0.09-0.44). CONCLUSION(S): The accurate prediction of response to CC would allow more rapid transfer of nonresponders to alternative treatments and may shorten the treatment to pregnancy interval. Although the current nomogram could identify 80% of nonresponders to CC, it was insufficiently accurate for use in the present clinical setting. Moreover, the nomogram could not identify the most appropriate dose to achieve ovulation. This nomogram should be tested on patients in other clinical settings, where it may perform better.

Does previous response to clomifene citrate influence the selection of gonadotropin dosage given in subsequent superovulation treatment cycles?

PURPOSE: To determine whether ovarian response to previous clomifene treatment could influence the selection of the starting dose of gonadotropins in subsequent in vitro fertilization (IVF) or intra uterine insemination (IUI). METHODS: Forty three anovular women who had received clomifene for ovulation induction followed by gonadotropins for IUI or IVF superovulation were reviewed retrospectively. Data on gonadotropin dose were compared between clomifene-resistant patients and clomifene responders. RESULTS: IVF patients who had had prior superovulation/IUI treatment received similar doses of gonadotropins regardless of response to clomifene (1610 IU versus 1560 IU, p = 0.74). In IVF patients not receiving prior IUI treatment, the clomifene-resistant women were given higher doses of gonadotropins than those responding to clomifene (2500 IU versus 1440 IU, p = 0.042). CONCLUSIONS: We found that, in our Unit, clinicians appeared to use prior non-response to clomifene as a reason for prescribing a higher starting dose of gonadotropins in IVF treatment, a practice that is not evidence-based.

Solid-phase microextraction: investigation of the metabolism of substances that may be abused by inhalation.

Purified liquefied petroleum gas (LPG), a mixture of butane, isobutane, and propane, is commonly abused by inhalation. Little is known about the mammalian metabolism of these substances. Metabolism of other hydrocarbons, including n-hexane and cyclohexane, has been studied in vitro using a range of liver preparations, with metabolites analyzed by static headspace techniques. Solid-phase microextraction (SPME) for sampling metabolites in the headspace of incubates of volatile compounds with activated rat liver microsomes is investigated. Cyclohexanol and cyclohexanone were formed from cyclohexane and 1-, 2-, and 3-hexanol and 2-hexanone from n-hexane as predicted. Secondary alcohols are found for the other compounds studied, except for propene and isobutane, together with 2-propanone and 2-butanone from propane and n-butane, respectively. Samples from three individuals who died following LPG abuse contained a range of putative n-butane metabolites: n-butanol, 2-butanol, 2,3-butanediol, 3-hydroxy-2-butanone, and 2,3-butanedione. To our knowledge, the last three compounds have not been proposed as metabolites of n-butane in man. These might be produced through similar metabolic pathways to those of n-hexane and n-heptane. The findings indicate the value of SPME for investigating the metabolism of volatile substances and for detecting and monitoring exposure to these compounds.

The impact of experimental design on assessing mechanism-based inactivation of CYP2D6 by MDMA (Ecstasy).

MDMA (3-4-methylenedioxymethamphetamine, commonly known as Ecstasy) is a potent mechanism-based inhibitor (MBI) of cytochrome P450 2D6 (CYP2D6), causing quasi-irreversible inhibition of the enzyme in vitro. An evaluation of the in vivo implications of this phenomenon depends on the accuracy of the estimates of the parameters that define the inhibition in vitro, namely k(inact) (the maximal inhibition rate) and KI (the inactivation constant). These values are determined in two steps, pre-incubation of the enzyme with the inhibitor (enzyme inactivation), followed by dilution and further incubation to measure residual enzyme activity with a probe substrate. The aim of this study was to assess the impact of different dilutions and probe substrate concentrations on the estimates of k(inact) and KI using recombinantly expressed CYP2D6. Enzyme activity was measured by the conversion of dextromethorphan (DEX) to dextrorphan (DOR). Dilution factors of 1.25, 2, 5, 10, 25 and 50 (DEX at 30 microM) gave mean (+/-SE) values of k(inact) (min-1) of 0.20+/-0.06, 0.21+/-0.05, 0.31+/-0.06, 0.37+/-0.11, 0.51+/-0.10 and 0.58+/-0.08, respectively, and KI (microM) values (after correction for non-specific microsomal binding) of 2.22+/-1.90, 2.80+/-1.34, 5.78+/-2.07, 6.36+/-2.93, 3.99+/-1.57 and 4.86+/-1.37, respectively. Accordingly, high (e.g. 50 fold) and low (e.g. 1.25 fold) dilutions were associated with statistically significant differences in kinetic values (p <0.05). Varying DEX concentration (10-100 microM) was not associated with significant changes in k(inact) and KI values when a five-fold dilution was used (with the exception of a lower KI at 10 microM DEX). High dilution was also shown to reduce non-specific microsomal binding of MDMA. The changes in the two kinetic parameters were dependent on the experimental procedure and shown to be unlikely to have a material influence on the maximum inhibition of CYP2D6 expected in vivo after typical recreational doses of MDMA (50-100 mg), since the potency of inhibition was high. The different values of the kinetic parameters were predicted to have a marginal influence on the time for recovery of enzyme activity following re-synthesis of CYP2D6.

Can saliva replace plasma for the monitoring of methadone?

The aims of this study were to determine the relationship between saliva and plasma methadone concentrations and the influence of variability in saliva pH. Saliva and plasma samples were taken before the daily dose of methadone in 60 patients undergoing methadone maintenance treatment (MMT). Saliva pH was measured immediately after sampling, and concentrations of (RS)-, (R)-, and (S)-methadone in saliva and plasma were assayed by LC/MS. In addition, unbound (R)- and (S)-methadone concentrations were measured in plasma samples by ultrafiltration. Plasma binding and pH differences between plasma and saliva were then used to estimate methadone saliva/plasma ratios and to compare them with observed values. Saliva pH ranged from 5.1 to 7.6 (mean +/- SD, 6.7 +/- 0.5). Plasma and saliva concentrations correlated weakly [(RS)-, r = 0.14, P = 0.007, n = 44; (R)-, r = 0.10, P = 0.04, n = 43; (S)-, r = 0.22, P = 0.002, n = 43], and the mean saliva-to-plasma methadone concentration ratios were 1.1 (+/-1.3 SD), 1.5 (+/-1.5), and 0.8 (+/-0.8), for (RS)-, (R)-, and (S)-methadone, respectively. Corresponding values based on unbound concentrations of methadone in plasma were 21 (+/-20.6, n = 31), 21 (+/-19, n = 34), and 17 (+/-15, n = 36). The salivary concentration-to-dose ratios showed statistically significant but weak inverse correlations with saliva pH [(RS)-, r = 0.27, P < 0.001; (R)-, r = 0.25, P < 0.001; (S)-, r = 0.29, P < 0.001, respectively]. There were significant correlations between predicted and observed saliva/plasma ratios [(RS)-, r = 0.44, P < 0.001, n = 31; (R)-, r = 0.58, P < 0.001, n = 32; (S)-, r = 0.10, P = 0.04, n = 34], but the mean predicted saliva concentrations were about 5 times lower than the mean observed values. The poor correlations between salivary and plasma methadone concentrations observed in this study are partly related to the effect of variable saliva pH. However, saliva pH explained only 10%-36% of the total variation. As a conclusion, monitoring methadone concentrations in saliva may not be a useful alternative to plasma concentration measurements. Correction for saliva pH measured immediately after collection improves the relationship between saliva and plasma methadone concentration, but most of the variation remains unexplained.

Monitoring plasma concentrations to individualize treatment with clomiphene citrate.

OBJECTIVE: Recent reports have indicated that a number of individual patient characteristics are responsible for the success or failure of clomiphene citrate treatment. However, a priori individualization of doses in different patients has not been investigated. We examined the thesis that wide variability in the metabolism of the active component (zuclomiphene) contributes to variability in response. METHODS: The dose-response relationship of clomiphene was established from a meta-analysis of data from 13 published reports. Limited data relating plasma drug concentrations to treatment outcome were examined to determine whether insufficient systemic exposure at a fixed dosage might contribute to therapeutic failure. RESULTS: A fixed-dosage regimen of 50 mg clomiphene per day is likely to cause ovulation in only 46% of patients; subsequent increment in dosage increases the number of responders but at the expense of considerable delay in individualization of treatment. Case reports indicated that dosage based on plasma drug concentration monitoring could improve patient management, and an algorithm is proposed to facilitate treatment. CONCLUSIONS: Prospective studies of clomiphene citrate should be performed to confirm the hypothesis that the monitoring of plasma zuclomiphene concentrations can significantly accelerate dose individualization and improve the therapeutic outcome with this "orphan" drug.

Metabolism of ethylbenzene by human liver microsomes and recombinant human cytochrome P450s (CYP).

The enzyme kinetics of the initial hydroxylation of ethylbenzene to form 1-phenylethanol were determined in human liver microsomes. The individual cytochrome P450 (CYP) forms catalysing this reaction were identified using selective inhibitors and recombinant preparations of hepatic CYPs. Production of 1-phenylethanol in hepatic microsomes exhibited biphasic kinetics with a high affinity, low Km, component (mean Km = 8 microM; V(max) = 689 pmol/min/mg protein; n = 6 livers) and a low affinity, high Km, component (Km = 391 microM; V(max) = 3039 pmol/min/mg protein; n = 6). The high-affinity component was inhibited 79%-95% (mean 86%) by diethyldithiocarbamate, and recombinant CYP2E1 was shown to metabolise ethylbenzene with low Km (35 microM), but also low (max) (7 pmol/min/pmol P450), indicating that this isoform catalysed the high-affinity component. Recombinant CYP1A2 and CYP2B6 exhibited high V(max) (88 and 71 pmol/min/pmol P450, respectively) and high Km (502 and 219 microM, respectively), suggesting their involvement in catalysing the low-affinity component. This study has demonstrated that CYP2E1 is the major enzyme responsible for high-affinity side chain hydroxylation of ethylbenzene in human liver microsomes. Activity of this enzyme in the population is highly variable due to induction or inhibition by physiological factors, chemicals in the diet or some pharmaceuticals. This variability can be incorporated into the risk assessment process to improve the setting of occupational exposure limits and guidance values for biological monitoring.

Influence of dose, cigarette smoking, age, sex, and metabolic activity on plasma clozapine concentrations: a predictive model and nomograms to aid clozapine dose adjustment and to assess compliance in individual patients.

The measurement of plasma clozapine concentrations is useful in assessing compliance, optimizing therapy, and minimizing toxicity. We measured plasma clozapine and norclozapine (N-desmethylclozapine) concentrations in samples from 3782 patients (2648 male, 1127 female). No clozapine was detected in 291 samples (227 patients, median prescribed dose 300 mg/d). In 4963 (50.2 %) samples (2222 patients); plasma clozapine concentration ranged from 10 to 350 ng/mL.Step-wise backward multiple regression analysis (37 % of the total samples) of log10 plasma clozapine concentration against log10 clozapine dose (mg/d), age (year), sex (male = 0, female = 1), cigarette smoking habit (nonsmokers = 0; smokers = 1), body weight (kg), and plasma clozapine/norclozapine ratio (clozapine metabolic ratio, MR) showed that these covariates explained 48% of the observed variation in plasma clozapine concentration (C = ng/mL x 10-3) (P < 0.001) according to the following equation: log 10 (C) = 0.811 log 10 (dose) + 0.332 (MR) + 69.42 X 10 (-3) (sex) + 2.263 x 10 (-3) (age) + 1.976 x 10(-3) (weight) - 0.171 (smoking habit) - 3.180. This model and its associated confidence intervals were used to develop nomograms of plasma clozapine concentration versus dose for male and female smokers and nonsmokers. Predicted plasma clozapine changes by +48% in nonsmokers, +17% in females, +/-8 % for every 0.1 change in MR (reference 1.32), +/-4% for every 5 years (reference 40 years), and +/-5 % for every 10 kg body weight (reference 80 kg). The nomograms can be used (i) to individualize dosage to achieve a given target plasma clozapine concentration, and (ii) for quantitative evaluation of adherence by estimating the likelihood of an observed concentration being achieved by a given dosage regimen. The model has been validated against published data.

Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: formation of the 4-hydroxy, 4'-hydroxy and N-desmethyl metabolites and isomerization of trans-4-hydroxytamoxifen.

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 microM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 microM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 microM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 microM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.