Nicotine dependence is associated with functional variation in FMO3, an enzyme that metabolizes nicotine in the brain.

A common haplotype of the flavin-containing monooxygenase gene FMO3 is associated with aberrant mRNA splicing, a twofold reduction in in vivo nicotine N-oxidation and reduced nicotine dependence. Tobacco remains the largest cause of preventable mortality worldwide. CYP2A6, the primary hepatic nicotine metabolism gene, is robustly associated with cigarette consumption but other enzymes contribute to nicotine metabolism. We determined the effects of common variants in FMO3 on plasma levels of nicotine-N-oxide in 170 European Americans administered deuterated nicotine. The polymorphism rs2266780 (E308G) was associated with N-oxidation of both orally administered and ad libitum smoked nicotine (P⩽3.3 × 10-5 controlling for CYP2A6 genotype). In vitro, the FMO3 G308 variant was not associated with reduced activity, but rs2266780 was strongly associated with aberrant FMO3 mRNA splicing in both liver and brain (P⩽6.5 × 10-9). Surprisingly, in treatment-seeking European American smokers (n=1558) this allele was associated with reduced nicotine dependence, specifically with a longer time to first cigarette (P=9.0 × 10-4), but not with reduced cigarette consumption. As N-oxidation accounts for only a small percentage of hepatic nicotine metabolism we hypothesized that FMO3 genotype affects nicotine metabolism in the brain (unlike CYP2A6, FMO3 is expressed in human brain) or that nicotine-N-oxide itself has pharmacological activity. We demonstrate for the first time nicotine N-oxidation in human brain, mediated by FMO3 and FMO1, and show that nicotine-N-oxide modulates human α4ß2 nicotinic receptor activity in vitro. These results indicate possible mechanisms for associations between FMO3 genotype and smoking behaviors, and suggest nicotine N-oxidation as a novel target to enhance smoking cessation.

Mechanism of ritonavir changes in methadone pharmacokinetics and pharmacodynamics: II. Ritonavir effects on CYP3A and P-glycoprotein activities.

Ritonavir diminishes methadone plasma concentrations, an effect attributed to CYP3A induction, but the actual mechanisms are unknown. We determined short-term (2-day) and steady-state (2-week) ritonavir effects on intestinal and hepatic CYP3A4/5 (probed with intravenous (IV) and oral alfentanil (ALF) and with miosis) and P-glycoprotein (P-gp) (fexofenadine), and on methadone pharmacokinetics and pharmacodynamics in healthy volunteers. Acute ritonavir increased the area under the concentration-time curve (AUC)(0-infinity)/dose ratio (ritonavir/control) for oral ALF 25-fold. Steady-state ritonavir increased the AUC(0-Infinity)/dose ratio for IV and oral ALF 4- and 10-fold, respectively; reduced hepatic extraction (from 0.26 to 0.07) and intestinal extraction (from 0.51 to 0); and increased bioavailability (from 37 to 95%). Acute ritonavir inhibits first-pass CYP3A > 96%. Chronic ritonavir inhibits hepatic CYP3A (> 70%) and first-pass CYP3A (> 90%). Acute and steady-state ritonavir increased the fexofenadine AUC(0-infinity) 2.8- and 1.4-fold, respectively, suggesting P-gp inhibition. Steady-state compared with acute ritonavir caused mild apparent induction of P-gp and hepatic CYP3A, but net inhibition still predominated. Ritonavir inhibited both intestinal and hepatic CYP3A and drug transport. ALF miosis noninvasively determined CYP3A inhibition by ritonavir.

Mechanism of ritonavir changes in methadone pharmacokinetics and pharmacodynamics: I. Evidence against CYP3A mediation of methadone clearance.

Ritonavir diminishes methadone plasma concentrations, an effect attributed to CYP3A induction, but the actual mechanisms are unknown. We determined ritonavir effects on stereoselective methadone pharmacokinetics and clinical effects (pupillary miosis) in healthy human immunodeficiency virus-negative volunteers. Subjects received intravenous plus oral (deuterium-labeled) racemic methadone after no ritonavir, short-term (3-day) ritonavir, and steady-state ritonavir. Acute and steady-state ritonavir, respectively, caused 1.5- and 2-fold induction of systemic and apparent oral R- and S-methadone clearances. Ritonavir increased renal clearance 40-50%, and stereoselectively (S > R) increased hepatic methadone N-demethylation 50-80%, extraction twofold, and clearance twofold. Bioavailability was unchanged despite significant inhibition of intestinal P-glycoprotein. Intestinal and hepatic CYP3A was inhibited > 70%. Ritonavir shifted methadone plasma concentration-miosis curves leftward and upward. Rapid ritonavir induction of methadone clearance results from increased renal clearance and induced hepatic metabolism. Induction of methadone metabolism occurred despite profound CYP3A inhibition, suggesting no role for CYP3A in clinical methadone metabolism and clearance. Ritonavir may alter methadone pharmacodynamics.

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.

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.

A pilot evaluation of alfentanil-induced miosis as a noninvasive probe for hepatic cytochrome P450 3A4 (CYP3A4) activity in humans.

OBJECTIVE: The opioid alfentanil is a CYP3A4 substrate whose plasma clearance depends exclusively on hepatic CYP3A4 activity. Alfentanil clearance is an excellent in vivo probe for hepatic CYP3A4 activity and drug interactions in humans. However, such pharmacokinetic studies are invasive and time-consuming, and they require extensive analytical effort. This investigation tested the hypothesis that alfentanil-induced miosis (drug effect) can be used as a surrogate measure for alfentanil plasma concentrations and that alfentanil effect clearance will reflect plasma clearance; thus alfentanil can serve as a noninvasive probe for hepatic CYP3A4. METHODS: Six healthy volunteers were studied in a 3-way randomized crossover design. Each volunteer received 1 mg intravenous midazolam, followed 1 hour later by 15 microg/kg intravenous alfentanil, after CYP3A4 induction (rifampin [INN, rifampicin]), CYP3A4 inhibition (troleandomycin), and control. Dark-adapted pupil diameter and dynamic light response were measured coincident with venous blood sampling for up to 8 hours. Midazolam and alfentanil were quantified by gas chromatography-mass spectrometry. Plasma concentrations of alfentanil and midazolam (an additional CYP3A4 probe) and pupil diameter versus time data were analyzed by use of noncompartmental modeling. Pupil diameter change was analyzed analogously to determine the area under the alfentanil effect (miosis)-time curve (AUEC), effect clearance (CL(miosis)), and effect half-time. RESULTS: Compared with control, CYP3A4 induction and inhibition significantly altered the clearances of alfentanil (2.8 +/- 1.4, 5.3 +/- 1.0, and 0.42 +/- 0.1 ml/kg/min, respectively; P <.05 versus control) and midazolam. Dark-adapted resting diameter (in millimeters) was the best measure of alfentanil pupil effects. Alfentanil-dependent miosis was significantly altered by CYP3A4 modulation, and log(diameter(0) - diameter(t)) versus time curves resembled alfentanil plasma disposition. AUEC(infinity) values after control, CYP3A4 induction, and inhibition were 280 +/- 150, 120 +/- 22, and 1030 +/- 240 mm x min, respectively (P <.05 versus control). Effect clearances (CL(miosis)) were 4.2 +/- 1.3, 8.8 +/- 2.4, and 1.2 +/- 0.8 microg/mm x min, respectively, and effect half-times were 62 +/- 23, 34 +/- 27, and 211 +/- 35 minutes, respectively (P <.05 versus control). CL(miosis) was significantly correlated with plasma clearances of alfentanil (r = 0.77, P <.001) and midazolam (r = 0.80; P <.001). CONCLUSIONS: Alfentanil effect (miosis) may be a sensitive and reliable surrogate for plasma alfentanil concentrations. Alfentanil effect kinetics may be used as a noninvasive surrogate for conventional pharmacokinetics. CL(miosis) appears to be a suitable noninvasive in vivo probe for hepatic CYP3A4 activity, and it merits further investigation.

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 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.

Contribution of CYP2E1 and CYP3A to acetaminophen reactive metabolite formation.

BACKGROUND: CYP2E1, 1A2, and 3A4 have all been implicated in the formation of N-acetyl-p-benzoquinone imine (NAPQI), the reactive intermediate of acetaminophen (INN, paracetamol), in studies in human liver microsomes and complementary deoxyribonucleic acid-expressed enzymes. However, recent pharmacokinetic evidence in humans has shown that the involvement of CYP1A2 is negligible in vivo. The purpose of this study was to evaluate the respective roles of CYP2E1 and 3A4 in vivo. METHODS: The involvement of CYP2E1 was assessed through pretreatment of adult human volunteers with disulfiram to inhibit the enzyme and the role of CYP3A4 through its induction in a second cohort of adults with rifampin (INN, rifampicin). Each of the respective studies was an open-label, balanced-randomized crossover design. Blood samples were obtained serially for 12 hours and urine was collected for 24 hours after acetaminophen administration. Acetaminophen was assayed in plasma, and acetaminophen and metabolites were assayed in urine. RESULTS: The recovery of the thiol metabolites formed by conjugation of NAPQI with glutathione was decreased by 69%, and the formation clearance of NAPQI was decreased by 74% (both P < .01) by pretreatment with disulfiram. Rifampin pretreatment had no effect on the formation of NAPQI or the recovery of thiol metabolites formed by conjugation of NAPQI with glutathione. CONCLUSIONS: CYP2E1 accounts for the formation of NAPQI in intact humans; the contribution of other isozymes of cytochrome P450 appears to be negligible. Under some conditions, disulfiram may be useful in diminishing the formation of NAPQI after acetaminophen overdose.

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.

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.

Fluvoxamine-theophylline interaction: gap between in vitro and in vivo inhibition constants toward cytochrome P4501A2.

OBJECTIVE: Several reports indicate that fluvoxamine decreases the clearance of cytochrome P4501A2 (CYP1A2) substrates. This study compared in vitro and in vivo inhibition potencies of fluvoxamine toward CYP1A2 with an approach based on inhibition constants (K(i)) determined in vitro and in vivo. METHODS: In vitro inhibition constant values were determined with human liver microsomes and complementary deoxyribonucleic acid-expressed CYP1A2 (supersomes). Fluvoxamine in vivo inhibition constants (K(i)iv) for CYP1A2 were obtained from an investigation of single-dose theophylline (250 mg) disposition in 9 healthy volunteers receiving steady-state (9 days) fluvoxamine at 3 doses (0, 25, or 75 mg/d) in a randomized crossover design. RESULTS: In vitro K(i) values based on total inhibitor concentrations were 177 +/- 56 nmol/L, 121 +/- 21 nmol/L, and 52 +/- 13 nmol/L in human liver microsomes with 1 mg/ml protein and 0.5 mg/ml protein and in supersomes with 0.3 mg/ml protein, respectively. The corresponding in vitro K(i) values based on unbound fluvoxamine concentrations were 35 nmol/L, 36 nmol/L, and 36 nmol/L. The ratio of 1-methyluric acid formation clearances (control/inhibited) in 8 subjects was positively correlated with fluvoxamine concentration (r (2) = 0.87; P <.001) with an intercept near 1. Mean values for K(i)iv based on total and unbound plasma concentrations at steady state were 25.3 nmol/L (range, 14-39 nmol/L) and 3.6 nmol/L (range, 2.4-5.9 nmol/L), respectively. CONCLUSION: Comparison of in vitro and in vivo K(i) values based on unbound fluvoxamine concentrations suggests that fluvoxamine inhibition potency is approximately 10 times greater in vivo than in vitro.

Inhibition of sulfamethoxazole hydroxylamine formation by fluconazole in human liver microsomes and healthy volunteers.

Sulfamethoxazole toxicity is putatively initiated by the formation of a hydroxylamine metabolite by cytochromes P450. If this reaction could be inhibited, toxicity may decrease. We have studied--in vitro and in vivo--fluconazole, ketoconazole, and cimetidine as potentially suitable clinical inhibitors of sulfamethoxazole hydroxylamine formation. Both fluconazole and ketoconazole in human liver microsomal incubations competitively inhibited sulfamethoxazole N-hydroxylation, with the inhibitory constant (Ki) values of 3.5 and 6 micromol/L, respectively. Cimetidine exhibited a mixed type of inhibition of sulfamethoxazole hydroxylamine formation in human liver microsomes, with IC 50 values (the concentration required to decrease hydroxylamine formation by 50%) of 80 and 800 micromol/L, the lower value being observed when cimetidine was preincubated with microsomes and reduced nicotinamide adenine dinucleotide phosphate. In an in vivo study in six healthy volunteers the inhibition of the cytochrome P450-mediated generation of the toxic metabolite in the presence of fluconazole was shown by a 94% decrease in the area under the plasma concentration-time curve of sulfamethoxazole hydroxylamine. In contrast, the recovery of hydroxylamine in urine decreased by only 60%. Total clearance of sulfamethoxazole was decreased by 26% by fluconazole, most likely because of the inhibition of unidentified P450 elimination pathways. There was close agreement between the predicted (87%) and observed inhibition (94%) of sulfamethoxazole hydroxylamine formation in vivo. Similarly, there was close agreement between in vivo and in vitro Ki values--1.6 and 3.5 micron/L, respectively.

Lack of effect of cimetidine on acetaminophen disposition in humans.

The effect of cimetidine administration on the disposition of acetaminophen was evaluated in seven men and six women. One gram of acetaminophen was administered to each volunteer after an overnight fast on two occasions in a balanced crossover design with and without cimetidine, 300 mg every 6 hours beginning 50 hours before acetaminophen administration and continuing for 22 hours after. N-Acetylcysteine was administered on both occasions when acetaminophen was ingested to protect against glutathione depletion. Blood samples were collected serially for 12 hours after acetaminophen administration, and total urine volume was collected for 24 hours. Fractional clearances of acetaminophen through renal and metabolic routes (sulfation, glucuronidation, 3-hydroxylation, and glutathione conjugate formation) were not altered by cimetidine administration. Studies in microsomes prepared from two human organ donors indicated that cimetidine inhibited acetaminophen reactive metabolite formation noncompetitively, with Ki values of 0.35 mmol/L and 0.32 mmol/L for the respective livers, which is 5 to 10 times the putative cimetidine concentration required for therapeutic effect.

Does ketamine-mediated N-methyl-D-aspartate receptor antagonism cause schizophrenia-like oculomotor abnormalities?

Evidence from histological and pharmacological challenge studies indicates that N-methyl-D-aspartate (NMDA) receptor hypofunction may play an important role in the pathophysiology of schizophrenia. Our goal was to characterize effects of NMDA hypofunction further, as related to schizophrenia-associated neuropsychological impairment. We administered progressively higher doses of ketamine (target plasma concentrations of 50, 100, 150, and 200 ng/ml) to 10 psychiatrically healthy young men in a randomized, single-blind, placebo-controlled design and assessed oculomotor, cognitive, and symptomatic changes. Mean ketamine plasma concentrations approximated target plasma concentrations at each infusion step. Verbal recall, recognition memory, verbal fluency, pursuit tracking, visually guided saccades, and fixation all deteriorated significantly during ketamine infusion; lateral gaze nystagmus explained some, but not all, of the smooth pursuit abnormalities. We concluded that ketamine induces changes in recall and recognition memory and verbal fluency reminiscent of schizophreniform psychosis. During smooth pursuit eye tracking, ketamine induces nystagmus as well as abnormalities characteristic of schizophrenia. These findings help delineate the similarities and differences between schizophreniform and NMDA-blockade-induced cognitive and oculomotor abnormalities.

Menstrual cycle variability in midazolam pharmacokinetics.

Activity of cytochrome P450 3A4 (CYP3A4), the most abundant human P450 isoform and responsible for metabolizing approximately half of all therapeutic agents, has been speculated to vary during the menstrual cycle. This investigation evaluated CYP3A4 activity during the menstrual cycle, using midazolam clearance as a metabolic probe. Midazolam (1 mg i.v.) was administered to nonsmoking, nonpregnant female volunteers (N = 11, age 26 +/- 5 years) with normal menstrual cycles on three separate occasions during the same cycle: days 2 (menstrual phase), 13 (estradiol peak), and 21 (progesterone peak). Venous plasma midazolam concentrations were determined by gas chromatography-mass spectrometry. Midazolam clearance was determined by noncompartmental and compartmental analysis. Midazolam plasma disposition did not differ between phases of the menstrual cycle. There was no significant difference in any measure of midazolam clearance. Noncompartmental clearances (mean +/- SD) were 7.36 +/- 2.73, 6.34 +/- 3.59, and 6.23 +/- 2.04 ml/kg/min, respectively, on days 2, 13, and 21 of the menstrual cycle. These results suggest no difference in hepatic CYP3A4 activity on menstrual cycle days 2, 13, and 21. Consideration of menstrual cycle variability in the metabolism of CYP3A4 substrates does not appear indicated in the dosing or design of clinical trials.

Intraindividual variability in male hepatic CYP3A4 activity assessed by alfentanil and midazolam clearance.

Clinical investigations using isoform-selective probes to phenotype cytochrome P450 activity and interaction studies using isoform-selective inhibitors to determine P450 involvement in drug metabolism assume minimal interday variability in P450 activity. CYP3A4 is the most abundant human P450 isoform and metabolizes approximately half of all therapeutic agents. This investigation evaluated interday variability in hepatic CYP3A4 activity in males, using the clearances of midazolam and alfentanil as metabolic probes. Midazolam (1 mg) followed 1 hour later by alfentanil (20 micrograms/kg) were administered by intravenous bolus to 9 nonsmoking male volunteers (ages 30 +/- 8 years). Drug administration was repeated 12 and 20 days later. Venous plasma midazolam and alfentanil concentrations were determined by gas chromatography/mass spectrometery. Drug clearances were determined by noncompartmental and multiexponential analysis. There were no significant interday differences in plasma drug concentrations or clearances (3.9 +/- 1.4, 3.9 +/- 1.7, and 4.2 +/- 1.7 ml/kg/min for alfentanil, respectively, and 6.6 +/- 2.0, 7.9 +/- 2.4, and 7.9 +/- 2.5 ml/kg/min for midazolam, respectively, on days 1, 13, and 21 [mean +/- SD]). Interday variability in clearance was 13% +/- 6% and 19% +/- 12% for alfentanil and midazolam, respectively. Interday variability in the clearance of these probes, and presumably hepatic CYP3A4 activity, was small compared with interindividual variability. Consideration of interday variability in the hepatic metabolism of CYP3A4 substrates does not appear significant in the design of clinical trials.

Acute tolerance and reversal of the motor control effects of midazolam.

The purpose of this study was to determine whether acute tolerance develops to the motor control effects of the short-acting benzodiazepine, midazolam. Using a bolus and constant infusion scheme, 40 healthy adults received a 70-min intravenous infusion of either saline (n = 20) or 6.1 (SE = 0.2) mg midazolam (n = 20). Following the 70-min infusion period, half of the subjects in each group (n = 10) received a 25-min intravenous infusion of flumazenil (benzodiazepine antagonist); the remainder of the subjects (n = 10/group) received a 25-min infusion of saline. Drug administration during both infusion periods was double blind. Prior to the infusions, subjects were trained in a motor control assessment battery. Throughout both infusions, repeated motor control testing and blood sampling were performed. The initial (10 min) midazolam plasma concentration was 52.0 (SE = 2.2) ng/ml. Plasma midazolam concentration rose gradually to 60.7 (SE = 2.1) ng/ml at the end of the infusion (70 min). Midazolam initially impaired performance on the motor control tasks. However, performance improved in subjects receiving midazolam despite the gradual increase in midazolam concentrations. This suggests that the recovery of motor task performance may be attributable to the development of acute tolerance rather than to waning drug concentrations. Flumazenil immediately reversed midazolam's effects on the visual tracking task. However, there was little evidence for precipitation of muscle force rebound, which has been hypothesized to result from the same underlying mechanism that is responsible for acute tolerance development.

Endotracheal lidocaine administration via an esophageal combitube.

The purpose of this study was to test the hypothesis that lidocaine is systemically absorbed after administration via a Combitube placed in the esophagus, and that therapeutically significant plasma lidocaine concentrations can be attained using this route with standard endotracheal doses (2.0 mg/kg). During general anesthesia, 27 elective surgical patients received 2.0 mg/kg lidocaine (diluted as necessary with 0.9% saline to a minimum total volume of 10 mL) via a Combitube (study group, n = 13) or an endotracheal tube (control group, n = 14). Venous blood samples were drawn for 3 h after lidocaine administration and plasma concentrations determined by gas chromatography using a nitrogen-phosphorus detector (NPD). Overall, average lidocaine concentrations were maximal after 5 min, reaching 0.8+/-0.7 and 1.7+/-0.7 microg/mL in the Combitube and endotracheal tube groups, respectively. Individual patient peak concentrations averaged 1.0+/-0.7 and 2.2+/-1.1 microg/mL in the same two groups, 19+/-16 and 10+/-15 min after lidocaine administration, respectively. No patients reported chest discomfort or dyspnea upon awakening, and no other side effects were noted. In support of the hypothesis, administration of lidocaine via an esophageal Combitube results in systemic drug uptake; however, at conventional endotracheal doses, plasma concentrations are subtherapeutic. It remains to be determined whether higher doses of lidocaine administered via an esophageal Combitube will result in therapeutic plasma concentrations.