Nefopam enantiomers: preclinical pharmacology/toxicology and pharmacokinetic characteristics in healthy subjects after intravenous administration.

Nefopam (NEF) is a potent analgesic compound administered as a racemic mixture. Previous in vitro and in vivo studies with nefopam enantiomers have shown that (+)nefopam [(+)NEF] is substantially more potent than (-)nefopam [(-)NEF]. Differences between enantiomers have also been suggested in metabolic studies in vitro. The impact of these differences in vivo is not known because there is little or no information on the relative plasma concentrations of the enantiomers or on their kinetics. In this study, individual enantiomers of nefopam were synthesized and examined for acute toxicity in male and female rats and mice. Pharmacologic properties of enantiomers were examined using in vitro binding assays and antinociceptive tests in rats and mice. Additionally, a pharmacokinetic study was conducted in human volunteers. Subjects were administered 20 mg nefopam as Acupan(R) either as a 5- or 20-min intravenous infusion. In a control phase, subjects were administered only vehicle. Blood samples were collected through the following 24 h. Plasma samples were analyzed for individual enantiomers using a chiral assay developed for this purpose. The pharmacologic differences of previous studies were confirmed in receptor binding assays and in the hot plate and the formalin tests in mice. Neither enantiomer demonstrated substantial activity in the tail flick test in rats. No significant differences were revealed between LD(50) values of nefopam enantiomers after oral or intravenous administration in male and female rats or mice. There were no significant differences in AUC(0-infinity), C(max), or half-life between enantiomers following intravenous administration. Based on these findings, there is currently no compelling rationale to justify administering or monitoring individual enantiomers.

Influence of stiripentol on cytochrome P450-mediated metabolic pathways in humans: in vitro and in vivo comparison and calculation of in vivo inhibition constants.

OBJECTIVE: The spectrum of cytochrome P450 inhibition of stiripentol, a new anticonvulsant, was characterized in vitro and in vivo. METHODS: Stiripentol was incubated in vitro with (R)-warfarin, coumarin, (S)-warfarin, (S)-mephenytoin, bufuralol, p-nitrophenol, and carbamazepine as probes for CYPs 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4, respectively. Caffeine demethylation and the 6 beta-hydroxycortisol/cortisol ratio were monitored in vivo before and after 14 days of treatment with stiripentol as measures of CYP1A2 and CYP3A4 activity, and dextromethorphan O- and N-demethylation were used to measure CYP2D6 and CYP3A4 activity, respectively. In vivo inhibition constants for CYP3A4 were calculated with use of data that previously documented the interaction between stripentol and carbamazepine. RESULTS: In vitro, stiripentol inhibited CYPs 1A2, 2C9, 2C19, 2D6, and 3A4, with inhibition constant values at or slightly higher than therapeutic (total) concentrations of stiripentol, but it did not inhibit CYPs 2A6 and 2E1 even at tenfold therapeutic concentrations. In vivo inhibition of caffeine demethylation and dextromethorphan N-demethylation were consistent with inhibition of CYP1A2 and CYP3A4, respectively. The 6 beta-hydroxycortisol/cortisol ratio did not provide a reliable index of CYP3A4 inhibition. Inhibition of CYP2D6-mediated O-demethylation was not observed in vivo. With use of carbamazepine, in vivo inhibition constants for CYP3A4 ranged between 12 and 35 mumol/L, whereas the corresponding in vitro value was 80 mumol/L. CONCLUSIONS: Stiripentol appears to inhibit several CYP450 enzymes in vitro and in vivo. In vivo inhibition constants show that stiripentol inhibition of CYP3A4 is linearly related to plasma concentration in patients with epilepsy.

Fentanyl metabolism by human hepatic and intestinal cytochrome P450 3A4: implications for interindividual variability in disposition, efficacy, and drug interactions.

The synthetic opioid fentanyl undergoes extensive metabolism in humans. Systemic elimination occurs primarily by hepatic metabolism. When administered as a lozenge for oral transmucosal absorption, swallowed fentanyl is subject to first pass metabolism in the liver and possibly small intestine. Little is known, however, about the identity and formation of human fentanyl metabolites. This investigation identified routes of human liver microsomal fentanyl metabolism and their relative importance, tested the hypothesis that fentanyl is metabolized by human duodenal microsomes, and identified the predominantly responsible cytochrome P450 isoforms. A GC/MS assay using deuterated internal standards was developed for fentanyl metabolites. Piperidine N-dealkylation to norfentanyl was the predominant pathway of liver microsomal metabolism. Amide hydrolysis to despropionylfentanyl and alkyl hydroxylation to hydroxyfentanyl were comparatively minor pathways. Hydroxynorfentanyl was identified as a minor, secondary metabolite arising from N-dealkylation of hydroxyfentanyl. Liver microsomal norfentanyl formation was significantly inhibited by the mechanism-based P450 3A4 inhibitor troleandomycin and the P450 3A4 substrate and competitive inhibitor midazolam, and was significantly correlated with P450 3A4 protein content and catalytic activity. Of six expressed human P450 isoforms (P450s 1A2, 2B6, 2C9, 2D6, 2E1, and 3A4), only P450 3A4 exhibited significant fentanyl dealkylation to norfentanyl. These results indicate the predominant role of P450 3A4 in the primary route of hepatic fentanyl metabolism. Human duodenal microsomes also catalyzed fentanyl metabolism to norfentanyl; the average rate was approximately half that of hepatic metabolism. Rates of duodenal norfentanyl formation were diminished by troleandomycin and midazolam, and were significantly correlated with P450 3A4 activity, suggesting a prominent role for P450 3A4. These results demonstrate that human intestinal as well as liver microsomes catalyze fentanyl metabolism, and N-dealkylation by P450 3A4 is the predominant route in both organs. The fraction of fentanyl lozenge that is swallowed likely undergoes significant intestinal, as well as hepatic, first-pass metabolism. Intestinal and hepatic first-pass metabolism, as well as systemic metabolism, may be subject to individual variability in P450 3A4 expression and to drug interactions involving P450 3A4.

Catalytic role of cytochrome P4503A4 in multiple pathways of alfentanil metabolism.

The synthetic opioid alfentanil (ALF) undergoes extensive metabolism via two major pathways: piperidine nitrogen dealkylation to noralfentanil (NA) and amide nitrogen dealkylation to N-phenylpropionamide (AMX). It is unknown whether AMX results from amide N-dealkylation of ALF directly, or indirectly from NA, the major metabolite of ALF. The major objectives of this investigation were to determine the metabolic origin of AMX and to identify the cytochrome P450 isoforms in human liver microsomes catalyzing ALF metabolism. Metabolites were quantitated by GC/MS. Significant amide N-dealkylation of ALF but not of NA by human liver microsomes was observed, indicating that AMX is derived directly from ALF and that there are two primary routes of ALF metabolism. Three strategies were used to identify the P450 isoform(s) catalyzing each of the two metabolic pathways: effect of isoform-selective inhibitors on metabolite formation catalyzed by human liver microsomes, correlation of metabolite formation rate with microsomal P450 isoform protein content and catalytic activity in a population of human livers, and metabolism by cDNA-expressed P450 isoforms. The mechanism-based P4503A4 inhibitor, troleandomycin, significantly inhibited formation of both NA and AMX. Other P4503A4 inhibitors, including midazolam, erythromycin, and ketoconazole, also diminished ALF metabolism to both metabolites. Formation rates of both NA and AMX were significantly correlated with microsomal P4503A4 protein content and catalytic activity. Of six expressed human P450 isoforms (P450s 1A2, 2A6, 2B6, 2D6, 2E1, and 3A4), only P4503A4 exhibited significant catalytic activity toward ALF dealkylation to NA and AMX. These results indicate the predominant role of P4503A4 in both major pathways of ALF metabolism.

Gas chromatographic-mass spectrometric analysis of alfentanil metabolites. Application to human liver microsomal alfentanil biotransformation.

The short-acting synthetic opioid alfentanil undergoes extensive biotransformation to several metabolites. A gas chromatographic-mass spectrometric assay, using selected-ion monitoring and deuterated internal standards, was developed for quantitating the predominant metabolites of alfentanil. Optimal extraction and derivatization conditions are described. The assay was applied to the analysis of metabolites formed during alfentanil metabolism in vitro by human liver microsomes. Formation of known alfentanil metabolites was confirmed, and formation of a metabolite, not previously detected in vitro, is described. The assay represents a significant improvement over existing methods of alfentanil metabolite analysis, which use HPLC and radiochemical detection.

Determination of alfentanil and noralfentanil in human plasma by gas chromatography-mass spectrometry.

This report describes a simple gas chromatographic-mass spectrometric (GC-MS) assay for the simultaneous analysis of alfentanil and its major metabolite, noralfentanil, in human plasma. The method facilitates the processing of numerous samples for pharmacokinetic analysis. Alfentanil and noralfentanil are extracted from plasma under basic conditions and noralfentanil is converted to the pentafluoropropionyl derivative. The extraction efficiencies for noralfentanil and alfentanil were > 99% and 70%, respectively. Standard curves were linear (r2 = 0.99) over the ranges of 5-500 ng/ml for alfentanil and 0.4-10 ng/ml for noralfentanil. Inter-day coefficients significant improvement over existing HPLC assays which require radiolabelled significant improvement over existing HPLC assays which require radiolabelled alfentanil. The simultaneous disposition of alfentanil and noralfentanil in plasma after intravenous administration in humans is described.

Ketamine as a probe for medetomidine stereoisomer inhibition of human liver microsomal drug metabolism.

Medetomidine (MED) is a novel, selective, alpha 2 adrenergic agonist with potent sedative, hypnotic, and analgesic properties, currently undergoing evaluation as an anesthetic adjuvant. The pharmacologic effects of MED are stereospecific, due entirely to the D-isomer (DMED), whereas the L-isomer (LMED) is essentially inactive. DMED, a 4(5)substituted imidazole, has been shown to inhibit adrenal steroidogenesis and human liver microsomal alfentanil metabolism, reactions mediated by cytochrome P-450. The mechanism of MED inhibition of cytochrome P-450 is unknown. The purpose of this investigation was to determine the mechanism of DMED inhibition of human cytochrome P-450-mediated microsomal metabolism, using ketamine as a probe. Ketamine undergoes extensive hepatic biotransformation and has been used previously to characterize the effects of imidazole anesthetics on human P-450-catalyzed drug metabolism. Ketamine N-demethylation by microsomes from three human livers was measured by gas chromatography-mass spectrometry with selected-ion monitoring. DMED was a potent, competitive inhibitor of S(+) ketamine N-demethylation, with a Ki of 0.11-0.18 microM for the high affinity ketamine demethylase. The IC50 for DMED inhibition of therapeutic concentrations of racemic ketamine (10 microM) was 0.15 +/- 0.02 microM. Preincubation of DMED with microsomes and an NADPH generating system prior to ketamine addition had no additional effect on the inhibition of ketamine demethylase activity, thereby implicating the parent compound rather than a DMED metabolite as the inhibitory species. LMED, although pharmacologically inactive, had a greater inhibitory effect than DMED on racemic ketamine and ketamine enantiomer demethylation at therapeutic concentrations. Spectral studies showed that DMED interacted with microsomal cytochrome P-450 to elicit a Type II binding spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of ketamine stereoisomers by human liver microsomes.

Ketamine is used clinically as a racemic mixture of optical isomers that differ in their analgesic properties and psychomimetic effects. Administered individually, or together as the racemate, ketamine enantiomers differ in their hepatic clearance and duration of anesthetic effect. S(+) ketamine exhibits a greater clearance and faster anesthetic recovery compared to the racemate and a greater clearance compared to R(-) ketamine. Ketamine undergoes extensive hepatic metabolism, primarily via N-demethylation to norketamine, yet little is known about the human metabolism of ketamine enantiomers. The purpose of this investigation therefore was to characterize ketamine racemate and enantiomer metabolism by human liver and to test the hypothesis that differences in hepatic ketamine enantiomer metabolism can account for observed differences in ketamine enantiomer pharmacokinetics. Ketamine N-demethylation by microsomes from three human livers was measured by gas chromatography-mass spectrometry. At ketamine concentrations typically achieved during anesthesia (5 microM), the rate of S(+) ketamine demethylation was 20% greater than that of R(-) ketamine and 10% greater than that of the racemate (P < .05). At all ketamine concentrations, the rate of racemate demethylation was less than the sum of the rates for the individual enantiomers, reflecting a metabolic enantiomeric interactin whereby one ketamine enantiomer inhibits the metabolism of the other enantiomer. N-demethylation of racemic ketamine and each enantiomer was catalyzed by two apparent enzymes, a high affinity-low capacity enzyme (Km1 30-50 microM, Vmax1 2-6 nmoles.min-1 x nmole-1) and a low affinity-high capacity enzyme (Km2 600-800 microM, Vmax2 9-15 nmoles.min-1 x nmole-1).(ABSTRACT TRUNCATED AT 250 WORDS)