Capillary electrophoresis to assess drug metabolism induced <b><i>in vitro</i></b> using single CYP450 enzymes (Supersomes™): Application to the chiral metabolism of mephenytoin and methadone

Prost, Francine; Thormann, Wolfgang

doi:10.1002/elps.200305493

Cited by 31 publications

(42 citation statements)

References 43 publications

(74 reference statements)

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“…Microsomal incubations were performed as described in detail elsewhere [26] and using acetonitrile as solvent to prepare the MQ stock solutions which should, if applied in small quantities, not significantly hamper the activity of most cDNA-expressed human CYPs [27]. Briefly, aliquots of the stock solutions were directly added to the incubation mixture thereby having acetonitrile concentrations 2.5% v/v.…”

Section: Microsomal Incubations and Extractionmentioning

confidence: 99%

Assessment of the stereoselective metabolism of methaqualone in man by capillary electrophoresis

Prost

Thormann

2003

Electrophoresis

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Methaqualone (MQ) and its hydroxylated metabolites are quinazoline derivatives that exhibit atropisomerism. As a continuation of our previous work with these compounds (Electrophoresis 2001, 22, 3270-3280), chiral capillary zone electrophoresis with hydroxypropyl-beta-cyclodextrin as buffer additive and multiwavelength absorbance detection is shown to be an effective tool to provide insight into the stereoselectivity of the MQ metabolism. The five major monohydroxy MQ metabolites formed during biotransformation do not show enantiomerization at temperatures up to 85 degrees C. Enzymatic and acidic hydrolysis of urines that were collected after concomitant administration of 250 mg of MQ and 25 mg diphenhydramine (DH) chloride are both shown to provide stereoselective metabolic patterns with 4'-hydroxymethaqualone, the major urinary metabolite, being excreted almost exclusively as a single enantiomer. A stereoselectivity in the formation of 2'-hydroxymethaqualone and 2-hydroxymethaqualone was also observed in vitro using human liver microsomes and preparations containing the cytochrome P450 enzyme (CYP) CYP3A4 only. The presence of DH during incubation with human liver microsomes did not reveal a difference in the metabolic pattern obtained. Furthermore, CYP2D6 and CYP2C19 do not significantly contribute to the metabolism of MQ. This was independently observed in vitro and via analysis of urines of individuals that are either efficient metabolizer phenotypes or poor metabolizer phenotypes for the two polymorphic enzymes. Although interindividual differences in the monitored metabolic patterns were noted, no marked difference could be related to a CYP2D6 or CYP2C19 polymorphism.

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Section: Microsomal Incubations and Extractionmentioning

confidence: 99%

Assessment of the stereoselective metabolism of methaqualone in man by capillary electrophoresis

Prost

Thormann

2003

Electrophoresis

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“…Identification of the diastereoisomers of 6OCOL and N6OCOL, however, was not possible. As there is very little information known about these keto-reduced metabolites [8,10,13,14,16], as there is no published instrumental method (e.g., HPLC or LC-MS) for their analysis, and as CE has been identified as an excellent technology to assess the stereoselectivity of drug metabolism [26][27][28], CE conditions for the separation of 6OCOL and N6OCOL diastereoisomers and their analysis in biological specimens were elucidated in our laboratory. In the present paper, CE assay conditions with UV detection that are suitable to identify the diastereoisomers of 6OCOL and N6OCOL in biological samples, including urine and in vitro incubations, are discussed.…”

Section: Introductionmentioning

confidence: 99%

Analysis of oxycodol and noroxycodol stereoisomers in biological samples by capillary electrophoresis

Baldacci¹,

Thormann²

2005

Electrophoresis

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A capillary electrophoresis (CE) method for the separation of the diastereoisomers of 6-oxycodol (6OCOL) and nor-6-oxycodol (N6OCOL), the 6-keto-reduced metabolites of oxycodone (OCOD) and noroxycodone (NOCOD), respectively, is reported and employed to assess the stereoselectivity of these metabolic steps in vivo, in vitro, and in chemical synthesis. CE in an untreated fused-silica capillary with acidic buffers containing 2-hydroxypropyl-beta-cyclodextrin, randomly sulfated beta-cyclodextrin, or single isomer heptakis(2,3-diacetyl-6-sulfato)-beta-cyclodextrin (HDAS-beta-CD) is shown to permit the simultaneous separation of the stereoisomers of 6OCOL and N6OCOL. A 100 mM phosphate buffer of pH 2.0 containing 2.05% w/v HDAS-beta-CD provides a medium for rapid analysis and unambiguous identification of these stereoisomers in solid-phase extracts of (i) urines stemming from patients under pharmacotherapy with OCOD, (ii) incubations of OCOD and NOCOD with human liver cytosol and the human liver S9 fraction, and (iii) after chemical synthesis from OCOD and NOCOD using NaBH(4). In all cases, alpha-N6OCOL is shown to be the predominant stereoisomer of N6OCOL. For 6OCOL, the same is true for in vitro formation and for chemical synthesis. In urine, however, beta-6OCOL is observed to be excreted in a higher amount than alpha-6OCOL. For the urinary alpha-/beta-isomer ratio of 6OCOL and N6OCOL, there are no differences between the data obtained for nonhydrolyzed and enzymatically hydrolyzed urines. The data document the stereoselectivity of the 6-keto-reduction of OCOD and NOCOD in man.

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“…In addition, in vitro and in vivo evidence suggests that methadone metabolism is stereoselective (13-16, 22, 24, 31, 35). CYP2B6 has been shown to preferentially metabolize the inactive L isomer, CYP2C19 has been shown to preferentially metabolize the active R isomer, and CYP3A4 has been shown to preferentially metabolize both isomers (16,31,35). Stereoselective metabolism may partly account for the fact that amprenavir reduces total methadone concentrations without provoking signs and symptoms of opioid withdrawal.…”

Section: Discussionmentioning

confidence: 99%

Nevirapine Significantly Reduces the Levels of Racemic Methadone and ( R )-Methadone in Human Immunodeficiency Virus-Infected Patients

Stocker

Kruse

Kreckel

et al. 2004

Antimicrob Agents Chemother

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Methadone is metabolized by various isoforms of the cytochrome P450 family, which can be induced by many drugs, including nevirapine. The objective of the present study was to determine the effects of coadministration of nevirapine and methadone on the dose-adjusted areas under the concentration-time curves (AUCs) of racemic and (R)-methadone. Twenty-five human immunodeficiency virus-infected subjects taking stable single daily doses of racemic methadone or (R)-methadone were included in this prospective, single-crossover trial. At the baseline, nevirapine was either started as part of a new regimen containing two nucleoside reverse transcriptase inhibitors (NRTIs) or added to an ongoing NRTI regimen. Patients could increase their methadone doses if withdrawal symptoms developed. Twelve-hour pharmacokinetic profiles were obtained before and 28 days after the start of nevirapine treatment. The total concentrations of methadone and its inactive metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in serum were determined by liquid chromatography-tandem mass spectrometry. Among the 20 evaluable patients, coadministration of nevirapine significantly decreased the mean dose-adjusted AUC of methadone by 41%. AUC reductions were similar for patients taking racemic methadone (37%; n ‫؍‬ 11) and (R)-methadone (44%; n ‫؍‬ 9). AUC changes ranged from mild increases in three patients to decreases of up to 70%. Fourteen of 20 patients required additional methadone due to withdrawal symptoms. However, the median dose increase was only 15%, which was less than that which would have been expected from the pharmacokinetic data. The AUC of EDDP increased significantly, by 35%. Methadone dose adjustments are justified when methadone is coadministered with nevirapine. Due to extensive variability, the adjustments must be tailored to the individual patient's needs.Human immunodeficiency virus (HIV)-infected patients who take methadone for the management of intravenous drug use frequently complain about symptoms of narcotic withdrawal after having started antiretroviral treatment containing nevirapine (NVP) (1,2,18,28,29). Symptoms rarely appear within the first few days, which suggests that the effect is caused by the induction of methadone metabolism by NVP (3, 11). One controlled trial involving eight patients has shown that concomitant NVP treatment substantially decreases the trough level and the area under the concentration-time curve (AUC) of methadone (11).Methadone is a racemic mixture of the pharmacodynamically inactive (S)-methadone and the active enantiomer (R)-methadone. It contains equal parts of both enantiomers. In some countries, methadone is frequently used in a formulation which contains only the active enantiomer, (R)-methadone. Both enantiomers are metabolized by members of the cytochrome P450 family. N-demethylation results in the formation of the inactive metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) (4, 30).NVP, a nonnucleoside inhibitor of the HIV reverse transcriptase,...

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Capillary electrophoresis to assess drug metabolism induced in vitro using single CYP450 enzymes (Supersomes™): Application to the chiral metabolism of mephenytoin and methadone

Cited by 31 publications

References 43 publications

Assessment of the stereoselective metabolism of methaqualone in man by capillary electrophoresis

Assessment of the stereoselective metabolism of methaqualone in man by capillary electrophoresis

Analysis of oxycodol and noroxycodol stereoisomers in biological samples by capillary electrophoresis

Nevirapine Significantly Reduces the Levels of Racemic Methadone and ( R )-Methadone in Human Immunodeficiency Virus-Infected Patients

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