Chlorzoxazone is metabolized by human CYP1A2 as well as by human CYP2E1

Ono, Satoshi; Hatanaka, T; Hotta, Hiroshi; Tsutsui, Michio; Satoh, Tetsuo; Gonzalez, Frank J.

doi:10.1097/00008571-199506000-00002

Cited by 102 publications

(48 citation statements)

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“…Studies with CYP2A6 inhibitory monoclonal antibody show that at substrate concentrations Յ10 M, more than 90% of the 7-hydroxylation of coumarin in HLMs is carried out by CYP2A6, demonstrating the unequivocal role of CYP2A6 in coumarin 7-hydroxylation (Li et al, 1997;Sai et al, 1999;Yang et al, 1999). Among nine cDNA-expressed enzymes, only CYP2A6 catalyzes this reaction (Ono et al, 1996). Consistently, kinetic studies in HLMs show monophasic formation of 7-hydroxy-coumarin at commonly used substrate concentrations of 0.1 to 10 M, with a K m value of 0.5 to 2 M (Shimada et al, 1996;Draper et al, 1997).…”

Section: Cyp1a2supporting

confidence: 55%

“…At 10 M substrate concentrations, the CYP2D6-mediated reaction proceeds at a rate 5-fold greater than that by CYP2C9 (Ono et al, 1996). But at 500 M, the CYP2D6 reaction rate is only one-sixth of the CYP2C9-based reaction rate.…”

Section: Evaluation Of In Vitro P450 Probe Reactionsmentioning

confidence: 95%

“…Tolbutamide 4Ј-hydroxylation is the initial and rate-limiting step of tolbutamide elimination. Studies with cDNA expressed enzymes show that at substrate concentrations Յ500 M, 90% of tolbutamide is hydroxylated by CYP2C9 and 10% by CYP2C8 (Minors et al, 1988;Relling et al, 1990;Ono et al, 1996). An immunoblotting assay with antibody developed against CYP2C9 provides further evidence to support the predominant role of CYP2C9 in tolbutamide 4Ј-hydroxylation (Edwards et al, 1998).…”

Section: Cyp1a2mentioning

confidence: 99%

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Evaluation of Cytochrome P450 Probe Substrates Commonly Used by the Pharmaceutical Industry to Study in Vitro Drug Interactions

Yuan

Madani²,

Wei³

et al. 2002

Drug Metab Dispos

295

191

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ABSTRACT:Pharmaceutical industry investigators routinely evaluate the potential for a new drug to modify cytochrome P450 (P450) activities by determining the effect of the drug on in vitro probe reactions that represent activity of specific P450 enzymes. The in vitro findings obtained with one probe substrate are usually extrapolated to the compound's potential to affect all substrates of the same enzyme. Due to this practice, it is important to use the right probe substrate and to conduct the experiment under optimal conditions. Surveys conducted by reviewers in CDER indicated that the most common in vitro probe reactions used by industry investigators include the following: phenacetin O-deethylation for CYP1A2, coumarin 7-hydroxylation for CYP2A6, 7-ethoxy-4-trifluoromethyl coumarin O-dealkylation for CYP2B6, tolbutamide 4-hydroxylation for CYP2C9, S-mephenytoin 4-hydroxylation for CYP2C19, bufuralol 1-hydroxylation for CYP2D6, chlorzoxazone 6-hydroxylation for CYP2E1, and testosterone 6␤-hydroxylation for CYP3A4. We reviewed the validation information in the literature on these reactions and other frequently used reactions, including caffeine N3-demethylation for CYP1A2, S-mephenytoin N-demethylation for CYP2B6, S-warfarin 7-hydroxylation for CYP2C9, dextromethorphan O-demethylation for CYP2D6, and midazolam 1-hydroxylation for CYP3A4. The available information indicates that we need to continue the search for better probe substrates for some enzymes. For CYP3A4-based drug interactions it may be necessary to evaluate two or more probe substrates. In many cases, the probe reaction represents a particular enzyme activity only under specific experimental conditions. Investigators must consider appropriateness of probe substrates and experimental conditions when conducting in vitro drug interaction studies and when extrapolating the results to in vivo situations.During the drug-candidate screening and development process, investigators often conduct two types of in vitro drug metabolism studies to assess the potential for P450 1 -based drug interactions. One type of study characterizes the metabolic pathway of the new drug and the potential for other drugs to modify the metabolism of the new drug. The other type of study evaluates the potential for the new drug to alter the metabolism of other drugs. Due to the availability of antibodies against specific P450 enzymes, cDNA-expressed enzymes, purified enzymes, and selective chemical inhibitors, the unequivocal identification of the major P450 isoform responsible for the metabolism of a new drug can be easily established. However, predicting the potential for the new drug to alter the metabolism of other drugs usually relies on the evaluation of the effect of the new drug on the rate of a probe reaction that represents a specific P450 enzyme activity. The second type of evaluation is more challenging and is the focus of this review.

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Section: Cyp1a2supporting

confidence: 55%

Section: Evaluation Of In Vitro P450 Probe Reactionsmentioning

confidence: 95%

Section: Cyp1a2mentioning

confidence: 99%

See 1 more Smart Citation

Evaluation of Cytochrome P450 Probe Substrates Commonly Used by the Pharmaceutical Industry to Study in Vitro Drug Interactions

Yuan

Madani²,

Wei³

et al. 2002

Drug Metab Dispos

295

191

View full text Add to dashboard Cite

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“…3), and IC 50 values of 8.94 and 17.1 M were measured for tranylcypromine (Table 8). The range of K m values in human liver microsomes reported in the literature is 39 to 157 M (Peter et al, 1990;Ono et al, 1995;Schmider et al, 1996;Court et al, 1997;Hickman et al, 1998;Yin et al, 2000;Lejus et al, 2002) (Fig. 4).…”

Section: Validated Assays For Human Cytochrome P450 Enzymesmentioning

confidence: 85%

Validated Assays for Human Cytochrome P450 Activities

Walsky

Obach²

2004

Drug Metab Dispos

465

380

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ABSTRACT:The measurement of the effect of new chemical entities on human cytochrome P450 marker activities using in vitro experimentation represents an important experimental approach in drug development. In vitro drug interaction data can be used in guiding the design of clinical drug interaction studies, or, when no effect is observed in vitro, the data can be used in place of an in vivo study to claim that no interaction will occur in vivo. To make such a claim, it must be assured that the in vitro experiments are performed with absolute confidence in the methods used and data obtained. To meet this need, 12 semiautomated assays for human P450 marker substrate activities have been developed and validated using approaches described in the GLP (good laboratory practices) as per the code of U.S. Federal Regulations. The assays that were validated are: phenacetin O-deethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), bupropion hydroxylase (CYP2B6), amodiaquine N-deethylase (CYP2C8), diclofenac 4-hydroxylase and tolbutamide methylhydroxylase (CYP2C9), (S)-mephenytoin 4-hydroxylase (CYP2C19), dextromethorphan O-demethylase (CYP2D6), chlorzoxazone 6-hydroxylase (CYP2E1), felodipine dehydrogenase, testosterone 6␤-hydroxylase, and midazolam 1-hydroxylase (CYP3A4 and CYP3A5). High-pressure liquid chromatography-tandem mass spectrometry, using stable isotope-labeled internal standards, has been applied as the analytical method. This analytical approach, through its high sensitivity and selectivity, has permitted the use of very low incubation concentrations of microsomal protein (0.01-0.2 mg/ml). Analytical assay accuracy and precision values were excellent. Enzyme kinetic and inhibition parameters obtained using these methods demonstrated high precision and were within the range of values previously reported in the scientific literature. These methods should prove useful in the routine assessments of the potential for new drug candidates to elicit pharmacokinetic drug interactions via inhibition of cytochrome P450 activities.Drug-drug interactions are of great interest to scientists involved in drug research, regulatory authorities who are responsible for public safety, physicians, and their patients. Since "polypharmacy," or the practice of simultaneous prescription of more than one drug to treat one or more conditions in a single patient, has become a more common practice, drug interactions have been cited as one of the major reasons for hospitalization and even death (Lazarou et al., 1998). Thus, a great deal of effort is expended by researchers engaged in new drug research in avoiding the development of compounds that will cause drug-drug interactions.The most common mechanism underlying drug-drug interactions is the inhibition of cytochrome P450 activities. Several drugs in common use cause large increases in exposure to other drugs. Examples include ketoconazole, itraconazole, erythromycin, clarithromycin, diltiazem, and nefazodone (CYP3A); quinidine, paroxetine, and terbinafine (CYP2D6); ticlopidine (CYP2C19); ...

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“…2) Whether changes in the CYP2E1 liver concentration parallel the changes in CZ metabolism in fasted rats. Although some studies demonstrated that the metabolism of CZ was determined by CYP2E1 level (Peter et al 1990;Lucas et al 1999), others suggested that other P450s such as CYP3A4 and CYP1A can play a role in chlorzoxazone 6-hydroxylation in in vitro studies (Ono et al 1995;Gorski et al 1997;Shimada et al 1999) and doubted the use of CZ as a specific probe for CYP2E1. Therefore, we also determined the correlation of in vivo CZ elimination with some other P450 enzyme activities to determine which P450s may also be related to CZ metabolism.…”

Section: Introductionmentioning

confidence: 99%

Chlorzoxazone metabolism is increased in fasted Sprague-Dawley rats

Wan

Ernstgård

Song

et al. 2006

Journal of Pharmacy and Pharmacology

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Earlier data showed that men fasted for 38 h have a reduced rate of chlorzoxazone (CZ) metabolism suggesting a decreased level of cytochrome P450 2E1 (CYP2E1). In contrast, CYP2E1 level in fasted rats has been shown to be elevated. In this study, we investigated whether CZ metabolism in fasted rats was changed by determining the pharmacokinetics of CZ and its metabolite 6-hydroxyCZ (6-OHCZ), as a CYP2E1 probe, and by measuring liver CYP2E1 using immunoblot techniques. CZ was administered both by gavage (50 mg/kg) and by iv (25 mg/kg) to control (9 for oral and 3 for iv) and 24 h-fasted (9 for oral and 4 for iv) male Sprague-Dawley rats. Following sampling of blood through a jugular vein cannula, CZ and 6-OHCZ plasma concentrations were measured by HPLC with ultraviolet detection. Pharmacokinetic parameters for CZ and 6-OHCZ in each treatment group were determined by model fitting and non-compartmental analysis. In parallel with the increased liver CYP2E1 level, the elimination of both CZ and 6-OHCZ was significantly increased in fasted rats in both the oral and iv study. A MANOVA covariance analysis and a multiple regression analysis revealed a significant correlation between 1/t ½ and CYP2E1 level and aniline hydroxylase activities. However, the correlations between 1/t ½ and pentoxyresorufin O-dealkylase, ethoxyresorufin Odealkylase and erythromycin N-demethylase were not significant. Therefore the contribution of other P450s to CZ metabolism seemed to be minor in the concentration range that we tested. In conclusion, fasting rats for 24 h causes a measurable induction of CYP2E1 that produces a significant increase in the rate of CZ metabolism and elimination. This is in contrast to the decreased metabolism of CZ reported for 38-hour fasted men (BJ prefers deletion of this sentence here but discussed in Discussion section?).

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Chlorzoxazone is metabolized by human CYP1A2 as well as by human CYP2E1

Cited by 102 publications

References 0 publications

Evaluation of Cytochrome P450 Probe Substrates Commonly Used by the Pharmaceutical Industry to Study in Vitro Drug Interactions

Evaluation of Cytochrome P450 Probe Substrates Commonly Used by the Pharmaceutical Industry to Study in Vitro Drug Interactions

Validated Assays for Human Cytochrome P450 Activities

Chlorzoxazone metabolism is increased in fasted Sprague-Dawley rats

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