Role of cDNA-Expressed Human Cytochromes P450 in the Metabolism of Diazepam

Yang, Taoxi; Shou, Magang; Korzekwa, Kenneth R.; Gonzalez, Frank J.; Gelboin, Harry V.; Yang, Shen K.

doi:10.1016/s0006-2952(97)00558-3

Cited by 44 publications

(19 citation statements)

References 21 publications

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“…Thus, the identification of CYP2C19 as the major P450 involved in the NCLB biotransformation is consistent with the accumulation of this metabolite in epileptic patients receiving felbamate cotherapy (Contin et al, 1999), since felbamate inhibits CYP2C19 and, then, the elimination of NCLB. These results are also consistent with studies concerning the metabolism of several 1,4benzodiazepines that have shown the major role mediated by the CYP3A4 and CYP2C19 isoforms: flunitrazepam (Hesse et al, 2001;Kilicarslan et al, 2001), diazepam Yang et al, 1998), and midazolam and triazolam (Perloff et al, 2000). Indeed, considering the structural analogy between the 1,4-and 1,5-benzodiazepines, the same P450 isoforms were likely to be involved.…”

Section: Discussionsupporting

confidence: 87%

In Vitro Characterization of Clobazam Metabolism by Recombinant Cytochrome P450 Enzymes: Importance of Cyp2c19

Giraud¹,

Tran²,

Rey³

et al. 2004

Drug Metab Dispos

105

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The specific cytochrome P450 (P450) isoforms mediating the biotransformations of clobazam (CLB) and those of its major metabolites, N-desmethylclobazam (NCLB) and 4-hydroxyclobazam were identified using cDNA-expressed P450 and P450-specific chemical inhibitors. Among the 13 cDNA-expressed P450 isoforms tested, CLB was mainly demethylated by CYP3A4, CYP2C19, and CYP2B6 and 4-hydroxylated by CYP2C19 and CYP2C18. CYP2C19 and CYP2C18 catalyzed the 4-hydroxylation of NCLB. The kinetics of the major biotransformations were studied: CYP3A4, CYP2C19, and CYP2B6 mediated the formation of NCLB with K m ‫؍‬ 29.0, 31.9, and 289 M, V max ‫؍‬ 6.20, 1.15, and 5.70 nmol/min/nmol P450, and intrinsic clearance (CL int ) ‫؍‬ 214, 36.1, and 19.7 l/min/nmol P450, respectively. NCLB was hydroxylated to 4-hydroxydesmethylclobazam by CYP2C19 with K m ‫؍‬ 5.74 M, V max ‫؍‬ 0.219 nmol/min/ nmol P450, and CL int ‫؍‬ 38.2 l/min/nmol P450 (Hill coefficient ‫؍‬ 1.54). These findings were supported by chemical inhibition studies in human liver microsomes. Indeed, ketoconazole (1 M) inhibited the demethylation of CLB by 70% and omeprazole (10 M) by 19%; omeprazole inhibited the hydroxylation of NCLB by 26%. Twentytwo epileptic patients treated with CLB were genotyped for CYP2C19. The NCLB/CLB plasma metabolic ratio was significantly higher in the subjects carrying one CYP2C19*2 mutated allele than in those carrying the wild-type genotype. CYP3A4 and CYP2C19 are the main P450s involved in clobazam metabolism. Interactions with other drugs metabolized by these P450s can occur; moreover, the CYP2C19 genetic polymorphism could be responsible for interindividual variations of plasma concentrations of N-desmethylclobazam and thus for occurrence of adverse events. Materials and MethodsChemicals and Reagents. Clobazam and N-desmethylclobazam were obtained from Laboratoires Roussel-Uclaf/Sanofi-Synthelabo France (Paris, France). 4Ј-Hydroxyclobazam and 4Ј-hydroxy-N-desmethylclobazam were synthesized and kindly provided by Laboratoires Biocodex (Montrouge, France).

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Section: Discussionsupporting

confidence: 87%

In Vitro Characterization of Clobazam Metabolism by Recombinant Cytochrome P450 Enzymes: Importance of Cyp2c19

Giraud¹,

Tran²,

Rey³

et al. 2004

Drug Metab Dispos

105

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“…Each P450 was expressed in Sf21 insect cells in the presence of hemin. The MOIs of the viruses (P450 versus OR) were optimized as a ratio of 5 to 10:1 to support maximally the P450-catalyzed reactions as previously reported (Shou et al, 1998). MOI exceeding 5:1 reduced the yield of P450 expression and hampered catalytic activities.…”

Section: Resultsmentioning

confidence: 93%

“…CYP3A12 was a phenobarbital-and rifampicin-inducible enzyme (Ciaccio and Halpert 1989;Nishibe et al, 1998). Substrate specificity characteristics of CYP3A forms in rat and human are comprised of testosterone 6␤-hydroxylation, diazepam 3-hydroxylation and dextromethorphan N-demethylation (Bertilsson et al, 1990;Hanioka et al, 1990;Hooper et al, 1992;Andersson et al, 1994;Yang et al, 1998). The same experimental approaches were utilized to probe the dog CYP3A.…”

Section: Discussionmentioning

confidence: 99%

Substrate Specificity and Kinetic Properties of Seven Heterologously Expressed Dog Cytochromes P450

Shou¹,

Norcross²,

Sandig³

et al. 2003

Drug Metab Dispos

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:Seven dog cytochromes P450 (P450s) were heterologously expressed in baculovirus-Sf21 insect cells. Of all enzymes examined, CYP1A1 exhibited high 7-ethoxyresorufin O-deethylase activity (low K m enzyme, 1 M). CYP2B11 and CYP3A12 effectively catalyzed the N 1 -demethylation and C 3 -hydroxylation of diazepam (and its derivatives), whereas CYP3A12 and CYP2D15 catalyzed exclusively the N-and O-demethylation, respectively, of dextromethorphan. However, no saturation velocity curves for the N-demethylation of dextromethorphan (up to 500 M) were achieved, suggesting a high K m for CYP3A12. In contrast to CYP3A12, the CYP2D15-dependent O-demethylation of dextromethorphan was a low K m process (K m ‫؍‬ 0.7 M), similar to that in dog liver microsomes (K m ‫؍‬ 2.3 M). CYP2D15 was also capable of metabolizing bufuralol (1-hydroxylation), with a K m of 3.9 M, consistent with that obtained with dog liver microsomes. CYP3A12 was shown to primarily oxidize testosterone at 16␣-, 2␣/2␤-, and 6␤-positions. Selectivity of CYP3A12 was observed toward testosterone 6␤-(K m ‫؍‬ 83 M) and 2␣/2␤-hydroxylations (K m ‫؍‬ 154 M). However, the 16␣-hydroxylation of testosterone was catalyzed by CYP2C21 also (K m ‫؍‬ 6.4 M for CYP2C21). Therefore, the 6␤-and 16␣-hydroxylation of testosterone can potentially be employed as markers of CYP3A12 and CYP2C21 (at low concentration), respectively. CYP2C21 was also capable of catalyzing diclofenac 4-hydroxylation, although some activity was detected with CYP2B11. Surprisingly, none of the P450s selectively metabolized (S)-mephenytoin 4-hydroxylation. The results described herein are a first step toward the systematic evaluation of a panel of dog P450s and the development of dog P450 isoenzyme-selective marker substrates, as well as providing useful information on prediction and extrapolation of the results from in vitro to in vivo and from dog to human.

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“…In comparison, CYP2C9 was shown to possess 3-to 6-fold lower intrinsic clearance for N-demethylation of diazepam. In a contrasting study, Yang et al (1998a) showed that CYP2B6 had the highest V max for desmethyl-diazepam formation (7.5 nmol/min/nmol-CYP) amongst a panel of various cDNA-expressed human CYP enzymes when incubated with 0.1 mm diazepam. The 3-hydroxylation of diazepam was catalyzed by CYP3A4 and 3A5 with the highest activity and CYP2B6 did not show this activity.…”

Section: Discussionmentioning

confidence: 99%

Pharmacokinetic study in pigs andin vitrometabolic characterization in pig- and human-liver microsomes reveal marked differences in disposition and metabolism of tiletamine and zolazepam (Telazol)

Kumar¹,

Mann

Beilman

et al. 2013

Xenobiotica

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1. An equal-dose combination of tiletamine and zolazepam (Telazol®) is used as a veterinary anesthetic. There also have been reports of human abuse of Telazol®. The pharmacokinetics and metabolic fate of tiletamine and zolazepam and the rationale for their administration as an equal-dose combination are unclear. 2. The single-dose pharmacokinetics of intramuscular tiletamine and zolazepam (3 mg/kg each) in 16 Yorkshire-crossbred pigs were determined. The metabolites of tiletamine and zolazepam in pig plasma and urine were identified by mass spectrometry. The metabolic stability of tiletamine and zolazepam and the kinetics of formation of their metabolites by pig- and human-liver microsomes were determined. 3. Higher concentrations of zolazepam were observed in pig plasma and it was cleared more slowly compared to tiletamine (apparent clearance: 11 versus 134 l/h; half-life: 2.76 versus 1.97 h). Three metabolites of zolazepam and one metabolite of tiletamine were identified in pig urine, plasma and in microsomal incubations. In vitro formation of each of these metabolites in microsomes was biphasic involving a high-affinity/low-capacity and a low-affinity/high-capacity enzyme. The in vitro metabolic stability of tiletamine was considerably lower compared to zolazepam. 4. These results collectively point to major pharmacokinetic and metabolic differences between the two components of this fixed-dose anesthetic combination.

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Role of cDNA-Expressed Human Cytochromes P450 in the Metabolism of Diazepam

Cited by 44 publications

References 21 publications

In Vitro Characterization of Clobazam Metabolism by Recombinant Cytochrome P450 Enzymes: Importance of Cyp2c19

In Vitro Characterization of Clobazam Metabolism by Recombinant Cytochrome P450 Enzymes: Importance of Cyp2c19

Substrate Specificity and Kinetic Properties of Seven Heterologously Expressed Dog Cytochromes P450

Pharmacokinetic study in pigs andin vitrometabolic characterization in pig- and human-liver microsomes reveal marked differences in disposition and metabolism of tiletamine and zolazepam (Telazol)

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