Metabolism of diazepam and related benzodiazepines by human liver microsomes

Hooper, W. D.; Watt, J. A.; McKinnon, GE; Reilly, Paul E.B.

doi:10.1007/bf03189988

Cited by 18 publications

(7 citation statements)

References 27 publications

(23 reference statements)

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“…The atypical formation kinetics for temazepam have been reported previously [12,30]. Similar atypical kinetics have been reported for other CYP3A mediated reactions including caffeine 8-oxidation [31] and progesterone 6f-hydroxylation [32].…”

Section: Discussionsupporting

confidence: 81%

“…The assay of Hooper et al [12] was used to measure for temazepam and N-desmethyldiazepam with minor modifications. A Model LC 1100 solvent-delivery system, a Model LC 1200 variable wavelength UV-VIS detector (ICI instruments, Melbourne, Australia), a Model 7125 Rheodyne injector and a Model SE 120 BBC Goetz Metrawatt recorder were used.…”

Section: Measurement Of Diazepam Metabolites In Human Liver Microsomesmentioning

confidence: 99%

“…Other in vitro studies report that the two major pathways for diazepam metabolism-the formation of temazepam and N-desmethyldiazepam-are catalysed by different cytochrome P450 (CYP) iso-forms, and the isoform responsible for the formation of N-desmethyldiazepam seems to be the same as that hydroxylating S-mephenytoin [9][10][11]. However, mephenytoin did not inhibit diazepam metabolism in vitro [12,13].…”

Section: Introductionmentioning

confidence: 99%

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Diazepam metabolism by human liver microsomes is mediated by both S‐ mephenytoin hydroxylase and CYP3A isoforms.

Andersson

Miners

Veronese

et al. 1994

Brit J Clinical Pharma

198

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Section: Discussionsupporting

confidence: 81%

Section: Measurement Of Diazepam Metabolites In Human Liver Microsomesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Diazepam metabolism by human liver microsomes is mediated by both S‐ mephenytoin hydroxylase and CYP3A isoforms.

Andersson

Miners

Veronese

et al. 1994

Brit J Clinical Pharma

198

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“…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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“…Hooper et al reported that propylene glycol (another glycol derivative) in combination with acetone inhibited in vitro human diazepam metabolism, which is mediated by a combination of P450 3A and 2C19 isoforms. 27 Acetone enhanced the formation of R-OH-ALP but not 4-OH-ALP. In previous studies, acetone increased the rate of aniline hydroxylation in rat 28,29 and human 30 microsomal systems and enhanced midazolam metabolism by human microsomes.…”

Section: Discussionmentioning

confidence: 98%

Influence of Polyethylene Glycol and Acetone on the in Vitro Biotransformation of Tamoxifen and Alprazolam by Human Liver Microsomes

Cotreau-Bibbo

Moltke

Greenblatt

1996

Journal of Pharmaceutical Sciences

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The use of in vitro hepatic microsomal models as a method of studying the biotransformation of xenobiotics can be complicated by the insolubility of a lipophilic substrate. Addition of solvent to an in vitro system is an approach used to increase solubility of such substrates, but solvents may interact with the microsomal enzymes and cause changes in the kinetic parameters. This study focused on the solvents polyethylene glycol-400 (PEG) and acetone and their influence on the human microsomal in vitro biotransformation of the antineoplastic agent tamoxifen (TAM). The antianxiety agent alprazolam (ALP), a verified P450 3A substrate, was also studied. TAM is a nonpolar drug that is metabolized to two oxidative metabolites, N-desmethyltamoxifen (DMT) and 4-hydroxytamoxifen (4-OH-TAM), by cytochrome P450 isoforms. DMT is formed primarily by 3A isoforms, and the pathway(s) responsible for 4-OH-TAM formation are unknown. Biotransformation of ALP is mediated by P450 3A isoforms, which form alpha-hydroxyalprazolam (alpha-OH-ALP) and 4-hydroxyalprazolam (4-OH-ALP). Both PEG and acetone at 5% of the total microsomal incubation volume were found to solubilize TAM in the in vitro system. However, both solvents had an effect on the P450 mediated metabolism of ALP and TAM. For ALP, PEG was a noncompetitive inhibitor of alpha-OH-ALP (mean Ki = 2.06%) and 4-OH-ALP (mean Ki = 2.37%) formation. Acetone stimulated the production of alpha-OH-ALP, but had no apparent influence on 4-OH-ALP formation. The solvent's influence on TAM metabolism varied. PEG decreased the amount of DMT formed by the microsomes as compared to the system containing no solvent (control); however, 4-OH-TAM formation in the presence of PEG was 146-226% of controls. Samples containing acetone produced smaller quantities of both DMT (39-63%) and 4-OH-TAM (45-69%) as compared to controls. Since PEG and acetone increased the solubility of TAM in the incubation buffer but inhibited or accelerated enzymatic reactions compared to buffer alone, actual solubility in buffer was not a determinant of the rate of TAM metabolism. TAM appeared to be taken up into the microsomal fraction, making it available for biotransformation by the P450 isoforms. Although solvents may increase the solubility of nonpolar agents in aqueous systems, careful evaluation of the effects of solvent on metabolite formation, as compared to buffer controls, is needed in order to properly evaluate an in vitro metabolic model.

show abstract

Metabolism of diazepam and related benzodiazepines by human liver microsomes

Cited by 18 publications

References 27 publications

Diazepam metabolism by human liver microsomes is mediated by both S‐ mephenytoin hydroxylase and CYP3A isoforms.

Diazepam metabolism by human liver microsomes is mediated by both S‐ mephenytoin hydroxylase and CYP3A isoforms.

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

Influence of Polyethylene Glycol and Acetone on the in Vitro Biotransformation of Tamoxifen and Alprazolam by Human Liver Microsomes

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