Expression of Four Rat CYP2D Isoforms inSaccharomyces cerevisiaeand Their Catalytic Specificity

Wan, Jie; Imaoka, Shingi; Chow, T. L.; Hiroi, Toyoko; Yabusaki, Yoshiyasu; Funae, Yoshihiko

doi:10.1006/abbi.1997.0402

Cited by 56 publications

(32 citation statements)

References 27 publications

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“…Recombinant CYP2D Enzymes Recombinant rat CYP2D2 9) and human CYP2D6 11) were expressed in yeast cells (Saccharomyces cerevisiae AH-22 strain) according to methods previously reported. Rat CYP2D2-G45V mutant protein, which has valine instead of glycine at position 45 of CYP2D2, was expressed in yeast cells as reported elsewhere.…”

Section: Methodsmentioning

confidence: 99%

“…7,8) If the epoxy metabolite(s) of IMI are responsible for the inactivation, DMI would also inactivate the rat CYP2D enzyme(s). Because several CYP2D enzymes are known to be expressed in the rat liver, 9) it is interesting to know what kind of CYP2D isoenzyme(s) are inhibited by IMI or DMI. Furthermore, there is a possibility that CYP2D6, a human functional CYP2D enzyme, is also inactivated by IMI and DMI in a similar manner.…”

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confidence: 99%

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Different Effects of Desipramine on Bufuralol 1"-Hydroxylation by Rat and Human CYP2D Enzymes

Isobe

Hichiya

Hanioka

et al. 2005

Biological & Pharmaceutical Bulletin

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Imipramine (IMI) and desipramine (DMI) are tricyclic antidepressants that are widely used clinically. As shown in Fig. 1, IMI is oxidized by cytochrome P450 (CYP) mainly via two pathways: side-chain N-demethylation and aromatic ring 2-hydroxylation, forming DMI and 2-hydroxyimipramine (2-OH-IMI), respectively. 1) DMI and 2-OH-IMI further undergo 2-hydroxylation and N-demethylation, respectively, forming 2-hydroxy-DMI as the common metabolite (Fig. 1). In the human liver, CYP2D6 is mainly responsible for IMI 2-hydroxylation whereas CYP2C19 and CYP1A2 are involved in N-demethylation. 2,3)It has been reported that repeated administration of IMI to rats changed hepatic CYP-dependent monooxygenase activities. [4][5][6] We have also found that repetitive oral administration of IMI to rats caused a decrease in hepatic microsomal CYP2D-dependent reactions such as debrisoquine 4-hydroxylation, bunitrolol 4-hydroxylation, lidocaine 3-hydroxylation and propranolol 4-, 5-and 7-hydroxylations. 7) We have proposed that binding of a reactive metabolite of IMI to rat CYP2D enzyme(s) resulted in the decreased enzyme activities.7) As a possible mechanism, we speculated that an epoxy metabolite of IMI (1,2-or 2,3-epoxide) was involved in the inactivation of rat CYP2D enzyme(s). 7,8) If the epoxy metabolite(s) of IMI are responsible for the inactivation, DMI would also inactivate the rat CYP2D enzyme(s). Because several CYP2D enzymes are known to be expressed in the rat liver, 9) it is interesting to know what kind of CYP2D isoenzyme(s) are inhibited by IMI or DMI. Furthermore, there is a possibility that CYP2D6, a human functional CYP2D enzyme, is also inactivated by IMI and DMI in a similar manner. The present study was thus conducted to examine these possibilities using radiolabeled and unlabeled DMI and recombinant human and rat CYP2D enzymes. Sciences, Fukuoka University; 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. Received November 15, 2004; accepted January 13, 2005 Inhibitory effects of desipramine (DMI) on rat and human CYP2D enzymes were studied using bufuralol (BF) 1؆-hydroxylation as an index. Inhibition was examined under the following two conditions: 1) DMI was coincubated with BF and NADPH in the reaction mixture containing rat or human liver microsomes or yeast cell microsomes expressing rat CYP2D1, CYP2D2 or human CYP2D6 (co-incubation); 2) DMI was preincubated with NADPH and the same enzyme sources prior to adding the substrate (preincubation). When either rat liver microsomes or recombinant CYP2D2 was employed, the preincubation with DMI (0.3 m mM) caused a greater inhibition of BF 1؆-hydroxylation than the co-incubation did, whereas BF 1؆-hydroxylation by rat CYP2D1 was not markedly affected under the same conditions. The inhibitory effect of DMI on BF 1؆-hydroxylation by human liver microsomal fractions or recombinant CYP2D6 was much lower than that on the hydroxylation by rat liver microsomes or CYP2D2. Kinetic studies demonstrated that the inhibition-type changed from competitive for the co-incu...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Different Effects of Desipramine on Bufuralol 1"-Hydroxylation by Rat and Human CYP2D Enzymes

Isobe

Hichiya

Hanioka

et al. 2005

Biological & Pharmaceutical Bulletin

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“…All experiments using animals were performed with the supervision and approval of the Institutional Animal Care and Use Committee of Hokkaido University. CYP2D1, CYP2D2, CYP2D3, and CYP2D4 proteins were expressed in yeast and purified (Wan et al, 1997).…”

Section: Methodsmentioning

confidence: 99%

“…In addition, antibodies generated against the purified P450 enzymes are often not specific. Despite the structural similarities among CYP2D isoforms, significant differences in the ability to metabolize drugs have been observed among these CYP2D isoforms (Wan et al, 1997;Chow et al, 1999;Hiroi et al, 2002). In this study, we identified the enzyme involved in diazepam p-hydroxylation to be CYP2D3 using yeast recombinant CYP2D isoforms.…”

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confidence: 90%

Importance of Cyp2d3 in Polymorphism of Diazepam P-Hydroxylation in Rats

Sakai

Saito²,

Kim³

et al. 2005

Drug Metabolism and Disposition

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ABSTRACT:Diazepam was metabolized to three primary metabolites, 3-hydroxy-diazepam, N-desmethyl-diazepam, and p-hydroxy-diazepam. Our previous studies reported metabolic position-specific inter-or intrastrain differences in diazepam metabolism among Sprague-Dawley, Brown Norway, Dark Agouti, and Wistar rats. Especially, there were marked (ϳ300 fold) inter-or intrastrain differences in diazepam p-hydroxylation activity at low concentration of substrate. In this study, we investigated the enzyme that catalyzes diazepam p-hydroxylation. The activity toward diazepam phydroxylation was inhibited by anti-cytochrome P450 2D (CYP2D) antibody, suggesting that this activity was catalyzed by CYP2D isoforms. Comparing the expression levels of the CYP2D subfamily in liver microsomes from various strains of rats using anti-CYP2D2 antibody, we found that there was a band of protein that was consistent with the phenotype of diazepam p-hydroxylation. Nterminal amino acid sequences of the specific protein exactly corresponded to those of CYP2D3, indicating that CYP2D3 might be involved in diazepam p-hydroxylation. Moreover, using rat CYP2D isoforms expressed in yeast, we tested CYP2Ds to catalyze diazepam p-hydroxylation. CYP2D1 and CYP2D2 practically did not participate in diazepam metabolism. On the other hand, diazepam p-hydroxylation was catalyzed by CYP2D3. CYP2D4 had high activity toward diazepam N-desmethylation, but not p-hydroxylation. In conclusion, the polymorphic expression of CYP2D3 caused the inter-or intrastrain differences in diazepam p-hydroxylation among rat strains or individuals.

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“…CYP2C9*1 and CYP2C9*3 cDNAs cut out with HindIII were each subcloned into the expression vector pGYR1. 20 The pGYR1 vectors containing CYP2C9 cDNAs were transformed into the Saccharomyces cerevisiae AH 22 strain. 21 …”

Section: Saccharomyces Cerevisiaementioning

confidence: 99%

CYP2C9*3 influences the metabolism and the drug-interaction of candesartan in vitro

et al. 2001

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Candesartan cilexetil is an angiotensin II receptor antagonist, and candesartan, its active metabolite, is metabolized by CYP2C9. However, the effect of CYP2C9*3 on candesartan metabolism is not established. We characterized the kinetics of candesartan by CYP2C9*1/*1 and CYP2C9*1/*3 in human liver microsomes. The difference between the two was not significant. Subsequently, CYP2C9*1 and CYP2C9*3 (Leu 359 ) were expressed in yeast, and the kinetics of candesartan were determined. The wild-type showed the lower K m (345 vs 439 M; 3/4) and higher V max /K m (1/3) than the Leu 359 variant. Also, we investigated potential interaction between candesartan and warfarin with both the wild-type and the Leu 359 variant. Candesartan had no effect on S-warfarin 7-hydroxylation. In contrast, S-warfarin inhibited candesartan metabolism by the wild-type (K i = 17 M) greater than by the Leu 359 variant (K i = 36 M). These findings suggest that CYP2C9*3 may change not only the metabolic activity but also the inhibitory susceptibility compared with

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Expression of Four Rat CYP2D Isoforms inSaccharomyces cerevisiaeand Their Catalytic Specificity

Cited by 56 publications

References 27 publications

Different Effects of Desipramine on Bufuralol 1"-Hydroxylation by Rat and Human CYP2D Enzymes

Different Effects of Desipramine on Bufuralol 1"-Hydroxylation by Rat and Human CYP2D Enzymes

Importance of Cyp2d3 in Polymorphism of Diazepam P-Hydroxylation in Rats

CYP2C9*3 influences the metabolism and the drug-interaction of candesartan in vitro

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