Substrate-Dependent Functional Alterations of Seven CYP2C9 Variants Found in Japanese Subjects

Maekawa, Keiko; Harakawa, Noriko; Sugiyama, Emiko; Tohkin, Masahiro; Kim, Su-Ryang; Kaniwa, Nahoko; Katori, Noriko; Hasegawa, Ryuichi; Yasuda, Kazuki; Kamide, Kei; Miyata, Toshiyuki; Saito, Yoshiro; Sawada, Jun‐ichi

doi:10.1124/dmd.109.027003

Cited by 57 publications

(64 citation statements)

References 33 publications

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“…1 and Table 1, the typical variant CYP2C9.3 exhibited approximately 8.6%-38.9% of the enzymatic activity of wild-type CYP2C9.1 toward tolbutamide, diclofenac, and losartan. When expressed in insect cell microsomes, previous reports revealed that typically defective variant CYP2C9.3 exhibited higher K m and/or decreased V max values than the wild-type enzyme for the hydroxylation of probe drugs diclofenac and losartan (Dickmann et al, 2001;Maekawa et al, 2009). Our data were quite consistent with those observed in previous studies.…”

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confidence: 83%

Identification and Functional Assessment of a New CYP2C9 Allelic Variant CYP2C959*

Dai

Wang

et al. 2015

Drug Metab Dispos

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CYP2C9, one of the most important drug-metabolizing enzymes, is responsible for metabolizing approximately 15% of clinically important drugs, including warfarin, diclofenac, and losartan. Similar to other CYP members, human CYP2C9 exhibits marked genetic polymorphisms among individuals of different ethnicities. In this study, a novel missense mutation (1300A>T) was identified in a warfarin-sensitive patient after a genetic screen of three candidate genes related to high variability in response to warfarin doses. This base transversion leads to an Ile-toPhe amino acid substitution at codon 434 within the CYP2C9 protein, and this new variant has been named a novel allele, CYP2C9*59, by the Human CYP Allele Nomenclature Committee (http://www.cypalleles.ki. se/cyp2c9.htm). The exogenous expression of CYP2C9.59 in insect cell microsomes revealed that, despite a similar protein expression level as wild-type CYP2C9, variant CYP2C9.59 exhibited significantly reduced maximal velocity, V max , and/or increased Michaelis constant, K m , values toward three CYP2C9-specific substrates. Our data suggest that the 1300A>T mutation can greatly decrease the enzymatic activity of the CYP2C9 protein both in vitro and in vivo. IntroductionCYP2C9 is the major CYP2C isoform in humans and is responsible for the metabolism of approximately 15% of all clinically important drugs (Samer et al., 2013;Chen et al., 2014). Similar to other CYP members, CYP2C9 is highly polymorphic across various racial and ethnic populations. These genetic polymorphisms have been shown to have clinical importance, leading to great inter-individual variability and serious adverse effects in the efficacies of many therapeutic drugs (Chaudhry et al., 2014). Alleles CYP2C9*2 (containing a R144C substitution) and CYP2C9*3 (containing an I359L substitution) have been well studied among different ethnic populations. They have been identified as the most common defective alleles and have exhibited reduced metabolic activities, both in vitro and in vivo, for the CYP2C9 substrates, such as warfarin. In addition to these two common alleles, other allelic variants, such as CYP2C9*4 ( Lee et al., 2007) Nahar et al., 2013), and CYP2C9*58 (Luo et al., 2014), were reported to affect warfarin sensitivity. In this study, we present the case of a Chinese warfarin-sensitive patient who requires only one-half of the normal oral warfarin dose. Genetic analysis in that patient revealed an absence of the common mutations in CYP2C9 (*2, *3), VKORC1 (rs9923231 and rs7294), and CYP4F2 (rs2108622) that are usually related to warfarin sensitivity. However, a novel missense mutation (1300A.T) was identified in exon 9 of the CYP2C9 gene. The in vitro functional assessment of this newly found CYP2C9 variant revealed that the substitution of an Ile to a Phe at codon 434 significantly reduced the enzymatic activity of this variant toward all of the tested substrates. Materials and MethodsStudy Subject. The study subject was a 47-year-old male Han Chinese patient who has taken warfarin...

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confidence: 83%

Identification and Functional Assessment of a New CYP2C9 Allelic Variant CYP2C959*

Dai

Wang

et al. 2015

Drug Metab Dispos

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“…b Dose adjustment ratios were predicted at the upper most value of estimated f m(CYP2C9) . dmd.aspetjournals.org features of CYP2C9 [i.e., a big active pocket allowing simultaneous binding of multiple ligands (Williams et al, 2003)] that potentially lead to highly substrate-dependent functionality of CYP2C9 variants (Maekawa et al, 2009). It will be of interest to examine whether the partition ratios, metabolite formation rates, and noscapine depletion rates are significantly different among these enzymes.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Inhibition Kinetics of (S)-Warfarin Hydroxylation by Noscapine: Implications in Warfarin Therapy

et al. 2013

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Noscapine is an antitussive and potential anticancer drug. Clinically significant interactions between warfarin and noscapine have been previously reported. In this study, to provide a basis for warfarin dosage adjustment, the inhibition kinetics of noscapine against warfarin metabolism was characterized. Our enzyme kinetics data obtained from human liver microsomes and recombinant CYP2C9 proteins indicate that noscapine is a competitive inhibitor of the (S)-warfarin 7-hydroxylation reaction by CYP2C9. Interestingly, noscapine also inhibited (S)-warfarin metabolism in a NADPHand time-dependent manner, and removal of unbound noscapine and its metabolites by ultrafiltration did not reverse inhibition of (S)-warfarin metabolism by noscapine, suggesting mechanism-based inhibition of CYP2C9 by noscapine. Spectral scanning of the reaction between CYP2C9 and noscapine revealed the formation of an absorption spectrum at 458 nm, indicating the formation of a metabolite-intermediate complex. Surprisingly, noscapine is a 2-to 3-fold more efficient inactivator of CYP2C9.2 and CYP2C9.3 variants than it is of the wild type, by unknown mechanisms. Based on the inhibitory kinetic data, (S)-warfarin exposure is predicted to increase up to 7-fold (depending on CYP2C9 genotypes) upon noscapine coadministration, mainly due to mechanism-based inactivation of CYP2C9 by noscapine. Together, these results indicate that mechanism-based inhibition of CYP2C9 by noscapine may dramatically alter pharmacokinetics of warfarin and provide a basis for warfarin dosage adjustment when noscapine is coadministered.

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“…Combined with the functional analysis data, our present data suggested that CYP2C9*16 was another important defective CYP2C9 allele for Chinese Han individuals, except for the commonly studied alleles *2, *3, and *13. Maekawa et al (2009) reported that recombinant microsome of CYP2C9*34 had no substantial effect on the metabolism of diclofenac, losartan, and glimepiride. However, in the present study, CYP2C9*34 exhibited decreased catalytic activity for tolbutamide in COS-7 cells, which is consistent with activity in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…When expressed in a baculovirus-insect cell system, CYP2C9*34 had no substantial effect. A study on substratedependent functional alteration of CYP2C9 demonstrated that CYP2C9*34 exhibited catalytic activities almost similar to those of the wild-type for diclofenac, losartan, and glimepiride (Maekawa et al, 2009). …”

Section: Introductionmentioning

confidence: 99%

In Vitro and In Vivo Characterization of 13 CYP2C9 Allelic Variants Found in Chinese Han Population

Pan

Wang

et al. 2015

Drug Metab Dispos

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Our previous study detected totally 35 CYP2C9 allelic variants in 2127 Chinese subjects, of whom 21 novel alleles were reported for the first time in Chinese populations. The aim of the present study was to characterize the 13 CYP2C9 allelic variants both in vitro and in vivo. Different types of CYP2C9 variants were highly expressed in COS-7 cells, and 50 mM tolbutamide was added as the probing substrate to evaluate their metabolic abilities in vitro. Subsequently, the concentrations of tolbutamide and its metabolite in the plasma and urine within individuals with different types of genotypes were determined by HPLC to evaluate the catalytic activity of the 13 mutant CYP2C9 proteins in vivo. Our results showed that compared with *1/*1 wild-type subjects, subjects with *1/*40 genotype showed increased oral clearance (CL/F), whereas individuals with

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Substrate-Dependent Functional Alterations of Seven CYP2C9 Variants Found in Japanese Subjects

Cited by 57 publications

References 33 publications

Identification and Functional Assessment of a New CYP2C9 Allelic Variant CYP2C959*

Identification and Functional Assessment of a New CYP2C9 Allelic Variant CYP2C959*

Characterization of Inhibition Kinetics of (S)-Warfarin Hydroxylation by Noscapine: Implications in Warfarin Therapy

In Vitro and In Vivo Characterization of 13 CYP2C9 Allelic Variants Found in Chinese Han Population

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Substrate-Dependent Functional Alterations of Seven CYP2C9 Variants Found in Japanese Subjects

Cited by 57 publications

References 33 publications

Identification and Functional Assessment of a New CYP2C9 Allelic Variant CYP2C9*59

Identification and Functional Assessment of a New CYP2C9 Allelic Variant CYP2C9*59

Characterization of Inhibition Kinetics of (S)-Warfarin Hydroxylation by Noscapine: Implications in Warfarin Therapy

In Vitro and In Vivo Characterization of 13 CYP2C9 Allelic Variants Found in Chinese Han Population

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Identification and Functional Assessment of a New CYP2C9 Allelic Variant CYP2C959*

Identification and Functional Assessment of a New CYP2C9 Allelic Variant CYP2C959*