Assessment of multiple cytochrome P450 activities in metabolically inactivated human liver microsomes and roles of P450 2C isoforms in reaction phenotyping studies

Murayama, Norie; Yajima, Kanako; Hikawa, Mikiko; Shimura, Kanami; Ishii, Yu; Takada, Masumi; Uno, Yasuhiro; Utoh, Masahiro; Iwasaki, Katsunori; Yamazaki, Hiroshi

doi:10.1002/bdd.2115

Cited by 13 publications

(19 citation statements)

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“…By applying the mechanism-based inactivation of P450 2C9 by tienilic acid, the inhibition ratios of S -warfarin oxidation in vitro were previously shown to be 68 and 76% at substrate concentrations of 1.0 and 10 μM, respectively . These in vivo values were consistent with the f m,P450 2C9 values for S -warfarin of 0.75 and 0.81 found in substrate depletion assays (after chiral separation) at 0.50 and 5.0 μM, respectively (in the presence of the same amount of R -warfarin) . However, while studying the roles of marmoset P450 enzymes in diclofenac oxidations, we recently noticed parallel roles of P450 3A and 2C19 enzymes in diclofenac hydroxylations.…”

Section: Introductionsupporting

confidence: 79%

“…We previously adopted the approach of selective P450 inhibition to evaluate the different roles of human P450 isoforms, especially P450 2C isoforms, in the oxidation of typical P450 probe substrates . By applying the mechanism-based inactivation of P450 2C9 by tienilic acid, the inhibition ratios of S -warfarin oxidation in vitro were previously shown to be 68 and 76% at substrate concentrations of 1.0 and 10 μM, respectively . These in vivo values were consistent with the f m,P450 2C9 values for S -warfarin of 0.75 and 0.81 found in substrate depletion assays (after chiral separation) at 0.50 and 5.0 μM, respectively (in the presence of the same amount of R -warfarin) .…”

Section: Introductionsupporting

confidence: 68%

Section: Introductionmentioning

confidence: 99%

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Different Roles of Human Cytochrome P450 2C9 and 3A Enzymes in Diclofenac 4′- and 5-Hydroxylations Mediated by Metabolically Inactivated Human Hepatocytes in Previously Transplanted Chimeric Mice

Miura

Uehara

Shimizu

et al. 2019

Chem. Res. Toxicol.

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To investigate the respective roles of cytochromes P450 2C9 and 3A in drug oxidation in human livers, the in vivo pharmacokinetics of S-warfarin and diclofenac were analyzed after intravenous administrations in chimeric mice that had been transplanted with human hepatocytes. P450 2C9 was metabolically inactivated in the humanized mice by orally pretreating them with tienilic acid. After intravenous administration of S-warfarin, a significant difference in the concentration–time profiles of the primary metabolite 7-hydroxywarfarin between untreated mice and mice treated with tienilic acid was observed. In contrast, there were no apparent differences in the profiles for S-warfarin between the treated and untreated groups. The mean values of the maximum concentrations (C max) and the areas under the plasma concentration versus time curves (AUCinfinity) for 7-hydroxywarfarin were significantly lower (22 and 16% of the untreated values, respectively) in the treated group. This presumably resulted from suppressed P450 2C9 activity in the primary oxidative metabolism in vivo in the treated group. After diclofenac administration, plasma levels of diclofenac, 5-hydroxydiclofenac, and diclofenac acylglucuronide were roughly similar in pretreated and untreated mice. However, the mean C max and AUCinfinity values for 4′-hydroxydiclofenac were significantly lower (38 and 53% of the untreated group, respectively) in the treated group. The reported value of ∼0.8 for the fraction of S-warfarin metabolized to 7-hydroxywarfarin mediated by P450 2C9 in in vitro systems was similar to the value implied by the present humanized-liver mouse model pretreated with tienilic acid in which the AUC of 7-hydroxywarfarin was reduced by 84%. In contrast, the fractions of diclofenac metabolized to 4′-hydroxydiclofenac in in vitro and in vivo experiments were inconsistent. These results suggested that humanized-liver mice orally treated with tienilic acid might constitute an in vivo model for metabolically inactivated P450 2C9 in human hepatocytes transplanted into chimeric mice. Moreover, diclofenac, a typical in vitro P450 2C9 probe substrate, was cleared differently in vitro and in humanized-liver mice in vivo.

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Section: Introductionsupporting

confidence: 79%

Section: Introductionsupporting

confidence: 68%

Section: Introductionmentioning

confidence: 99%

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Different Roles of Human Cytochrome P450 2C9 and 3A Enzymes in Diclofenac 4′- and 5-Hydroxylations Mediated by Metabolically Inactivated Human Hepatocytes in Previously Transplanted Chimeric Mice

Miura

Uehara

Shimizu

et al. 2019

Chem. Res. Toxicol.

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“…The inactivated HLM is then frozen for storage and ready to be used. Silensomes™ provide a convenient (‘thaw‐and‐go’) and more consistent source for reaction phenotyping without the need of adding inhibitors for each experiment (Murayama et al, ). Silensomes™ was used for reaction phenotyping of AZD6738 (Jones, Markandu, Gu, & Scarfe, ) to demonstrate that CYP2C8 played a dominant role in the oxidation of AZD6738 to its sulfoxide metabolite, AZ8982.…”

Section: Reaction Phenotyping To Identify Victim Ddi Riskmentioning

confidence: 99%

In vitro and in vivo methods to assess pharmacokinetic drug– drug interactions in drug discovery and development

Lu¹,

2020

Biopharm & Drug Disp

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Drug-drug interactions (DDIs) caused by the co-administration of multiple drugs are major safety concerns in the clinic. Several drugs have been withdrawn from the market due to perpetrator or victim DDIs. Strategies have been developed to assess DDI risks early in drug discovery to reduce DDI liabilities. High-to-medium throughput assays are available to identify undesirable scaffolds and to guide structural modifications to minimize DDIs. Definitive methods are used at later stages of drug discovery and development to provide a more accurate measurement of DDI parameters and to enable clinical translations. Physiologically based pharmacokinetic modeling and simulations are powerful tools to accurately predict DDIs and to assess risks in the clinic. Although significant advances have been made over the years, many challenges remain for clinical DDI translations. This includes DDIs involving non-cytochrome P450 enzymes, transporters, enzyme-transporter interplay, indirect effects from biologics, and pharmacodynamic based DDI. This review focuses on methods that are used to assess hepatic DDIs caused by enzyme inhibition and induction.

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“…It was found that desethylamiodarone inactivates CYP1A1, CYP1A2, 2B6 and 2D6 but not CYP3A4. Finally, it is noteworthy that while rCYPs may not be useful for DDI prediction and IVIVE, they are a very useful tool for deducing the mechanism of inactivation of specific CYP isoforms (Murayama, et al, 2018;Orr, et al, 2012).…”

Section: In Vitro Experimental Approaches and Assay Designmentioning

confidence: 99%

Time-dependent enzyme inactivation: Numerical analyses of in vitro data and prediction of drug-drug interactions

Yadav

Paragas

Korzekwa

et al. 2020

Pharmacology & Therapeutics

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Cytochrome P450 (CYP) enzyme kinetics often do not conform to Michaelis-Menten assumptions, and time-dependent inactivation (TDI) of CYPs displays complexities such as multiple substrate binding, partial inactivation, quasi-irreversible inactivation, and sequential metabolism. Additionally, in vitro experimental issues such as lipid partitioning, enzyme concentrations, and inactivator depletion can further complicate the parameterization of in vitro TDI. The traditional replot method used to analyze in vitro TDI datasets is unable to handle complexities in CYP kinetics, and numerical approaches using ordinary differential equations of the kinetic schemes offer several advantages. Improvement in the parameterization of CYP in vitro kinetics has the potential to improve prediction of clinical drug-drug interactions (DDIs). This manuscript discusses various complexities in TDI kinetics of CYPs, and numerical approaches to model these complexities. The extrapolation of CYP in vitro TDI parameters to predict in vivo DDIs with static and dynamic modeling is discussed, along with a discussion on current gaps in knowledge and future directions to improve the prediction of DDI with in vitro data for CYP catalyzed drug metabolism.

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Assessment of multiple cytochrome P450 activities in metabolically inactivated human liver microsomes and roles of P450 2C isoforms in reaction phenotyping studies

Cited by 13 publications

References 14 publications

Different Roles of Human Cytochrome P450 2C9 and 3A Enzymes in Diclofenac 4′- and 5-Hydroxylations Mediated by Metabolically Inactivated Human Hepatocytes in Previously Transplanted Chimeric Mice

Different Roles of Human Cytochrome P450 2C9 and 3A Enzymes in Diclofenac 4′- and 5-Hydroxylations Mediated by Metabolically Inactivated Human Hepatocytes in Previously Transplanted Chimeric Mice

In vitro and in vivo methods to assess pharmacokinetic drug– drug interactions in drug discovery and development

Time-dependent enzyme inactivation: Numerical analyses of in vitro data and prediction of drug-drug interactions

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