Modification of single-nucleotide polymorphism in a fully humanized CYP3A mouse by genome editing technology

Abe, Satoshi; Kobayashi, Kaoru; Oji, Asami; Sakuma, Tetsushi; Kazuki, Kanako; Takehara, Shoko; Nakamura, Kazuomi; Okada, Azusa; Tsukazaki, Yasuko; Senda, Naoto; Honma, Kazuhisa; Yamamoto, Takashi; Ikawa, Masahito; Chiba, Kazuhiro; Oshimura, Mitsuo; Kazuki, Yasuhiro

doi:10.1038/s41598-017-15033-0

Cited by 28 publications

(23 citation statements)

References 36 publications

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“…To further investigate quetiapine metabolism pathways, we used quetiapine metabolism inhibitors. Ketoconazole is well known to be a CYP3A inhibitor, including CYP3A4 and CYP3A5, having high inhibitory potency and producing reversible inhibition through competitive and non-competitive mechanisms [31][32][33][34][35][36][37] . In this way, in vitro studies have shown that 10 µM ketoconazole inhibits specific activity of CYP3A4 on HepaRG cells 27 .…”

Section: Discussionmentioning

confidence: 99%

New insights into quetiapine metabolism using molecular networking

Daré¹,

Ferron²,

Allard³

et al. 2020

Sci Rep

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Metabolism is involved in both pharmacology and toxicology of most xenobiotics including drugs. Yet, visualization tools facilitating metabolism exploration are still underused, despite the availibility of pertinent bioinformatics solutions. Since molecular networking appears as a suitable tool to explore structurally related molecules, we aimed to investigate its interest in in vitro metabolism exploration. Quetiapine, a widely prescribed antipsychotic drug, undergoes well-described extensive metabolism, and is therefore an ideal candidate for such a proof of concept. Quetiapine was incubated in metabolically competent human liver cell models (HepaRG) for different times (0 h, 3 h, 8 h, 24 h) with or without cytochrom P450 (CYP) inhibitor (ketoconazole as CYP3A4/5 inhibitor and quinidine as CYP2D6 inhibitor), in order to study its metabolism kinetic and pathways. HepaRG culture supernatants were analyzed on an ultra-high performance liquid chromatography coupled with tandem mass spectrometry (LC-HRMS/MS). Molecular networking approach on LC-HRMS/MS data allowed to quickly visualize the quetiapine metabolism kinetics and determine the major metabolic pathways (CYP3A4/5 and/or CYP2D6) involved in metabolite formation. In addition, two unknown putative metabolites have been detected. In vitro metabolite findings were confirmed in blood sample from a patient treated with quetiapine. This is the first report using LC-HRMS/MS untargeted screening and molecular networking to explore in vitro drug metabolism. Our data provide new evidences of the interest of molecular networking in drug metabolism exploration and allow our in vitro model consistency assessment.

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Section: Discussionmentioning

confidence: 99%

New insights into quetiapine metabolism using molecular networking

Daré¹,

Ferron²,

Allard³

et al. 2020

Sci Rep

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“…In these cases, chromosomal aberrations should be fully evaluated. 37 A new technology such as gene modification without genomic cleavage (DNA modification without DSB such as Dead Cas9, deaminase, or DNA methylation/demethylation). To prevent chromosomal breaks, translocations, and large deletions associated with genome editing, genome-editing technologies without DSB have been developed [38][39][40] as a new approach to genome editing that enables the direct irreversible conversion of one target DNA base into another in a programmable manner, such as the conversion of C to T or A to G with a deaminase.…”

Section: Classification By Purpose Of Genome Editingmentioning

confidence: 99%

Aspects of Gene Therapy Products Using Current Genome-Editing Technology in Japan

Yamaguchi

Uchida²,

Okada

et al. 2020

Human Gene Therapy

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The development of genome-editing technology could lead to breakthrough gene therapy. Genome editing has made it possible to easily knock out or modify a target gene, while current gene therapy using a virus vector or plasmid hampering modification with respect to gene replacement therapies. Clinical development using these genome-editing tools is progressing rapidly. However, it is also becoming clear that there is a possibility of unintended gene sequence modification or deletion, or the insertion of undesired genes, or the selection of cells with abnormalities in the cancer suppressor gene p53; these unwanted actions are not possible with current gene therapy. The Science Board of the Pharmaceuticals and Medical Devices Agency of Japan has compiled a report on the expected aspects of such genomeediting technology and the risks associated with it. This article summarizes the history of that discussion and compares the key concepts with information provided by other regulatory authorities.

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“…Because hCYP3A-MAC/ hPXR mice have a 6986A.G mutation in the CYP3A5*3 allele, the expression level of CYP3A5 protein is negligible. Recently, we generated CYP3A-MAC mice with CYP3A5*1 by genome editing technology (Abe et al, 2017). Because CYP3A5 could be induced by RIF (Burk et al, 2004), further studies for comparison of induction between CYP3A4 and CYP3A5 in vivo may be accomplished by generation of a mouse model containing CYP3A5*1 and hPXR.…”

Section: Groupmentioning

confidence: 99%

CYP3A4 Induction in the Liver and Intestine of Pregnane X Receptor/CYP3A-Humanized Mice: Approaches by Mass Spectrometry Imaging and Portal Blood Analysis

Kobayashi

Kuze

Abe

et al. 2019

Molecular Pharmacology

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Induction of cytochrome P450 enzyme 3A (CYP3A) in response to pregnane X receptor (PXR) activators shows species-specific differences. To study the induction of human CYP3A in response to human PXR activators, we generated a double-humanized mouse model of PXR and CYP3A. CYP3A-humanized mice generated by using a mouse artificial chromosome (MAC) vector containing the entire genomic human CYP3A locus (hCYP3A-MAC mouse line) were bred with PXR-humanized mice in which the ligand-binding domain of mouse PXR was replaced with that of human PXR, resulting in double-humanized mice (hCYP3A-MAC/hPXR mouse line). Oral administration of the human PXR activator rifampicin increased hepatic expression of CYP3A4 mRNA and triazolam (TRZ) 19-and 4-hydroxylation activities, CYP3A probe activities, in the liver and intestine microsomes of hCYP3A-MAC/hPXR mice. The plasma concentration of TRZ after oral dosing was significantly decreased by rifampicin treatment in hCYP3A-MAC/hPXR mice but not in hCYP3A-MAC mice. In addition, mass spectrometry imaging analysis showed that rifampicin treatment increased the formation of hydroxy TRZ in the intestine of hCYP3A-MAC/hPXR mice after oral dosing of TRZ. The plasma concentration of 19-and 4-hydroxy TRZ in portal blood was also increased by rifampicin treatment in hCYP3A-MAC/hPXR mice. These results suggest that the hCYP3A-MAC/hPXR mouse line may be a useful model to predict human PXR-dependent induction of metabolism of CYP3A4 substrates in the liver and intestine. SIGNIFICANCE STATEMENT We generated a double-humanized mouse line for CYP3A and PXR. Briefly, CYP3A-humanized mice generated by using a mouse artificial chromosome vector containing the entire genomic human CYP3A locus were bred with PXR-humanized mice in which the ligand-binding domain of mouse PXR was replaced with that of human PXR. Expression of CYP3A4 and metabolism of triazolam, a typical CYP3A substrate, in the liver of CYP3A/PXR-humanized mice were enhanced in response to rifampicin, a typical human PXR activator. Enhancement of triazolam metabolism in the intestine of CYP3A/PXR-humanized mice was firstly shown by combination of mass spectrometry imaging of sliced intestine and liquid chromatography with tandem mass spectrometry analysis of metabolite concentration in portal blood after oral dosing of triazolam.

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Modification of single-nucleotide polymorphism in a fully humanized CYP3A mouse by genome editing technology

Cited by 28 publications

References 36 publications

New insights into quetiapine metabolism using molecular networking

New insights into quetiapine metabolism using molecular networking

Aspects of Gene Therapy Products Using Current Genome-Editing Technology in Japan

CYP3A4 Induction in the Liver and Intestine of Pregnane X Receptor/CYP3A-Humanized Mice: Approaches by Mass Spectrometry Imaging and Portal Blood Analysis

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