Evaluation of Cynomolgus Monkey Pregnane X Receptor, Primary Hepatocyte, and in Vivo Pharmacokinetic Changes in Predicting Human CYP3A4 Induction

Kim, Sean; Dinchuk, Joseph; Anthony, Monique; Orcutt, Tami; Zoeckler, Mary; Sauer, Mary B.; Mosure, Kathleen W.; Vuppugalla, Ragini; Grace, James E.; Simmermacher, Jean; Dulac, Heidi; Pizzano, Jennifer; Sinz, Michael

doi:10.1124/dmd.109.029637

Cited by 40 publications

(34 citation statements)

References 25 publications

(25 reference statements)

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“…The EC 50 values for the two hepatocyte preparations were 2.4 M (95% confidence interval, 1.1-5.2 M) and 1.7 M (95% confidence interval, 1.0 -3.0 M). These results were comparable with the previously reported EC 50 values (0.67-2.34 M) for the induction of MDZ 1Ј-hydroxylase activity by RIF in cynomolgus monkey hepatocytes (Kim et al, 2010).…”

Section: Apz Metabolism In Liver and Smallsupporting

confidence: 82%

“…However, concentration-response relationships could vary among inducers, nuclear receptors, or the species examined. Kim et al (2010) investigated PXR transactivation profiles of 30 compounds, including known CYP3A4 inducers, in reporter assays, demonstrating that EC 50 values correlated well between cynomolgus and human PXRs, although there were notable species differences in EC 50 and E max values for reserpine. Thus, further studies using other inducers are required for a better understanding of similarities and differences in CYP3A induction in vivo between humans and monkeys.…”

Section: Discussionmentioning

confidence: 99%

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Alprazolam as an In Vivo Probe for Studying Induction of CYP3A in Cynomolgus Monkeys

Ohtsuka¹,

Yoshikawa²,

Kozakai³

et al. 2010

Drug Metab Dispos

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ABSTRACT:Induction of the cytochrome P450 (P450) enzyme is a major concern in the drug discovery processes. To predict the clinical significance of enzyme induction, it is helpful to investigate pharmacokinetic alterations of a coadministered drug in a suitable animal model. In this study, we focus on the induction of CYP3A, which is involved in the metabolism of approximately 50% of marketed drugs and is inducible in both the liver and intestine. As a marker substrate for CYP3A activity, alprazolam (APZ) was selected and characterized using recombinant CYP3A enzymes expressed in Escherichia coli. Both human CYP3A4 and its cynomolgus P450 ortholog predominantly catalyzed APZ 4-hydroxylation with sigmoidal kinetics. When administered intravenously and orally to cynomolgus monkeys, APZ had moderate clearance; its first-pass extraction ratio after oral dosing was estimated to be 0.09 in the liver and 0.45 in the intestine. Pretreatment with multiple doses of rifampicin (20 mg/kg p.o. for 5 days), a known CYP3A inducer, significantly decreased plasma concentrations of APZ after intravenous and oral administrations (0.5 mg/kg), and first-pass extraction ratios were increased to 0.39 in the liver and 0.63 in the intestine. The results were comparable to those obtained in clinical drug-drug interaction (DDI) reports related to CYP3A induction, although the rate of recovery of CYP3A activity seemed to be slower than rates estimated in clinical studies. In conclusion, pharmacokinetic studies using APZ as a probe in monkeys may provide useful information regarding the prediction of clinical DDIs due to CYP3A induction.

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Section: Apz Metabolism In Liver and Smallsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Alprazolam as an In Vivo Probe for Studying Induction of CYP3A in Cynomolgus Monkeys

Ohtsuka¹,

Yoshikawa²,

Kozakai³

et al. 2010

Drug Metab Dispos

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“…7) It was previously reported that cmCYP3A4 (3A8) is induced by RIF through the transcription factor PXR. 29) Our study showed that PXR is expressed in cmESC-derived hepatic cells. These results suggest that PXR is active in these hepatocytes.…”

Section: Discussionmentioning

confidence: 96%

Differentiation of Monkey Embryonic Stem Cells to Hepatocytes by Feeder-Free Dispersion Culture and Expression Analyses of Cytochrome P450 Enzymes Responsible for Drug Metabolism

Maruyama

Matsunaga

Yamaori

et al. 2013

Biological & Pharmaceutical Bulletin

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We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.Key words embryonic stem cell; differentiation; hepatocyte; monkey; cytochrome P450; feeder-free dispersed culture Investigation of drug metabolism with human hepatocytes is important in the early stages of drug development. However, primary human hepatocytes are short-lived and cannot be maintained in culture over the long term. In addition, there are large donor-dependent variations in drug metabolism. On the other hand, human embryonic stem cells (ESCs) are able to replicate infinitely and differentiate into various types of somatic cells including germ cells.1) Thus, they represent an attractive source to provide large numbers of cells that can be utilized for the development of candidate drug-screening strategies in place of primary cells.2) However, ethical and legal restrictions have limited the availability of human ESCs. The phenotype of human ESCs is known to closely resemble that of monkey ESCs but not mouse ESCs with regard to morphology, leukemia inhibitory factor responsiveness, gene expression profiles, and some disease models.1,3-6) Thus, monkey ESCs are a more suitable model for preclinical research of drug development. In particular, hepatocytes derived from monkey ESCs may be useful for pharmacokinetic studies, such as investigation of drug-drug interactions and the inducibility of drug-metabolizi...

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“…Macaque CYP3A8(4) metabolizes typical human CYP3A4 substrates such as midazolam and nifedipine (Iwasaki et al, 2010;Uno et al, 2010) and is induced by the typical human CYP3A4 inducer, rifampicin (Kim et al, 2010), indicating that CYP3A8(4) has drug-metabolizing enzyme properties similar to human CYP3A4. Induction of cynomolgus macaque CYP3A8(4) and human CYP3A4 by rifampicin is mediated via PXR (Kim et al, 2010), which binds to the regulatory element in the upstream region of CYP3A4, suggesting that the regulatory region of cynomolgus macaque CYP3A8(4) and human CYP3A4 is conserved well between macaque and human. Induction of CYP3A8(4) expression in macaque liver by estradiol, therefore, raises the possibility that CYP3A4 is also regulated by estradiol in human liver.…”

Section: Estradiol-regulated Drug-metabolizing Enzymes In Macaquementioning

confidence: 99%

Effect of Estradiol on Gene Expression Profile in Cynomolgus Macaque Liver: Implications for Drug-Metabolizing Enzymes

Uno

Kito²

2011

Drug Metab Dispos

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ABSTRACT:Estrogen regulation of gene expression is essential for physiological function of estrogen-responsive tissues, such as mammary glands, ovaries, and the uterus. In the liver, estrogen is responsible for sex-dependent gene expression of drug-metabolizing enzymes in rodents. However, the influence of estrogen on hepatic gene expression has not been fully investigated in primates, including human. Macaque, including cynomolgus macaque, is an important species for comparative studies aimed at understanding human physiology due to its evolutionary closeness to human. To identify estrogen-responsive genes in primate liver, therefore, hepatic gene expression was compared, by microarray analysis, in ovariectomized cynomolgus macaques treated with estradiol or solvent (control). The analysis identified 98 estradiol-responsive genes; 47 and 51 were up-and down-regulated by estradiol, respectively (>2.0-fold, P < 0.05). Expression of drug-metabolizing enzyme genes was also influenced by estradiol treatment; estradiol enhanced expression of GSTM5 (3.8-fold, P < 0.05) and CYP3A8(4) (2.7-fold, P < 0.01), but lowered expression of CYP4F12 (2.2-fold, P < 0.01), as verified by quantitative polymerase chain reaction. In particular, CYP3A8(4), orthologous to human CYP3A4, is an essential drug-metabolizing enzyme in cynomolgus macaque liver. These results suggest that expression of hepatic genes, including drug-metabolizing enzyme genes, is at least partly regulated by estradiol in cynomolgus macaque.

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Evaluation of Cynomolgus Monkey Pregnane X Receptor, Primary Hepatocyte, and in Vivo Pharmacokinetic Changes in Predicting Human CYP3A4 Induction

Cited by 40 publications

References 25 publications

Alprazolam as an In Vivo Probe for Studying Induction of CYP3A in Cynomolgus Monkeys

Alprazolam as an In Vivo Probe for Studying Induction of CYP3A in Cynomolgus Monkeys

Differentiation of Monkey Embryonic Stem Cells to Hepatocytes by Feeder-Free Dispersion Culture and Expression Analyses of Cytochrome P450 Enzymes Responsible for Drug Metabolism

Effect of Estradiol on Gene Expression Profile in Cynomolgus Macaque Liver: Implications for Drug-Metabolizing Enzymes

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