Development of a method to determine cytochrome P450 1A2, 2C9, 2D6 and 3A4 activity sheep hepatic microsomes

Grace M McBride; Jia Yin Soo; Tamara Varcoe; Janna L Morrison; Michael D Wiese

doi:10.1016/j.vascn.2020.106934

Development of a method to determine cytochrome P450 1A2, 2C9, 2D6 and 3A4 activity sheep hepatic microsomes

J Pharmacol Toxicol Methods. 2020 Nov-Dec:106:106934. doi: 10.1016/j.vascn.2020.106934. Epub 2020 Oct 18.

Authors

Grace M McBride¹, Jia Yin Soo¹, Tamara Varcoe¹, Janna L Morrison¹, Michael D Wiese²

Affiliations

¹ Early Origins of Adult Health Research Group, Australia; UniSA: Clinical and Health Sciences, University of South Australia, Adelaide 5000, Australia.
² UniSA: Clinical and Health Sciences, University of South Australia, Adelaide 5000, Australia. Electronic address: Michael.Wiese@unisa.edu.au.

PMID: 33080390
DOI: 10.1016/j.vascn.2020.106934

Abstract

Introduction: Ex vivo studies of human fetal hepatic drug metabolism are uncommon as it requires access to functional liver tissue and therefore raises practical and ethical concerns. Large animal models provide an alternative opportunity to study changes in cytochrome P450 (CYP) activity in the mother and fetus during pregnancy. We aimed to develop methods to determine the activity of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in sheep hepatic microsomes.

Methods: We identified optimal conditions to determine the activity of CYP1A2 (using the probe drug phenacetin), CYP2C9 (diclofenac), CYP2D6 (dextromethorphan) and CYP3A4 (midazolam) by varying techniques for microsome extraction, probe drug concentration, incubation time and microsome concentration. The specificity of each probe drug was assessed by determining the rate of metabolism when specific CYP enzyme inhibitors were included in the reaction.

Results: The optimum incubation time and probe drug concentration was six hours with 5 μM phenacetin (CYP1A2), four hours with 10 μM diclofenac (CYP2C9), 30 min with 1 μM of midazolam (CYP3A4) and 10 min with 1 μM dextromethorphan (CYP2D6). For both CYP2D6 and CYP3A4 reactions required 20 μg of microsomal protein, whereas for CYP1A2 and CYP2C9, reactions required 40 μg of microsomal protein. Metabolism of phenacetin, dextromethorphan and midazolam was reduced by specific enzyme inhibitors, but the specific CYP2C9 inhibitor sulfaphenazole did not substantially inhibit diclofenac metabolism.

Discussion: This study identifies the optimal conditions for determining CYP activity in maternal sheep hepatic microsomes. In doing so, we have developed a standardised protocol for assessment of microsomal activity of CYP3A4, CYP1A2 and CYP2D6, but we were unable to optimise conditions for assessment of CYP2C9. This approach can be applied to investigate the impact of pregnancy complications on maternal and fetal hepatic drug metabolism.

Keywords: CYP1A2; CYP2C9; CYP2D6; CYP3A4; Cytochrome P450; Drug metabolism; Fetal; Microsomes; Sheep.

MeSH terms

Animals
Cell Fractionation / methods
Cytochrome P-450 Enzyme Inhibitors / pharmacology*
Cytochrome P-450 Enzyme System / analysis
Cytochrome P-450 Enzyme System / metabolism*
Dextromethorphan / pharmacokinetics
Diclofenac / pharmacokinetics
Dose-Response Relationship, Drug
Enzyme Assays / methods*
Feasibility Studies
Female
Maternal-Fetal Exchange
Microsomes, Liver / drug effects
Microsomes, Liver / enzymology*
Midazolam / pharmacokinetics
Phenacetin / pharmacokinetics
Pregnancy
Pregnancy Complications / drug therapy
Pregnancy Complications / metabolism*
Sheep

Substances

Cytochrome P-450 Enzyme Inhibitors
Diclofenac
Dextromethorphan
Cytochrome P-450 Enzyme System
Phenacetin
Midazolam