Application of a Higher Throughput Approach to Derive Apparent Michaelis-Menten Constants of Isoform-Selective P450-Mediated Biotransformation Reactions in Human Hepatocytes

Title:Application of a Higher Throughput Approach to Derive Apparent Michaelis-Menten Constants of Isoform-Selective P450-Mediated Biotransformation Reactions in Human Hepatocytes

Volume: 8 Issue: 1

Author(s): Albert P. Li and Kari E. Schlicht

Affiliation:

Keywords: Amodiaquine, bupropion, cryopreservation, dextromethorphan, diclofenac, high throughput mass spectrometry, human hepatocytes, michaelis-menton constants, midazolam, P450 isoforms, P450 metabolism, RapidFire, tacrine.

Abstract: A higher throughput platform was developed for the determination of KM values for isoformselective P450 substrates in human hepatocytes via incubation of the hepatocytes with substrates in 384- well plates and metabolite quantification by RapidFire™ mass spectrometry. Isoform-selective P450 substrates were incubated at 8 concentrations in triplicate with cryopreserved human hepatocytes from 16 donors. The metabolic pathways examined were the CYP1A2-catalyzed tacrine 1-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C9- catalyzed diclofenac 4'-hydroxylation, CYP2D6-catalyzed dextromethorphan O-demethylation, and CYP3A4-catalyzed midazolam 1'-hydroxylation. Typical saturation enzyme kinetics was observed for all the pathways evaluated. Individual differences in the apparent V_max and K_M values were observed among the human hepatocytes from each of the 16 individual donors, with no statistically significant gender- or age-associated differences. A “composite” K_M value was calculated for each of the pathways via normalizing the individual activities to their respective V_max values to develop “relative activities” followed by Michaelis-Menten analysis of the mean relative activities of the 16 donors at each of the 8 substrate concentrations. The resulting “composite” KM values for the P450 substrates may be used to guide in vitro P450 inhibition and induction studies and kinetic modeling of in vivo drug-drug interaction.

Biography: Dr. Albert Li (Ph. D., Biomedical Sciences) is CEO and President of In Vitro ADMET Laboratories, Columbia, MD and Malden, MA. He is a pioneer in hepatocyte cryopreservation and application of hepatocytes in the assessment of ADMET drug properties. He has published over 160 scientific articles and edited 6 books.

Cite this article as:

Li P. Albert and Schlicht E. Kari, Application of a Higher Throughput Approach to Derive Apparent Michaelis-Menten Constants of Isoform-Selective P450-Mediated Biotransformation Reactions in Human Hepatocytes, Drug Metabolism Letters 2014; 8 (1) . https://dx.doi.org/10.2174/1872312808666140606101902