Application of a Higher Throughput Approach to Derive Apparent Michaelis-Menten Constants of Isoform-Selective P450-Mediated Biotransformation Reactions in Human Hepatocytes
Albert P. Li,
Kari E. Schlicht.
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 Vmax and KM 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” KM
value was calculated for each of the pathways via normalizing the individual activities to their respective Vmax 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.
Keywords: Amodiaquine, bupropion, cryopreservation, dextromethorphan, diclofenac, high throughput mass spectrometry,
human hepatocytes, michaelis-menton constants, midazolam, P450 isoforms, P450 metabolism, RapidFire, tacrine.
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