Differential modulation of cytochrome P450 activity and the effect of 1-aminobenzotriazole on hepatic transport in sandwich-cultured human hepatocytes.
Sandwich-cultured human hepatocytes (SCHH) have been widely used for in vitro assessments of biliary clearance. However, the modulation of metabolism enzymes has not been fully evaluated in this system. The present study was therefore undertaken to determine the activity of cytochrome P450 (P450) 1A2, 2C8, 2C9, 2C19, 2D6, and 3A and to evaluate the impact of 1-aminobenzotriazole (ABT) on hepatic uptake and biliary excretion in SCHH. The SCHH maintained integrity and viability as determined by lactate dehydrogenase release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays conducted over the culture period. Although all assessed P450 activity decreased in day 2 SCHH, the extent of the decrease and the subsequent rebound in activity varied across the different isoforms. Day 5 CYP1A2 activity was approximately 2.5-fold higher than day 1 activity, whereas the CYP3A and CYP2C9 activities were 90 and 60% of the day 1 levels, respectively. In contrast, the initial CYP2C8, CYP2C19, and CYP2D6 activity losses did not rebound over the 5-day culture period. Furthermore, ABT was not found to have an effect, whether directly or indirectly as a P450 inactivator, with respect to the hepatic transport of rosuvastatin, atrovastatin, and midazolam in SCHH. Taken together, these results suggest that the SCHH model is a reliable tool to characterize hepatic uptake and biliary excretion. Due to the differential modulation of P450 activity, SCHH may not be considered a suitable tool for metabolic stability assessments with compounds predominantly cleared by certain P450 enzymes.
Pfizer Global Research and Development, Groton, Connecticut, USA.
Emi
E
Kimoto
Robert
R
Walsky
Hui
H
Zhang
Yi-an
YA
Bi
Kevin M
KM
Whalen
Young-Sun
YS
Yang
Collette
C
Linder
Yongling
Y
Xiao
Ken
K
Iseki
Katherine S
KS
Fenner
Ayman F
AF
El-Kattan
Yurong
Y
Lai
D014151:Anti-Anxiety Agents / Q000378:metabolism; D000924:Anticholesteremic Agents / Q000378:metabolism; D000069059:Atorvastatin; D001646:Bile / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002470:Cell Survival; D002478:Cells, Cultured; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005464:Fluorobenzenes / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D006538:Heptanoic Acids / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D008874:Midazolam / Q000378:metabolism; D008954:Models, Biological; D011743:Pyrimidines / Q000378:metabolism; D011758:Pyrroles / Q000378:metabolism; D000068718:Rosuvastatin Calcium; D013449:Sulfonamides / Q000378:metabolism; D013997:Time Factors; D014230:Triazoles / Q000494:pharmacology*
D014151:Anti-Anxiety Agents; D000924:Anticholesteremic Agents; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D005464:Fluorobenzenes; D006538:Heptanoic Acids; D007527:Isoenzymes; D011743:Pyrimidines; D011758:Pyrroles; D013449:Sulfonamides; D014230:Triazoles; C033020:1-aminobenzotriazole; D000068718:Rosuvastatin Calcium; D003577:Cytochrome P-450 Enzyme System; D000069059:Atorvastatin; D008874:Midazolam