title: Effect of cryopreservation on the activity of OATP1B1/3 and OCT1 in isolated human hepatocytes.
abstract: Drug metabolism in liver is the major pathway for xenobiotic elimination from the body. Access to intracellular metabolising enzymes is possible through passive diffusion of lipophilic drugs through cell membrane or active uptake of more polar drugs by specific uptake transporters. Organic Anion Transporting Polypeptides (OATP/SLCO) and Organic Cation Transporters (OCT/SLC22A) are among the most important transporters involved in xenobiotic transport into hepatocytes. Isolated hepatocytes are the model of choice for drug metabolism and drug transport investigations. These primary cells are used either as fresh directly after isolation from liver biopsies, or after subsequent cryopreservation in liquid nitrogen. While cryopreserved hepatocytes are a more convenient and flexible tool for in vitro investigations, information on the functionality of transporter activity after cryopreservation is still sparse. The present study investigated the effect of cryopreservation of human hepatocytes on the uptake of [(3)H]-estradiol-17β-glucuronide (E(2)17βG, substrate of OATP1B1/3/SLCO1B1/3) and [(3)H]-1-methyl-4-phenylpyridinium (MPP+, substrate of OCT1/SLC22A1) into hepatocytes from 6 and 5 human donors, respectively. The results showed that cryopreserved human hepatocytes display carrier-mediated uptake of E(2)17βG and MPP+. While the affinity of E(2)17βG for OATP1B1/3/SLCO1B1/3 was not affected by cryopreservation (Km unchanged, the Wilcoxon signed pair t test gave p=1), V(max) and CL(uptake) values decreased in average by 47% (p=0.06). The passive diffusion of E(2)17βG decreased significantly after cryopreservation (p=0.03). Cryopreservation did not affect Km, V(max) or the passive diffusion of MPP+ in human hepatocytes. In conclusion, the present study showed that cryopreserved human hepatocytes are useful tool to investigate hepatic uptake mediated by OATP1B1/3/SLCO1B1/3 or OCT1/SLC22A1, two of the most important hepatic uptake transporters.
authors.affiliation: Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Denmark. laba@lundbeck.com
authors.forename: Lassina
authors.initials: L
authors.lastname: Badolo
authors.forename: Marie Michèle
authors.initials: MM
authors.lastname: Trancart
authors.forename: Lena
authors.initials: L
authors.lastname: Gustavsson
authors.forename: Christophe
authors.initials: C
authors.lastname: Chesné
mesh_terms: D015655:1-Methyl-4-phenylpyridinium / Q000378:metabolism; D000328:Adult; D000368:Aged; D002478:Cells, Cultured; D015925:Cryopreservation; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism; D005260:Female; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D027381:Liver-Specific Organic Anion Transporter 1; D008297:Male; D008875:Middle Aged; D027361:Organic Anion Transporters / Q000378:metabolism*; D029362:Organic Anion Transporters, Sodium-Independent / Q000378:metabolism*; D027702:Organic Cation Transporter 1 / Q000378:metabolism*; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3
chemical_list: D027381:Liver-Specific Organic Anion Transporter 1; D027361:Organic Anion Transporters; D029362:Organic Anion Transporters, Sodium-Independent; D027702:Organic Cation Transporter 1; C503999:SLCO1B1 protein, human; C421209:SLCO1B3 protein, human; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; C025483:estradiol-17 beta-glucuronide; D004958:Estradiol; D015655:1-Methyl-4-phenylpyridinium