Title: Determination of OATP-, NTCP- and OCT-mediated substrate uptake activities in individual and pooled batches of cryopreserved human hepatocytes
Authors: De Bruyn, Tom
Ye, Zhiwei
Peeters, Annelies
Sahi, Jasminder
Baes, Myriam
Augustijns, Patrick
Annaert, Pieter # ×
Issue Date: 2011
Publisher: American Society for Pharmacology and Experimental Therapeutics
Series Title: European Journal of Pharmaceutical Sciences vol:43 issue:4 pages:297-307
Abstract: While the utility of cryopreserved human hepatocyte suspensions (CHHS) for in vitro drug metabolism assays has been established, less is known about the effects of cryopreservation on transporter activity in human hepatocytes. In the present study, the activities of NTCP (sodium taurocholate co-transporting polypeptide; SLC10A1), as well as of the hepatic OATP (organic anion transporting polypeptide; SLCO gene family) and OCT (organic cation transporter; SLC22A) isoforms were assessed in 14 individual and four pooled batches of CHHS. For comparative purposes, substrate accumulation rates were also measured in sandwich-cultured human hepatocytes.
In CHHS, the mean accumulation clearance of the NTCP substrate taurocholate (1 lM) was 27.5 (±15.0) µl/min/million cells and decreased by 10-fold when extracellular sodium was replaced by choline. The accumulation clearance of digoxin and of the OATP substrates estrone-3-sulfate and estradiol-17b-D-glucuronide (E2-17b-G; 1µM) amounted to 9.5 (±4.9), 99 (±67) and 5.2 (±2.6)µl/min/ million cells, respectively. Presence of the known OATP inhibitor rifampicin (25µM) significantly (p < 0.01) decreased the accumulation of estrone-3-sulfate and E2-17b-G to 48% and 70% of the control value, respectively, while no significant effect on digoxin accumulation was observed. The mean accumulation clearance of the OCT substrate 1-methyl-4-phenylpyridinium amounted to 19.8 (±10.9) µl/min/million cells. Co-incubation with the OCT1 inhibitor prazosin (3 lM) and the OCT3 inhibitor corticoste- rone (1 µM) resulted in a significant (p < 0.01) decrease to 72% and 85% of the accumulation in control conditions, respectively. Experiments in pooled CHHS generally showed accumulation values that were comparable with the mean of the individual batches. A good correlation (R2 = 0.93) was observed between estrone-3-sulfate accumulation values and OATP1B3 mRNA levels, as determined in five batches of CHHS. Compared to substrate accumulation measured in sandwich-cultured human hepatocytes, accu- mulation values in CHHS were comparable (taurocholate and digoxin) to slightly higher (estrone-3-sul- fate). Our data indicate that cryopreserved human hepatocyte suspensions are a reliable in vitro model to study transporter-mediated substrate uptake in the liver. Systematic characterization of multiple batches of CHHS for transporter activity supports rational selection of human hepatocytes for specific applications.
ISSN: 0928-0987
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Drug Delivery and Disposition
Cell Metabolism
× corresponding author
# (joint) last author

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