Abstract
During drug development, in vivo human biliary drug clearances (CL) are usually predicted using human sandwich-cultured hepatocytes (SCH). To do so, SCH are pre-incubated with Ca2+-containing or Ca2+-free buffer to maintain or disrupt canalicular tight junctions (CTJ), respectively. Drug uptake into SCH is then conducted in the presence of Ca2+ (up to 20 min). Under this standard protocol, two key assumptions are made: first, that the CTJ are not reformed during the uptake phase when Ca2+ is repleted, and second, disruption of CTJ by the Ca2+-free buffer does not affect the activity of any of the transporters present in the sinusoidal or canalicular membrane. Here we investigated the validity of these assumptions using rosuvastatin (RSV) and taurocholic acid (TCA) as our model drugs. In human SCH, the disrupted CTJ were “reformed” with just 10-min Ca2+ repletion as reflected in a significant increase in TCA cell accumulation. To avoid CTJ reformation and cell toxicity, the standard SCH protocol was modified by conducting the uptake in the absence of Ca2+ for 10 min. Surprisingly, using this protocol, RSV uptake into SCH, plated hepatocytes, and transporter-expressing cells confirmed that Ca2+ depletion substantially decreased NTCP and not OATP1B1 activity. Collectively, this study provides the first evidence of reformation of CTJ in human SCH with 20-min Ca2+ repletion, whereas Ca2+ depletion, during the uptake phase, leads to a significant reduction in NTCP uptake. Thus, the entire SCH protocol needs to be re-examined and optimized to correctly estimate hepatobiliary CL of drugs including those that are NTCP substrates.
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Abbreviations
- BCA:
-
Bicinchoninic acid assay
- BCRP:
-
Breast cancer resistance protein
- CL:
-
Clearance
- HBSS:
-
Hank’s balanced salt solution
- LC-MS/MS:
-
Liquid chromatography tandem mass spectrometry
- LDH:
-
Lactate dehydrogenase
- NTCP:
-
Sodium-taurocholate co-transporting polypeptide
- OATP:
-
Organic anion transporting polypeptide
- RSV:
-
Rosuvastatin
- SCH:
-
Sandwich-cultured hepatocytes
- SDS:
-
Sodium dodecyl sulfate
- TCA:
-
Taurocholic acid
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Acknowledgments
The authors thank Tot Bui Nguyen for her support in human hepatocytes and transporter-expressing cell line culture. We thank BioIVT for providing the human hepatocytes.
Funding
This work was supported by University of Washington Research Affiliate Program on Transporters (UWRAPT) through funding from Gilead Sciences, Amgen, Biogen, Genentech, Merck, Pfizer, and Takeda. VK was supported in part by the Simcyp Grant & Partnership Scheme.
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EK and ZG are employees of SOLVO Biotechnology, Budapest, Hungary. KI is an employee of Gilead Sciences.
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Kumar, V., Li, C.Y., Ishida, K. et al. Pitfalls in Predicting Hepatobiliary Drug Transport Using Human Sandwich-Cultured Hepatocytes. AAPS J 22, 110 (2020). https://doi.org/10.1208/s12248-020-00496-3
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DOI: https://doi.org/10.1208/s12248-020-00496-3