Pitfalls in Predicting Hepatobiliary Drug Transport Using Human Sandwich-Cultured Hepatocytes
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Research Article Pitfalls in Predicting Hepatobiliary Drug Transport Using Human Sandwich-Cultured Hepatocytes Vineet Kumar,1 Cindy Yanfei Li,1 Kazuya Ishida,1 Emese Kis,2 Zsuzsanna Gáborik,2 and Jashvant D. Unadkat1,3
Received 24 April 2020; accepted 1 August 2020 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 preincubated 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. KEY WORDS: hepatobiliary clearance; NTCP; rosuvastatin; sandwich-cultured hepatocytes; uptake transport.
INTRODUCTION Biliary drug clearance (CL) is a major route of drug elimination for many drugs (1). Accurate estimation of biliary drug clearance is important for predicting first in human dose, systemic clearance, and potential drug-drug interactions (2). Vineet Kumar and Cindy Yanfei Li contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1208/s12248-020-00496-3) contains supplementary material, which is available to authorized users. 1
Department of Pharmaceutics, University of Washington, P.O. Box 357610, Seattle, Washington 98195, USA. 2 SOLVO Biotechnology, Budapest, Hungary. 3 To whom correspondence should be addressed. (e–mail: [email protected]) 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 polypepti
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