Pitfalls in Predicting Hepatobiliary Drug Transport Using Human Sandwich-Cultured Hepatocytes

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.