Cell-to-Medium Concentration Ratio Overshoot in the Uptake of Statins by Human Hepatocytes in Suspension, but Not in Monolayer: Kinetic Analysis Suggesting a Partial Loss of Functional OATP1Bs

Abstract

Suspended human hepatocytes (SHH) have long been used in assessing hepatic drug uptake, while plated human hepatocytes in short-term monolayer culture (PHH) have gained use in recent years. This study aimed to cross-evaluate SHH and PHH in measuring the hepatic uptake mediated by organic anion transporting polypeptide 1Bs (OATP1Bs). We compared the time courses of cell-to-medium (C/M) concentration ratios and initial uptake clearance values of the OATP1B substrates (pitavastatin, rosuvastatin, cerivastatin, pravastatin, dehydropravastatin, and SC-62807) between SHH and PHH. For all compounds except cerivastatin, the C/M ratios in SHH displayed an apparent overshoot (an initial increase followed by a decrease) during the 180-min uptake experiment, but not in PHH. Based on the literature evidence suggesting the possible internalization of OATP1Bs in primary hepatocytes, separate experiments measured the drug uptake after varying lengths of pre-incubation in the drug-free medium. The initial uptake clearances of pitavastatin and rosuvastatin declined in SHH beyond an apparent threshold time of 20-min drug-free pre-incubation, but not in PHH. Kinetic modeling quantitatively captured the decline in the active uptake clearance in SHH, and more than half of the active uptake clearances of pitavastatin and rosuvastatin were prone to loss during the 180-min uptake experiment. These results suggested a partial, time-delayed loss of the functional OATP1Bs in SHH upon prolonged incubation. Our results indicate that PHH is more appropriate for experiments where a prolonged incubation is required, such as estimation of unbound hepatocyte-to-medium concentration ratio (Kp,uu) at the steady-state.

Evaluation of Hepatic Uptake of OATP1B Substrates by Short Term-Cultured Plated Human Hepatocytes: Comparison With Isolated Suspended Hepatocytes

“…For the evaluation of drug uptake, drug concentrations (0.1 mM for CRV, PTV, and RSV, 2 mM for PRV and DHP, and 0.2 mM for SC-62807) were selected to be lower than the reported Michaelis constant values in the literature; 6e18.3, 1.3e11.5, 0.8e15.3, 27.0e76.8, 6.5e17.1, and 19.8e260 mM for CRV, PTV, RSV, PRV, DHP, and SC-62807, respectively. 18 The uptake was initiated by the addition of 0.5 mL of the cassette dosing solution to the plated cells and co-incubated for varying length of time. The reactions were terminated by washing the cells three times with ice-cold Hank's balanced salt solution buffer.…”