A Modified Procedure for Estimating the Impact of the Uptake on the Overall Biliary Clearance in Sandwich Culture of Rat Hepatocytes
Sandwich culture of hepatocytes is commonly applied for the prediction of in vivo biliary
clearance (CLbil). In this paper, we present a modified procedure for the determination of in vitro CLbil
in sandwich culture of rat hepatocytes, which allows the estimation of the impact of uptake processes
on the overall CLbil. The main point of this modification is the separation of uptake and efflux
processes. Ten drugs from four biopharmaceutics drug disposition classification system classes were chosen in order to
demonstrate the advantages of this method: 1) the uptake is performed identically before the canaliculi are opened, thus
the efflux starts at the same intracellular concentration of the drugs and the effect of Ca2+/Mg2+ depletion on the uptake is
excluded; 2) exact intracellular concentrations can be measured at the start and at the end of the efflux; 3) the biliary
clearance can be determined irrespective of the uptake; 4) the canalicular and the sinusoidal transport can be measured
simultaneously; 5) drug-drug interactions concerning uptake and efflux transporters can be estimated independently.
Depending on the degree of uptake, CLbil,app (calculated using the concentration of drugs in the medium) was significantly
higher (sulfasalazine, fluvastatin, rosuvastatin, atorvastatin) or lower (pravastatin, procainamide) than CLbil,int (calculated
using the intracellular concentration of drugs). When the uptake had no impact on the CLbil, the apparent and intrinsic
CLbil did not differ significantly (lovastatin, rifampicin, quetiapine). Our results confirm that transporters may play a
significant role in the uptake of drugs both with high and poor permeability and solubility.
Keywords: Efflux, hepatic clearance, hepatocyte sandwich culture, in vitro, transport proteins, uptake.
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