Application of a Loading Wash-out Method for Investigating the Hepatocellular Efflux of a Hepatically-generated Metabolite, Morphine-3-glucuronide

Abstract

Previous studies using the rat isolated perfused liver demonstrated that the hepatic disposition of morphine-3-glucuronide is membrane permeability-rate limited, and that the movement of the metabolite across hepatic sinusoidal and canalicular membranes is partly via carrier-mediated transport systems. As a consequence of the membrane permeability-limitation, the biliary excretion of hepatically-generated morphine-3-glucuronide is much more efficient than that of morphine-3-glucuronide reaching the liver via the vasculature. We have quantitated the cellular efflux kinetics (cell-to-blood and cell-to-bile) of morphine-3-glucuronide in the rat isolated perfused liver using a loading wash-out design. In the ‘loading’ phase, morphine was infused into the liver (2.7 μM) and the biliary excretion and sinusoidal efflux of morphine-3-glucuronide was assessed under steady-state conditions. Subsequently, the infusion was stopped and the concentration vs time profile of morphine-3-glucuronide in outflow perfusate (the wash-out phase) was determined. A physiologically-based pharmacokinetic model was used to determine the rate-constants for the movement of hepatically-generated morphine-3-glucuronide into the sinusoidal and canalicular spaces of the liver, and the associated membrane permeability terms.The mean (±s.d.) rate constants for the biliary excretion and sinusoidal efflux of morphine-3-glucuronide were determined to be 0.160±0.043 and 0.169 ± 0.068 min−1, respectively, and the corresponding membrane permeability parameters were 1.12 and 1.18 mL min−1, respectively. The sinusoidal membrane permeability term was significantly less than hepatic blood flow in the rat. The volume of distribution of hepatically-generated morphine-3-glucuronide (207.5 ± 74.8 mL) was found to be approximately 50-times the intracellular space of the rat liver, suggesting that hepatically-generated morphine-3-glucuronide accumulates within hepatocytes. The results indicate that hepatically-generated morphine-3-glucuronide undergoes intracellular accumulation, probably as a consequence of poor membrane permeability.