Received for publication December 27, 2005.
Revised February 2, 2006.
Accepted for publication February 2, 2006.

Transport Is Not Rate-Limiting in Morphine Glucuronidation In the Single Pass Perfused Rat Liver Preparation

Abstract

Binding, transport and metabolism are factors that influence morphine (M) removal in the rat liver. For M and the morphine 3-glucuronide metabolite (M3G), modest binding existed with 4% BSA (unbound fractions of 0.89 ± 0.07 and 0.98 ± 0.09, respectively), and there was partitioning of M into red blood cells. Transport studies of M (<750 µM) showed similar, concentration-independent uptake clearances of 1.5 ml.min^-1.g^-1 among zonal and homogeneous, isolated rat hepatocytes. Transport of M3G, ascertained in multiple indicator dilution (MID) studies at various steady-state M3G concentrations (10-262 µM), uncovered a low and concentration-independent influx clearance (< 10% of flow rate). The outflow dilution curve of [³H]M3G was superimposable onto that of [¹⁴C]sucrose, the extracellular reference, displaying similarity in transit times (23.5 s and 22.2 s), negligible biliary excretion, and almost complete dose-recovery from perfusate. In contrast, M3G appeared abundantly in both perfusate and bile in single pass perfusion studies of the precursor, M, and revealed a biliary clearance of formed M3G that was 12.3-fold that of preformed M3G, suggesting a sinusoidal, diffusional barrier for M3G. With increasing concentrations of M (9 to 474 µM), clearance decreased, and metabolism and biliary excretion displayed concentration-dependent kinetics. Fitting of the data to a physiologically-based liver model revealed that M removal mechanisms were saturable, with a K_m,met of 52.2 µM and V_max,met of 58.8 nmole.min^-1.g^-1 for metabolism, and a K_m,ex, of 41.2 µM and V_max,ex of 8.1 nmole.min^-1.g^-1 for excretion. Sinusoidal transport was not rate-limiting for M removal.

Key words: glucuronidation, liver model, liver perfusion, morphine, rat hepatocytes, transport

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