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% bovine serum albumin (unbound fractions of 0.89 Ϯ 0.07 and 0.98 Ϯ 0.09, respectively), and there was partitioning of M into red blood cells. Transport studies of M (Ͻ750 M) showed similar, concentration-independent uptake clearances (CLs) of 1.5 ml min Ϫ1 g Ϫ1 among zonal and homogeneous, isolated rat hepatocytes. Transport of M3G, ascertained in multiple indicator dilution studies at various steady-state M3G concentrations (10 -262 M), uncovered a low and concentration-independent influx clearance (Ͻ10% of flow rate). The outflow dilution curve of [ 3 H]M3G was superimposable onto that of [ 14 C]sucrose, the extracellular reference, displaying similarity in transit times (23.5 and 22.2 s), negligible biliary excretion, and almost complete dose recovery from perfusate. In contrast, M3G occurred 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 -474 M), 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 M and V max,met of 58.8 nmol min Ϫ1 g Ϫ1 for metabolism, and a K m,ex of 41.2 M and V max,ex of 8.1 nmol min Ϫ1 g Ϫ1 for excretion. Sinusoidal transport was not rate-limiting for M removal.

Metabolite Kinetics: The Segregated Flow Model for Intestinal and Whole Body Physiologically Based Pharmacokinetic Modeling to Describe Intestinal and Hepatic Glucuronidation of Morphine in Rats In Vivo

“…Several assumptions were made: deglucuronidation of M was absent and reabsorption of MG was absent (Doherty et al, 2006) but not through the intestine membrane for secretion .…”

“…MG is excreted from formation tissues; enterohepatic circulation in rats has been noted (Dahlström and Paalzow, 1978;Horton and Pollack, 1991) but not for the rat with an open bile fistula. The influx permeability clearance of MG through the liver (0.1 ml·min 21 ·g 21 liver) was estimated to be 5%-10% of the flow rate, suggesting the existence of a diffusional barrier for MG to enter the hepatocyte (Doherty et al, 2006). Intestinally formed MG undergoes luminal secretion via the multiple drug resistance-associated protein 2 (Mrp2) and is effluxed into the circulation via the multiple drug resistance-associated protein 3 (Mrp3), in the rat (van de Wetering et al, 2007).…”

“…We studied M, which enters the cell by passive diffusion (Doherty et al, 2006) and is primarily glucuronidated at the 3-position to form morphine 3b-glucuronide (MG) by the rat UDP-glucuronosyltransferase 2 family, polypeptide b1, Ugt2b1, in the rat intestine and liver (Rane et al, 1985). Morphine is also known to be metabolized by cytochrome P450 (Projean et al, 2003) and undergoes excretion via P-glycoprotein (Böerner 1975;Iwamoto and Klaassen, 1977;Letrent et al, 1999;Wandel et al, 2002) to minor extents.…”

The “Albumin Effect” and in Vitro-in Vivo Extrapolation: Sequestration of Long-Chain Unsaturated Fatty Acids Enhances Phenytoin Hydroxylation by Human Liver Microsomal and Recombinant Cytochrome P450 2C9

“…Nonspecific microsomal binding, variability in experimental conditions and physiological scaling factors, and inappropriate kinetic modeling all affect the reliability of in vitro-in vivo extrapolation , but significant underprediction remains even when these factors are taken into account. Similarly, involvement of hepatic uptake transporters potentially accounts for the improved predictivity of kinetic data generated with hepatocytes, although recent data suggest that hepatic uptake is not rate-limiting in the clearance of lipophilic drugs (Doherty et al, 2006;Halifax and Houston, 2006).The addition of bovine serum albumin (BSA) to incubations of HLMs has been reported to increase the rate of metabolism of CYP2C9 substrates (Ludden et al, 1997;Carlile et al, 1999;Tang et al, 2002;Zhou et al, 2004) and microsomal CL int values of UGT2B7 substrates (Uchaipichat et al, 2006;Rowland et al, 2007 19885/3327892 DMD 36:870-877, 2008 Printed in U.S.A. …”

Prediction of Altered Bile Acid Disposition Due to Inhibition of Multiple Transporters: An Integrated Approach Using Sandwich-Cultured Hepatocytes, Mechanistic Modeling, and Simulation

“…However, albumin at physiologic concentrations [e.g., 4% bovine serum albumin (BSA)] (Doherty et al, 2006;Wolf et al, 2008) has not been added routinely into in vitro experimental systems, such as membrane vesicles, to study transporter-based interactions and assess IC 50 or K i values. In addition, according to the free drug hypothesis, the inhibitory effect is driven by the local unbound concentration of inhibitor, which is the cytosolic unbound inhibitor concentration ([I] u,cyt ) for efflux transporters, and the medium unbound inhibitor concentration ([I] u,med ) for uptake transporters (Smith et al, 2010).…”