Mathematical Modeling of the in Vitro Hepatic Disposition of Mycophenolic Acid and Its Glucuronide in Sandwich-Cultured Human Hepatocytes

Abstract

In recent years, it has become increasingly important to test the safety of circulating metabolites of novel drugs as part of drug discovery and development programs. Accordingly, it is essential to develop suitable methods for identifying the major metabolites and their disposition in animal species and in humans. Mycophenolic acid (MPA), a selective inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitor, is metabolized by glucuronidation and enterohepatic circulation of MPA-glucuronides is an important factor in the continuous systemic exposure of MPA. In humans, about 90% of the administered MPA dose is finally excreted as MPA phenyl-glucuronide (MPAG) in urine. Notably, the plasma concentration of MPAG is much higher than that of MPA. These factors suggest that, after its formation in hepatocytes, MPAG is excreted into bile and is also transported across the basolateral membrane to enter the circulation. In the present study, we performed metabolic/hepatobiliary transport studies of MPA and MPAG using sandwich-cultured human hepatocytes (SCHH) and constructed mathematical models of their hepatic disposition. We also performed vesicular transport studies to identify which human multidrug resistance-associated proteins (MRPs) are involved in the transport of MPAG from hepatocytes. MPAG was a preferred substrate for the biliary excretion transporter MRP2 and the hepatic basolateral transporters MRP3 and MRP4 in conventional and metabolic/hepatobiliary transport studies using SCHH and vesicular transport studies using human MRP-expressing membrane vesicles. The resulting mathematical model suggested that the basolateral transport plays an important role in the hepatic disposition of MPAG formed in hepatocytes. Our findings suggest that mathematical modeling of metabolic/hepatobiliary transport studies using SCH will provide useful information for determining the fate of metabolites formed in hepatocytes.

Role of Multidrug Resistance–Associated Protein 4 in the Basolateral Efflux of Hepatically Derived Enalaprilat

“…Recently, Pfeifer et al (2013a,b) and Matsunaga et al (2014) demonstrated the importance of the interplay between basolateral and canalicular efflux in the systemic and hepatic disposition of rosuvastatin and mycophenolic acid glucuronide, respectively. Extensive biliary excretion of rosuvastatin and mycophenolic acid glucuronide required mathematical modeling to deconvolute the contribution of basolateral versus canalicular transport.…”

Analysis of the Metabolic Pathway of Bosentan and of the Cytotoxicity of Bosentan Metabolites Based on a Quantitative Modeling of Metabolism and Transport in Sandwich-Cultured Human Hepatocytes

“…Cryopreserved human hepatocytes were thawed, plated, and cultured as described in our previous study (Matsunaga et al, 2014), and human SCHs on day 4 were used. Modulation of Ca 2+ in the incubation buffer was applied to maintain or disrupt the canalicular networks between cells sealed by tight junctions.…”

“…Using this approach, the masses of compounds in the bile canalicular lumen can be measured with Ca 2+ -containing and Ca 2+ -free buffers (B-CLEAR technology; Qualyst Transporter Solutions, Durham, NC) (Brouwer et al, 2013). Metabolism and hepatobiliary transport studies of human SCHs were performed as described in our previous report (Matsunaga et al, 2014). In brief, human SCHs were incubated with Ca 2+ -containing or Ca 2+ -free buffer containing bosentan at an initial concentration of 1 mM at 37°C for 3, 5, 10, 15, 20, 30, or 45 minutes.…”

“…Furthermore, model-based concepts have recently become widespread in various research fields, including drug discovery and development. Several model-based analyses have been undertaken for the hepatic disposition of drugs and their metabolites in human SCHs to predict intracellular concentration changes by alterations in hepatobiliary transport, to investigate rate-limiting transport processes, and to evaluate drug-drug interaction at clinically relevant concentrations (Lee et al, 2010;Matsunaga et al, 2014Matsunaga et al, , 2015.…”

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

“…To overcome this limitation, mechanistic pharmacokinetic modeling coupled with data from sandwichcultured hepatocytes has been used to deconvolute the relative contribution of various clearance (CL) pathways to the disposition of rosuvastatin, mycophenolic acid, and 3 H-taurocholic acid (TCA) (Pfeifer et al, 2013c;Matsunaga et al, 2014;Yang et al, 2015). Transporters are expressed and properly localized in the sandwich-cultured hepatocyte system, which can be used to assess the function of multiple transporters (Yang et al, 2016).…”