Species differences in sinusoidal and canalicular efflux transport of mycophenolic acid 7‐O‐glucuronide in sandwich‐cultured hepatocytes

Abstract

Metabolism and sinusoidal/canalicular efflux of mycophenolic acid (MPA) was investigated using sandwich-cultured hepatocytes (SCHs). After applying MPA to SCHs from humans, wild-type rats, and multidrug resistance-associated protein (Mrp) 2-deficient rats, the MPA metabolites 7-O-glucuronide (MPAG) and acyl glucuronide (AcMPAG) were detected in the intracellular compartment of the SCHs. Sinusoidal efflux of MPAG was detected in all SCH preparations including Mrp2-deficient rat SCHs, whereas canalicular efflux of MPAG was observed in wild-type rat and human SCHs but not in Mrp2-deficient rat SCHs. The ratio of canalicular efflux to net (canalicular plus sinusoidal) efflux was 37 ± 8% in wild-type rat SCHs, while the ratio in human SCHs was significantly lower (20 ± 2%, P < 0.05), indicating species differences in the direction of hepatic MPAG transport. This 20% ratio in human SCHs corresponds to a high sinusoidal MPAG efflux (80%) that can in part account for the urine-dominated recovery of MPAG in humans. Both sinusoidal and canalicular MPAG efflux in rat SCHs shows a good correspondence to urinary and biliary recovery of MPAG after MPA dosing. The sinusoidal efflux of AcMPAG in human SCHs was detected from one out of three donors, suggesting donor-to-donor variation. In conclusion, this study demonstrates the predictive value of SCHs for elucidating the interplay of metabolism and efflux transport, in addition to demonstrating a species difference between rat and human in sinusoidal and canalicular efflux of MPAG.

Glucuronidation and Subsequent Biliary Excretion of Mycophenolic Acid in Rat Sandwich-cultured Hepatocytes

“…In this regard, we have measured sinusoidal efflux of both MPA and MPAG in SCH. 31) Contrary to findings with MPAG, CysA did not change the AcMPAG BEI ( Table 2). This suggests that unique or additional transporter(s) are involved in biliary excretion of AcMPAG.…”

Recent Progress in Hepatocyte Culture Models and Their Application to the Assessment of Drug Metabolism, Transport, and Toxicity in Drug Discovery: The Value of Tissue Engineering for the Successful Development of a Microphysiological System

“…As an in vitro methodology for investigating the kinetic aspect of the sinusoidal efflux pathway, Pfeifer et al 65,91 developed a protocol for investigating the basolateral efflux of a compound in SCH and reported the significant contribution of this pathway in the hepatic disposition of rosuvastatin. In addition, Tetsuka et al 66 expanded this assay to allow the detection of continuous events of metabolism followed by basolateral and canalicular efflux of MPA. They showed both sinusoidal efflux and canalicular efflux of MPAG are detectable in rat SCH and human SCH, while canalicular efflux of the metabolite in SCH prepared from MRP2 knockout rat was not detectable (Fig.…”

“…Sinusoidal (basolateral) versus canalicular efflux of 7-O-glucuronide of MPA (MPAG) after applying MPA to sandwich-cultured hepatocytes. Adopted from Tetsuka et al 66 MPA was incubated with rat (a), Mrp2 knockout rat (b), and human (c) SCHs, and then the dosing solution was washed and replaced with fresh buffer with or without calcium. Adopted from Tetsuka et al 66 Assay shown in Figure 1 was further conducted in SCHs prepared from different donors.…”

“…Adopted from Tetsuka et al 66 MPA was incubated with rat (a), Mrp2 knockout rat (b), and human (c) SCHs, and then the dosing solution was washed and replaced with fresh buffer with or without calcium. Adopted from Tetsuka et al 66 Assay shown in Figure 1 was further conducted in SCHs prepared from different donors. The relative canalicular efflux of MPAG in rat SCHs ranged from 30%-45% and was significantly higher than that in human SCHs (19%-22%, p ¼ 0.019 by Student t-test), resulting in sinusoidal-oriented (78%-81%) MPAG efflux in human SCH.…”

Challenges and Opportunities with Predicting In Vivo Phase II Metabolism via Glucuronidation From In Vitro Data

“…And as shown in hepatocytes, the canalicular efflux of MPAG was mainly mediated by MRP2 (68). Furthermore, it had been found that MRP2 is the major transporter involved in the urinary excretion of MPAG (69).…”