Evaluating variations in bilirubin glucuronidation activity by protease inhibitors in canine and human primary hepatocytes cultured in a 3D culture system.

Bilirubin is excreted into the bile from hepatocytes, mainly as monoglucuronosyl and bisglucuronosyl conjugates, reflecting bilirubin glucuronidation activity. However, there is limited information on the in vitro evaluation of liver cell lines or primary hepatocytes. This study aimed to investigate variations in the bilirubin metabolic function of canine and human hepatocyte spheroids formed in a three-dimensional (3D) culture system indicated by the formation of bilirubin glucuronides when protease inhibitors such as atazanavir, indinavir, ritonavir, and nelfinavir were treated with bilirubin. The culture supernatant was collected for bilirubin glucuronidation assessment and the cells were used to evaluate viability. On day 8 of culture, both canine and human hepatocyte spheroids showed high albumin secretion and distinct spheroid formation, and their bilirubin glucuronidation activities were evaluated considering cell viability. Treatment with atazanavir and ritonavir remarkably inhibited bilirubin glucuronide formation, wherein atazanavir showed the highest inhibition, particularly in human hepatocyte spheroids. These results may reflect the effects on cellular uptake of bilirubin and its intracellular metabolic function. Thus, primary hepatocytes cultured in a 3D culture system may be a useful in vitro system for the comprehensive evaluation of bilirubin metabolic function and risk assessment in bilirubin metabolic disorders for drug development.

Comparative analysis of bilirubin glucuronidation activity in canine and human primary hepatocytes using a 3D culture system.

Species differences in bilirubin glucuronidation activity are observed between humans and dogs through liver microsomes and recombinant UDP-glucuronosyltransferase 1A1. Humans exhibit higher activity than that of dogs. In this study, bilirubin glucuronidation activity was examined in canine and human primary hepatocyte spheroids formed using a 3D culture system. When spheroid development in canine and human primary hepatocytes was evaluated on days 7 and 14 after the start of culture, canine primary hepatocyte spheroids had a more distinct spherical shape than human hepatocyte spheroids, irrespective of the culture period. Furthermore, mono- and di-glucuronide generation detected in spheroids were significantly higher (P < 0.05) in human primary hepatocytes than in canine primary hepatocytes after 24 h of incubation with bilirubin for each culture period. These results suggest that there are species differences in the bilirubin glucuronidation activity of primary hepatocytes with spheroid formation between humans and dogs, with the activity being higher in humans than in dogs.

Effect of dimethylnitrosamine-induced liver dysfunction on the pharmacokinetics of 5-fluorouracil after administration of S-1, an antitumour drug, to rats.

OBJECTIVES: The anti-tumour agent S-1 comprises tegafur (a prodrug of 5-fluorouracil; 5-FU), gimeracil (2-chloro-2,4-dihydroxypyridine (CDHP); a competitive inhibitor of 5-FU metabolism) and oteracil potassium. The effect of hepatic dysfunction induced by dimethylnitrosamine (DMN) on the pharmacokinetics of 5-FU after administration of S-1 to rats was investigated. METHODS: S-1 (5 mg/kg) was administered intravenously and orally to rats with DMN-induced liver dysfunction. Plasma concentrations of S-1 components and 5-FU were measured by HPLC and LC/MS-MS. Blood tests and in-vitro enzymatic investigations were also conducted. KEY FINDINGS: DMN treatment induced hepatic dysfunction and decreased the conversion of tegafur to 5-FU in the liver without altering renal function or dihydropyrimidine dehydrogenase activity. Following intravenous administration of S-1, the blood concentration-time profiles of CDHP were similar between control rats and rats with hepatic dysfunction, but the half-life of tegafur was significantly prolonged. The maximum plasma concentration (C(max)) of 5-FU was significantly reduced and the area under the blood concentration-time curve (AUC) was reduced by 22%. Following oral administration, the C(max) of tegafur, 5-FU and CDHP were significantly decreased and half-lives significantly increased. Hepatic dysfunction had a less pronounced effect on the AUC of 5-FU (13.6% reduction). CONCLUSIONS: The pharmacokinetic profiles of tegafur, 5-FU and CDHP were altered by changes in the elimination rate of tegafur induced by a decrease in the conversion of tegafur to 5-FU. However, hepatic dysfunction had less of an effect on the AUC of 5-FU, which correlates with anti-tumour effect, after the oral administration of S-1.