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3-Hydroxybutyric Acid/metabolism; Biotransformation; Cells, Cultured; Cytochrome P-450 Enzyme System/metabolism; Diclofenac/administration & dosage/pharmacokinetics/toxicity; Drug-Related Side Effects and Adverse Reactions; Galactose/metabolism; Glucose/metabolism; Hepatocytes/drug effects/metabolism; Humans; Lactic Acid/metabolism; Liver/drug effects/metabolism; Metabolic Detoxication, Phase I/physiology; Metabolic Detoxication, Phase II/physiology; Metabolome/drug effects
[en] In vitro repeated dose testing for the assessment of chronic drug-induced effects is a huge challenge in preclinical pharmaceutical drug development. Chronic toxicity results in discontinuation of therapy or post-marketing withdrawal of drugs despite in vivo preclinical screening. In case of hepatotoxicity, due to limited long term viability and functionality of primary hepatocytes, chronic hepatic effects are difficult to detect. In this study, we maintained primary human hepatocytes in a serum-free cultivation medium for more than 3 weeks and analyzed physiology, viability and drug metabolizing capacities of the hepatocytes. Moreover, we assessed acute (24 h) diclofenac toxicity in a range of (10-1000 muM) concentrations. The chronic (9 repeated doses) toxicity at one clinically relevant and another higher concentration (6.4 and 100 muM) was also tested. We investigated phase I and II metabolism of diclofenac upon repeated dose exposure and analyzed effects on the cellular exometabolome. Acute 24 h assessment revealed toxicity only for the highest tested concentration (1 mM). Upon repeated dose exposure, toxic effects were observed even at a low, clinically relevant concentration (6.4 muM). Biotransformation pathways were active for 3 weeks and diclofenac-acylglucuronide was detected as the predominant metabolite. Dose dependent diclofenac-induced effects on exometabolome, such as on the production of lactate and 3-hydroxybutyric acid as well as glucose and galactose metabolism, were observed upon nine repeated doses. Summarizing, we show that repeated dose testing on long-term functional cultures of primary human hepatocytes may be included for the assessment of long term toxic effects in preclinical screening and can potentially help replace/reduce in vivo animal testing.