Diosmin and silibinin (SIL) are polyphenolic compounds which are the active components of several drugs and dietary supplements. After the oral administration of diosmin (flavonoid glycoside), only its aglycone diosmetin (DIO) reaches the systemic circulation. Both DIO and SIL form complexes with serum albumin and are able to inhibit several cytochrome P450 enzymes. Therefore, it is reasonable to hypothesize that these polyphenols may displace some drugs from serum albumin and inhibit their biotransformation, potentially leading to the disruption of drug therapy. In this study, the inhibitory action of DIO and SIL on CYP2C9-catalyzed metabolism of diclofenac to 4'-hydroxydiclofenac was examined, using warfarin as a positive control. Furthermore, interaction of DIO and SIL with human and bovine serum albumins as well as the displacement of warfarin from albumin by DIO and SIL were tested, employing steady-state fluorescence spectroscopy, fluorescence anisotropy, ultrafiltration, and molecular modeling. It is demonstrated that DIO and SIL are potent inhibitors of CYP2C9 enzyme and are able to displace the Site I ligand warfarin from human serum albumin. Because DIO and SIL may interfere with the pharmacokinetics of several drugs through both ways, we need to consider the potentially hazardous consequences of the consumption of diosmin or SIL together with other drugs.
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Liver biology and function, drug-induced liver injury (DILI) and liver diseases are difficult to study using current in vitro models such as primary human hepatocyte (PHH) monolayer cultures, as their rapid de-differentiation restricts their usefulness substantially. Thus, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in chemically-defined, serum-free conditions. Using whole proteome analyses, we found that PHH spheroids cultured this way were similar to the liver in vivo and even retained their inter-individual variability. Furthermore, PHH spheroids remained phenotypically stable and retained morphology, viability, and hepatocyte-specific functions for culture periods of at least 5 weeks. We show that under chronic exposure, the sensitivity of the hepatocytes drastically increased and toxicity of a set of hepatotoxins was detected at clinically relevant concentrations. An interesting example was the chronic toxicity of fialuridine for which hepatotoxicity was mimicked after repeated-dosing in the PHH spheroid model, not possible to detect using previous in vitro systems. Additionally, we provide proof-of-principle that PHH spheroids can reflect liver pathologies such as cholestasis, steatosis and viral hepatitis. Combined, our results demonstrate that the PHH spheroid system presented here constitutes a versatile and promising in vitro system to study liver function, liver diseases, drug targets and long-term DILI.