Regular Article
Evaluation of Human Hepatocytes Cultured by Three-dimensional Spheroid Systems for Drug Metabolism

https://doi.org/10.2133/dmpk.DMPK-13-RG-105Get rights and content

Summary:

We investigated the utility of three-dimensional (3D) spheroid cultures of human hepatocytes in discovering drug metabolites. Metabolites of acetaminophen, diclofenac, lamotrigine, midazolam, propranolol and salbutamol were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) to measure enzyme activities in this system cultured for 2 and 7 days. Sequential metabolic reactions by Phase I and then Phase II enzymes were found in diclofenac [CYP2C9 and UDP-glucuronyltransferases (UGTs)], midazolam (CYP3A4 and UGTs) and propranolol (CYP1A2/2D6 and UGTs). Moreover, lamotrigine and salbutamol were metabolized to lamotrigine-N-glucuronide and salbutamol 4-O-sulfate, respectively. These metabolites, which are human specific, could be observed in clinical studies, but not in conventional hepatic culture systems as in previous reports. Acetaminophen was metabolized to glucuronide and sulfate conjugates, and N-acetyl-p-benzo-quinoneimine (NAPQI) and its metabolites were not observed. In addition, mRNA of drug-metabolism enzymes [CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, UGT1A1, UGT2B7, sulfotransferase 1A1 (SULT1A1) and glutathione S-transferase pi 1 (GSTP1)], which were measured by qRT-PCR, were expressed in the human hepatocyte spheroids. In conclusion, these results suggest that human hepatocyte spheroids are useful in discovering drug metabolites.

References (37)

  • Y. Miyamoto et al.

    Preconditioned cell array optimized for a three-dimensional culture of hepatocytes

    Cell Transplant

    (2009)
  • K. Kusumoto et al.

    A new high-throughput analysis for drug metabolism profiling using liquid chromatography coupled with tandem mass spectrometry

    Drug Res (Stuttg)

    (2013)
  • R. Bort et al.

    Diclofenac toxicity to hepatocytes: a role for drug metabolism in cell toxicity

    J. Pharmacol. Exp. Ther

    (1999)
  • W.W. Wang et al.

    Assessment of a micropatterned hepatocyte coculture system to generate major human excretory and circulating drug metabolites

    Drug Metab. Dispos

    (2010)
  • S. Anderson et al.

    Predicting circulating human metabolites: how good are we?

    Chem. Res. Toxicol

    (2009)
  • W. Tang

    The metabolism of diclofenac-enzymology and toxicology perspectives

    Curr. Drug Metab

    (2003)
  • M. Darnell et al.

    In vitro evaluation of major in vivo drug metabolic pathways using primary human hepatocytes and HepaRG cells in suspension and a dynamic three-dimensional bioreactor system

    J. Pharmacol. Exp. Ther

    (2012)
  • D. Dalvie et al.

    Assessment of three human in vitro systems in the generation of major human excretory and circulating metabolites

    Chem. Res. Toxicol

    (2009)
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