Evaluation of human hepatocytes cultured by three-dimensional spheroid systems for drug metabolism.

Drug metabolism and pharmacokinetics (2014-04-04)
Takako Ohkura, Kunihiro Ohta, Takuya Nagao, Kumiko Kusumoto, Akiko Koeda, Tadayoshi Ueda, Tomoko Jomura, Takeshi Ikeya, Emiko Ozeki, Kazuki Wada, Kazushi Naitoh, Yukiko Inoue, Naoki Takahashi, Hisakazu Iwai, Hiroshi Arakawa, Takuo Ogihara

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.

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Salbutamol, European Pharmacopoeia (EP) Reference Standard
Acetaminophen solution, 1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®
Acetaminophen, analytical standard
Acetaminophen, meets USP testing specifications, 98.0-102.0%, powder
Acetaminophen, BioXtra, ≥99.0%
Acetaminophen, Pharmaceutical Secondary Standard; Certified Reference Material
Salbutamol, VETRANAL®, analytical standard
Paracetamol, European Pharmacopoeia (EP) Reference Standard