Evaluation of human hepatocytes cultured by three-dimensional spheroid systems for drug metabolism.
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.
Spheroid Working Group in Safety Evaluation Forum.
Takako
T
Ohkura
Kunihiro
K
Ohta
Takuya
T
Nagao
Kumiko
K
Kusumoto
Akiko
A
Koeda
Tadayoshi
T
Ueda
Tomoko
T
Jomura
Takeshi
T
Ikeya
Emiko
E
Ozeki
Kazuki
K
Wada
Kazushi
K
Naitoh
Yukiko
Y
Inoue
Naoki
N
Takahashi
Hisakazu
H
Iwai
Hiroshi
H
Arakawa
Takuo
T
Ogihara
D000082:Acetaminophen / Q000378:metabolism; D000420:Albuterol / Q000378:metabolism; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004008:Diclofenac / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D000077213:Lamotrigine; D008874:Midazolam / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011433:Propranolol / Q000378:metabolism; D018874:Spheroids, Cellular; D053719:Tandem Mass Spectrometry; D014227:Triazines / Q000378:metabolism
D004364:Pharmaceutical Preparations; D014227:Triazines; D004008:Diclofenac; D000082:Acetaminophen; D003577:Cytochrome P-450 Enzyme System; D011433:Propranolol; D014453:Glucuronosyltransferase; D000420:Albuterol; D008874:Midazolam; D000077213:Lamotrigine