Regional Distribution of Drug-metabolizing Enzyme Activities in the Liver and Small Intestine of Cynomolgus Monkeys

The endoderm, differentiated from human induced pluripotent stem cells (iPSCs), can differentiate into the small intestine and liver, which are vital for drug absorption and metabolism. The development of human iPSC-derived enterocytes (HiEnts) and hepatocytes (HiHeps) has been reported. However, pharmacokinetic function-deficiency of these cells remains to be elucidated. Here, we aimed to develop an efficient differentiation method to induce endoderm formation from human iPSCs. Cells treated with activin A for 168 h expressed higher levels of endodermal genes than those treated for 72 h. Using activin A (days 0–7), CHIR99021 and PI−103 (days 0–2), and FGF2 (days 3–7), the hiPSC-derived endoderm (HiEnd) showed 97.97% CD−117 and CD−184 double-positive cells. Moreover, HiEnts derived from the human iPSC line Windy had similar or higher expression of small intestine-specific genes than adult human small intestine. Activities of the drug transporter P-glycoprotein and drug-metabolizing enzyme cytochrome P450 (CYP) 3A4/5 were confirmed. Additionally, Windy-derived HiHeps expressed higher levels of hepatocyte- and pharmacokinetics-related genes and proteins and showed higher CYP3A4/5 activity than those derived through the conventional differentiation method. Thus, using this novel method, the differentiated HiEnts and HiHeps with pharmacokinetic functions could be used for drug development.

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Generation of tetracycline-controllable CYP3A4-expressing Caco-2 cells by the piggyBac transposon system

Scientific Reports ◽

10.1038/s41598-021-91160-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Moe Ichikawa ◽

Hiroki Akamine ◽

Michika Murata ◽

Sumito Ito ◽

Kazuo Takayama ◽

...

Keyword(s):

Small Intestine ◽

Drug Absorption ◽

Cell Model ◽

Negative Effects ◽

Piggybac Transposon ◽

Drug Metabolizing Enzyme ◽

Metabolizing Enzyme ◽

Cyp3a4 Expression

AbstractCaco-2 cells are widely used as an in vitro intestinal epithelial cell model because they can form a monolayer and predict drug absorption with high accuracy. However, Caco-2 cells hardly express cytochrome P450 (CYP), a drug-metabolizing enzyme. It is known that CYP3A4 is the dominant drug-metabolizing enzyme in human small intestine. In this study, we generated CYP3A4-expressing Caco-2 (CYP3A4-Caco-2) cells and attempted to establish a model that can simultaneously evaluate drug absorption and metabolism. CYP3A4-Caco-2 cells were generated by piggyBac transposon vectors. A tetracycline-controllable CYP3A4 expression cassette (tet-on system) was stably transduced into Caco-2 cells, thus regulating the levels of CYP3A4 expression depending on the doxycycline concentration. The CYP3A4 expression levels in CYP3A4-Caco-2 cells cultured in the presence of doxycycline were similar to or higher than those of adult small intestine. The CYP3A4-Caco-2 cells had enough ability to metabolize midazolam, a substrate of CYP3A4. CYP3A4 overexpression had no negative effects on cell proliferation, barrier function, and P-glycoprotein activity in Caco-2 cells. Thus, we succeeded in establishing Caco-2 cells with CYP3A4 metabolizing activity comparable to in vivo human intestinal tissue. This cell line would be useful in pharmaceutical studies as a model that can simultaneously evaluate drug absorption and metabolism.

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Cisplatin-induced changes in cytochrome P-450, lipid peroxidation and drug-metabolizing enzyme activities in rat kidney cortex

Toxicology Letters ◽

10.1016/0378-4274(89)90174-4 ◽

1989 ◽

Vol 48 (2) ◽

pp. 193-199 ◽

Cited By ~ 25

Author(s):

G. Bompart

Keyword(s):

Lipid Peroxidation ◽

Enzyme Activities ◽

Rat Kidney ◽

Kidney Cortex ◽

Rat Kidney Cortex ◽

Drug Metabolizing Enzyme ◽

Metabolizing Enzyme ◽

Induced Changes ◽

Cytochrome P 450

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The Influence of Phenobarbitone on Maternal and Perinatal Hepatic Drug-metabolizing Enzymes in the Rat

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y75-160 ◽

1975 ◽

Vol 53 (6) ◽

pp. 1147-1157 ◽

Cited By ~ 14

Author(s):

J. U. Bell ◽

M. M. Hansell ◽

D. J. Ecobichon

Keyword(s):

Endoplasmic Reticulum ◽

Enzyme Activities ◽

Morphological Changes ◽

Smooth Endoplasmic Reticulum ◽

Transplacental Passage ◽

Hepatic Drug ◽

Rat Pups ◽

Drug Metabolizing Enzyme ◽

Metabolizing Enzyme ◽

Conjugating Enzyme

Phenobarbitone (PB) (75 mg/kg) was administered orally for three consecutive days to pregnant or lactating rats at different pre- and postnatal stages in order that the perinatal animals would receive the agent either by transplacental passage or via the milk. Control animals received equivalent volumes of saline. The dams, fetuses, and pups were killed 24 h after the last dose. Hepatic p-nitroanisole O-demethylase (OD), carboxylesterase (CE), and bromosulfophthalein–glutathione (BSP–GSH) conjugating enzyme activities in a 12 100 g – 20 min supernatant of a 20% w/v homogenate were measured. The morphology of the developing rat liver in the absence and presence of PB was examined by electron microscopy.The results demonstrated that the transplacental passage of PB to rat fetuses at term or 3 days prepartum had no effect on either the hepatic drug-metabolizing enzyme activities or on the ultrastructural appearance of the liver. Increased hepatic OD activity was observed in the pregnant animal but no effect was observed in the lactating dam. Phenobarbitone received by the suckling rat had two distinct effects. Compared to control activities, twofold increases in hepatic OD activity were observed in rat pups as early as 4 days after birth, associated with a marked proliferation in hepatic smooth endoplasmic reticulum. In contrast, PB-related significant increases in neonatal hepatic CE and BSP–GSH conjugating enzyme activities were not observed until 21 days of age. In the 4-day-old treated pups, characteristic morphological changes included numerous small membrane whorls in addition to increased smooth endoplasmic reticulum and microbodies in the liver.

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