Characterization of FUJIFILM human iPS cell-derived Small … · 2020-04-07 · 100 80 60 40 20 0...
Transcript of Characterization of FUJIFILM human iPS cell-derived Small … · 2020-04-07 · 100 80 60 40 20 0...
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Characterization of FUJIFILM human iPS cell-derived Small Intestinal Epithelial like Cell
(F-hiSIEC™): Applications and possibilities as a pharmacokinetics prediction tool
〇Shinji Mima1, Yuki Imakura1, Izumi Ogura1, Shun Goto1, Chihaya Kakinuma1, Takahiro Iwao2, Tamihide Matsunaga2, Tadanori Yamada1, Ken-ichiro Hata1 1 Bio Science & Engineering Laboratory, FUJIFILM Corporation 2 Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University
[Purpose] Bioavailability is the most important factor of drug development on the point of efficacy and toxicity. For
oral drugs, intestinal permeability has great impact to the bioavailability. Caco-2 cells - a human colon carcinoma cells
have been widely used as a model system for prediction of intestinal epithelial permeability. However, the apparent
permeability coefficients acquired using Caco-2 cell system shows poor correlation with the intestinal absorption and
availability of drugs in humans. Thus we to develop the alternative method to predict intestinal absorption and
availability. In this investigation, we report on the potential of FUJIFILM human iPS cell-derived Small Intestinal
Epithelial like Cells (F-hiSIEC™), especially about functional characteristics of pharmacokinetics prediction.
[Methods] We developed differentiation protocol from iPS cells to small intestinal epithelial cells, which a method is
adding a new small molecular compound to the previous report (Iwao, et al. Drug Metab. Dispos. 43:603-610, 2015).
[Results and Discussion] The activity of CYP3A4 was equal to the primary small intestinal cells. The expression
levels of pharmacokinetics-related genes (CYP2C9, UGT1A1, ABCB1/MDR1, ABCG2/BCRP, SLC15A1/PEPT1)
and intestinal markers (VIL1, FABP2, CDX2, ISX, GATA4) in F-hiSIEC™ were similar to the adult small intestine.
We tested the relationship between Papp and observed intestinal absorption for clinical drugs with cell culture insert
system using F-hiSIEC™ or Caco-2 cells. As a result, the evaluation model using F-hiSIEC™ showed higher
correlation than Caco-2 cells. These results suggested that physiological characteristics of F-hiSIEC™ and the cell
sheet are close to the primary small intestinal cells.
[Conclusions] F-hiSIEC™ is expected as an ideal material for the alternative method to predict intestinal absorption
and availability.
Endoderm Intestinal stem cells Intestinal epithelial cells
F-hiSIEC showed major cytochrome P450 enzymes activities.
Characteristics of F-hiSIEC™
Gene expression
(metabolizing enzymes, transporters and cell type markers)
Each cells were incubated with 5 µM midazolam, 5 µM diclofenac, 45.7 µM (S)-mephenytoin, 5 µM bufuralol, 40 µM phenacetin for 2 h at 37℃.
*CYP3A4/5 in F-hiSIEC was measure with F-hiSIEC assay medium. The supernatants were collected and the metabolites were measured by UPLC–
MS/MS. All data are presented as mean ± standard deviation (n = 3–6). Human Enterocytes (purchased from In Vitro ADMET Laboratories, Inc.)
were used as primary small intestinal cells.
[Conclusion] F-hiSIEC showed characteristics which are close to in vivo human small intestine.
F-hiSIEC is expected as a ideal material for the alternative method to predict intestinal absorption and bioavailability.
Applications as pharmacokinetics evaluation tools
P-148
Abstract
We developed cryopreserved small intestinal epithelial cell and evaluated that characteristics.
How to use F-hiSIEC
Cryopreservation
Methods Day0 Day30
Drug-metabolizing enzyme (cytochrome P450) activities
Lot-to-lot variation in CYP3A4 and MDR1 (P-gp) activities
CYP3A4 activities and P-gp activities using F-hiSIEC showed little variation between lots
to lot and could be experimented with good reproducibility.
At terminal differentiation, the differentiated cells were incubated with F-hiSIECTM Assay Medium containing 5 mM midazolam for 2 h at 37℃.
10 µM digoxin was used as a substrate . Additionally, HBSS containing 10 mM HEPES (pH 7.4) was used as a transport buffer. After the preincubation,
the transport buffer containing the substrate was added to the apical or basal chambers, and the cells were incubated at 37℃ for 60 min.
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AI Caco-2 Diff. I
CYP3A4
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AI Caco-2 Diff. I
CYP2C9
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AI Caco-2 Diff. I
CYP2C19
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AI Caco-2 Diff. I
CES1
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CES2
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UGT1A1
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SULT1B1
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SI
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UGT2B7
F-hiSIEC F-hiSIEC F-hiSIEC F-hiSIEC F-hiSIEC
F-hiSIEC F-hiSIEC F-hiSIEC F-hiSIEC
pmol/2h/mg protein
CYP3A4/5 CYP2C9 CYP2C19 CYP2D6 CYP1A2
Human primary enterocytes 848 ± 148 1330 ± 780 20 ± 11 15 ± 2 N.D.
Caco-2 cells 26 ± 3 N.D. N.D. 3 ± 1 225 ± 94
F-hiSIEC 840± 50* 130 ± 43 76 ± 18 3 ± 0 364 ± 76
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ABCB1 / MDR1
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AI Caco-2 Diff. I
ABCC1 / MRP1
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ABCC2 / MRP2
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ABCC3 / MRP3
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ABCG2 / BCRP
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SLC15A1 / PEPT1
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SLC22A1 / OCT1
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SLCO2B1 / OATP2B1
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VIL1
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MUC2
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LYZ
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REG4
F-hiSIEC F-hiSIEC F-hiSIEC F-hiSIEC F-hiSIEC
F-hiSIEC F-hiSIEC F-hiSIEC
F-hiSIEC F-hiSIEC F-hiSIEC F-hiSIEC F-hiSIEC
<Transporters> Morphology, purity and barrier function
- Almost all F-hiSIECs were differentiated to epithelial cells and the purity was more than 90%.
- Cell sheet formed on cell culture insert indicated appropriate barrier function.
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Villin 1+
92.5%
Villin - Alexa Fluor 488
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50 μm 50 μm
Occludin / DAPI
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SLC15A1 / PEPT1
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Evaluation of drug permeability using cell culture insert system
The drug permeability assay was carried out on the cell culture system, correlation
was observed between Papp of the drug and Human intestinal absorption.
1.Antipyrine , 2.Caffeine, 3.Cephalexin, 4.Acebutolol, 5.Ribavirin, 6.Metformin, 7.Hydrochlorothiazide, 8.Indinavir,
9.Verapamil, 10.Enalapril, 11.Erythromycin, 12.Midazolam, 13.Sulpiride, 14.Lisinopril, 15.Tacrolimus, 16.Lucifer yellow.
*R2 scores were determined exclude CYP3A4 substrates (9, 12 and 15).
Standard Transporter CYP3A4 Others
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CYP3A4 activities MDR1 (P-gp) activities
At the end of differentiation (Day 9), (A) cells were stained with villin 1 (green) and occuludin (red). Transmission electron microscopy
images of microvilli. (B) Ratio of villin 1 positive cells was determind with FACS analysis. (C) Barrier funtion of cell sheet on cell
culture insert by TEER (blue bars) and Papp of 110 μM lucifer yellow (red bars).
ER=14.3 ER=8.6 ER=15.8
<Metabolizing enzymes>
<Cell type markers>
F-hiSIEC expressed major intestinal metabolic enzymes, transporters, and cell type
markers much as the in vivo small intestine.
Various gene expression of F-hiSIECTM were measured by RT-PCR and compared
with Adult Intestine (AI) and Caco-2.(Y-axis: relative mRNA expression)
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SLC5A1 / SGLT1
F-hiSIEC 1
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TRPV6 / CaT1
F-hiSIEC
ex. 1 Seeding MC MC MC MC MC MC
ex. 2 Seeding MC MC MC MC
Thu Fri Sat Sun Mon Tue Wed Thu Fri Sat Sun Mon Tue Wed
Usable term for a test
Caco-2 (R2 = 0.37*) F-hiSIEC (R2 = 0.76*)
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