100

80

60

40

20

0

0.01 0.1 1 10 100

%h

um

an

in

tes

tin

al a

bs

orp

tio

n

Papp (x10-6 cm/s)

100

80

60

40

20

0

0.01 0.1 1 10 100

%h

um

an

in

tes

tin

al a

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orp

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Papp (x10-6 cm/s)

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.

1

10

100

1000

AI Caco-2 Diff. I

CYP3A4

1

10

100

1000

AI Caco-2 Diff. I

CYP2C9

1

10

100

1000

AI Caco-2 Diff. I

CYP2C19

1

10

100

1000

AI Caco-2 Diff. I

CES1

1

10

100

1000

AI Caco-2 Diff. I

CES2

1

10

100

1000

AI Caco-2 Diff. I

UGT1A1

1

10

100

1000

AI Caco-2 Diff. I

SULT1B1

1

10

100

1000

AI Caco-2 Diff. I

SI

1

10

100

1000

AI Caco-2 Diff. I

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

1

10

100

1000

AI Caco-2 Diff. I

ABCB1 / MDR1

1

10

100

1000

AI Caco-2 Diff. I

ABCC1 / MRP1

1

10

100

1000

AI Caco-2 Diff. I

ABCC2 / MRP2

1

10

100

1000

AI Caco-2 Diff. I

ABCC3 / MRP3

1

10

100

1000

AI Caco-2 Diff. I

ABCG2 / BCRP

1

10

100

1000

AI Caco-2 Diff. I

SLC15A1 / PEPT1

1

10

100

1000

AI Caco-2 Diff. I

SLC22A1 / OCT1

1

10

100

1000

AI Caco-2 Diff. I

SLCO2B1 / OATP2B1

1

10

100

1000

AI Caco-2 Diff. I

VIL1

1

10

100

1000

AI Caco-2 Diff. I

MUC2

1

10

100

1000

AI Caco-2 Diff. I

LYZ

1

10

100

1000

AI Caco-2 Diff. I

GP2

1

10

100

1000

AI Caco-2 Diff. I

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.

Co

un

t

Villin 1+

92.5%

Villin - Alexa Fluor 488

0

400

800

1200

F-hiSIEC Lot #1

F-hiSIEC Lot #2

F-hiSIEC Lot #3

TE

ER

(Ω・c

m2)

(A)

(B) (C)

0

1

2

3

4

5

F-hiSIEC Lot #1

F-hiSIEC Lot #2

F-hiSIEC Lot #3

Pa

pp

(x

10

-6 c

m/s

)

Villin 1 / DAPI

50 μm 50 μm

Occludin / DAPI

2.0 μm

1

10

100

1000

AI F-hiSIEC Lot #1

F-hiSIEC Lot #2

F-hiSIEC Lot #3

SLC15A1 / PEPT1

1

10

100

1000

AI F-hiSIEC Lot #1

F-hiSIEC Lot #2

F-hiSIEC Lot #3

CDH17 / HPT1

1

10

100

1000

AI F-hiSIEC Lot #1

F-hiSIEC Lot #2

F-hiSIEC Lot #3

GLUT2 / SLC2A2

0

2

4

6

8

10

12

F-hiSIEC Lot #1

F-hiSIEC Lot #2

F-hiSIEC Lot #3

Pap

p (

x1

0-6

cm

/s)

A to B

B to A

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

0

200

400

600

800

1000

1200

1400

F-hiSIEC Lot #1

F-hiSIEC Lot #2

F-hiSIEC Lot #3

F-hiSIEC Lot #4

F-hiSIEC Lot #5

CY

P3

A4

ac

tivit

y

(pm

ol/2

h/m

g p

rote

in)

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)

1

10

100

1000

AI Caco-2 Diff. I

SLC5A1 / SGLT1

F-hiSIEC 1

10

100

1000

AI Caco-2 Diff. I

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*)

1 2

9

4 5

6

8

10

16

3

7

11

12 13

14

15

1 2

9

4 5

6

8

10

16

3

7

11

12 13

14

15

MC