Oral Presentation 1

日時： 2020 年 10 月 29 日 15:45-17:15
チャンネル： １

口頭発表１『臨床応用（その他）/ レギュラトリサイエンス』
Clinical application/Regulatory Science
座長： 石田 誠一 （崇城大学）
Seiichi Ishida (Sojo University)
O1-01"Evaluation of the expression profile of diarrhea induced by irinotecan using theJapanese Adverse Drug Event Report Database"Mashiro Okunaka(Meiji Pharmaceutical University / National Cancer Center Hospital East)
O1-02"Development of in vitro Blood Brain Barrier model reproducing microglia-induced
cytokine/chemokine dynamics"
Kaoru Sato(Lab Neuropharmacol, Div Pharmacol, NIHS)
O1-03"Establishment of culture protocol for application of human iPS cell-derived
hepatocytes to cholestasis toxicity test"
Shinichiro Horiuchi(National Institute of Health Sciences)
O1-04"Long term culture using hepatocytes from chimeric mice with humanized livers for
toxicity tests"
Yukie Kuroda(National Institute of Health Sciences)O1-01

Evaluation of the expression profile of diarrhea induced byirinotecan using the Japanese Adverse Drug Event Report
Database

12

Mashiro Okunaka 1, 2

Daisuke Kano 2

mokunaka@east.ncc.go.jp.

dkano@east.ncc.go.jp.

Reiko Matsui 2

Yoshihiro Uesawa 1

rmatsui@east.ncc.go.jp.

uesawa@my-pharm.ac.jp

Department of Medical Molecular Informatics, Meiji Pharmaceutical University, 2 -522-1
Noshio, Kiyose, Tokyo 204-8588, Japan
Department of Hepatobiliary & Pancreatic Oncology, National Cancer Center Hospital East,
6-5-1 Kashiwanoha, Kashiwa Chiba 277-8577, Japan

Keywords:Irinotecan; Diarrhea; Chemotherapy; JADER
Background Irinotecan (CPT-11) has been used in chemotherapy for advanced and recurrent
colorectal cancer and diarrhea is one of the dose -limited toxicities of CPT-11 [1-4]. Currently, thedifferences in time of onset of diarrhea expression among CPT-11-containing regimens have notbeen fully clarified. Therefore, we investigated th e tendency for CPT-11-induced diarrhea using
the Japanese Adverse Drug Event Report (JADER) database published by the Pharmaceuticals andMedical Devices Agency [5].
Method The drugs selected for this investigation were CPT-11, CPT-11 plus fluorouracil (5-FU),
and CPT-11 plus S-1, which are among the drugs listed in the JADER. We calculated the period
starting from the beginning of onset of the adverse events. Moreover, we performed a time -to-onset
analysis using the Weibull distribution, and examined the e xpression profile of adverse events foreach drug.
Results A total of 15,563 reports on adverse events with CPT-11 as the suspected drug were found.
Furthermore, 1,069 cases of diarrhea were reported for all drugs. In the time -to-onset analysis,
CPT-11 was the earliest expressed diarrhea, with a median onset of 5 days. On the other hand,diarrhea developed more slowly with CPT-11 plus S-1 than with the other treatment, with a median
onset of 14 days (p < 0.05).
Discussion These results, which show that the time of onset of diarrhea varies greatly dependingon the CPT-11-containing regimen, are expected to contribute to the management of toxicity incancer chemotherapy.
[1] National Comprehensive Cancer Network. NCCN clinical practic e guidelines in oncology
colon cancer. Version 4. 2020.
[2] Japanese Society for Cancer of the Colon and Rectum (JSCCR) guidelines 2019 for thetreatment of colorectal cancer.
[3] Van Cutsem E, Cervantes A, Nordlinger B, Arnold D, et al. Metastatic colorectal cancer: ESMOClinical Practice Guidelines for diagnosis, treatment and follow -up. Ann Oncol. 2014;25(3):1-9.
[4] http://www.mochida.co.jp/dis/interview/iri_n7.pdf[5] https://www.pmda.go.jp/safety/info-services/drugs/adr-info/suspected-adr/0003.html

O1-02Development of in vitro Blood Brain Barrier model reproducing
microglia-induced cytokine/chemokine dynamics
Kaoru Sato1

kasato@nihs.go.jp

Kazue Hoshikawa1

hoshikawa@nihs.go.jp

Yukari Shigemoto-Mogami1, 2
shigemoto@nihs.go.jp

Laboratory of Neuropharmacology, Division of Pharmacology, National Institute of Health Sciences, 3-25-26 Tonomachi,
Kawasaki-ku, Kawasaki city Kanagawa 210-9501, Japan2
Department of Neuropharmacology, Interdisciplinary Graduate School of Medicine, University of Yamanashi, 1110
Shimokato, Chuo, Yamanashi 409-3898, Japan
1

Keywords: BBB, Microglia, Cytokine, Chemokine
The blood-brain barrier (BBB) restricts the transfer of substances between blood and brain tissue thereby
protecting the brain from harmful substances. In the drug development, the BBB barrier function is important interms both of the drug delivery and the diagnosis of the pathological changes. We therefore attempted to establish
the in vitro BBB model reflecting the pathological permeability changes. We here focused on microglia, the cells
playing key roles in neuroinflammation.
In this study, we employed in vitro BBB model composed of vascular endothelial cells (EC), pericytes (Peri), andastrocytes (Ast), and then investigated the contribution of microglia to BBB barrier function via cytokine/chemokines(C/C). When non-stimulated microglia (non-MG) or LPS-activated microglia (LPS-MG) was added to theabluminal side of the in vitro BBB model, the BBB barrier function was disrupted only when activated microglia wasadded. LPS-MG caused decreases in the trans-endothelial electrical resistance (TEER) and in the expression levelsof tight junction (TJ) proteins. Under these conditions, 19 C/Cs were markedly increased on the abluminal side.
Unexpectedly, although LPS-MG alone released 10 of the 19 C/Cs, their concentrations were much lower than those
detected on the abluminal side of the BBB model with LPS-MG. Co-culture of LPS-MG with Ast caused marked
increases in 12 of the 19 C/Cs, while co-culture of LPS-MG with EC and Peri resulted in a significant increase in only
1 of the 19 C/Cs (fractalkine). These results suggest that C/C dynamics in this experiment model are not only causedby activated microglia but also are due to the interaction between activated microglia and astrocytes. Taken together,microglia is indispensable to in vitro BBB model reproducing the complex interactions among neurovascular unit
(NVU) cells and pathological changes via C/Cs in the progressive process of neuroinflammation.
[1] Shigemoto-mogami Y., et al.: Frontiers in Cellular Neuroscience, 12: 494, 2018[2] Shigemoto-mogami Y. and Sato K.: Folia Pharmacologica Japonica Nippon Yakurigaku Zasshi, (in press).

O1-03

Establishment of culture protocol for application ofhuman iPS cell-derived hepatocytes tocholestasis toxicity test
Shinichiro Horiuchi 1

Yukie Kuroda 1

Su-Ryang Kim 1

hori542@nihs.go.jp

Ryuya Fujii 1

Seiichi Ishida 1,2

1

Division of Pharmacology, National Institute of Health Sciences,
3-25-26 Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa, 210-9501, Japan
2
Sojo University, Faculty of Biotechnology and Life Science
4-22-1 Ikeda, Nishi-ku, Kumamoto, Kumamoto, 860-0082, Japan

Keywords: human iPS cell-derived hepatocytes, drug induced cholestasis toxicity,
sandwich culture, bile canaliculi, drug metabolism[Purpose] Drug-induced liver injury (DILI) is one of the primary reasons for the failure ofpharmaceutical agents during drug development. 40% of DILI cases in japan had either
cholestatic or mixed cholestatic hepatic toxicity (1) and fatality rate of the case reached 10- 50% (2). Accordingly, evaluation for the drugs that cause cholestasis is important in drugdevelopment. Primary/cryo-preserved human hepatocytes is used to test the toxicity of candidate
compounds in the early stages of drug development. However, primary/cryo-preserved humanhepatocytes is not considered suitable for the evaluation of cholestasis toxicity because theextended bile canaliculi formation is difficult. In our previous experiments, the extended bile
canaliculi were observed on a part of cell culture surface when hiPS cell-derived hepatocytes(hiPSC-hep) were culture for long term. Therefore, we studied better culture conditions forfunctional extended bile canaliculi formation, aiming the application of hiPSC-hep to evaluation
for cholestasis toxicity.
[Method] hiPSC-hep from vendor A was used in this study. hiPSC-hep were sandwich-cultured
after long term culture for formation of extended bile canaliculi. Bile canaliculi were observed
using fluorogenic substrates of biliary efflux transporters MRP2 or BSEP. In addition, localizationof MRP2 and BSEP, effect of biliary efflux transporter inhibitor and expression of major
cytochrome P450 (CYPs) were observed.
[Results and Discussion] Extended bile canaliculi were evenly formed on the whole cell culture
surface by examining culture condition. The localizations of MRP2 and BSEP in the canalicularmembrane were confirmed by immunostaining and t he excretion of model substrates to bile
canaliculi were inhibited by corresponding inhibitors. These results suggested that functional bile
canaliculi were formed. Expressions of major CYPs were same level as or slightly lower thancryopreserved human hepatocyte. From above results, culture protocol that was established in thisstudy is expected to apply to drug induced cholestasis toxicity test.
[1] Takikawa, H., Murata, Y., Horiike, N., Fukui, H., Onji, M., Drug-induced liver injury in Japan:
An analysis of 1676 cases between 1997 and 2006 , Hepatol Res., 39(5):427-431 , 2009.
[2] Navarro, VJ., Senior, JR., Drug-related hepatotoxicity, N Engl J Med ., 354(7):731-739,
2006

O1-04

Long term culture using hepatocytes from chimeric mice withhumanized livers for toxicity tests
Yukie Kuroda 1

y-kuroda@nihs.go.jp

Su-Ryang Kim 1
Hiroshi Suemizu 2Shinichiro Horiuchi 1

Ryuya Fujii 1

Seiichi Ishida 1,31

Division of Pharmacology, National Institute of Health Sciences, 3-25-26 Tonomachi,Kawasaki-ku, Kawasaki city Kanagawa 210-9501, Japan2
Department of Laboratory Animal Research, Central Institute for Experimental Animals,
3-25-12 Tonomachi, Kawasaki-ku, Kawasaki city Kanagawa 210-0821, Japan
3
SOJO University, Faculty of Biotechnology and Life sciences, 4-22-1 Ikeda, Nishi-ku
Kumamoto city Kumamoto 860-0082, Japan

Keywords: Chimeric mice, Hepatocyte, Gene expression, Cytochrome P450, Transporter[Purpose] In the assessment on acute and chronic influence of drug or chemical exposure to human
body, in vitro test has been required continuous improvement for pharmacokinetic prediction in thebody. Chimeric mice with humanized hepatocytes are constructed by transplanting humanhepatocytes to severely immunodeficient hepatopathy mice. Hepatocytes isolated from chimericmice expressed human albumin, human cytochrome P450 and human transporter, and expression
levels of P450 subfamily mRNA correlated to those of human hepatocytes [1]. Thus, hepatocytes
from chimeric mice with humanized liver are thought to be effective cell resource for metabolic
activity evaluation and for toxicity assays.
In this study, we tried to culture hepatocytes from chimeric mice with humanized livers for thelonger period to apply them to repetitive toxicity tests in the future.
[Materials and Methods] Hepatocytes from chimeric mice with humanized livers (Hu-Liver cells)
prepared at Central Institute for Experimental Animals were cultured with hiPS-HEP LTM Medium(LTMM) (Takara Bio Inc., Shiga, Japan), Hepatocyte maintenance medium (HMM) (Thermo Fisher
Scientific, Waltham, MA), or mixed medium of these two on the collagen coated plate or on 3dimensional cell culture plate Cell-able ® (Toyo Gosei Co., Ltd, Tokyo, Japan).
[Results and Discussion] Gene expression level of transporters in Cell-able ® or HMM culture
conditions were equal to those in the cells right after the preparation from chimeric mice. CYP3A4
activity and drug metabolizing enzyme gene expression level showed higher in LTMM condition
than in others. Cell polarity was observed in HMM and LTMM with collagen plate conditions. So,
these cell culture conditions using Hu-Liver were able to improve or maintain cell functions. In thispresentation, we are planning to report the results of cell culture condition improvement for thetoxicity tests.
[1]

S. Uehara, et al. Expression and inducibility of cytochrome P450s in human hepatocytes isolated fromchimeric mice with humanised livers. Xenobiotica, 49.6: 678-687, 2019.