日本薬物動態学会　第34回大会抄録集

Abstracts

抄　録

SC・年会合同特別招待講演

（Honor Invited Lecture）

招待講演

（Invited Lecture）

会長講演

（President's Lecture）

新会長講演

（New President's Lecture）

シンポジウム

（Symposium）

宇宙創薬シンポジウム

（Space Symposium）

アジア４か国合同シンポジウム

（Asia Joint Symposium）

学生・若手企業研究者シンポジウム

（The Predoctoral Researcher-Initiative Session (PRIS) 2019）

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Seminar

Oral

Poster

SIL

IL

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NPL

Symposium

SIL

ヒューマン・オルガノイドで拓く未来創薬

Next-gen drug development with human organoid technology

武部　貴則

東京医科歯科大学 Takanori Takebe

Tokyo Medical and Dental University

Organoids hold great promise to dissect human specific developmental processes in a tractable way coupled with stem cells, otherwise inaccessible. Recently, we have developed a novel culture principle, named self-condensation culture, whereby mesenchymal progenitors initiated organoid (or organ bud) formation within multiple progenitor cell mixtures. Recently, we have developed a method to develop 3D, transplantable organ buds (or organoids) from diverse tissues including kidney, pancreas and cartilage (Cell Stem Cell, 2015). By organoid transplant model, we showed iPSC-derived human hepatic endoderm with supportive mesenchymal and endothelial cells results in three-dimensional liver bud (LB)-like organoid that becomes vascularized upon transplantation into mice, extending life in a mouse model of liver failure (Nature, 2013). To dissect how heterotypic interactions impact lineage identity, we used single-cell transcriptomics to reconstruct hepatic, stromal, and endothelial lineage progression in organoids from pluripotency. We find that liver bud hepatoblasts diverge from the two-dimensional lineage, and express epithelial migration signatures characteristic of organ budding. Our molecular dissection at single cell level reveals interlineage communication regulating organoid development, and illuminates previously inaccessible aspects of human liver development and disease (Nature, 2017: Cell Metabolism, 2019: Nature, in press). In this talk, I will summarize the state-of-art of these hepatobiliary organoid based approaches, discuss their mechanistic basis and potential future applications.

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Symposium

IL1

Pharmacometrics and Systems Modeling in Hematological Malignancies

Department of Pharmaceutical Sciences, University at Buffalo, SUNY, Buffalo, NY 14214, USA

Cancer continues to represent a major source of morbidity and mortality around the world despite significant advancements in the development of precision chemotherapy, biologics, and cell-based therapies. Compounds designed to treat cancer continue to exhibit one of the highest rates of attrition in drug development, primarily owing to a lack of efficacy and unanticipated adverse drug effects in later phase trials. The use of clinically approved drugs is also challenged by tumor heterogeneity and known and unknown factors that result in innate and acquired resistance. Substantial innovations in the computational modeling of cancer therapeutics have been made in pharmacometrics (Pmx) and quantitative systems pharmacology (QSP). Pmx is grounded in the basic principles of pharmacokinetics (PK), statistics, and pharmacodynamics (PD), and can be readily extended to diverse data types. Traditional PK/PD models contain a minimal number of identifiable parameters to describe temporal profiles of therapeutic and adverse drug effects. Coupled with nonlinear mixed effects modeling and relatively large clinical trials, a covariate analysis can be used to identify patient-specific characteristics that explain the variability in model parameters and clinical outcomes. This approach can be limited by study designs and is rarely sufficient for recapitulating multiple, complex genotype-phenotype relationships. A major opportunity for pharmacometrics is the extension of pharmacostatistics to QSP models, which recognize that both drugs and disease processes give rise to complex and dynamic phenotypes by altering natural interconnected biochemical networks. Multi-scale QSP models that combine physiological PK/PD principles and signaling networks can serve as a platform for integrating factors that regulate drug effects and therapeutic outcomes. This presentation will discuss basic principles of translational Pmx and QSP modeling in hematological malignancies and highlight approaches to identify or qualify drug targets, design and evaluate combinatorial drug regimens, explore the impact of tumor heterogeneity, and identify factors influencing anti-cancer drug action.

Invited Lecture

IL2

Integration of innovative mechanistic test systems for chemical safety testing: the EU-ToxRisk strategy

1_{Leiden University, Leiden, The Netherlands，}2_{Konstanz University, Konstanz, Germany，}3_{BASF, Ludwigshafen, Germany，}4_Fraunhofer

Institute, Germany

There is a need for a paradigm shift in toxicology towards an animal-free, mechanism-based integrated approach to human chemical safety assessment. The aim of the EU-ToxRisk project is to unite all relevant disciplines and stakeholders to establish: i) pragmatic, solid read-across procedures incorporating mechanistic and toxicokinetic knowledge; and ii) ab initio hazard and risk assessment strategies of chemicals with little background information. The project is focused on repeated dose systemic toxicity (liver, kidney, lung and nervous system) as well as developmental/reproduction toxicity. Different human tiered test systems are integrated to balance speed, cost and biological complexity. Advanced technologies, including high throughput transcriptomics, RNA interference, and high throughput microscopy, will provide quantitative and mechanistic underpinning of AOPs and key events (KE). The project combines in silico tools and in vitro assays by computational modelling approaches to provide quantitative data on the activation of KE of AOP. This information, together with detailed toxicokinetics data, and in vitro-in vivo extrapolation algorithms forms the basis for improved hazard and risk assessment. The EU-ToxRisk work plan is structured along a broad spectrum of case studies, driven by the cosmetics, (agro)-chemical, pharma industry together with regulators. The approach involves iterative training, testing, optimization and validation phases to establish fit-for-purpose integrated approaches to testing and assessment with key EU-ToxRisk methodologies. The proof-of-concept for the new mechanism-based testing strategy makes EU-ToxRisk the flagship in Europe for animal-free chemical safety assessment. The presentation will provide a brief overview of the project exemplified by a few real life case studies that make advantage of the various innovative approaches.

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IL3（SY08-04）

Merging human microphysiological systems with quantitative systems pharmacology for in vitro in vivo

translation (IVIVT)

Murat Cirit

Javelin Biotech, Inc, Woburn, MA USA

A large percentage of drug candidates fail at the clinical trial stage due to a lack of efficacy and unacceptable toxicity, primarily because of translational gap between human physiology and preclinical models including both in vitro culture and animal models. This need for more human-physiology relevant in vitro systems for preclinical efficacy and toxicity testing has led to a major effort to develop “Microphysiological Systems (MPS)”, aka tissue chips (TC) or organs on chips (OOC), based on engineered human tissue constructs. MPS technologies are the best in vitro representation of human physiology and provide high-content, human-relevant information about drug-physiology interaction, which can be used to accelerate various aspects of preclinical drug discovery, such as evaluation of pharmacokinetic properties of xenobiotics, biotransformations, mechanism of action of drugs, diagnostic & predictive biomarker discovery and safety profiles.

We believe that the full impact of MPS technologies to bridge the gap preclinical and clinical gap will be realized only when robust approaches for in vitro–in vivo (MPS-to-human) translation are developed and utilized. The IVIVT approaches potentially can be used as a clinical pharmacology tool to estimate, FIH starting doses, NOAEL and MABEL, dosing regimen adjustment for sub-populations, such as renally or hepatically impaired patient population, pediatrics.

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PL

JSSX会長講演：生理学的薬物動態モデルのトピックス

Message from the JSSX president: Current topics in societal impact of physiologically based kinetic modeling

in cutting-edge sciences

山崎　浩史

昭和薬科大学 Hiroshi Yamazaki

Showa Pharmaceutical University

The 34th Annual Meeting of the Japanese Society for the Study of Xenobiotics (JSSX) has the overall theme of intersection of the cutting-edge sciences - Quantitative elucidation of pharmacokinet-ics-pharmacodynamics by integrating pathophysiology-based modeling and bio-mimetic engineering -. Current methods to estimate health risks of industrial/food functional chemicals and pharmaceutical medicines require guideline animal testing studies. This has resulted in only a small fraction of chemicals with adequate data for assessing potential risks or hazards and highlights the urgent need to develop more efficient and informative determination tools. In vitro high throughput screening assays with bio-mimetic engineering combined with computational modeling might provide an alternative to traditional animal testing studies. It should be noted that relationship between chemical doses (mg/kg) outside bodes and toxico/ pharmaco-kinetic concentrations (ng/mL) inside bodes by physiologically based kinetic (PBK) modeling. However, there are various jurisdictions in these cutting-edge sciences. In PBK modeling, blood flow rates in animals and humans are some of key factors to develop good models in allometry scaling. A greater understanding amongst wide members would be important to support decision-making in the intersection of regulatory frameworks. How are the data generated by these new methods used to support decision-making for quantitative risk characterization? What are our specific regulatory data requirements and with what novel test methods are these met? The broader context should be shared and discussed.

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NPL

新会長からのメッセージ

Message from the new president

斎藤　嘉朗

国立医薬品食品衛生研究所 Yoshiro Saito

National Institute of Health Sciences

JSSX 16th new steering committee with 19 council members for next 2 years starts from this annual meeting at Tsukuba. As the new president of JSSX, I would like to emphasize the following 3 issues to run the Society. First is to research on new drug modalities. Modalities in the currently developing drugs are diverse; from small molecule drugs and protein therapeutics to oligonucleotides, cell therapeutics, and gene therapeutics. According to the variety of their chemical/biological characteristics, their evaluation methods for pharmacokinetics are also diverse and still developing. For example, measurement methods, and necessity of their metabolites and impurity evaluation of oligonucleotides are not fully established. An AMED research group has been discussing non-clinical evaluation, including pharmacokinetics, of peptide drugs with non-natural structures. In line with this movement, research trend survey working group of JSSX did a survey to company/academia researches in 2018, and presents the summary results as a poster at this annual meeting. In addition to raise these topics in annual meetings, please consider to choose and pick a topic up as your new research target. Second issue is international alliance. In the 15th steering committee headed by Prof. Yamazaki, JSSX started to collaborate with the counterpart academic societies in China and Thai, in addition to Korea, and we have an international session at this annual meeting. Collaborations with these Asian societies are increasingly important. We should know academic achievements each other and get together to proceed. In addition, collaboration with ISSX is also a priority of our JSSX.Last topic is to foster new generation including bachelor/ graduate students and young scientists. Thanks to Prof. Terasaki, JSSX established funds for young scientists to assist presentation at the international meeting. In addition, we should assist bachelor students to understand the importance of new research and its presentation at this academic meeting. Also, it may be a good time to start discussion on certified pharmacist of pharmacokinetics. Please participate in the activities of JSSX and enjoy them!

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SY-01-01

チロシンキナーゼ阻害薬のケラチノサイトにおける分布・毒性と皮膚障害との関連

The interaction of tyrosine kinase inhibitors with keratinocytes and its dermal toxicity in humans

増尾　友佑、加藤　将夫

金沢大学医薬保健研究域薬学系 Yusuke Masuo, Yukio Kato

Faculty of Pharmacy, Kanazawa University

[Purpose] Hand-foot skin reaction (HFSR) is one of common and severe adverse effects provoked by tyrosine kinase inhibitors (TKIs). Although HFSR symptom itself is not life threatening, the occurrence of HFSR often leads to dose reduction and/or discontinuation of therapy, limiting anti-tumor effects of TKIs in patients. While molecular mechanisms of HFSR is largely unknown, histological analysis showed the occurrence of toxicity in keratinocytes, leading to hypothesis that the cytotoxic effects of TKIs in keratinocytes is related to HFSR. The aim of the present study is to characterize cytotoxicty of TKIs in keratinocytes to evaluate its possible relevance to HFSR. [Methods] Mouse keratinocytes isolated from newborn mice and commercially obtained human epidermal keratinocytes were exposed to TKIs. Cell viability was evaluated using ATP luminescence assay to assess IC50 of each TKI. The ratio of the maximum plasma concentration in humans (Cmax) to the IC50 values (Cmax/IC50) were then compared with the reported incidence of HFSR in humans. Uptake of TKIs in keratinocytes were also evaluated after treatment with siRNA against transporters.

[Results and Discussion] Exposure to most of the TKIs caused concentration-dependent decrease in viability of both mouse and human keratinocytes, the Cmax/IC50 values of 8 TKIs being more than unity. Among them, the incidence of HFSR by 4 drugs was more than 5%, suggesting that the risk of HFSR might be at least partially associated with the cytotoxicity in keratinocytes. Especially, high Cmax/IC50 value (~2) and incidence of HFSR (>50%) were observed for regorafenib. Cellular uptake of TKIs were inversely correlated with IC50 values, suggesting high uptake of TKIs may cause cytotoxicity in keratinocytes. Single knockdown of ABCC1, ABCC3, and ABCC4 did not increase the uptake of regorafenib, implying that other transporters and/or multiple efflux transporters may be involved in exposure of regorafenib to keratinocytes.

[Conclusion] The cytotoxic effect of TKIs in keratinocytes may be at least partially associated with dermal toxicity although further characterization of this relationship should be evaluated.

Symposium 1

（Drug Metabolism and Drug Induced Toxicity DIS 1）

SY-01-02

薬剤性光線過敏症のリスク予測と戦略的回避

Drug-induced phototoxicity: prediciction and de-risking

尾上　誠良

静岡県立大学 Satomi Onoue

University of Shizuoka

Chemical photosensitivity can be elicited by exposure of the skin to various pharmaceutical substances, foods, cosmetics and other environmental chemicals, followed by exposure to sunlight. There are at least three types of chemical photosensitivity, i.e., photoirritancy (narrowly defined as phototoxicity), photogenotoxicity and photoallergenicity, and their clinical characteristics and mechanisms are quite different. Concerns about chemical phototoxicity is increasing, and various studies have been made to clarify the photobiochemical characteristics of phototoxins and the mechanisms involved. Various methodologies, including in silico prediction models, photochemical assay systems, and in vitro phototoxicity prediction tools, have been developed to predict the phototoxic potential of chemicals. Our group developed the reactive oxygen species (ROS) assay as in chemico photosafety testing tool, and ROS assay was successfully adopted as ICH S10 guideline (2014) and OECD test guideline 495 (2019). These in vitro photosafety assessments might also be effective for clarifying the mechanism of phototoxicity for tested chemicals in detail; however, the results from in vitro photosafety evaluations would not always reflect clinical observations of drug-induced phototoxicity. Herein, further characterization of pharmacokinetics with a focus on skin distribution might ensure prediction of the in vivo photosafety of tested chemicals, since phototoxic reactions mainly occur in the skin after topical and/or systemic administration. Our previous study also demonstrated that the combined use of in vitro photobiochemical/phototoxic and pharmacokinetic data might enable evaluation of in vivo phototoxic risk with high clinical relevance.

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SIL

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SY-01-03

SGLT2阻害薬による重篤な皮膚障害と皮膚組織への移行性

Susceptibility to serious skin and subcutaneous tissue disorders and skin tissue distribution of

sodium-dependent glucose co-transporter type 2 (SGLT2) inhibitors

栄田　敏之

京都薬科大学薬物動態学分野 Toshiyuki Sakaeda

Department of Pharmacokinetics, Kyoto Pharmaceutical University

Objectives: In Japan, sodium-glucose co-transporter type 2 (SGLT2) inhibitors have been reported to be associated with serious skin and subcutaneous tissue disorders. A preliminary post-marketing surveillance (PMS) study suggested that the association was specific for ipragliflozin and, to a lesser extent for dapagliflozin. These studies were performed to confirm the association of 6 SGLT2 inhibitors with serious skin disorders in a clinical setting and to understand the underlying mechanisms. Methods: The latest PMS records were retrieved from the Japanese Adverse Drug Event Report (JADER) database, and the associations were analyzed by data mining techniques. The skin tissue distribution of SGLT2 inhibitors was evaluated using albino rats after oral administration at clinical doses. Reconstructed 3-D human skin model consisting of keratinocyte and fibroblast was exposed to SGLT2 inhibitors, and alterations of gene expression were assessed by microarray. Results: The adjusted reporting odds ratio (95% confidential limit) was 1.667 (1.415, 1.963) for ipragliflozin, 0.514 (0.317, 0.835) for dapagliflozin, 0.149 (0.048, 0.465) for tofogliflozin, 0.624 (0.331, 1.177) for luseogliflozin, 0.590 (0.277, 1.257) for canagliflozin and 0.293 (0.073, 1.187) for empagliflozin, when drugs other than the SGLT2 inhibitors were referred, and the association was detected only for ipragliflozin in clinical use. The skin tissue-to-plasma concentration ratio of ipragliflozin was 0.46 ± 0.20 (±SD) at 1 hr after administration and increased in a time-dependent manner to 5.82 ± 3.66 at 24 hr (p<0.05), but not in case of other SGLT2 inhibitors, suggesting the interaction of ipragliflozin with skin tissue components. Microarray analysis suggested that signal transducer and activator of transcription 3, STAT3 was key factor in ipragliflozin-specific serious skin disorders.

SY-01-04

マルチキナーゼ阻害薬による手足皮膚反応におけるSTAT3の役割

Role of STAT3 in hand–foot skin reaction induced by multi-targeted tyrosine kinase inhibitors

山本　和宏

神戸大学医学部附属病院薬剤部 Kazuhiro Yamamoto

Department of Pharmacy, Kobe University Hospital

Multi-targeted tyrosine kinase inhibitors (TKIs) can cause serious and characteristic side effects that may lead to treatment interruption or dosage decrease. Hand–foot skin reaction (HFSR), which is a common side effect of multi-targeted TKIs, is characterized by inflammation and keratinization of the palms or soles and results in functional problems, such as difficulty in walking and grasping, thereby remarkably reducing the quality of life (QOL) of patients. Considering that the development of HFSR correlates with the therapeutic outcomes of TKIs in patients with cancer, the management of HFSR is linked to a prolonged cancer treatment period. Therefore, the clarification of the mechanism underlying the presentation of HFSR is crucial for the development of effective prophylactic strategies aimed at improving the QOL and prognosis of these patients.

We performed a basic and clinical study to clarify the mechanism of HFSR by focusing on the signal transducer and activator of transcription 3 (STAT3), which is a regulator of homeostasis in the skin tissue and is correlated to several skin diseases. We found that STAT3 activity regulated the multi-targeted TKI-induced keratinocyte growth inhibition. Moreover, our findings suggested an association between the development of HFSR and STAT3 polymorphism in patients with cancer. Based on these perceptions, we searched for a novel mechanism-based prophylaxis and are currently verifying its effectiveness in treating HFSR in a clinical study.

In this symposium, I will describe our findings pertaining to the molecular biological mechanism of HFSR and discuss the role of STAT3 in the development of HFSR. Finally, I will address future perspectives.

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SY-02-01

中枢作用薬開発を促進する条件的不死化細胞と生体模倣によるヒト血液脳関門モデル

Development of a conditionally immortalized cell-based human blood-brain barrier model for CNS drug

development

降幡　知巳

東京薬科大学 Tomomi Furihata

Tokyo University of Pharmacy and Life Sciences

In vitro blood-brain barrier (BBB) models are expected to be among the most important experimental tools in central nervous system drug development in order to identify a drug or a carrier that can go beyond the BBB. To develop a BBB model, we have focused on human immortalized cells by taking an advantage of their simultaneous possession of cell type-specific functions and high proliferation abilities. Accordingly, we aim to develop two- and three-dimensional in vitro human BBB models using three originally created human immortalized cell lines, which are HBMEC/ci18 (brain microvascular endothelial cells), HASTR/ci35 (astrocytes), and HBPC/ci37 (pericytes). To setup a two-dimensional model, we prepare a transwell culture system, where HBMEC/ci18 and HBPC/ci37 cells are seeded on an inner and an outer side of the insert membrane, respectively, and HASTR/ci35 cells are placed on the bottom well. This tri-culture BBB model shows a maximum 134 (Ω × cm2_{) and efflux transporter functions. In drug permeability analyses, the average}

permeability coefficient of the BBB-permeable drugs (propranolol, etc.) is 960 × 10-6 _{(cm/s), but that of the non-BBB-permeable drugs}

(desloratadine, etc.) is 73 × 10-6 _{(cm/s). Therefore, it is considered likely that the immortalized cell-based human BBB model has a power}

to discriminate between BBB-permeable and non-permeable drugs to a certain extent.Meanwhile, we have also worked on development of spheroid BBB model, in which HASTR/ci35 and HBPC/ci37 cells form a core structure and HBMEC/ci18 cell monolayer covers it. HBMEC/ci18 cells in the spheroid BBB model clearly show the barrier functions against 5 kDa dextran and rhodamine123 penetration into the core region. In transcytosis assays, the permeability rate of MEM-189 (a BBB permeable antibody) is significantly higher than that of control IgG, suggesting that its transcytosis routes are active in the spheroidal BBB model.Based on these findings, we believe that the immortalized cell-based human BBB models have a great potential for providing opportunities to identify BBB-permeable drug candidates as well as strategies by which therapeutic molecules can be delivered into the brain.

Symposium 2

（Microphysiological System DIS）

SY-02-02

iPS細胞を用いたOrgans-on-chipsの開発と小腸機能再現への応用

Engineering of iPS cell-based Organs-on-Chips to mode intestinal function

鳥澤　勇介

京都大学

Yu-suke Torisawa

Kyoto University

Recent advances in microsystems technology and tissue engineering have led to the development of organs-on-chips, which reconstitute key functional units of organs in vitro by mimicking natural tissue arrangements and microenvironmental chemical and physical cues within microfluidic devices. Since the physiological microenvironments in living systems are mostly microfluidic in nature, microfluidic systems facilitate engineering of cellular microenvironments. In this presentation, I will show two microfluidic approaches to model intestinal function using human induced pluripotent stem cell (iPSC)-derived cells. 1) A microfluidic device to replicate the intestinal tissue-tissue interface. The device consists of two layers of microchannels separated by a thin porous membrane lined with opposing layers of intestinal epithelial cells and vascular endothelial cells. This device permits application of dynamic mechanical forces including fluid shear stress and cyclic mechanical strain that are crucial for the development and function of organs. We show that this device can be used to test permeability of chemical compounds as well as drug transport through the intestinal epithelium generated from human iPS cells. 2) A microfluidic method to engineer vascularized 3D cell constructs to model cellular interactions through blood vessels. Vascular networks are essential to maintain cellular viability and functions; however, current 3D culture models lack a vascular system. Engineering perfusable vascular networks that can deliver nutrients and blood cells to 3D cell constructs could be a useful platform to recapitulate cellular microenvironments. We show a microfluidic device to form 3D vascular networks generated from iPSC-derived endothelial cells that can be perfused with blood cells including immune cells. Application of physiological cyclic strain promoted the formation and maintenance of vascular networks, suggesting that mechanical forces play an important role in the maintenance of 3D blood vessels. We use these microfluidic devices to recapitulate intestinal microenvironment including tissue architecture and physicochemical signals.

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SY-02-03

コラーゲンビトリゲル膜を利用した上皮および内皮の関門組織モデルの構築と化学物質の動態および

毒性を予測するシステムへの応用

Fabrication of epithelial and endothelial barrier tissue models utilizing a collagen vitrigel membrane and

their application to the system for predicting ADME/Tox of chemicals

竹澤　俊明

国立研究開発法人農業・食品産業技術総合研究機構 生物機能利用研究部門 Toshiaki Takezawa

Institute of Agrobiological Sciences, National Agriculture and Food Research Organization

Extracellular matrix in vivo provides the microenvironment functioned as not only the scaffold for epithelial, mesenchymal and endothelial cells but also the pathway of chemicals. It can be interpreted that the barrier function of epithelial and endothelial tissues in vivo is maintained by the microenvironment of epithelial and endothelial cells, respectively. A collagen vitrigel membrane (CVM) we developed provides such microenvironment in a culture system in vitro because it is composed of high density-collagen fibrils equivalent to connective tissues in vivo. The CVM possesses excellent transparency and protein permeability, and consequently it functions well as a scaffold that can facilitate the fabrication of various 3d-culture models reflecting both structure and function in tissues and organs in vivo. Here, the cross-talking between different types of cells via the CVM is essential for fabricating organoids composed of two or more types of cells. Also, we developed a CVM chamber useful for fabricating epithelial or endothelial barrier tissue models and subsequently measuring transepithelial or transendothelial electrical resistance (TEER), respectively. From the viewpoint of extrapolating ADME/Tox in vivo of chemicals, we have developed vitrigel-EIT (Eye Irritancy Test), CPT (Corneal Permeability Test), VPT (Vascular Permeability Test), and LMET (Liver Metabolism and Excretion Test) methods using corneal epithelium, cornea, microvascular endothelium and hepatic lobule models fabricated in the CVM chamber, respectively. Here, the vitrigel-EIT is a test method for determining the presence or absence of eye irritation by analyzing the profile on the time course of TEER in the measurement for 3 minutes, after exposing chemicals to a human corneal epithelial model prepared by culturing HCE-T cells (a human corneal epithelial cell line) in a CVM chamber. The vitrigel-CPT is a test method to evaluate drug permeability in a cornea (epithelium-acellular stroma-endothelium) model prepared by co-culturing HCE-T cells and BCE C/D-1b cells (a bovine corneal endothelial cell line) via the CVM of chamber. The vitrigel-VPT is a test method to evaluate drug permeability in normal and inflammatory (histamine-treated) skin-reflected microvascular endothelium models prepared by co-culturing HMVECs (human microvascular endothelial cells) and HDFs (human dermal fibroblasts) via the CVM of chamber. The vitrigel-LMET is a test method to excrete the hepatic metabolites of drugs accumulated in bile canaliculus-like structures (BCLS) in a hepatic lobule model, prepared by co-culturing “HepG2-NIAS cells (a human hepatoma cell line) with BCLS formation by the oxygenation culture via the CVM of chamber” on “the monolayer of TFK-1 cells (a human bile duct carcinoma cell line)”.

SY-02-04

Nephrotoxicity, kidney transport, and disease modelling on organotypic kidney-on-a-chip models

MIMETAS BV, J.H. Oortweg 19, NL-2333CH Leiden, The Netherlands

The proximal tubule of the kidney is the primary site of the nephron for drug clearance which makes it a target for drug-induced toxicity. Current 2D and animal models generally fail to recapitulate key renal aspects, resulting in low translatability to the human situation. The lack of reliable models hampers the development of efficient drugs and therapies. There is an unmet need for robust and predictive in vitro kidney models that recapitulate physiological relevant renal characteristics and assess both tubular transport and toxicity. To overcome the current limitations, we have used a standardized high-throughput microfluidic organ-on-a-chip platform, the OrganoPlate®_,

to develop perfused and precisely controlled kidney models. To this end, kidney cells from different cell sources such as cell lines, primary material or organoids derived from stem cells were used to build the model. An extracellular matrix (ECM) gel can be pattered into one of the membrane-free microchannels of each chip. Additionally, renal epithelial cells were cultured against the ECM and upon perfusion flow grew into tubular structure with clear apical and basolateral access. To increase physiological relevance, endothelial or interstitial cells were added to obtain a more complex co-culture kidney model.

Kidney epithelial cells cultured in the OrganoPlate® _{form leak-tight confluent tubular structures and were used to investigate}

drug-induced toxicity, drug-drug interactions or altered drug-drug-induced transporter functionality. The tubules showed expected polarization as assessed by immunofluorescent staining of relevant kidney markers such as acetylated tubulin or ezrin. The effect of compounds on the barrier integrity was assessed by measuring the permeability of a fluorescent dye added to the lumen of the tube or through transepithelial electrical resistance (TEER). Additionally, the viability, expression of kidney relevant biomarkers, transport activity and release of proteins was assessed in the model.

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SY-02-05

Proximal tubule-on-a-chipを用いたMATE2-K発現メカニズムの解明

Development of proximal tubule-on-a-chip: fluid shear stress stimulates MATE2-K expression via Nrf2

pathway activation

福田　保則

サノフィ株式会社希少疾患メディカル部 Yasunori Fukuda

Rare Disease Medical, Sanofi K.K.

Renal toxicity has been reported for various types of drugs such as antibiotics and anticancer agents. In drug development, accurate prediction of drug-induced nephrotoxicity is critical to obtain safer drugs efficiently and to reduce the costs arising from high attrition rates. Therefore, great efforts have been directed towards the improvement of predictive models for nephrotoxicity. Drugs are considered to exert toxic effects against various sites within the kidney. Among them, the proximal tubule, where drug excretion, reabsorption, and accumulation occur, is considered the main target of nephrotoxicity.Cellular assay systems that recapitulate physiologically relevant microenvironments have been proposed for correct estimation of drug responses in the human body. However, establishment of such assay systems for accurate prediction of renal toxicity is still challenging because of the lack of readily available in vitro assay systems. In this study, we investigated the cellular response to fluid shear stress, which is a characteristic of the environment in the kidney proximal tubules, using microfluidic devices and primary human proximal tubule epithelial cells. The global gene expression profiles under the fluidic conditions revealed upregulation of MATE2-K and activation of Nrf2 signaling in response to fluid shear stress. Network and cell biological analysis additionally showed that expression of MATE2-K is regulated by Nrf2 signaling. These results strongly suggest that fluid shear stress is involved in the expression and maintenance of functions of tissue-specific drug transporters in the proximal tubule, where the cells are exposed to continuous shear stress by primary urine. Furthermore, the microfluidic culture of human proximal tubules, proximal tubule-on-a-chip, was demonstrated to be a useful system to analyze biomolecular functions in physiologically relevant cell conditions.In my presentation, I will also mention challenges and expectations of organ-on-a-chips technology based on my experience in a pharmaceutical company. This study was conducted when I was a researcher in Takeda Pharmaceutical Company Ltd.

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SY-03-01

腫瘍ターゲティングとEPR効果

Passive tumor targeting via EPR effect

石田　竜弘

徳島大学大学院医歯薬学研究部 Tatsuhiro Ishida

Graduate School of Biomedical Sciences, Tokushima University

The enhanced permeability and retention (EPR) effect was first reported by Matsumura and Maeda in 1986. They showed via EPR effect macromolecules larger than 40 kDa selectively leak out from tumor vessels and accumulate in tumor tissues, while this EPR effect-driven drug delivery does not occur in normal tissues. This unique phenom-enon in solid tumors—the EPR effect—is thus considered to be a landmark principle in tumor-targeting chemo-therapy and became a promising paradigm for anticancer drug development. For example, Doxil, which is a PEGylated (polyethylene glycol-coated) liposome-encapsulated formulation of doxorubicin, was approved for treat-ment of Kaposi sarcoma and other cancers. Many other polymeric or micellar drugs are in clinical stage development (phases I and II). Compared with conventional anticancer drugs, most of which are small molecular drugs, these macromolecular drugs have superior in vivo pharmacokinetics (e.g., a prolonged plasma half-life) and, more im-portant, greater tumor selectivity, so that they produce improved antitumor effects with no or less adverse reactions. The EPR effect is still the “gold standard” in anticancer drug design and anticancer strategies using macromolecu-lar agents, including gene delivery, molecular imaging, antibody therapy, micelles, liposomes, and protein-polymer conjugates. However, recent results from preclinical studies examining the EPR effect have been mixed, with some reports supporting preferential, EPR-mediated accumulation of nanomedicines within tumors, while others show that the EPR effect is highly dependent on the tumor model. In this talk, I would like to overview the history, fea-ture, and limitation of the EPR effect.

Symposium 3

（DDS DIS）

SY-03-02

腫瘍血管の多様性

Heterogeneity of tumor blood vessels

樋田　京子

北海道大学大学院歯学研究院口腔病態学分野 血管生物分子病理学教室 Kyoko Hida

Vascular Biology and Molecular Pathology, Graduate School of Dental Medicine, Hokkaido University

Tumor growth and metastasis are dependent on angiogenesis, which is the formation of new blood vessels. The balance between angiogenic stimulators and inhibitors regulates angiogenesis in the tumor microenvironment. Tumor blood vessels, especially the endothelial cells lining tumor blood vessels (tumor endothelial cells [TECs]), are important targets in cancer therapy. As newly formed tumor blood vessels originate from pre ‐ existing normal vessels, tumor blood vessels and TECs have traditionally been considered to be the same as normal ones. However, tumor blood vessels have a distinctively abnormal phenotype, including morphological alterations. Recently, it has been revealed that TECs constitute a heterogeneous population, exhibiting characteristics that are induced by tumor microenvironmental factors. Furthermore, TECs contribute to cancer progression through metastasis. For example, TECs in highly metastatic tumors aberrantly express angiocrine factors which stimulates cancer cell intravasation, in turn they instigate tumor cells to metastasize. Also, TEC intracellular adhesion molecule, VE-cadherin expression was downregulated by tumor-extracellular vesicles (EVs), causing in tumor metastasis. Besides, we have found that TECs express ABCB1, a drug transporter molecule and support cancer cells even during chemotherapy. Our experimental results have shown that targeting such abnormal TECs could show the anti-tumor effects in the mouse model. TEC abnormalities related to cancer progression will be to provide insight into new anticancer therapies.

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SY-03-03

内因性アルブミン輸送システムを介した膵臓がんへのアルブミン標的化

Targeting of albumin to pancreatic cancer via endogenous albumin transport system

異島　優

_{、木下　遼}

_{、丸山　徹}

_{、小田切　優樹}

_{、石田　竜弘}

1_{徳島大学大学院医歯薬学研究部、}2_{熊本大学大学院薬学教育部、}3_{崇城大学薬学部}

Yu Ishima1_{, Ryo Kinoshita}2_{, Toru Maruyama}2_{, Masaki Otagiri}3_{, Tatsuhiro Ishida}1

1_{Institute of Biomedical Sciences, Tokushima University，}2_{Graduate School of Pharmaceutical Sciences, Kumamoto University，}3_Faculty

of Pharmaceutical Sciences, Sojo University

Human serum albumin (HSA), which is the most abundant protein in human serum, possesses many functions, such as transport carriers of ligands including fatty acids, and antioxidant action. In particular, many DDS studies that utilize the superiority of HSA in blood retention have been reported and are being clinically applied to improve retention of peptides with short blood half-lives. Additionally, HSA is known as one of the endogenous proteins that receive enhanced permeability and retention (EPR) effect and is used as a DDS carrier for cancer tissues. This EPR effect is a phenomenon in which macromolecules are more likely to leak out from blood vessels and accumulate due to increased vascular permeability in cancer tissues and immature lymphatic system, which is known as one of the “passive targeting”. Abraxane® _{is an example of an HSA-containing anticancer agent that has already been used clinically.}

Although HSA used in this Abraxane® _{is not intended for the EPR effect but is intended to aid dissolution of paclitaxel, superiority to}

conventional paclitaxel preparations has also been confirmed since the launch of Abraxane®_{. These clinical findings suggest that HSA is}

not limited to the role as a solubilizer of paclitaxel. In fact, it has been reported that not only “passive targeting” of the EPR effect but "Active targeting" by gp60 and Secreted Protein Acidic and Rich in Cysteine (SPARC) could be involved in the delivery of HSA to cancer tissues. The mechanism of HSA uptake by such cancer cells is considered to be one of the survival tools that use HSA as a “nutrition source”. It has been clarified that this phenomenon is particularly observed in pancreatic ductal adenocarcinoma with poor angiogenesis. We propose that this phenomenon is called “Endogenous Albumin Transport (EAT) System”. The advantage of this EAT system is that it is possible to actively deliver what was delivered to the cancer tissue by the EPR effect to “cancer stroma” and further to “intracellular”. In this symposium, I would like to introduce the usefulness of albumin as a carrier for pancreatic cancer with our latest findings.

SY-03-04

血液脳関門透過性タンパク質と脳血管内皮細胞における輸送特性

Blood-brain barrier-permeable proteins and transport characteristics in brain endothelial cells

立川　正憲

_{、寺崎　哲也}

1_{徳島大学大学院医歯薬学研究部(薬学域)、}2_{東北大学大学院薬学研究科}

Masanori Tachikawa1_{, Tetsuya Terasaki}2

1_{Graduate School of Biomedical Sciences, Tokushima University，}2_{Graduate School of Pharmaceutical Sciences, Tohoku University}

The central nervous system (CNS)-acting proteins such as transferrin and insulin undergo the blood-to-brain transport via specific receptor-mediated endocytosis at the blood-brain barrier (BBB). It has also been shown that certain proteins such as inflammatory cytokines, which are released from injured or diseased tissues into the circulating blood, are transported to the brain and contribute to CNS pathology. These prompt us to hypothesize that the BBB possesses specific blood-to-brain transport systems for proteins derived from peripheral tissues although the BBB restricts transport of proteins like albumin. The purpose of this study was to identify the in vivo BBB permeable protein(s) derived from the mouse liver, which could be the major tissue producing blood-circulating proteins, by comprehensive and quantitative proteomics (SWATH) and to clarify their transport characteristics at the BBB using human brain microvessel endothelial cells (hCMEC/D3 cells). Cytosolic proteins were extracted from the mouse liver and then labeled with biotin. After intravenous administration of the biotin-labeled proteins to mice in vivo, the labeled proteins were collected from the cerebrum by streptavidin beads and identified by SWATH method. We found 9 proteins derived from the liver, including creatine kinase (CK), in the cerebrum. In vivo integration plot analysis revealed that CK-MM was readily detected in the fraction of brain parenchyma after its intravenous administration in mice. The hCMEC/D3 cells exhibited preferential uptake of muscle-type CK (CK-MM) over brain-type CK (CK-BB). The uptake of CK-MM by hCMEC/D3 cells was temperature-dependent and concentration-dependent. The uptake was significantly inhibited in the presence of clathrin- and caveolin-dependent endocytosis inhibitors. These results suggest that entry of the liver-derived CK into the brain from the circulating blood is mediated by a receptor-mediated transport system(s) at the BBB. The BBB would play a role in proteins-mediated transduction between the brain and peripheral tissues and complementation of the CNS functional architecture by supplying the peripheral tissues-derived proteins.

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SY-03-05

生体内微小環境を再現する血管3次元構築マイクロ流体デバイス

Microfluidic device for reproduction of in vivo microenvironment with three-dimensional vascular structure

船本　健一

東北大学流体科学研究所 Kenichi Funamoto

Institute of Fluid Science, Tohoku University

In an in vivo microenvironment, cells are exposed to spatial and temporal heterogeneity of oxygen tension, and cyclic hypoxic stress possibly damages vascular network. Therefore, it is essential to understand cell behaviors under oxygen-controlled conditions for elucidation of mechanisms of vascular diseases. Microfluidic device is a potential tool which enables high-resolution, real-time observation of three-dimensionally cultured cells. My research group has developed microfluidic devices with controllability of oxygen tension to reproduce hypoxic microenvironments. Inside the devices, a central gel channel, in which hydrogel is placed to mimic extracellular matrix, is flanked by media channels, to which cell culture media are supplied. Cells can be seeded in either the gel channel or media channels. To control oxygen tension, two gas channels, to which gas mixtures are supplied, are positioned next to or above the other channels. The device was fabricated from polydimethylsiloxane, and the bottom was covered with a glass cover slip. In addition, a gas-impermeable polycarbonate film was embedded to prevent oxygen diffusion from the atmosphere. First, permeability change of vascular endothelial monolayer by hypoxic exposure was investigated. A monolayer of human umbilical vein endothelial cells was formed in the media channel, and it was exposed to a hypoxic condition. Permeability was measured by quantifying diffusion of fluorescence-labelled dextrans passing through the monolayer. Hypoxic exposure increased the permeability of the vascular endothelial monolayer, and impaired size-selective barrier function. Next, microvascular network with blood brain barrier was modeled by culturing human induced pluripotent stem cell-derived endothelial cells, brain pericytes, and astrocytes in fibrin gel infused in the gel channel. Permeability of the microvascular network was lower than that of the above-mentioned vascular endothelial monolayer and represented little variations by hypoxic exposure. Thus, cellular experiments using microfluidic devices give us new insights on vascular permeability.

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SY-04-01

Systems modeling development over the past 10 years

Department of Pharmaceutical Sciences, University at Buffalo, SUNY, Buffalo, NY 14214, USA

The fields of systems biology and pharmacokinetic/pharmacodynamic (PK/PD) modeling had evolved largely in parallel; however, there was an emerging consensus that an effective integration of these disciplines was needed in order to fully realize the promise of each in bringing new therapeutic molecules and combination regimens to the bedside. Significant insights were realized from the recognition that both drugs and disease processes give rise to complex and dynamic clinical phenotypes by altering natural interconnected biochemical networks and supported the emergence of systems pharmacology models of drug action. Multi-scale models that combine PK/PD principles and signaling networks have the potential to serve as a platform for integrating molecular factors that regulate drug effects and clinical outcomes. There are many challenges to the development of such models, which include (but are not limited to): analytical and computational limitations, system complexity, unknown robust and redundant processes, emergent systems properties, biological noise, physiological control systems, wide spatiotemporal scales, and difficulties translating across in vitro, preclinical, and human studies. Over the past decade, major advances have been made to address these challenges, and the goal of this presentation is to highlight advances in systems modeling of drug action. Despite substantial progress, much more is needed to fully realize the potential of systems modeling, and future efforts might focus on more complete, unbiased, curated and readily available interactomes, along with analytical advances in proteomics and unbiased computational approaches for network development and hybrid multi-scale architectures.

Symposium 4

（Systems Pharmacology DIS）

SY-04-02

臨床エビデンスに基づくドラッグリポジショニングと創薬標的の発見

Drug repositioning and target finding based on clinical evidence

金子　周司

京都大学大学院薬学研究科生体機能解析学分野 Shuji Kaneko

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University

Adverse drug reactions have been utilized to make animal models of human disease, and many drugs have been found using these models. Therefore it is reasonable to consider that there may be a common mechanism underlying the pathology of human disease and adverse drug reactions. In this context, accumulating case reports of human adverse events would be attractive clinical evidence, if we could statistically identify a concomitant medication that reduces the occurrence of an adverse event. This simply enables ‘drug repositioning’ that proposes a practical relief of adverse reaction. Moreover, the drug-drug interaction may provide important clues to the discovery of new molecular mechanism of adverse effects, and hopefully, of human diseases. The FDA Adverse Event Reporting System (FAERS) is a public database that accumulates huge self-reports of adverse events. In nearly half of the cases, multiple drugs are prescribed, so that potential drug-drug interactions are to be analyzed. Focusing on adverse reactions relating to diabetes mellitus (DM) caused by an atypical anti-schizophrenic drug, quetiapine, we found that concomitant use of vitamin D analogs significantly suppresses the occurrence of the quetiapine-induced DM in FAERS. Experimental validation using mice revealed that quetiapine acutely caused insulin resistance, which was mitigated by dietary supplementation with cholecalciferol. In a gene expression database, several genes downstream of insulin receptor were downregulated by quetiapine in the liver. Further experiments clarified that a PI3K regulatory protein gene, pik3r1, was downregulated by quetiapine, which was reversed by cholecalciferol in mouse skeletal muscle. In addition, the insulin-stimulated glucose uptake into cultured myotubes was inhibited in the presence of quetiapine, which was reversed by pretreatment with calcitriol in a PI3K-dependent manner. Taken together, these results suggest that vitamin D prevents the atypical antipsychotic-induced hyperglycemia and insulin resistance by upregulation of PI3K function. This new strategy will pave the way for drug repositioning and clarifying unknown disease mechanisms.

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SY-04-03

QSPモデルを用いた仮想臨床試験とその適用事例

Virtual clinical trial using QSP model and its case study

宮野　拓也

第一三共株式会社 Takuya Miyano

DAIICHI SANKYO CO., LTD.

[Purpose] Systematic understanding of linkage from a target molecule to a disease is crucial to improve the probability of success in

drug development. It is difficult, however, to establish such linkage, especially when there exist no clinically relevant animal disease models. Quantitative systems pharmacology (QSP) is a framework that visualizes mechanistic understanding of pharmacological effects in the context of pathogenesis and simulates clinical outcomes following drug treatments. This presentation shows a case study, where QSP-based virtual clinical trials gave substantial insights into the potential of anti-protein C antibody as a drug candidate in hemophilia A before proceeding to clinical development.

[Methods] The QSP model was built with in vitro experimental data of anti-protein C antibody, public data of emicizumab which is an

approved antibody drug for hemophilia A mimicking the cofactor function of factor VIII, public QSP models of the coagulation cascade, and natural history of the disease. Pharmacokinetics (PK) parameters of anti-protein C antibody were assumed to be the same as emicizumab. The model generated virtual patients with different PK parameters and levels of coagulation factors.

[Results and Discussion] The virtual clinical trials suggested that the anti-protein C antibody under body weight-based weekly dosing

has a high risk of thrombosis, which could be associated with excessive reduction of protein C. This implies the necessity of dose titration according to hemostatic activity and safety marker of individuals to achieve better efficacy than emicizumab with sufficient safety.

[Conclusions] This hemophilia case demonstrated the usefulness of QSP modeling to organize a vast amount of relevant knowledge,

estimate clinical outcomes, and support designing rational clinical development plans that maximize the value of the product concept while minimizing associated risks. Thus, QSP can improve systematic understanding of diseases and target molecules, which should be one of the key concepts to increase the probability of success in drug development.

SY-04-04

Integrated use of QSP (PBPK) and MPS; Current status, issues and future perspectives

Certara, Sheffield UK and Ceratar rinceton, New Jersey, USA.

The term “Organ on Chip” has appeared in over 10,000 publications. These refer typically, but not exclusively, to microphysiological systems (MPS) which mimic one or several biological elements of biological functions in an in vivo system. There is a debate as to whether they should always contain some form of flows or whether they should be void of dynamics related to circulation.

Interestingly a very small proportion of these publications (<3%) have focused on pharmacodynamics or pharmacokinetics containing terms that demonstrate quantitative translation of the data (PBPK <0.1%, QSP <0.01%). Many people have questioned whether the push for the use of MPS is the result of some “hype” or there is genuine “hope” for their added value. With a projected market of over $100M in 2022, the pharmaceutical industry are a main target for the use of these systems though the chemical industry are also assumed to be part of such market on the back of the move by many of the environmental agencies to reduce the use of animal studies - see as an example USA EPA announcement on 10th _September:

https://www.epa.gov/newsreleases/administrator-wheeler-signs-memo-reduce-animal-testing-awards-425-million-advance

Our vision for the use of these tools has aligned with the following points and they heavily depend on quantitative “translatability” of the data that are obtained from the systems:

　 • Short-term: MPS can be complementary to animal in IND

　 • Long-term: MPS will replace some of the in vitro/in vivo data in IND

We also believe that these systems play a major role in precision medicine at point of care (virtual triplet) by adding to the power of diagnostic tools.

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SY-05-01

ヒト凍結肝細胞を用いたin vitro CYP1A2ダウンレギュレーション評価

Evaluation of in vitro CYP1A2 down-regulation using cryopreserved human hepatocytes

石田　千尋

_{、佐能　正剛}

_{、古武　弥一郎}

1_{広島大学大学院 医系科学研究科、}2_{日本新薬株式会社 創薬研究所 安全性・動態研究部}

Chihiro Ishida1,2_{, Seigo Sanoh}1_{, Yaichiro Kotake}1

1_{Graduate School of Biomedical and Health Sciences, Hiroshima University，}2_{Pharmacokinetics and Safety Assessment Dept., Discovery}

Research Labs, Nippon Shinyaku Co., Ltd.

CYP down-regulation, the suppression of CYP activity at the transcriptional level, is the opposite of CYP induction and is a relatively new area in DDI studies. To date there are few published observations of drug-induced down-regulation by small molecules in vitro, and the underlying mechanism of down-regulation is not fully understood. Therefore, our ability to establish in vitro models for assessing down-regulation is significantly limited, in contrast to in vitro induction studies for which recommended criteria using cryopreserved human hepatocytes exist. The FDA currently has no recommendations on the evaluation of CYP down-regulation. Both the EMA and the PMDA recommend attempting to understand the potential clinical relevance of CYP mRNA down-regulation by carrying out in vitro studies using hepatocytes through the evaluation of CYP induction. We focused on obeticholic acid (OCA), because a clinical study of OCA with caffeine suggested that OCA may be a useful positive control to establish a method to evaluate CYP1A2 down-regulation and to investigate the mechanism of its down-regulation. We investigated the ability of OCA to down-regulate CYP1A2 in vitro using the testing method of CYP induction studies, which 1-100μM OCA was incubated with cryopreserved human hepatocytes for 72 hrs. OCA suppressed CYP1A2 mRNA expression and CYP1A2 enzyme activity without causing direct inhibition of CYP1A2 or cytotoxicity at 1 and 10μM. The decreases in CYP1A2 mRNA expression were correlated with the decreases in activity. These results suggest that standard in vitro induction study can detect CYP1A2 down-regulation by OCA and OCA would be a suitable positive control for studies of CYP1A2 down-regulation.

This study confirmed that the effect of OCA on CYP1A2 in vitro at least qualitatively reflected in vivo findings. Although changes in pharmacokinetics caused by enzyme inhibition and induction can be predicted quantitatively, further clinical reports of NCEs which cause down-regulation in vivo are needed for quantitative prediction of changes in pharmacokinetics caused by down-regulation. References: Ishida C et al., J Pharm Sci. (2019) in press.

Symposium 5

（Drug Metabolism and Drug Induced Toxicity DIS 2）

SY-05-02

創薬段階で認められた酵素誘導とダウンレギュレーション

Cytochrome P450 induction and down-regulation in human hepatocytes at the lead-optimization stage of

drug development

永井　美香

科研製薬株式会社 Mika Nagai

Kaken Pharmaceutical Co., LTD.

The induction of cytochrome P450s (P450s) is one of the important mechanisms causing drug-drug interactions (DDIs), which can affect the pharmacokinetic, pharmacologic and toxicological effects of co-administrated drugs. To reduce the DDI risk via induction, in vitro assays using cryopreserved human hepatocytes are carried out at drug discovery stage. In some cases, however, it is difficult to evaluate P450 induction using human hepatocytes due to the insufficient physicochemical properties and cytotoxicity of test compounds. Moreover, we sometimes face the dose-dependent decrease of P450 mRNA levels in the induction assays. The aims of the current study were to establish the in silico prediction models of P450 induction which can be used at the drug discovery stage and to investigate the impact of CYP3A4 down-regulation on metabolic activity.

Cryopreserved human hepatocytes were treated with the learning set of compounds and mRNA levels were determined. To obtain a well predictable in silico model, the compounds were selected using principal component analysis with the fold-induction data. The obtained multiple regression model for CYP3A4 induction were represented by a few physicochemical parameters. Next, the down-regulation of CYP3A4 was evaluated using several lots of human hepatocytes. To elucidate the effects of the down-down-regulation on the metabolic activity, we evaluated the time-dependent CYP3A activity measured by 1’-hydroxymidazolam formulation. The hepatocytes were treated with 3-methylcholanthrene, sulforaphane, our compounds, and erythromycin for 48 hours. After removal of the test compounds, the recovery of metabolic activity was measured. The recovery rates of sulforaphane and our compounds were much slower than that of erythromycin.

In this presentation, we will introduce the application of our in silico prediction model for CYP3A4 induction in the lead-optimization. In addition, we will show our in-house criteria for determination of the down-regulation at drug discovery stage.

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SY-05-03

三次元培養細胞を用いた薬物代謝酵素の発現低下予測および機序解明ツールとしての有用性

Prediction and analysis for down-regulation of drug metabolizing enzymes: Utility of 3D-cultured cells

小林　カオル

千葉大学大学院薬学研究院薬物学研究室 Kaoru Kobayashi

Laboratory of DDS Design and Drug Disposition, Graduate School of Pharmaceutical Sciences, Chiba University

Cytochrome P450 (CYP) expression is known to be decreased under several pathological conditions such as inflammation. IL-1β, IL-6 and tumor necrosis factor (TNF)-α, cytokines induced during inflammation, have been shown to decrease the expression of various CYP isoforms in primary human hepatocytes. In addition, decreases in enzyme activities by new chemical entities can often observed during routine in vitro induction studies using human hepatocytes. Although human hepatocytes are now recognized as the most relevant and practical in vitro model, it is hard to fully understand the underlying mechanism of down-regulation because of the limited availability of donated human materials.

Recently, we have established a three-dimensionally cultured FLC-4 cell model. Its stable phenotype and relatively high level of CYP3A4 expression make this model suitable for thorough and extended research of the effects of CYP3A4 suppression by protein stimulants and new chemical entities. Our study using this cell model demonstrated modulation of CYP3A4 expression by cytokines, increased toxicity of gefitinib and paclitaxel by IL-6, and restoration of CYP3A4 mRNA expression by anti-cytokine agents. These results obtained by using three-dimensionally cultured FLC-4 cells are consistent with results obtained by using primary human hepatocytes and results of clinical studies. Next, we found that a compound decreased expression level of CYP3A4 mRNA under screening small-molecules to decrease covalently closed circular DNA in hepatocytes infected with hepatisis B virus. The decrease of CYP3A4 mRNA was also found in three-dimensionally cultured FLC-4 cells. Consistent with CYP3A4 expression, CYP3A protein and the formation of metabolites from triazolam were decreased. The decreased expression of CYP3A4 mRNA was restored by co-treatment with a JAK inhibitor.

In conclusion, three-dimensionally cultured FLC-4 cell model may be a promising cellular tool to assess the effects of cytokines and new chemical entities on CYP3A4 expression.

SY-05-04

転写後修飾A-to-I RNA編集ならびにアデノシンメチル化によるヒト薬物代謝酵素発現制御

Regulation of human drug metabolizing enzymes by two prevalent post-transcriptional modifications,

A-to-I editing and methylation of adenosine

中野　正隆、中島　美紀

金沢大学 薬学系 薬物代謝安全性学研究室 Masataka Nakano, Miki Nakajima

Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University

There are large interindividual differences in the expression and activity of cytochrome P450 (P450, CYP), causing variability in therapeutic efficacy or adverse events of drugs. Understanding the factors causing the interindividual differences in drug metabolism potencies would lead to the practice of personalized medicine and promotion of efficient drug development. As one of the mechanisms of the variability in the P450 expression, nuclear receptors-mediated transcriptional regulations, most of which are upregulation, are well known. Our recent studies have uncovered that two prevalent post-transcriptional modifications, A-to-I editing and methylation of adenosine, significantly affect the expression of drug-metabolizing enzymes.

Hydrolytic deamination at C6 _{position of adenosines on RNA, catalyzed by adenosine deaminase acting on RNA (ADAR) enzyme, results}

in conversion to inosine (A-to-I editing). Inosine forms a base pair with cytidine as if it was guanosine; thus, the conversion may affect the amino acid sequence, splicing, and microRNA targeting. Our recent study revealed that ADAR downregulates CYP1A2 and CYP3A4 expression in human liver cells. These regulations are mediated by ADAR-dependent repression of aryl hydrocarbon receptor and pregnane X receptor expression, respectively. The underlying mechanisms will be introduced. In addition, our latest finding that ADAR expression is decreased by exposure to cigarette smoke will be presented.

In addition to A-to-I editing, adenosine is enzymatically methylated at its N6 _{position (m}6_{A modification). Unlike A-to-I editing, this}

modification does not result in base conversion, but splicing, translation and decay can be changed by recognitions of m6_{A by reader}

proteins. We found that the m6_{A modification in the 3’-UTR of CYP2C8 facilitates degradation of CYP2C8 mRNA, leading to decreases}

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SY-05-05

胎児の薬物動態学的解析からみた妊娠中の薬物摂取の影響

The influence of drug administration during pregnancy evaluated from the pharmacokinetic analysis of

embryo

落合　和

星薬科大学 薬動学研究室 Wataru Ochiai

Department of Clinical Pharmacokinetics, School of Pharmacy and Pharmaceutical Sciences, Hoshi University

【PURPOSE】 Breast cancer is the most common cancer during pregnancy. Breast cancer during pregnancy is known to be fast prognosis and poor prognosis. Therefore, it is necessary to start treatment with anti-cancer drug as soon as possible. Administration of anticancer drugs during pregnancy starts from mid-pregnancy (3 months pregnancy) in which fetal organs are almost completed. However, the middle stage of pregnancy corresponds to the time when neural stem cells differentiate into neurons and astrocytes. That is, the brains of the fetus in the middle of pregnancy may be strongly affected by anticancer drugs.Against this backdrop, our laboratory has analyzed the effect of anticancer drugs on the brains of the fetus during the middle of pregnancy. Among them, we clarified that doxorubicin used for pregnant breast cancer will promptly migrate from mother to fetal brain. In addition, the localization of doxorubicin transferred to the fetal brain was consistent with the localization of neural stem cells. These findings indicate the possibility that doxorubicin has some influence on fetal neurogenesis. Therefore, in this study, in order to clarify the influence of doxorubicin on fetal neurogenesis, it was examined from the viewpoint of toxicology.【METHODS】Neural stem cells were isolated from embryonic mouse cerebrum at embryonic day 14.5. Subsequently, the effect of doxorubicin on the differentiation of neural stem cells into neurons and astrocytes was analyzed by immunostaining.【Results / Discussion】 When doxorubicin was added at concentrations of 100 and 1000 nM, most of the neural stem cells had been killed on the fourth day. On the other hand, even when doxorubicin was added at a concentration of 25 nM, significant cell death was not confirmed. However, compared to the control cells, the addition of doxorubicin affected the differentiation process of neural stem cells. In particular, it was suggested that doxorubicin may specifically promote neuronal differentiation from neural stem cells. We are currently investigating the effects of doxorubicin on developing brain.