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カニクイザル新鮮及び凍結肝細胞における薬物動態関連遺伝子発現の経時変動並びに誘導プロファイルの比較

小枝 暁子 Koeda Akiko 名古屋市立大学

2020.03.25

概要

医薬品開発は,新規化合物の創製に始まり,候補化合物のスクリーニングなどの基礎研究を経て,非臨床試験において有効性及び安全性を確認した後に,初めてヒトを対象とした臨床治験へと移る.少数の健常人を対象とした第I相,少数の患者を対象とした第II相,多くの患者を対象とした第III相の治験により安全性と有効性が確認できた医薬品は厚生労働省へ承認申請され,独立行政法人医薬品医療機器総合機構(PMDA)により審査される.申請が認められれば市場での販売となり,さらに発売後の安全性や使用法についても調査される.

非臨床安全性試験には単回・反復投与毒性試験,遺伝毒性試験,がん原性試験,生殖発生毒性試験,局所刺激性試験,依存性試験等があり,PMDAによって調査・認定された医薬品GLP適合施設において実験動物を用いた試験が実施される.厚生労働省が定めた医薬品毒性試験ガイドライン(平成元年9月11日薬審1第24号,一部改正:平成5年8月10日薬新薬第88号)において,単回及び反復毒性試験においては2種以上の動物を使用することとし,一種はマウスやラットなどのげっ歯類,もう一種はウサギ以外の非げっ歯類を用いることとしている.このため非げっ歯類の動物として,イヌ(ビーグル)やサル(カニクイザル,アカゲザル)が広く用いられ,背景データも豊富である.

欧米では動物試験に代わる代替法の確立が盛んであり,国内でも動物愛護の観点から3Rの原則(Replacement,Reduction,Refinement)の遵守が強く求められるようになってきている.新規医薬品の安全性を確認するのに動物実験は有効ではあるが,動物を用いない新規代替法の開発,最小限の動物数で多くの結果を得られる評価系の構築,苦痛の軽減や飼育環境の整備などが必要とされる.近年,invivoの細胞に機能を近づけた新たな細胞の作出,生体内の環境を再現する培養環境の開発などが盛んに行われている.

薬物は体内に取り込まれた後,主に肝臓で代謝が行われる.肝臓から実質細胞を分離した初代肝細胞は,様々な動物種で市販されており,生存率や接着効率の高い良質なヒト初代肝細胞も入手可能である.肝臓中の薬物代謝酵素には種差があり,ヒト初代肝細胞がinvitro薬物代謝試験ではゴールドスタンダードであるが,ヒトの個人差は大きく,ロットの選定によって結果が大きく異なる可能性がある.

カニクイザルは動物実験として背景データを豊富に有しているのに加え,近年,生息地によって遺伝的背景が異なることが分かってきた.東南アジアに生息するカニクイザルは,大陸産(中国,カンボジア,ベトナム),島国産(インドネシア,フィリピン)で系統が異なることがミトコンドリアDNAのDループ領域の解析により明らかにされている1).また免疫にかかわるMHC分子に着目すると,フィリピン産のカニクイザルはMHC遺伝子領域の多型の頻度が低く,遺伝的に均質であることが報告されている2).このため,MHCの型がマッチしたカニクイザル間におけるiPS細胞由来の心筋細胞の移植実験にも応用されている3).

本研究では,カニクイザルの初代肝細胞を試験材料として選択した.初代肝細胞は1頭分の肝臓あるいは1葉程度の肝臓組織から十分量を調製することが可能で,最低限の動物数で複数の試験が実施できる利点がある.同一個体の肝細胞を用いた複数の化合物のスクリーニングや,また凍結肝細胞での評価が可能であれば,個体差を考慮せずに複数回の試験に用いることができる利点がある.本実験では遺伝的に均質な背景を有するフィリピン産のカニクイザルを選択し,12頭から初代肝細胞を調製し,6頭分の新鮮肝細胞と6頭分の凍結肝細胞を用いて,薬物代謝酵素関連遺伝子のmRNA発現レベルを指標に,肝細胞の機能について評価した.初めに培養中のmRNA発現レベルの推移を追うことで細胞の状態をモニタリングし,次に酵素誘導剤によるmRNA発現レベルの変動を検証し,カニクイザル肝細胞の非臨床試験におけるinvitro薬物代謝試験への有用性について検証を試みた.

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参考文献

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