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Systematic chemical screening identifies disulfiram as a repurposed drug that enhances sensitivity to cisplatin in bladder cancer: a summary of preclinical studies

Kita, Yuki 京都大学 DOI:10.14989/doctor.k23565

2021.11.24

概要

ゲムシタビン+シスプラチン(GC)療法は進行性膀胱癌の標準療法だが、初期不応や治療中の薬剤耐性獲得により効果は限定的である。近年、既存の承認薬を他の疾患の治療薬として適応させる薬剤再利用の概念が注目を集めている。そこでGC療法の作用を増強するまたは薬剤耐性を克服する既存の化合物を同定するため、化合物スクリーニングを行った。

 2種の膀胱癌細胞株(UMUC-3、J82)に2,098種の既存薬.機能既知化合物をIC20相当のGCと72時間共投与し、細胞生存率をWST-8アッセイで測定した。GC単独と比較して併用投与により50%以上の増殖抑制を認める、かつ無治療と比較して化合物単独で30%以上の増殖抑制を認めない化合物を探索した。1次スクリーニングで8化合物がヒットし、2次スクリーニングでより詳細な感受性試験を行った結果、最終的に抗酒薬ジスルフィラム(disulfiram、以下DSF)を候補薬として同定した。DSFは複数の膀胱癌細胞株でGCとの併用効果を示し、Combination index assayにてジェムシタビンではなくシスプラチンと相乗効果があることを確認した。相乗効果の機序として、DSFとGCの共投与により活性酸素種増大とともにアポトーシスが誘導されることをウェスタンブロットとフローサイトメトリーで明らかにした。さらにDSFとシスプラチンの共投与により白金のDNA架橋量が増加することをICP-MSによる測定で明らかにした。そこでシスプラチンが銅輸送体により細胞内輸送および排泄されることに着目し、銅輸送体の局在を免疫蛍光細胞染色で確認した。銅排泄輸送体の一つであるATP7Aが核周囲のゴルジ体に集積している細胞数の割合は、シスプラチン単独投与時よりもDSFの共投与時の方が有意に低下していた。続いて臨床への応用を念頭に置いて難水溶性のDSFを未代謝な状態で腫瘍へ送達させるために、ミセル化ナノ粒子(DSF-NP)をエマルション溶媒拡散法により作製し、マウス投与実験を行った。UMUC-3およびT24皮下移植マウスモデルにおいて、シスプラチン+DSF-NP併用投与はシスプラチン単独投与よりそれぞれ45%、40%の腫瘍増殖抑制効果を示した。併用投与された腫瘍における白金のDNA架橋量とアポトーシス細胞の割合はともに増加していた。併用による体重減少や腎機能低下は認めなかった。さらに患者由来腫瘍組織皮下移植マウスモデルの1系統でシスプラチンとDSF-NPの併用投与が単独投与よりも腫瘍増殖抑制効果を示すこと、およびその腫瘍から作製したCancer-tissue originated spheroid(CTOS)の3次元培養の系でもin vivoと同様の併用効果を示すことが確認できた。そこで、経尿道的膀胱腫瘍切除術から得られた新鮮な腫瘍組織からCTOSを作製し、シスプラチンとDSF-NPの併用効果を症例毎に検討した。15症例中6例で薬剤感受性試験を行えるだけの十分な数のCTOSが得られ、そのうち4例で併用による増殖抑制効果の増強が確認された。

 既知化合物ライブラリーを用いた化合物スクリーニングで、膀胱癌におけるシスプラチン増感剤としてDSFを同定した。DSFは抗酒薬として長年使用されてきた薬剤であり、比較的安価かつ安全性に関するデータの蓄積もある。DSFを化学療法増感剤として再利用することは、臨床への導出可能性の高い新規治療法と考えられた。

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