JP7116252B2 - 難治性小児固形腫瘍に対するハロゲン化キサンテンのインビトロ(in vitoro)および異種移植片での抗腫瘍活性 - Google Patents
難治性小児固形腫瘍に対するハロゲン化キサンテンのインビトロ(in vitoro)および異種移植片での抗腫瘍活性 Download PDFInfo
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Description
本出願は、2018年5月16日に出願された米国仮特許出願第62/672,373号に基づく優先権を主張し、当該米国仮特許出願の開示内容を援用するものである。
特に好適であるローズベンガル(4,5,6,7-テトラクロロ-2’,4’,5’,7’-テトラヨードフルオレセイン)等の企図されるハロゲン化キサンテン、または、エリスロシンB、フロキシンB、4,5,6,7-テトラブロモ-2’,4’,5’,7’-テトラヨードフルオレセイン、2’,4,5,6,7-ペンタクロロ-4’,5’,7’-トリヨードフルオレセイン、4,4’,5,6,7-ペンタクロロ-2’,5’,7’-トリヨードフルオレセイン、2’,4,5,6,7,7’-ヘキサクロロ-4’,5’-ジヨードフルオレセイン、4,4’,5,5’,6,7-ヘキサクロロ-2’,7’-ジヨードフルオレセイン、2’,4,5,5’,6,7-ヘキサクロロ-4’,7’-ジヨードフルオレセイン、4,5,6,7-テトラクロロ-2’,4’,5’-トリヨードフルオレセイン、4,5,6,7-テトラクロロ-2’,4’,7’-トリヨードフルオレセイン、4,5,6,7-テトラブロモ-2’,4’,5’-トリヨードフルオレセイン、および4,5,6,7-テトラブロモ-2’,4’,7’-トリヨードフルオレセインを含むその他のハロゲン化キサンテンは、適切な医薬組成物中に溶解または分散して存在する。
小分子(非タンパク性、約1000グラム/モル未満)またはより大きなタンパク性分子である全身性抗がん剤は、ハロゲン化キサンテンの病巣内投与によって行われる局所投与と比較して、薬剤が治療対象の哺乳動物の全身に行きわたるように哺乳動物に投与される。例示的な小分子抗がん剤には、本明細書で使用したドキソルビシン、エトポシド、ビンクリスチン、シスプラチン、イリノテカン、およびシタラビンが含まれ、また、例示的なタンパク性分子はアスパラギナーゼである。これらの薬剤のうち、ドキソルビシン、エトポシド、およびビンクリスチンは、致死量以下の用量のPV-10による治療と相乗作用するようであり、好ましい。
本明細書で報告された結果は、PV-10による治療を電離放射線と組み合わせることでも、治療全体の細胞毒性を増強したことを示している。このインビトロ研究では、まず最初に神経芽細胞腫細胞を致死量以下のPV-10と4時間接触させ、次に0.5、1、または2グレイの電離放射線を照射した。
さらに別の併用治療レジメンでは、PV-10の投与と、特別な全身性抗がん剤と見なすことができるチェックポイント抗体阻害剤の投与とを利用する。有用なチェックポイント抗体阻害剤は、投与されることで、免疫系ががん細胞を異物として認識し、このがん細胞を体から排除する手助けを行うことを可能にする、ヒト化モノクローナル抗体である。
PV-10併用全身性薬剤は、受容対象者によって必要とされるかまたは耐容される回数だけ投与することができる。小分子薬は通常、インビボ半減期が比較的短く、数分から数日である。一方、チェックポイント阻害抗体のインビボ半減期は、多くの場合1週間~3週間である。
PV-10は小児固形腫瘍細胞株の成長を阻害する
ユーイング肉腫、神経芽細胞腫、骨肉腫、横紋筋肉腫、正常線維芽細胞株、および正常初代骨髄サンプルを、様々な濃度のPV-10(3.125~400μM)で96時間処理し、alamarBlue(登録商標)を使用して細胞生存率を測定することで(図1A~1E)、小児固形腫瘍に対するPV-10の効果を判定した。PV-10は、試験を行ったすべての細胞株で濃度依存的に細胞生存率を低下させた。検査したすべての固形腫瘍細胞株についてIC50値を算出した。以下の表1は、処理後96時間のPV-10処理小児固形腫瘍細胞株の値を示す。表1に示される通り、値は45~108μMの範囲であり、平均は70μMであった。
PV-10が小児固形腫瘍細胞株に対して細胞毒性であることを特定した後、小児で最も一般的な頭蓋外がんであることから、神経芽細胞腫に着目した。PV-10が神経芽細胞腫細胞株に対して細胞毒性または細胞増殖抑制性であるかを調べた。次の4つの異なる細胞株を研究のために選択した;すなわち、異なる変異を有するとともに、IC50値に基づいてPV-10に対する感受性が異なる3つの神経芽細胞腫細胞株[SK-N-AS、SK-N-BE(2)、およびIMR5]と、IC50値に基づいてPV-10に対する感受性が非常に高い神経上皮腫細胞株[SK-N-MC]1つとを選択した。
経時的顕微鏡動画解析を使用して、100μMのPV-10による処理の12時間後、24時間後、36時間後、および48時間後にプレートに付着していた細胞の割合を定量化することで、4つの細胞株(SK-N-AS、SK-N-BE(2)、IMR5、およびSK-N-MC)のPV-10に対する感受性の違いを調べた。処理後に付着していた細胞の数は、ゼロ時間での細胞数に対して正規化された(図2B)。
以前に、PV-10はリソソームの完全性の喪失を誘導することが示されていた[非特許文献6]。したがって、SK-N-AS細胞、SK-N-BE(2)細胞、およびIMR5細胞を、PBS(コントロール)または100μMのPV-10のいずれかで16時間処理した。酸性の細胞小器官に濃縮されて蛍光を発する核酸染色剤であるHoechst 33342およびLysoTracker(登録商標)Green DND-26で生細胞を染色し、これらの細胞を蛍光顕微鏡解析により観察した(図3)。
細胞周期に対するPV-10の効果をフローサイトメトリにより解析し(図4A)、PV-10による標的調節をさらに解明した。最も耐性のある神経芽細胞腫細胞株(SK-N-AS)および最も感受性の高い神経芽細胞腫細胞株(IMR5)を、PBS(コントロール)、50μMのPV-10、または100μMのPV-10のいずれかで16時間または24時間処理した。
次に、ウエスタンブロット分析を実施して、PV-10処理細胞がアポトーシスを起こしているかどうかを調べた。SK-N-AS細胞、SK-N-BE(2)細胞、IMR5細胞、およびSK-N-MC細胞を、PBS(コントロール)、75μMのPV-10、または100μMのPV-10のいずれかで24時間処理した。全細胞抽出物をウエスタンブロットで分析して、総および切断ポリADPリボース(PARP)、総および切断カスパーゼ3、総および切断カスパーゼ7、およびアクチン(ローディングコントロール)のレベルを検出した(図4B)。
一般的に使用される小分子全身性抗がん剤のうち、PV-10と組み合わせることで細胞毒性を高めることができるものを解明するために、まず、神経芽細胞腫細胞株SK-N-AS、SK-N-BE(2)、およびIMR5、神経上皮腫細胞株SK-N-MC、ならびに正常線維芽細胞株BJを、異なる作用機序を有する7つの従来の化学療法剤のパネルに対してスクリーニングした(図5)。すべての薬剤は、0.1μMの単独で、および細胞毒性以下の濃度である50μMのPV-10との併用でスクリーニングされた。処理の96時間後に、alamarBlue(登録商標)アッセイを使用して細胞生存率を判定した。
一般的に使用される化学療法に加えて、PV-10がSK-N-AS細胞(図6A)およびIMR5細胞(図6B)において電離放射線(IR)による治療の効果を増強するかどうかを調べた。細胞をPBS(コントロール)または50μMのPV-10のいずれかで4時間前処理し、次に0.5、1、または2グレイ(Gy)のいずれかの放射線を細胞に照射した。最初の処理の96時間後に、細胞生存率をalamarBlue(登録商標)で測定した。
PV-10がインビボでも活性であるかどうかを判断するために、CB17 SCIDマウスの皮下SK-N-AS腫瘍および皮下IMR腫瘍に対するPV-10腫瘍内注射の効果を特徴づけた。25μlまたは50μlのPV-10を腫瘍に1回注射し[非特許文献8]、毎日観察した。
小児固形腫瘍の小児の全生存率は、血液悪性腫瘍の小児の全生存率よりも低い[非特許文献1]。再発または転移性のユーイング肉腫、神経芽細胞腫、骨肉腫、および横紋筋肉腫の小児の全生存率は、30%未満である[非特許文献1]。
細胞株および組織の培養
細胞株(SK-N-AS、SK-N-BE(2)、IMR5、LAN1、SK-N-MC、SK-N-SH、SHEP、BJ、BJ hTERT、WI38、WI38 hTERT、Hs68 hTERT、RD、RH30、143B、HOS、SK-ES、およびSK-PN-DW)は、5%(v/v)熱不活性化ウシ胎仔血清(FBS)(Gibco、オンタリオ州、カナダ)、100ユニット/mlペニシリン、および100ユニット/mlストレプトマイシン(Gibco)を添加したダルベッコ改変イーグル培地(DMEM)(Gibco)で培養した。細胞培養物は、5%CO2の加湿インキュベータ内で37℃に維持した。初代骨髄サンプルは、地域の研究倫理委員会(REB)による承認および書面によるインフォームドコンセントを得た後に採取した(倫理ID#17184)。これまでに記載されているように[非特許文献17]、Ficoll-Paque Plus(GEヘルスケアライフサイエンス、オンタリオ州、カナダ)を使用した密度勾配遠心分離により、骨髄サンプルからリンパ球を分離した。
PV-10(0.9%生理食塩水中のローズベンガル二ナトリウムの10%溶液)は、プロヴェクタス バイオファーマスーティカルズ インク.(ノックスヴィル、テネシー州、USA)から提供を受けて、室温で暗所に保管した。ドキソルビシン、エトポシド、ビンクリスチン、シスプラチン、PEGアスパラギナーゼ、イリノテカン、およびシタラビンのストック溶液は、アルバータ州立小児病院の薬局(カルガリー、アルバータ州、カナダ)から入手し、室温で暗所に保管した。その後の研究のために、薬品は添加剤含有DMEMで適切な濃度に希釈した。
96ウェルプレート(Greiner Bio-One、ノースカロライナ州、USA)の各ウェルに、100μlのDMEM中の5×103細胞を播種し、24時間培養した。単独のPV-10またはリン酸緩衝生理食塩水(PBS;137mM NaCl、2.7mM KCl、10mM Na2HPO4、1.8mM KH2PO4、pH 7.25)(コントロール)をDMEMで希釈し、100μlを各ウェルに添加して処理を行った。各処理で添加した薬剤の最終濃度は3.125~400μMの範囲であり、各処理は3回ずつ行われた。
6ウェルプレート(Corning Inc.、ニューヨーク州、USA)の各ウェルに2×105細胞を播種し、24時間培養した。細胞をPBS(コントロール)またはPV-10のいずれかで処理し、96時間培養した。Zeiss AxioVision Se64ソフトウェアを使用して、Zeiss AxioCam MRm Rev.3 FireWireカメラを備えたZeiss Axiovert 200M顕微鏡で位相差画像を撮像した。Adobe Photoshop(Adobe Creative Cloud 2017)を使用して画像の処理を行った。
96ウェルプレート(Greiner Bio-One)の各ウェルに5×103細胞を播種し、24時間培養した。細胞をPBS(コントロール)またはPV-10のいずれかで処理した。IncuCyte(登録商標)Zoom顕微鏡およびIncuCyte(登録商標)Zoomソフトウェア(エッセンバイオサイエンス、ミシガン州、USA)を使用して、37℃、5%CO2の加湿インキュベータ内で、ウェルあたり3枚の画像を30分ごとに48時間撮像した。ImageJソフトウェアを使用して各ウェルの細胞数を計測し、0時間での細胞数に対して正規化した。各実験の各処理ごとに、少なくとも350個の細胞を計測した。
6ウェルプレート(Corning)内の滅菌カバースリップ上に、未処理細胞の場合は2×105細胞/ウェル、処理細胞の場合は6×105細胞/ウェルで細胞を播種し、24時間培養した。細胞をPBS(コントロール)またはPV-10のいずれかで16時間処理した。 ウェルをPBSで2回洗浄し、2.5μg/mlのHoechst 33342染色剤(Invitrogen)を含む2mlのDMEMを各ウェルに添加した。細胞を37℃で10分間インキュベートした後、LysoTracker(登録商標)Green DND-26(Invitrogen)を最終濃度500nMで培地に添加した。細胞を37℃で15分間インキュベートし、撮像時にカバースリップをスライドガラス上に載せ、Zeiss AxioVision Se64ソフトウェアを使用して、Zeiss AxioCam MRm Rev.3 FireWireカメラを備えたZeiss Axiovert 200M顕微鏡で撮像を行った。Adobe Photoshop(Adobe Creative Cloud 2018)を使用して画像の処理を行った。
細胞周期の変化を分析するために、細胞を100mmディッシュ(Corning)に播種して、処理後に最低2×106細胞を収集できるようにした。細胞を24時間培養し、PBS(コントロール)またはPV-10のいずれかで処理し、16時間または24時間培養した。細胞をトリプシン処理によって収集し、PBSで洗浄し、40μmナイロンセルストレーナ(Falcon、Corning、ニューヨーク州、USA)でろ過し、トリパンブルー染色を使用して血球計算盤により細胞数を計測し、0.9%(w/v)滅菌NaClに再懸濁し、氷冷90%(v/v)エタノールで固定した。サンプルを室温で30分間インキュベートした後、-20℃で保管した。
細胞を100mmディッシュ(Corning)に1×106で播種し、24時間培養した。次に、細胞をPBS(コントロール)またはPV-10のいずれかで処理し、24時間培養した。細胞培養物から培地を回収し、細胞をPBSで洗浄して、トリプシン処理後に収集した。細胞を氷冷PBSで洗浄し、1200rpm、4℃で5分間遠心分離した。上清を除去し、1%(v/v)ホスファターゼ阻害剤(Sigma)および1%(v/v)プロテアーゼ阻害剤(Sigma)を添加した放射性免疫沈降アッセイ(RIPA)緩衝液(50mM Tris-HCl(pH8)、150mM NaCl、1%(v/v)NP-40、0.5%(w/v)デオキシコール酸ナトリウム、0.1%(w/v)ドデシル硫酸ナトリウム(SDS))にペレットを再懸濁した。サンプルを1.5mlチューブに移し、氷上で10分間インキュベートし、ボルテックスし、12,000rpmで10分間遠心分離した。上清を全細胞溶解液として収集し、すぐに使用するか、または-20℃で保管した。
これまでに記載された通りの方法で[非特許文献17]ウエスタンブロットを行った。簡単に説明すると、Trans-Blot(登録商標)Turbo(商標)Transfer System(BioRad、ケベック州、カナダ)を使用してタンパク質をニトロセルロース膜に転写し、ポンソーS染色液(5%(v/v)酢酸中0.1%(w/v))を使用して転写を確認し、0.1%(v/v)Tween(登録商標)-20を含むトリス緩衝生理食塩水(TBS-T;50mM Tris-HCl(pH7.5)、150mM NaCl、0.1%(v/v)Tween(登録商標)-20)中の5%脱脂粉乳で、膜を室温で2時間ブロッキングした。次に、TBS-T中の5%(w/v)脱脂粉乳で希釈した下記の一次抗体とともに、膜を4℃で一晩(約18時間)インキュベートした:抗PARP抗体(1:3000、Cell Signaling、9542S)、抗カスパーゼ3抗体(1:500、Cell Signaling、9662S)、抗カスパーゼ7抗体(1:1000、Cell Signaling、9492S)、抗カスパーゼ9抗体(1:1000、Cell Signaling、9502S)、および抗βアクチン抗体(1:5000、Cell Signaling、8457L)。膜をTBS-Tで3回洗浄し、抗ウサギ二次抗体(1:3000、Cell Signaling、7074S)と共にインキュベートし、TBS-Tで3回洗浄し、Western Lightning Plus-ECL試薬(Perkin-Elmer、MA、USA)で2分間インキュベートし、ChemiDoc MP Imaging System(BioRad)の化学発光設定を使用して現像した。
細胞毒性アッセイの項目で記載した方法にて細胞を培養した。試験薬剤(ドキソルビシン、エトポシド、ビンクリスチン、シスプラチン、PEGアスパラギナーゼ、イリノテカン、シタラビン)を、PBS(コントロール)またはPV10(最終50μM)のいずれかを含む培地で最終濃度0.1μMにて調製した。細胞に対し3回処理を行った。細胞毒性アッセイの項目で記載した方法にて、プレートを培養し、洗浄し、細胞生存率をalamarBlue(登録商標)で解析した。
細胞毒性アッセイの項目で記載した方法にて細胞を培養した。3つの試験薬剤(ドキソルビシン、エトポシド、ビンクリスチン)の希釈系列を、PBS(コントロール)またはPV10(最終50μM)のいずれかを含むDMEMで調製し、細胞に3回添加した。細胞毒性アッセイの項目で記載した方法にて、プレートを培養し、洗浄し、細胞生存率をalamarBlue(登録商標)で解析した。CompuSynソフトウェア(ComboSyn Inc.)を使用して、50μMのPV-10と組み合わせた試験薬剤のIC50に基づく組み合わせ指数(CI)を算出した。CI値を以下の基準に従って評価した:CI<1は相乗的活性を示し、CI=1は相加的活性を示し、CI>1は相乗的活性を示した[非特許文献18]。
細胞を60mmディッシュ(Corning)に5×104で播種し、24時間インキュベートし、PBS(コントロール)または50μMのPV-10のいずれかで処理し、37℃で4時間インキュベートした。Gammacell(登録商標)1000 Elite(MDS Nordion、オンタリオ州、カナダ)を使用して、細胞に0.5、1、または2グレイ(Gy)のいずれかの放射線を照射し、92時間培養した。処理は3回行った。細胞毒性アッセイの項目で記載したように、ディッシュをPBSで2回洗浄し、細胞生存率をalamarBlue(登録商標)で解析した。
動物に対するすべての処置は、カナダ動物管理協会のガイドライン、および実験動物の管理および使用に関するNIHガイドラインに従って行われた。すべての実験計画は、カルガリー大学の動物管理委員会によって検討および承認された(計画承認番号:AC16-0243)。
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Claims (10)
- 腫瘍細胞のアブレーションを誘発する量のローズベンガルまたはその薬学的に許容される塩を含む、哺乳動物対象の小児がん性固形腫瘍を治療するための医薬組成物であって、前記ローズベンガルまたはその薬学的に許容される塩は病巣内に投与され、前記小児がん性固形腫瘍は、ユーイング肉腫、神経芽細胞腫、骨肉腫、神経上皮腫、および横紋筋肉腫からなる群の1つまたは複数から選択される、医薬組成物。
- 前記ローズベンガルの前記薬学的に許容される塩はローズベンガル二ナトリウムである、請求項1に記載の医薬組成物。
- 前記哺乳動物はヒトである、請求項1に記載の医薬組成物。
- ドキソルビシン、エトポシド、ビンクリスチン、シスプラチン、イリノテカン、およびシタラビンからなる群から選択される、腫瘍抑制に有効な量の小分子全身性抗がん剤を更に含む、請求項1に記載の医薬組成物。
- (1)腫瘍細胞のアブレーションを誘発する量のローズベンガルまたはその薬学的に許容される塩、および(2)前記ローズベンガルと相乗的な細胞毒性を提供する、腫瘍抑制に有効な量の全身性抗がん剤を含む、哺乳動物対象の小児がん性固形腫瘍を治療するための医薬組成物であって、前記ローズベンガルまたはその薬学的に許容される塩は病巣内に投与され、前記全身性抗がん剤は、ドキソルビシン、エトポシド、およびビンクリスチンの1つまたは複数から選択され、前記小児がん性固形腫瘍は、ユーイング肉腫、神経芽細胞腫、骨肉腫、神経上皮腫、および横紋筋肉腫からなる群の1つまたは複数から選択される、医薬組成物。
- 前記ローズベンガルはローズベンガル二ナトリウムである、請求項5に記載の医薬組成物。
- 前記哺乳動物はヒトである、請求項5に記載の医薬組成物。
- (1)腫瘍細胞のアブレーションを誘発する量のローズベンガルまたはその薬学的に許容される塩、および(2)電離放射線による、腫瘍抑制に有効な量の全身性抗がん剤を含む、哺乳動物対象の小児がん性固形腫瘍を治療するための医薬組成物であって、前記ローズベンガルまたはその薬学的に許容される塩は病巣内に投与され、前記小児がん性固形腫瘍は、ユーイング肉腫、神経芽細胞腫、骨肉腫、神経上皮腫、および横紋筋肉腫からなる群の1つまたは複数から選択される、医薬組成物。
- 前記ローズベンガルはローズベンガル二ナトリウムである、請求項8に記載の医薬組成物。
- 前記哺乳動物はヒトである、請求項8に記載の医薬組成物。
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2018 ASCO ANNUAL MEETING ,10557,2018年06月02日,DOI:10.1200/JCO.2018.36.15_supppl.10557 |
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PROVECTUS BIOPHARMACEUTICALS AND PEDIATRIC ONCOLOGY EXPERIMENTAL THERAPEUTICS INVESTIGATORS’ CONSORTIUM (POETIC) ANNOUNCE ACCEPTANCE OF PV-10 POSTER PRESENTATION AT AMERICAN SOCIETY OF CLINICAL ONCOLOGY (ASCO) ANNUAL MEETING,2018年04月25日 |
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