JP6830132B2 - 非小細胞肺がんを治療するための方法及び組成物 - Google Patents
非小細胞肺がんを治療するための方法及び組成物 Download PDFInfo
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- JP6830132B2 JP6830132B2 JP2019132291A JP2019132291A JP6830132B2 JP 6830132 B2 JP6830132 B2 JP 6830132B2 JP 2019132291 A JP2019132291 A JP 2019132291A JP 2019132291 A JP2019132291 A JP 2019132291A JP 6830132 B2 JP6830132 B2 JP 6830132B2
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Description
本明細書で言及されるすべての出版物、特許及び特許出願は、各々の出版物、特許、または、特許出願が、特異的にかつ個別に参照することによって組み込まれると意図されるのと同じ程度まで、参照によって本明細書に組み込まれる。
1)風邪(風):病気の急な発生、固定しない病気、かゆみ、鼻炎、「流動的」脈拍、揺れ、麻痺、けいれん。
2)寒邪(寒):寒気、悪寒、熱による症状緩和、水気/透明な***物、強い痛み、腹痛、筋肉の凝り/収縮、(粘膜性が強い)舌苔、「深い」/「隠れた」又は「糸のような」脈、または遅い脈。
3)火/熱邪(熱):熱っぽさ、高熱、喉の渇き、濃度が高い尿、赤顔、赤い舌、黄色の舌苔、高速の脈。
4)湿邪(湿):体の重さ、だるさ、脾臓の機能不全の症状、油っぽい舌苔、「滑りやすい」脈。
5)乾燥邪(燥):乾燥した咳、ドライマウス、乾燥した喉、乾燥した唇、鼻血、乾燥肌、乾燥した便。
6)暑邪(暑):暑さ又は蒸し暑さとの混合による症状。
上記の六気に基づき、医者はる漢方薬組成物を処方し、上記の1以上の気を治療する。
一般的に、疾患を治療するにあたり、漢方では一種類以上の漢方薬を処方する。
一実施形態において、本願明細書に記載される本発明の組成物は、IL−1β発現の生成を増加する。一実施形態において、本発明の組成物はTNF−α発現の生成を増加する。一実施形態において、本発明の組成物は血管新生又はABCG2を阻害する。一実施形態において、本発明の組成物はホスホール−EGFR受容体シグナルを低減し、又は脂肪分解を予防し、又はゲフィチニブ抵抗性を克服する。
別途排除されていない限り、本願の明細書並びに請求項全体にわたり、以下の用語は以下に定義する意味を持つ。本明細書及び添付の請求項において使用される、単数形「a」、「an」および「the」は、文脈で他のことを明確に指示されていない限り、複数の指示対象を含む。別途記載されていない限り、質量分析、NMR、HPLC、タンパク質化学、生化学、組換えDNA技法および薬理学の従来の方法が利用される。本願において、「又は(or)」または「及び(and)」という用語は、別途明確に指示されている場合を除き、および/または、を意味する。さらに、用語「含む(including)」並びに「含む(include)」、「含む(includes)」および「含まれる(included)」など、他の形態の使用は限定的ではない。本明細書で使用される表題は、単に形式的な目的のためのものであり、記載されている対象を限定するものと解釈されてはならない。
適した投与経路は、限定的ではないが、経口、静脈内、直腸、エアロゾル、非経口、眼、肺、経粘膜、経皮、膣、耳、鼻、および局所へ投与が含まれる。さらに一例として、非経口送達には、筋肉内、皮下、静脈内、髄内への注射、並びに髄腔内、直接的な脳室内、腹腔内、リンパ管内、および鼻腔内注射が含まれる。
一般的に、本明細書で説明されている組成物、並びに本明細書で開示されている実施様式に基づき併用療法を行う実施例において、他の製剤は同じ医薬組成物として投与する必要は無く、一実施形態では、それらの物理的特性及び化学的特性が異なることから、異なる経路で投与される。一実施形態において、当初の投与は確立されたプロトコールで投与され、その後、確認された効果に基づき、治療を行う者が投与量、投与形式、投与回数を変形させる。
<複数の実施例>
本調査で使用される組成物1の煎じ薬は、以下の原料の水抽出物を凍結乾燥粉末にすることによって用意した:オウギの根(Huang Qi)12.50%、ショウマ(Cimicifuga foetida Rhizoma)12.50%、麦門冬の根(Ophiopogon Radix)8.33%、蒼朮の根茎(Atractylodes Lancea Rhizoma)8.33%、高麗人参6.25%、白朮の根茎(Atractylodes Rhizoma-White)6.25%、神麹(Massa Medicata Fermentata)4.17%、青皮(Citrus Reticulata-Viride)4.17%、マンダリンオレンジ(Citrus Reticulata)4.17%、甘草の根(Glycyrrhizae Radix)4.17%、五味子(Schisandra Fructus)4.17%、カラトウキの根(Angelica Sinensis Radix)4.17%、黄柏の樹皮(Phellodendron Cortex)4.17%、沢潟の根茎(Alisma Rhizoma)4.17%、カッコンの根(Pueraria Radix)4.16%、タイソウ(Ziziphus Fructus-Red)4.16%、ショウガの根(Zingiber Officinale Radix)4.16%。組成物1は、中医薬においては暑邪を払い、気を増強し、陰に栄養を与え、水分を発生させる。
全ての肺がん細胞株を、ウシ胎児血清(FBS, Invitrogen)(10%)及びL−グルタミン(Invtrogen)(2mM)、ペニシリン/ストレプトマイシン/アムホテリシンB溶液(1%)、NEAA(非必須アミノ酸)(1%)を含むRPMI培地において、摂氏37度、CO2濃度5%の状態で維持した。
<細胞生存アッセイ>
本実験において、様々ながん細胞株、例えば肺腺癌細胞(A549、CL141、CL97(EGFR−G719A−T790M変異を持つ)、H441、H23、HCC827、PC−9、H1650、H1975)、肺上皮がん細胞(CL152、H226、H1299)及び非小細胞性肺癌(H460)を、25、50、100、200、400μg/mlのAM−煎じ薬−DMSO、組成物2、組成物3を用いて治療した。コロニーは少なくとも50細胞を含むと定義された。コロニーは、メタノールと酢酸との混合物(3:1)で固定し、0.5%クリスタルバイオレットで着色した。組成物3のIC50値は、全ての細胞株において、組成物2よりも低い。従って、組成物3はコロニー形成を効率的に阻害できる(表2、左側)。さらに、組成物2及び組成物3の細胞毒性を調査するために、様々な細胞を組成物2、組成物3で、24時間、48時間、72時間治療し、その後スルホローダミンB比色分析法(SRB)アッセイ法を行った。データによると、組成物3は、72時間以内に、A549の細胞生存性を約50%低減した(IC50値は約294±43μg/mlであった)(表2、右側)。組成物2及び組成物3は、異なる肺がん細胞において、コロニー形成能力を低減した。SRBアッセイにおいて、組成物3は低い細胞毒性を有するが、濃度が低い状態においてコロニー形成を抑制する能力が著しく高いことが判明した。インビトロデータは、組成物3は組成物2よりも、肺がん抗がん剤として優れていることを示している。
肺がん細胞に対し、組成物3にシスプラチン又はゲフィチニブ(チロシンキナーゼ阻害剤)を併用して治療した場合の細胞障害性を調査した。細胞を96ウェルプレートに1日植え付けた。細胞を植え付けてから24時間後、試薬を添加し、48時間又は72時間のインキュベーションを行った。生存細胞の数は、スルホローダミンBアッセイ(SRBアッセイ)を使用して計算した。10%トリクロロ酢酸(TCA)を1時間使用し、細胞を固定した。その後TCAを取り除き、スルホローダミンB(SRB)を1時間使用して着色した。プレートを1%酢酸でリンスした後、20mMのトリス溶液を各ウェルに添加してタンパク質着色を溶解し、マイクロプレートリーダーを使用して、540nmにおいて色彩強度測定を行った。DMSO又は対応する溶媒(vehicle)細胞を対照とみなし、100%と定めた。
6個のウェルプレートに、ウェル毎に800個の細胞を14日間植え付けた。細胞を植え付けてから24時間後、細胞に試薬を添加した。溶媒及び試薬を4日ごとに取り替えた。治療後、細胞をPBSで洗い流し、コロニーを固定液で固定し(メタノール:酢酸=3:1)、メタノールに0.5%クリスタルバイオレットを添加した溶液で着色した。クリスタルバイオレットを注意して取り除いて水道水で洗い流した後、各コロニーを目視で数えた。実験は少なくとも3回繰り返して行い、細胞毒性は中央±SD値で計算した。
細胞を6cmディッシュに、各ディッシュあたり5x105細胞を1日間植え付けた。細胞を植え付けてから24時間後、試薬を添加し、48時間のインキュベーションを行った。トリプシン−EDTA(Invitrogen)によって、ディッシュから細胞の単細胞浮遊液を取り除き、3%ウシ胎児血清及び10mMのHEPESを含むHBSS培地で、各細胞1x106細胞/mLで浮かせた。これらの細胞は、20μg/mlのHoechst33342(Sigma Chemical社 セントルイス)で、摂氏37度において90分間のインキュベーションを行い、単独又は50μmol/Lのベラパミル(Sigma社)を含んでもよく、これはベラパミルに反応を示すABC輸送体である。90分のインキュベーションの後、細胞は300g及び摂氏4度において、5分間の遠心分離をすぐに行い、再度氷のように冷たいHBSSで遊離した。細胞は氷を使用して保管し、Hoechst着色の流出を防止し、1μg/mlの核染色液(BD)を添加して死滅細胞を取り除いた。最後に、これらの細胞は40μmのセルストレナー(BD)でフィルターし、単体の遊離細胞を確保した。デュアルレーザーFACS Vantage SE(BD)を使用し、細胞の二重波長分析及び精製を行った。Hoechst33342は、450/20バンドパスフィルタ(BP)による355nmのUV光での青色蛍光、並びに675nmエッジフィルターロングパス(EFLP)の赤色蛍光を照射した。放出された波長を分離するために、610nmのダイクロイックミラー(DMSP)を使用した。分析から、PI−ポシティブ(死滅)細胞を取り除いた。
EZH2(enhancer of zeste homologue 2)は細胞サイクル、DNA修復、細胞分化を規制するために重要な遺伝子である。EZH2は、複数の種類のガン幹細胞と関連すると報告されている。組成物3による幹細胞マーカー及びWntターゲット発現の調査を行った。
ATP結合カセットサブファミリーGメンバー2であるAGCG2(乳癌耐性蛋白、BCRPとしても知られている)、これは多剤耐性阻害薬フェノタイプと関連するプラズマ膜製剤流出ポンプであり、肺がんのCSCを特定するサイドポピュレーションマーカーとして、組成物3及び組成物2の活動を調査した。R482−HEK293細胞(HEK293細胞は野生型ヒトABCG2蛋白に安定してトランスフェクトされている)において、組成物2ではなく、組成物3が輸送活動を適度に阻害することが判明した。Ko143はABCG2のベンチマーク阻害化合物であり、ここではポジティブ対照(control)として使用された(図7A、7B)。
単球であるTHP−1の分化は、ホルボールミリステートアセテート(PMA)によって誘発した。簡単には、細胞は1時間、薬品で事前に治療した。治療後、細胞を培養した。上澄みサンプルにおいて、酵素結合免疫吸着法(ELISA)キットを使用し、全体のヒトIL−1β及びTNF−α水準を特定した(eBioscience 88-7010-88及びeBioscience 88-7346-88)。サンプル(50μL)を96ウェルプレートに添加し、それぞれ特定したサイトカインモノクローナル抗体でプレコーティングを行い、室温で2時間インキュベーションを行った。PBSTで洗い流した後、第2の抗体を添加し、プレートを室温で1時間インキュベートした。洗い流した後、100μlのアジビン−HRP溶液を添加し、プレートを室温で30分インキュベートした。洗い流した後、100μlの基質溶液を添加し、プレートを室温かつ暗い状態でインキュベートした。リン酸(H3PO4)を添加することによって反応が止まり、450nmにおいて吸光度(OD)を測定した。
分解された3T3−L1脂肪細胞を無血清培地で6時間インキュベートし、24時間の治療後、培地のグリセロール濃度を測定した。脂肪細胞の分解及び脂肪細胞の脂肪酸結合蛋白(AP2)発現時における、組成物2、3の使用に伴うトリグリセリド蓄積の変化は、下記の表に示す。
AP2発現>1、又はトリグリセリド含有量>1:これらの製剤は脂肪細胞の分解を向上するポテンシャルを持つ。
がんの微小環境は、免疫細胞、サイトカイン、血管及び腫瘍細胞からなる。がんの微小環境における複雑な相互作用は、腫瘍発生を促進する。
代表的なイメージは図9A〜9Cに示す。図9Aは、対照群と比較し、組成物3は投与量に比例した血管新生を引き起こした旨を示す。各ウェルにおける五角形または六角形のリングループ数を算出し、溶媒対照(DMSO)を100%として使用した場合において、組成物3(600μg/ml)はチューブ形成を著しく阻害することが判明した(図9B)。全てのサンプルに対し、アッセイを3回実施した。96ウェルプレートにおいて、各プレートにて4枚のイメージを10倍拡大で撮影した。これらのデータは、各ウェルにおける五角形または六角形のリングループ数を算出し、溶媒対照(DMSO)を100%として使用した場合で数値化した。
組成物1〜3の抗腫瘍効果を評価するにあたり、H441ゼノグラフトマウスモデルを使用した。
ヒト肺がん細胞株CL97(Dr. Pan-Chyr Yangのラボより購入、注射あたり100万細胞)をNOD/SCIDマウス(雌、生後4〜6週間)の右脇腹に皮下注射し、1〜2週間かけて腫瘍を成長させた。注射から1週間後、腫瘍移植マウスを対照群(DMSO溶媒(vehicle))及び中医薬治療群(組成物1:1g/kg、5日/一週間あたり;組成物2:600mg/kg、5日/一週間あたり;組成物3:150mg/kg、5日/一週間あたり 経口投与 ゲフィチニブ:100mg/kg、2回/一週間あたり、経口投与)にランダムに振り分けた。キャリパを使用して、両方の群で発生した腫瘍を毎週、10週間にわたり測定した。腫瘍の大きさの変化を倍率変化で表し、プロットした。
研究者は、第一選択療法としてシスプラチン及びドデタキセル(腺癌の場合はペメトレキセド)ダブレット化学療法を受け、第二選択療法としてエルロチニブによる標的治療を受けた、進行した非小細胞肺がん患者に対し、組成物1が免疫変化をリバースする役割を調査した。研究者は、進行型の非小細胞肺がん患者に対し、組成物1が免疫抑制を調節及びリバースするメカニズムの可能性を調査した。
調査のデザイン:allocation: randomized
分類のエンドポイント:安全性/有効性 調査
介入モデル(Intervention model): parallel assignment
マスキング:single blind (治療成績)
主な目的:治療
主要評価項目測定基準:
全体的な生存率[時間枠:3年]
[安全性の問題としての指定:あり]
副次的評価項目:
・無進行インターバル、他の結果判定
・生活の質(Quality of Life)
・安全プロフィール(Safety profile)
選択基準:
1.ステージIIIB又はIVの非小細胞肺がんの病理診断を受けた患者であること。
2.年齢:18歳以上
3.書面によるインフォームド・コンセント
4.ECOG:0−1
除外基準
1.炎症性、伝染性又は免疫性障害を持つ患者、例えば結核、エイズ、肺炎、皮膚筋炎、全身性エリテマトーデス、リューマチ。
2.全身性の臓器障害を持つ患者、例えば心不全、慢性腎臓炎、肝炎、肝硬変。
3.非小細胞肺がん以外の悪性腫瘍を持つ患者。
4.抗炎症剤又は免疫抑制治療を受けている患者、例えばステロイド(化学療法を除く経口又は吸入型)、NASID。
5.書面によるインフォームド・コンセントに署名しない患者
図12A〜12Gは、臨床試験結果を示し、それぞれ、無増悪生存期間(12A)、全体生存期間(12B)、腺癌における全体生存期間(12C)、非腺癌での全体生存期間(12D)、野生型肺腺癌の全体生存期間(12E)、EGF変異を有する肺腺癌の全体生存期間(12F)及び第一選択治療後の全体生存期間を調査した(RECIST−PD生存)(12G)。
細胞は6cmディッシュに、各ディッシュあたり5x105細胞を1日間植え付けた。細胞を植え付けてから24時間後、試薬を添加し、24時間又は48時間のインキュベーションを行った。細胞は100%エタノールを使用し、摂氏4度で固定し、細胞ディッシュは20μg/mLのヨウ化プロピジウム、0.2mg/mLのRNase及び0.1%のTriton X−100を含む溶液を使用し、室温で15分間培養した。細胞は、フローサイトメトリーによる分析が終わるまで、氷上で管理された。
図13を参照。
Claims (11)
- 非小細胞肺がんを治療する組成物であって、抽出された又は粉末状の薬草混合物と抗がん剤とを含み、前記薬草混合物は、キバナオウギ、ショウマの根茎、麦門冬の根、蒼朮の根茎、高麗人参、白朮の根茎、神麹、青皮、マンダリンオレンジ、甘草の根、五味子、カラトウキの根、黄柏の樹皮、沢潟の根茎、カッコンの根、タイソウ及びショウガの根を含み、
前記抗がん剤は、チロシンキナーゼ(RTKs)受容体を妨害する薬剤、微小管安定化剤またはプラチナベースの化学療法である、組成物。 - 前記プラチナベースの化学療法は、カルボプラチン、ネダプラチン、オキサリプラチン、ヘプタプラチン、ロバプラチン、サトラプラチン、cis−アミンジクロロ(2−メチル−ピリジン)白金、または(trans、trans、trans)−ビス−mu−(ヘキサン−1,6−ジアミン)−mu−[ジアミン−白金(II)]ビス[ジアミン(クロロ)白金(II)]テトラクロリドの群から選択される、請求項1に記載の組成物。
- チロシンキナーゼ受容体(RTKs)を妨害する薬剤は、アファチニブ、エルロチニブ、オシメルチニブおよびAZD3759(4−(3−クロロ−2−フルオロアニリノ)−7−メトキシキナゾリン−6−イル)−(2R)−2,4−ジメチルピペラジン−1−カルボキシレート)の群から選択される、請求項1に記載の組成物。
- 前記組成物は、(A)シスプラチンを基礎する化学療法の効率を改善し、(B)IL−1β発現の生成を増加し、(C)TNF−α発現の生成を増加し、(D)血管新生又はABCG2を阻害し、又は(E)ホスホール−EGFR受容体シグナルを低減し、又は脂肪分解を予防し、又はゲフィチニブ抵抗性を克服する、請求項1に記載の組成物。
- 治療上有効な量の抽出された又は粉末状の薬草混合物と、任意で抗がん剤とを含み、非小細胞肺がんを治療するために用いられる医療法組成物であって、
前記薬草混合物は、キバナオウギ、ショウマの根茎、麦門冬の根、蒼朮の根茎、高麗人参、白朮の根茎、神麹、青皮、マンダリンオレンジ、甘草の根、五味子、カラトウキの根、黄柏の樹皮、沢潟の根茎、カッコンの根、タイソウ及びショウガの根を含み、
前記抗がん剤は、アファチニブ、エルロチニブ、オシメルチニブ、AZD3759(4−(3−クロロ−2−フルオロアニリノ)−7−メトキシキナゾリン−6−イル)−(2R)−2,4−ジメチルピペラジン−1−カルボキシレート)、グリベック、パクリタキセル、カルボプラチン、パゾパニブ、GSK1120212、AZD2171、チピファルニブ、またはこれらの組み合わせの群から選択される、医薬組成物。 - 前記非小細胞肺がんは腺がんである、請求項5に記載の医薬組成物。
- 前記非小細胞肺がんは大細胞がんである、請求項5に記載の医薬組成物。
- 非小細胞肺がんの腫瘍成長率を低減し、又は非小細胞肺がんの腫瘍の大きさまたは容積を減らす、請求項5に記載の医薬組成物。
- 前記抗がん剤はエルロチニブである、請求項5に記載の医薬組成物。
- 前記薬草混合物の抽出物又は粉末並びに前記抗がん剤は、別々に、同時に、又は順次投与される、請求項5に記載の医薬組成物。
- 前記薬草混合物の抽出物又は粉末、並びに前記抗がん剤は、経口投与または非経口投与される、請求項5に記載の医薬組成物。
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