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Description
近年、ＡＭＬを治療するために５つのさらなる医薬が米国食品医薬品局に承認されている：ミドスタウリン、エナシデニブ、ＣＰＸ－３５１、ゲムツズマブ・オゾガマイシン（Bloomfield CD, et al., Blood Revs. 2018; 32: 416-425）、およびイボシデニブ（Megias-Vericat J, et al., Blood Lymph. Cancer: Targets and Therapy 2019; 9: 19-32）である。ＡＭＬを患うおよそ６０％～７０％の成人が適切な導入療法後に完全寛解（ＣＲ）状態を達成することを期待することができ、ＡＭＬを患う成人の２５％以上（ＣＲを達成する患者の約４５％）は３年以上生存することを期待することができ、治癒できる可能性もある。 Five additional drugs have been approved by the US Food and Drug Administration to treat AML in recent years: midostaurin, enasidenib, CPX-351, and gemtuzumab ozogamicin (Bloomfield CD, et al., Blood Revs. 2018; 32: 416 -425), and ivosidenib (Megias-Vericat J, et al., Blood Lymph. Cancer: Targets and Therapy 2019; 9: 19-32). Approximately 60% to 70% of adults with AML can be expected to achieve complete remission (CR) status after appropriate induction therapy, and more than 25% of adults with AML (approximately (45%) can be expected to survive for three years or more, and may even be cured.
しかしながら、ＩＤＨ１耐性変異が７～１４％のＡＭＬ対象に観察され、付随する高２－ＨＧレベルはエピジェネティックな過剰メチル化表現型および分化の遮断の原因となり得、白血病誘発の原因となり得る（Megias-Vericat J, et al., Blood Lymph. Cancer: Targets and Therapy 2019; 9: 19-3）。加えて、Ｆｌｔ３キナーゼの変異がおよそ３分の１のＡＭＬ対象に観察されている（Lee HJ, et al., Oncotarget 2018; 9: 924-936）。 However, IDH1 resistance mutations are observed in 7-14% of AML subjects, and the concomitant high 2-HG levels may cause an epigenetic hypermethylation phenotype and a block in differentiation, which may contribute to leukemogenesis (Megias -Vericat J, et al., Blood Lymph. Cancer: Targets and Therapy 2019; 9: 19-3). Additionally, mutations in Flt3 kinase are observed in approximately one-third of AML subjects (Lee HJ, et al., Oncotarget 2018; 9: 924-936) .
別の実施態様では、癌は血液悪性腫瘍である。別の実施態様では、血液悪性腫瘍は急性骨髄性白血病、骨髄異形成症候群 骨髄増殖性腫瘍、血管免疫芽球性Ｔ細胞リンパ腫、Ｔ細胞性急性リンパ芽球性白血病、真性赤血球増加症、本態性血小板血症、原発性骨髄線維症、または慢性骨髄性白血病である。別の実施態様では、血液悪性腫瘍は急性骨髄性白血病である。 In another embodiment, the cancer is a hematological malignancy. In another embodiment, the hematological malignancy is acute myeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasm, angioimmunoblastic T-cell lymphoma, T-cell acute lymphoblastic leukemia, polycythemia vera, essential Thrombocythemia, primary myelofibrosis, or chronic myeloid leukemia. In another embodiment, the hematological malignancy is acute myeloid leukemia.
用語「血液悪性腫瘍対象」は、血液悪性腫瘍と診断された対象を意味する。１つの実施態様では、血液悪性腫瘍対象はＡＭＬ対象である。用語「ＡＭＬ対象」は、ＡＭＬと診断された対象を意味する。ＡＭＬを診断する方法は当業者に知られており、例えばDohner H, et al., Blood 2017; 129: 424-447に記載されている。 The term "hematologic malignancy subject" refers to a subject diagnosed with a hematologic malignancy. In one embodiment, the hematologic malignancy subject is an AML subject. The term "AML subject" refers to a subject diagnosed with AML. Methods for diagnosing AML are known to those skilled in the art and are described, for example, in Dohner H, et al., Blood 2017; 129: 424-447 .
「血液悪性腫瘍（例えば、ＡＭＬ）治療に対する反応性」には、全生存の改善、治療に対する部分反応、長期にわたる安定した疾患の改善、または完全寛解（骨髄中５％未満の骨髄芽球もしくは循環芽球の欠如で決定される）もしくは血液学的回復（赤血球輸血を必要とせず、末梢血好中球絶対数が１，０００細胞／μＬを超え、血小板数が１００，０００／μＬを超え、髄外疾患がないことで証明される）を特徴とする長期生存の改善を含む（Bloomfield CD, et al., Blood Revs. 2018; 32: 416-425）。 “ Responsiveness to hematologic malignancy (e.g., AML) treatment” includes improved overall survival, partial response to treatment, long-term stable disease improvement, or complete remission (less than 5% myeloblasts in the bone marrow or circulating (determined by absence of blasts) or hematological recovery (without the need for red blood cell transfusion, absolute peripheral blood neutrophil count greater than 1,000 cells/μL, platelet count greater than 100,000 cells/μL, (Bloomfield CD, et al., Blood Revs. 2018; 32: 416-425) .
材料および方法
２－ＨＧ阻害アッセイ
細胞株構成
ＭＯＬＭ１４野生型ヒト白血病細胞およびＭＯＬＭ１４＿Ｒ１３２コンストラクト細胞株は、ＩＤＨ１ ＷＴ（ドキシサイクリン誘導性） ｐＳＬＩＫ－ＩＤＨ１－ＦＬＡＧ（Ａｄｄｇｅｎｅプラスミド ＃６６８０２）およびＩＤＨ１ Ｒ１３２Ｈ（ドキシサイクリン誘導性） ｐＳＬＩＫ－ＩＤＨ１－Ｒ１３２Ｈ－ＦＬＡＧ（Ａｄｄｇｅｎｅプラスミド ＃６６８０３）を用いてＭＯＬＭ１４細胞のレンチウイルストランスフェクションによって作成し、ジャン－エマヌエル・サリー博士（トゥールーズ癌研究センター（Centre de Recherches en Cancerologie de Toulouse）, UMR1037, Inserm, トゥールーズ第３ポール・サバティエ大学（Universite de Toulouse 3 Paul Sabatier, Toulouse）, フランス; bioRxiv 749580; doi.org/10.1101/749580）の厚意によって提供される。ＭＯＬＭ１４－ＷＴおよびＭＯＬＭ１４－Ｒ１３２変異細胞は、１０％ＦＢＳ（シグマ社）および１ｘＰｅｎ／Ｓｔｒｅｐ（シグマ社）を含有する５ｍＬの完全ＲＰＭＩ培地内の６ウェルプレートに播種される（１ｘ１０６／ウェル）。２μｇ／ｍｌのドキシサイクリンで誘導した後、細胞を３７℃で４日間インキュベートする。
Materials and Methods 2 - HG Inhibition Assay Cell Line Construction MOLM14 wild type human leukemia cells and MOLM14_R132 construct cell lines were IDH1 WT (doxycycline inducible) pSLIK-IDH1-FLAG (Addgene plasmid #66802) and IDH1 R132H (doxycycline inducible) pSLIK-IDH1-R132H-FLAG (Addgene plasmid #66803) was created by lentiviral transfection of MOLM14 cells and was produced by Dr. Jean-Emmanuel Sally (Centre de Recherches en Cancerologie de Toulouse, UMR1037). Kindly provided by Inserm, Universite de Toulouse 3 Paul Sabatier, Toulouse, France; bioRxiv 749580; doi.org/10.1101/749580). MOLM14-WT and MOLM14-R132 mutant cells are seeded in 6-well plates (1×10 6 /well) in 5 mL of complete RPMI medium containing 10% FBS (Sigma) and 1×Pen/Strep (Sigma). After induction with 2 μg/ml doxycycline, cells are incubated at 37° C. for 4 days.
以下に記載の通り、ＤＭＳＯ（３連で）、または０、１２５、２５０、５００、もしくは１０００ｎＭの化合物Ａ（各３連で）で細胞を処理する。上記の最初のインキュベーション後、１ｘドキシサイクリン＋１ｘ化合物ＡまたはＤＭＳＯを含む０．５ｍＬの培地を加え、３日間インキュベートする。３日後、１Ｘドキシサイクリン＋１Ｘ化合物ＡまたはＤＭＳＯを含む別の０．５ｍＬ分量の新鮮な培地を加え、さらに３日間インキュベートする。次いで、ベネトクラクス（０、２５、５０、１００、または２００ｎＭ）を単剤として、または１Ｘ化合物Ａと組み合わせて加える。２日後、フローサイトメトリー濾過チューブ内で細胞を収集し、フローサイトメトリー用に処理する。アネキシンＶバインディングバッファー（ＡＢＢ、２ｍＬ）［１Ｍ Ｈｅｐｅｓバッファー（１０ｍＬ）、５Ｍ ＮａＣｌ（２８ｍＬ）、１Ｍ ＣａＣｌ２（５ｍＬ）、Ｈ２Ｏ（９５７ｍＬ）］で細胞を洗浄し、次いで１５００ｒｐｍで５分間遠心分離する。細胞をＡＢＢ（５０μＬ）で染色し、ＡＰＣアネキシンＶ抗体（１μＬ、バイオレジェンド社 カタログ番号６４０９４１）で染色し、２０分間暗闇状態に置く。ＡＢＢ（２ｍＬ）で細胞を洗浄し、１５００ｒｐｍで５分間遠心分離する。ＤＡＰＩ（インビトロジェン社 カタログ番号Ｄ３５７１）およびカウンティングビーズ（インビトロジェン社 カタログ番号Ｃ３６９５０）をアネキシンＶバインディングバッファーに加え［ＤＡＰＩ（５μＬ、２ｇ／ｍＬ）＋カウンティングビーズ（５μＬ（５２０，０００ビーズ／５０μＬ））＋ＡＢＢ（１５０μＬ）］、全容量１６０μＬ／チューブとなるようにし、次いでＧａｌｌｉｏｓフローサイトメーター（ベックマン・コールター社）で分析する。カウンティングビーズが２５０ビーズ／サンプルに達したときに細胞収集を停止する。分析にＫａｌｕｓａソフトウェアを用いる。ゲートされた（ｇａｔｅｄ）アネキシンＶネガティブ／ＤＡＰＩネガティブ細胞のパーセンテージは％生存細胞を構成し、一方アネキシンＶポジティブ細胞のパーセンテージは％アポトーシスを構成する。 Cells are treated with DMSO (in triplicate) or 0, 125, 250, 500, or 1000 nM Compound A (in triplicate each) as described below . After the initial incubation described above, add 0.5 mL of medium containing 1x doxycycline + 1x Compound A or DMSO and incubate for 3 days. After 3 days, add another 0.5 mL aliquot of fresh medium containing 1X doxycycline + 1X Compound A or DMSO and incubate for an additional 3 days. Venetoclax (0, 25, 50, 100, or 200 nM) is then added as a single agent or in combination with 1X Compound A. After 2 days, cells are collected in flow cytometry filtration tubes and processed for flow cytometry. Wash cells with Annexin V binding buffer (ABB, 2 mL) [1 M Hepes buffer (10 mL), 5 M NaCl (28 mL), 1 M CaCl 2 (5 mL), H 2 O (957 mL)], then centrifuge at 1500 rpm for 5 min. do. Cells are stained with ABB (50 μL) and APC Annexin V antibody (1 μL, Biolegend Cat. No. 640941) and left in the dark for 20 minutes. Wash cells with ABB (2 mL) and centrifuge at 1500 rpm for 5 minutes. Add DAPI (Invitrogen Cat. No. D3571) and counting beads (Invitrogen Cat. No. C36950) to Annexin V binding buffer [DAPI (5 μL, 2 g/mL) + counting beads (5 μL (520,000 beads/50 μL)) + ABB ( 150 μL)] to a total volume of 160 μL/tube and then analyzed on a Gallios flow cytometer (Beckman Coulter). Stop cell collection when counting beads reach 250 beads/sample. Kalusa software is used for analysis. The percentage of gated Annexin V negative/DAPI negative cells constitutes % viable cells, while the percentage of Annexin V positive cells constitutes % apoptosis.
表２Ａ～２Ｃの結果は、いずれかの化合物単独で処理した細胞の白血病細胞生存率のレベルと比較して、ベネトクラクスと化合物Ａの組み合わせが白血病細胞生存率のさらなる減少をもたらすことを立証している。
ＡＭＬ－ＰＤＸモデル
オスのＮＳＧマウス（６～９週齢、ジャクソン・ラボラトリー社）に２５０ｃＧｙを照射し、翌日、ＡＭＬ－ＰＤＸ（１ｘ１０６細胞／１００μＬ）を静脈内注射する。後眼窩経路で末梢血液を収集し、処理し、フローサイトメトリーによってｈＣＤ４５＋細胞を測定し、白血病の発症を確認する。
AML-PDX Model Male NSG mice (6-9 weeks old, Jackson Laboratory) are irradiated with 250 cGy, and the next day AML-PDX (1×10 6 cells/100 μL) is intravenously injected . Peripheral blood is collected via the retroorbital route, processed, and hCD45+ cells are measured by flow cytometry to confirm the development of leukemia.
Claims (19)
Ｒ１は－ＣＨ２ＣＨ（ＣＨ３）２、－ＣＨ２ＣＨ３、－ＣＨ２ＣＨ２ＯＣＨ３、または－ＣＨ２－シクロプロピルであり；
Ｒ２は－ＣＨ３または－ＣＨ２ＣＨ３であり；かつ
ＸはＮまたはＣＨである］
の化合物またはその医薬的に許容される塩を含む医薬組成物。 For use in combination with a Bcl-2 inhibitor or a pharmaceutically acceptable salt thereof in the treatment of cancer in a human subject with an IDH mutation, the formula:
R 1 is -CH 2 CH(CH 3 ) 2 , -CH 2 CH 3 , -CH 2 CH 2 OCH 3 , or -CH 2 -cyclopropyl;
R 2 is -CH 3 or -CH 2 CH 3 ; and X is N or CH]
or a pharmaceutically acceptable salt thereof.
７－［［（１Ｓ）－１－［４－［（１Ｒ）－２－シクロプロピル－１－（４－プロプ－２－エノイルピペラジン－１－イル）エチル］フェニル］エチル］アミノ］－１－エチル－４Ｈ－ピリミド［４，５－ｄ］［１，３］オキサジン－２－オン；
７－［［（１Ｓ）－１－［４－［（１Ｓ）－２－シクロプロピル－１－（４－プロプ－２－エノイルピペラジン－１－イル）エチル］フェニル］エチル］アミノ］－１－エチル－４Ｈ－ピリミド［４，５－ｄ］［１，３］オキサジン－２－オン；もしくは
１－エチル－７－［［（１Ｓ）－１－［４－［１－（４－プロプ－２－エノイルピペラジン－１－イル）プロピル］フェニル］エチル］アミノ］－４Ｈ－ピリミド［４，５－ｄ］［１，３］オキサジン－２－オン
またはその医薬的に許容される塩である、請求項１～５のいずれか１項に記載の医薬組成物。 The compound is:
7-[[(1S)-1-[4-[(1R)-2-cyclopropyl-1-(4-prop-2-enoylpiperazin-1-yl)ethyl]phenyl]ethyl]amino]-1 -ethyl-4H-pyrimido[4,5-d][1,3]oxazin-2-one;
7-[[(1S)-1-[4-[(1S)-2-cyclopropyl-1-(4-prop-2-enoylpiperazin-1-yl)ethyl]phenyl]ethyl]amino]-1 -Ethyl-4H-pyrimido[4,5-d][1,3]oxazin-2-one; or 1-ethyl-7-[[(1S)-1-[4-[1-(4-prop- 2-enoylpiperazin-1-yl)propyl]phenyl]ethyl]amino]-4H-pyrimido[4,5-d][1,3]oxazin-2-one
or a pharmaceutically acceptable salt thereof , the pharmaceutical composition according to any one of claims 1 to 5 .
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