AZD0156

ATM

体外活性：AZD0156 被确定为同类首个口服 ATM 抑制剂，IC50 为 0.58 nM，在基于细胞的 ATM 抑制测定中显示出亚纳摩尔效力。此外，AZD0156 的选择性比 PIKK 家族酶的其他成员高出 1000 倍以上。激酶测定：AZD0156 抑制 ATM 的激酶活性和 ATM 介导的信号传导，防止 DNA 损伤检查点激活，破坏 DNA 损伤修复，诱导肿瘤细胞凋亡，并导致 ATM 过表达的肿瘤细胞死亡。细胞测定：将HT29细胞以6000个细胞/孔的密度接种到含有1% L谷氨酰胺和10% FBS的40 μL EMEM培养基中的384孔测定板中，并使其粘附过夜。第二天早晨，通过声学分配将100% DMSO中的式(I)化合物添加到测定板中。在37°C和5% CO 2 下孵育1小时后，通过声学分配将40 nL 3 mM 4NQO的100% DMSO溶液添加到所有孔中，除了未用4NQO处理以产生无效响应对照的最小对照孔之外。将板放回到培养箱中再培养lh。然后通过添加 20 μL 3.7% 甲醛的 PBS 溶液并在室温下孵育 20 分钟来固定细胞。然后添加 20 μL 0.1% Triton XI 00 的 PBS 溶液并在室温下孵育 10 分钟，以透化细胞。然后使用 Biotek EL405 洗板机，用 50 μL/孔 PBS 将板洗涤一次。

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AZD0156对人ADPKD囊肿的影响[2]
由于MDCK细胞没有PKD致病基因突变，我们使用WT 9-12细胞进一步评估了ATM抑制在人类ADPKD囊肿模型中的作用，WT 9-12具有纯合PKD1变体[36,37,41]。在该模型中，与MDCK细胞衍生的囊肿相比，囊肿的形成较慢（需要18天才能达到最大尺寸），并且更高比例的囊肿呈偏心形状（图3A）。与MDCK囊肿一致，AZD0156也以剂量依赖的方式在WT 9-12衍生的囊肿中减少了囊肿生长，到第18天减少了4.1倍（图3B）。此外，如图3C、D所示，AZD0156没有改变WT 9-12单层中的细胞增殖，并降低了LDH泄漏百分比。

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AZD0156是一种强效、选择性、生物可利用的共济失调毛细血管扩张突变（ATM）蛋白抑制剂，ATM蛋白是一种参与DNA损伤反应的信号激酶。我们提供了临床前数据，证明低剂量AZD0156可以消除辐射诱导的ATM信号传导，这是通过ATM底物的磷酸化来测量的。AZD0156是一种体外强放射增敏剂。由于ATM缺乏会导致PARP抑制剂敏感性，在临床前，我们评估了AZD0156与PARP抑制剂olaparib联合使用的效果。使用ATM同源FaDu细胞，我们证明AZD0156阻碍了olaparib诱导的DNA损伤的修复，导致DNA双链断裂信号升高、细胞周期停滞和凋亡。临床前，AZD0156在体外增强了奥拉帕尼对一组肺癌、胃癌和乳腺癌癌症细胞系的作用，并提高了奥拉帕利在两种患者来源的癌症三阴性乳腺异种移植物模型中的疗效。AZD0156目前正在I期研究中进行评估（NCT02588105）[3]。

在动物研究中，AZD0156 显示出优异的临床前 PK 特性，包括口服生物利用度。此外，在小鼠异种移植模型中，AZD0156 与双链断裂 (DSB) 诱导剂联合口服后显示出强大的功效。

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AZD1390和AZD0156对MDCK囊肿模型的影响[2]
为了进一步验证ATM抑制对体外囊肿生长的影响，并选择体内测试的候选者，我们接下来评估了两种口服生物可利用类似物的疗效：（i）AZD1390（IC50，0.78 nM，ATM选择性>10000倍），和（ii）AZD0156（IC50 0.58 nM），ATM选择性>1000倍[30]。这两种ATM抑制剂都处于I期临床试验中，而KU-60019仅限于体外研究。如图2所示，AZD1390在1μM的浓度下仅在第8天减少了MDCK囊肿的生长（1.4倍），而AZD0156更有效，将囊肿直径减少了4.4倍，与西罗莫司的疗效相当（图2A-C）。有趣的是，与KU-60019不同，AZD0156没有改变BrdU的合成，而是与载体相比，增加了MDCK细胞单层中LDH泄漏的百分比（图2D，E）。

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AZD0156降低Pkd1RC/RC小鼠肾细胞增殖并增加p53[2]
接下来，我们评估了AZD0156对Pkd1RC/RC小鼠囊性肾病生物标志物的短期体内影响。AZD0156治疗共10天对Pkd1RC/RC小鼠的一般健康或体重没有不良影响（从第0天到第10天的体重变化：赋形剂：0.18±0.63，AZD0156 5 mg/kg：-0.38±0.48，AZD0156:20 mg/kg：-0.18±0.53 g；p>0.05）。在Pkd1RC/RC小鼠中，与赋形剂相比，AZD0156降低了肾细胞增殖（通过Ki-67测量）（图4A，B）。相比之下，尽管DNA损伤标志物γ-H2AX在治疗组之间没有差异（图4A，C），但AZD0156增加了肾脏p53（图4D，E）。由于ATM在细胞凋亡中的重要作用，还研究了切割的半胱氨酸天冬氨酸蛋白酶-3（Asp175）。裂解型半胱氨酸蛋白酶-3在远端小管中表达，但未被AZD0156改变（图4A）。此外，由于本研究持续时间短，正如预期的那样，AZD0156治疗后没有观察到肾脏增大和囊肿面积百分比的变化（图5）。

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AZD0156改善奥拉帕尼治疗患者来源的TNBC异种移植物的反应[3]
为了确定AZD0156是否可以在体内增强奥拉帕尼，选择了两种对奥拉帕尼具有不同敏感性的患者来源的TNBC异种移植物模型，HBCx-10和HBCx-9（29）。在每种模型中，使用两种耐受剂量和方案，方案1:AZD0156以5mg/kg的剂量每周连续3天给药，方案2:AZD0156每周以2.5mg/kg的剂量连续5天给药。在这两种方案中，奥拉帕尼均以50mg/kg的剂量持续给药。先前发表的数据表明，已知HBCx-10模型对单独使用奥拉帕尼治疗敏感，并使用方案1与AZD0156联合使用时出现消退（28）。在该HBCx-10模型中使用方案2，单独使用奥拉帕尼可诱导10个肿瘤中的一个出现消退，但与AZD0156肿瘤联合使用时，8只接受治疗的小鼠中有3只出现消退，另外，其余5只小鼠中有4只出现肿瘤停滞（图6A）。尽管在奥拉帕尼不敏感的HBCx-9模型中，单独使用奥拉帕尼治疗对肿瘤生长几乎没有影响，但使用方案1添加AZD0156后，肿瘤生长抑制得到改善（图6B）。有趣的是，该模型中的附表2并没有增强奥拉帕尼单独反应以外的肿瘤生长抑制作用（补充图S4）。AZD0156单药治疗对两种模型的肿瘤生长都没有影响。

在过度表达 ATM 的肿瘤细胞中，AZD0156 通过抑制 ATM 的激酶活性和 ATM 介导的信号传导、阻断 DNA 损伤检查点激活、破坏 DNA 损伤修复并诱导肿瘤细胞凋亡来导致细胞死亡。

使用补充有 10% FBS 和 1% L 谷氨酰胺的 40 μL EMEM 培养基，在 384 孔测定板中以 6000 个细胞/孔的密度培养 HT29 细胞，并使其粘附整晚。第二天早上，使用声学分配法将式(I)化合物的100％DMSO溶液填充到测定板中。在 37°C 和 5% CO₂ 下孵育 1 小时后，所有孔均通过声学分配接收 40 nL 100% DMSO 中的 3 mM 4NQO，但最小对照孔除外，这些孔为未用 4NQO 进行处理，以产生无效响应对照。将板放回培养箱中继续lh。随后，向细胞中加入20μL 3.7%甲醛的PBS溶液，并在室温下孵育20分钟以固定细胞。之后，添加 20 μL 0.1% Triton XI 00 的 PBS 溶液，并将细胞在室温下孵育 10 分钟以进行透化。随后，使用 Biotek EL405 洗板机和 50 μL/孔 PBS 将板清洗一次。

In Vivo Mouse Experiments [2]
Two experiments were performed to investigate the in vivo effects of reducing ATM. In the first study, to determine the effect of pharmacological ATM kinase inhibition, groups of Pkd1RC/RC mice (n = 4 per group; aged 2–3 months) were randomized to receive either: (i) vehicle (0.5% (w/v) hydroxypropyl methylcellulose (HPMC) and 0.1% (v/v) Tween 80 in ultrapure water), (ii) low dose AZD0156 (5 mg/kg body weight/day), or (iii) high dose AZD0156 (20 mg/kg body weight/day). Mice were dosed via oral gavage on Monday, Wednesday, and Friday for 2 consecutive weeks for a total study duration of 10 days. Mice were sacrificed 4 h after dosing on day 10. The vehicle composition, drug dose, and frequency of administration were based on previous studies.

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Xenograft-targeted irradiation study [3]
NCI-H441 cells for in vivo xenograft implant were cultured in RPMI1640 with 10% v/v FCS and 1% v/v l-glutamine at 37°C, 7.5% CO2. Cells were implanted subcutaneously in serum-free media with 50% Matrigel at 5 × 106 per mouse. Male nude mice at greater than 18 g had tumor size monitored twice weekly by bilateral caliper measurements. Treatments were started when tumor volumes reached an average of approximately 0.26 cm3. Targeted irradiation of 2 Gy was delivered over 2 minutes daily over the first 5 days. AZD0156 (10 mg/kg) was given orally for the duration of the study.

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Patient-derived xenograft studies [3]
Triple-negative breast cancer (TNBC) HBCx-10 and HBCx-9 (29) patient-derived xenograft studies were carried out at XenTech, France in accordance with French regulatory legislation concerning the protection of laboratory animals. Female athymic nude mice greater than 18 g were implanted with HBCx-10 or HBCx-9 tumor derived from a primary ductal adenocarcinoma. Donor mice were sacrificed to provide tumor fragments, which were surgically implanted subcutaneously. Tumor size was monitored twice weekly by bilateral caliper measurements. Mice body weights were recorded at the same time. For efficacy studies, treatment started when tumor volume averaged approximately 0.15 cm3 and for pharmacodynamic studies treatment started at approximately 0.5 cm3. In efficacy and pharmacodynamic studies, control mice were dosed orally with vehicle, AZD0156, or olaparib alone or in combination using different dosing schedules.

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Oral

Mouse xenograft models

[1]. Abstract 4859: Identifying high quality, potent and selective inhibitors of ATM kinase: Discovery of AZD0156. Cancer Res (2016) 76 (14_Supplement): 4859.

[2]. Effect of Reducing Ataxia-Telangiectasia Mutated (ATM) in Experimental Autosomal Dominant Polycystic Kidney Disease. Cells. 2021 Mar 3;10(3):532.

[3]. Pharmacology of the ATM Inhibitor AZD0156: Potentiation of Irradiation and Olaparib Responses Preclinically . Mol Cancer Ther (2020) 19 (1): 13–25.

ATM Kinase Inhibitor AZD0156 is an orally bioavailable ataxia telangiectasia mutated (ATM) kinase inhibitor, with potential chemo-/radio-sensitizing and antineoplastic activities. Upon oral administration, AZD0156 targets and binds to ATM, thereby inhibiting the kinase activity of ATM and ATM-mediated signaling. This prevents DNA damage checkpoint activation, disrupts DNA damage repair, induces tumor cell apoptosis, and leads to cell death of ATM-overexpressing tumor cells. In addition, AZD0156 sensitizes tumor cells to chemo- and radiotherapy. ATM, a serine/threonine protein kinase, is upregulated in a variety of cancer cell types; it is activated in response to DNA damage and plays a key role in DNA-strand repair.

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Ataxia telangiectasia mutant (ATM) is a serine/threonine protein kinase from the phosphatidylinositol 3-kinase-related kinase (PIKK) family of protein kinases (also comprising ATR, DNA-PKcs, mTOR etc.) and plays a crucial role in the cellular DNA damage response signalling activated by DNA double strand breaks (DSB). Activated ATM promotes DNA repair and S/G1-cell cycle checkpoints to prevent premature mitosis, maintain genomic integrity and promote appropriate cell survival or death pathways. DSBs arise intrinsically through the collapse of stalled replication forks, which are induced by a wide range of chemotherapies, or extrinsically through exposure to ionizing radiation. Therefore, ATM inhibition represents an exciting clinical opportunity as a target to hyper-sensitize tumors to chemo/radiotherapy. Herein, we describe our efforts to identify multiple, novel series of ATM inhibitors. We will describe our optimization efforts with particular attention given to improving the potency of the compounds in cellular systems and the selectivity of the compounds over other closely related proteins (e.g. ATR, mTOR etc.). We will also describe our efforts to optimize both the physicochemical properties of the molecules as well as the pharmacokinetic profile to enable low dose, oral administration in the clinic. These efforts culminated in the discovery of the clinical candidate AZD0156, a first in class orally available ATM inhibitor. AZD0156 shows sub-nanomolar potency in cell based assays of ATM inhibition with selectivities of greater than 1000 fold over other members of the PIKK family of enzymes. AZD0156 is a permeable, highly soluble compound with excellent preclinical pharmacokinetic properties including oral bioavailability. AZD0156 shows robust efficacy in mouse xenograft models after oral administration when combined with DSB inducing agents. AZD0156 is currently undergoing early clinical assessment.[1]

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Clinical efficacy has been seen with olaparib in patients with tumors containing BRCA mutations, and our studies using TNBC patient-derived xenograft models demonstrate that AZD0156 enhances the antitumor activity of olaparib irrespective of intrinsic olaparib sensitivity and DDR deficiencies. Here we present data demonstrating that intermittent dosing of AZD0156 enhances olaparib activity in vivo, which should prove valuable in facilitating the development of well-tolerated combination regimes in the clinic. Our data provides proof-of-concept for the assessment of AZD0156 and olaparib combinations in the clinic. Furthermore, AZD0156 provides a valuable tool for preclinical target validation and research into the roles of ATM in the DDR and noncanonical pathways.[3]

C26H31N5O3

461.56

461.242

C, 67.66; H, 6.77; N, 15.17; O, 10.40

1821428-35-6

118502708

White to off-white solid powder

1.2±0.1 g/cm3

628.3±55.0 °C at 760 mmHg

333.8±31.5 °C

0.0±1.8 mmHg at 25°C

1.626

2.4

71Ų

0

6

7

34

687

0

O1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N1C(N(C([H])([H])[H])C2=C([H])N=C3C([H])=C([H])C(C4=C([H])N=C(C([H])=C4[H])OC([H])([H])C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H])=C([H])C3=C12)=O

AOTRIQLYUAFVSC-UHFFFAOYSA-N

InChI=1S/C26H31N5O3/c1-29(2)11-4-12-34-24-8-6-19(16-28-24)18-5-7-22-21(15-18)25-23(17-27-22)30(3)26(32)31(25)20-9-13-33-14-10-20/h5-8,15-17,20H,4,9-14H2,1-3H3

8-[6-[3-(dimethylamino)propoxy]pyridin-3-yl]-3-methyl-1-(oxan-4-yl)imidazo[4,5-c]quinolin-2-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: 0.83 mg/mL (1.80 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 8.3 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 0.83 mg/mL (1.80 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 8.3 mg/mL 澄清 DMSO 储备液加入 900 μL 20% SBE-β-CD 生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: 0.83 mg/mL (1.80 mM) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.
例如，若需制备1 mL的工作液，可将 100 μL 8.3 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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