AZD1390   中文名称: AZD-1390

ATM ( IC50 = 0.78 nM )

体外活性：AZD1390 是一种新型、有效、选择性、一流的口服生物利用度和 CNS 渗透性 ATM 抑制剂，在细胞测定中 IC50 为 0.78 nM。它的选择性比 PIKK 酶家族密切相关的成员高出 10,000 倍以上，并且对广泛的激酶具有出色的选择性。 AZD1390具有穿过血脑屏障（BBB）的能力，这使其适合治疗颅内恶性肿瘤。 AZD1390 对神经胶质瘤和肺癌细胞系具有放射敏感性，p53 突变型神经胶质瘤细胞通常比野生型细胞具有更高的放射敏感性。 AZD1390 目前正处于早期临床开发阶段，用作中枢神经系统恶性肿瘤的放射增敏剂。激酶测定：AZD1390 是一种新型、有效、选择性、一流的口服生物利用度和 CNS 渗透性 ATM 抑制剂，在细胞测定中 IC50 为 0.78 nM。它的选择性比 PIKK 酶家族密切相关的成员高出 10,000 倍以上，并且对广泛的激酶具有出色的选择性。细胞测定：将3000个细胞接种到384孔板的每个孔中，并置于含有10%胎牛血清的RPMI中。 24小时后，用每种化合物的半对数剂量稀释液对板进行回波给药，最高浓度为1250 nM。化合物给药后一小时，用 0、2.5 或 4 Gy 照射板。照射后 1、6、24 和 48 小时，通过将 1:1 体积的 8% PFA 直接添加到培养基中来固定板，得到 4% PFA 的终浓度，并在洗涤前在室温下孵育 30 分钟用磷酸盐缓冲盐溶液（PBSA）三次。

AZD1390 在临床前物种中表现出优异的口服生物利用度（大鼠为 66%，狗为 74%）。在非人类灵长类动物 PET 研究中，它可以有效地穿过 BBB。与单独放疗相比，在 AZD1390 与放疗联合治疗 2 或 4 天后，在脑癌原位异种移植模型中观察到显着的肿瘤消退和动物存活率的增加（> 50 天）。在体内同基因和患者来源的神经胶质瘤以及原位肺脑转移模型中，与单独的 IR 治疗相比，AZD1390 与每日部分 IR（全脑或立体定向放射治疗）联合给药可显着诱导肿瘤消退并增加动物存活率。 AZD1390 具有良好的物理、化学、PK 和 PD 特性，适合需要中枢神经系统内暴露的临床应用

AZD1390属于与临床开发化合物AZD0156（图1）相同的强效ATM抑制剂系列。然而，AZD1390是在一系列旨在筛选（i）ATM自磷酸化活性的体外试验后发现的；（ii）对密切相关的PIKK家族激酶ATR、DNA-PK和mTOR活性的选择性，以及（iii）更广泛的激酶组；以及（iv）在新型双转染人MDR1和BCRP外排转运体检测中缺乏底物活性。AZD1390针对ATM[谷胱甘肽S-转移酶（GST）-p53 Ser15]进行了筛选，其活性定义为≥50%[中位抑制浓度（IC50）]为0.00009μM（0.00004μM校正为紧密结合）。对密切相关和纯化的PIKK家族酶的IC50活性从未超过1μM。在更广泛的纯化激酶筛选小组中，AZD1390在1和0.1μM两种浓度下与赛默飞世尔科技公司的激酶小组进行了测试。在1μM的极高浓度下，AZD1390对3个靶点（CSF1R、NUAK1和SGK）显示出≥50%的抑制作用，对其余118个测试靶点没有活性。在0.1μM时，对354种激酶没有发现活性（抑制率<50%）。我们还测试了AZD1390对Eurofins Panlabs运行的一组激酶的活性和选择性。AZD1390对1种激酶FMS显示出活性（在1μM时抑制率>50%），对来自该组的124种其他激酶没有活性（1μM下抑制率<50%）（表1）。[2]

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AZD1390的脑和血浆结合[2]
如Fridén等人所述，使用大鼠脑切片结合法测定大鼠脑结合（fubrain）。使用快速平衡装置通过平衡透析测定血浆结合（大鼠、小鼠、狗、猴子和人类）。血浆中1或0.1μM的化合物用pH 7.4和37°C的缓冲液透析16小时。孵育后，在离心和UPLC-MS/MS分析上清液之前，将血浆和缓冲液的等分试样分别加入等体积的空白缓冲液和血浆中，然后用乙腈沉淀。Fuplasma是通过将缓冲室中的浓度除以血浆室内的浓度来测定的。

在 RPMI 格式中，每孔使用 10% 胎牛血清在 384 孔格式中接种 3000 个细胞。24 小时后，从顶部开始，用每种化合物的半对数剂量稀释液对板进行回波给药浓度为1250nM。化合物给药后，将板暴露于 0、2.5 或 4 Gy 的辐射下一小时。辐射后1、6、24和48小时固定板后，在室温下孵育30分钟，然后用磷酸盐缓冲盐溶液（PBSA）孵育3次。这是通过直接向培养基中添加 1:1 体积的 8% PFA 来完成的，最终浓度为 4% PFA。

In vivo H2228 model efficacy[2]
Bioluminescence signaling of implanted 3 × 105 NCI-H2228-Luc cells was measured using an IVIS Xenogen imaging machine to monitor tumor growth. When the signal reached the range of 107 to 108, the mice were randomized into different treatment groups and treated orally with either vehicle or AZD1390 QD or BID + IR at 2.5 Gy daily for four consecutive days. AZD1390 or vehicle was dosed at 1 hour before IR on each dosing day. The bioluminescence signals and body weight of the mice were measured once weekly, and the raw data were recorded according to their study number and measurement date in the in vivo database. TGI from the start of treatment was assessed by comparison of the mean change in bioluminescence intensity for the control and treated groups and presented as % of TGI. The calculation of inhibition and regression was based on the geometric mean of relative tumor volume (RTV) in each group. “CG” means the geometric mean of RTV of the control group, whereas “TG” means the geometric mean of RTV of the treated group. On specific day, for each treated group, the inhibition value was calculated using the following formula: Inhibition% = (CG − TG) * 100/(CG − 1). CG should use the corresponding control group of the treated group during calculation. If inhibition was >100%, then regression was calculated using the following formula: Regression = 1 – TG. Statistical significance was evaluated using a one-tailed t test. Survival benefit was measured by Kaplan-Meier plots at the end of the study.[2]

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In vivo efficacy studies in syngeneic glioma model[2]
GL261_Luc cells (1.6 × 105) were implanted into mice through ICB injection, as described above. An IVIS Xenogen imaging machine used to monitor tumor growth measured the bioluminescence signals. When the signals reached the range of 107 to 108, the mice were randomized into treatment groups and treated orally with either vehicle, AZD1390, IR at 2.5 Gy per day for four consecutive days, IR + AZD1390, or AZD1390 + TMZ. AZD1390, TMZ, or vehicle was dosed 1 hour before IR on each dosing day. The bioluminescent signals and body weight of the mice were measured once a week. TGI from the start of treatment was assessed by comparison of the mean change in bioluminescence intensity for the control and treatment groups, and data are presented as % of TGI. The calculation of inhibition and regression was based on the geometric mean of RTV in each group. CG means the geometric mean of RTV of the control group, whereas TG means the geometric mean of RTV of the treated group. On specific day, for each treated group, inhibition value was calculated using the following formula: Inhibition% = (CG − TG) * 100/(CG − 1). CG should use the corresponding control group of the treated group during calculation. If Inhibition was >100%, then regression was calculated using the following formula: Regression = 1 − TG. Statistical significance was evaluated using a one-tailed t test. A Kaplan-Meier curve was generated to calculate the survival benefit of mice treated with compounds.[2]

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In vivo PDX efficacy studies[2]
Human tumor tissue fragments were taken from TMZ-resistant or TMZ-sensitive GBM patients, derived from START (http://startthecure.com/preclinical_services_research.php), and implanted subcutaneously in female NMRI nude mice (Janvier Labs) between 7 and 11 weeks of age to establish the GBM PDX models. Animals were enrolled into the study when their tumor volume was approximately 200 mm3 and randomized into four groups: vehicle, 0.5% (w/v) HPMC, and 0.1% (w/v) Tween 80 given QD for 5 days by oral gavage; 2-Gy XRT given QD for 5 days; AZD1390 (20 mg/kg) given QD for 5 days by oral gavage; and AZD1390 + XRT given QD for 5 days. XRT was performed with X-RAD 320 (Precision X-Ray) to the whole head, and AZD1390 was administered 1 hour before XRT in the combination group. Animals were observed daily, and tumor volume and body weight were measured twice per week. Tumor volumes were calculated using the following formula: 0.52 (width × length2). All animal experiments were performed under a protocol approved by the Danish Animal Experiments Inspectorate. [2]

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Bioluminescent imaging (BLI) is performed after intracranial implantation of mouse GL261 glioma (p53 mutant) cells into immunocompetent, syngeneic C57/bl6 mice, before the mice are randomly assigned. Before receiving several fractions of 2-3 Gy of radiation over the course of two to four days, AZD1390 is given orally via gavage. Radiation therapy is applied to the tumor site using a 5 x 5 mm lateral field using the Small Animal Radiation Research Platform (SARRP).

BBB penetration[2]
The endothelial cells of the BBB contain efflux transporters MDR1 (Pgp) and BCRP, which serve to actively exclude the compound from the brain (32). In vitro efflux assays were set up using Madin-Darby canine kidney (MDCK) cells dual-transfected with human MDR1 and BCRP efflux transporters to identify compounds without substrate activity. In vitro MDCK_MDR1_BCRP studies at both 1 and 0.1 μM suggest that AD1390 is not a substrate for the human Pgp and/or BCRP efflux transporters (efflux ratio, <2); however, it does have a higher efflux rate in rodent species, as lower Kp,uu values were observed in rat and mouse (0.17 and 0.04, respectively). This reflects that, in rodents, AZD1390 is seen to be an efflux substrate with increased brain exposure (Kp,uu, 0.85 and 0.77) on administration of the chemical efflux transporter knockout elacridar (Fig. 1C) and an efflux ratio of 3.2 in the rat transporter-transfected in vitro LLC-PK1-rMdr1a assay at 1 μM. In contrast, AZD0156 at 0.1 μM has an efflux ratio of 23, indicating that it is a human efflux transporter substrate (Fig. 1, B and C). This BBB permeability difference is also reflected in vivo with AZD1390 rat and mouse brain Kp,uu values six- and sevenfold higher, respectively, than AZD0156.

Cynomolgus macaque positron emission tomography (PET) images for the two compounds (Fig. 1D) show that only AZD1390 gives significant brain penetration with a Cmax (%ID) of 0.68 ± 0.078 (n = 5) [compare AZD0156 Cmax %ID 0.15 ± 0.036 (n = 3, P < 0.01)]. Two-tissue compartment (2-TC) modeling of AZD1390 PET data yielded a VT (equivalent to Kp) of 5.8 ± 1.2 (n = 5) and a calculated Kp,uu of 0.33 ± 0.068 (n = 5). It was not possible to accurately determine Kp for AZD0156 in cynomolgus macaques. Here, the 2-TC model showed poor identifiability with very high SEs in VT. We observed lower Kp,uu values in rat and mouse for AZD1390 (0.17 and 0.04, respectively). This reflects that, in rodents, AZD1390 appears to be an efflux substrate with increased brain exposure (Kp,uu, 0.85 and 0.77) on administration of the chemical efflux transporter knockout elacridar (Fig. 3B) and an efflux ratio of 3.2 in the rat transporter-transfected in vitro LLC-PK1-rMdr1a assay at 1 μM. Despite the lower rodent Kp,uu values in mouse at 2 to 20 mg/kg, free brain exposure is achieved, with pATM inhibition and efficacy observed.[2]

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PD and PK of AZD1390 in vivo[2]
Researchers performed an extensive assessment of the relationship between PK and PD of AZD1390 in plasma, brain, and tumor samples from our orthotopic brain tumor model, NCI-H2228, implanted in the brain. The data show that the combination of pharmacologically active doses of AZD1390 from the in vitro and cell potency assays inhibited the IR-induced PD biomarkers pATM (Ser1981) and phospho-Rad50 (pRad50) (Ser635) in vivo in a dose- and time-dependent manner (Fig. 3, A and B). The antibody used to detect the latter is being used in clinical trials, and the data in Fig. 4B show the staining levels correlating with PK observations in Fig. 2A. The combination of AZD1390 with IR also significantly increased the apoptotic marker CC3 (cleaved caspase-3) compared to IR alone in NCI-H2228 lung cancer brain metastasis (LC-BM) model, suggesting that the combination is inducing tumor cell death (Fig. 3C). The data reveal a correlation between PK and PD modulation, with AZD1390 free brain levels peaking within 1 hour of dosing and dissipating over a 24-hour period, correlating with ATM inhibition activity (see fig. S4, A and D, for further details on PK and PD analyzed).

The IC50 against the cardiac ion channel hERG was also confirmed as minimal for both AZD0156 and AZD1390: >33.3 and 6.55 μM, respectively. (A similar IC50 for AZD1390 of 7.99 μM against hERG was generated using an alternative assay with improvements in compound handling and data processing).[2]

[1]. AACR Mol Cancer Ther. 2018, 17(1 Suppl):Abstract nr A124.

[2]. Science Advances. 2018, 4(6): eaat1719.

ATM Kinase Inhibitor AZD1390 is an orally bioavailable inhibitor of ataxia telangiectasia mutated (ATM) kinase, with potential antineoplastic activity. Upon oral administration, AZD1390 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 in ATM-overexpressing tumor cells. AZD1390 hypersensitizes tumors to chemo/radiotherapy. In addition, AZD1390 is able to cross the blood-brain barrier (BBB). ATM, a serine/threonine protein kinase belonging to the phosphatidylinositol 3-kinase-related kinase (PIKK) family of protein kinases, is upregulated in a variety of cancer cell types. It is activated in response to DNA double-strand breaks (DSB) and plays a key role in DNA repair.

C27H32FN5O2

477.5737

477.25

C, 67.90; H, 6.75; F, 3.98; N, 14.66; O, 6.70

2089288-03-7

126689157

White to off-white solid powder

4.2

61.8

0

6

7

35

720

0

VQSZIPCGAGVRRP-UHFFFAOYSA-N

InChI=1S/C27H32FN5O2/c1-18(2)33-26-21-14-20(22(28)15-23(21)29-17-24(26)31(3)27(33)34)19-8-9-25(30-16-19)35-13-7-12-32-10-5-4-6-11-32/h8-9,14-18H,4-7,10-13H2,1-3H3

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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));
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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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