ATR; PI3Kδ

AZD6738在存在遗传毒性应激的情况下，通过对循环CLL细胞中的ATM/p53通路的补偿激活，提供了对ATR信号传导的有效和特异性抑制。在p53或ATM缺陷细胞中，AZD6738处理导致复制分叉停滞和未修复DNA损伤的积累，如γH2AX和53BP1焦点形成所证明的，其被带入有丝分裂，导致有丝分裂突变导致细胞死亡。AZD6738对ATM或p53缺陷的CLL细胞显示出选择性细胞毒性，并且与细胞毒性化疗联合具有高度协同作用。这一发现在DDR缺陷CLL的原代异种移植物模型中得到了证实，其中AZD6738治疗导致肿瘤负荷减少，并选择性减少具有ATM或TP53改变的CLL亚克隆[1]。

了解药物剂量和时间安排的治疗效果对于临床试验的设计和实施至关重要。单一疗法，特别是联合疗法的复杂性日益增加，在肿瘤学药物开发的临床阶段提出了重大挑战。使用系统药理学方法，我们用细胞周期的机制模型扩展了现有的肿瘤生长的PK-PD模型，从而能够模拟ATR抑制剂AZD6738和电离辐射的单次和联合治疗。使用AZD6738，我们开发了基于细胞的多参数测定法，测量DNA损伤和细胞周期转变，提供了适合模型校准的定量数据。我们的体外校准细胞周期模型可以预测在体内小鼠异种移植物研究中观察到的肿瘤生长。该模型正在用于AZD6738的I期临床试验设计，目的是通过定量剂量和计划预测来改善患者护理[2]。

DNA损伤反应（DDR）缺陷，特别是TP53和双等位基因共济失调毛细血管扩张突变（ATM）畸变，与慢性淋巴细胞白血病（CLL）的基因组不稳定性、克隆进化和化学耐药性有关。缺乏能够为患有DDR缺陷的CLL患者提供长期疾病控制的治疗方法。使用新型ATR抑制剂AZD6738，我们研究了ATR通路抑制作为靶向具有这些缺陷的CLL细胞的综合致死策略[1]。

AZD6738的作用通过关键DDR蛋白的蛋白质印迹和免疫荧光来评估。细胞毒性通过CellTiter发光测定法（Promega，Madison，WI，USA）和碘化丙啶排除法进行评估。将具有双等位基因TP53或ATM失活的原代CLL细胞异种移植到NOD/Shi-scid/IL-2Rγ小鼠中。在用AZD6738或载体处理后，通过渗透脾脏的流式细胞术分析测量肿瘤负荷，并通过荧光原位杂交测量17p（TP53）或11q（ATM）缺失的亚克隆组成[1]。

Female athymic nude (Foxn1nu) mice, 6–7 weeks old, were purchased from Harlan Laboratories. H23 (3 × 106 cells) or H460 (7 × 105 cells) were injected subcutaneously into the right hind flank in a volume of 100 μL (equal parts 1x PBS and Matrigel). Cells were tested for mycoplasma prior to inoculation in mice. Mice began receiving treatment once tumors reached approximately 220 mm3 (± 15%) for H23 or 180 mm3 (± 15%) for H460. Tumor volume was calculated as (L × W2)/2. AZD6738 was administered by oral gavage (qd × 14) at 25 mg/kg (H23) or 50 mg/kg (H460). Cisplatin was administered intraperitoneally (q7d × 2) at 3 mg/kg. The dosing volume was 10 mL/kg. Growth curves depict mean (± SEM) tumor volume over time. Mean tumor growth inhibition was calculated as TGI = (1–(Tf–T0)/(Cf–C0))*100, where Tf and T0 represent final and initial mean tumor volumes in the treatment arm, respectively, and Cf and C0 represent final and initial mean tumor volumes in the vehicle control arm, respectively. Mean tumor regression was calculated as % Regression = ((T0–Tf)/T0)*100. For H460 xenografts, the experimental endpoint was defined as the day on which any single tumor within the treatment arm reached 2000 mm3. Tumor growth delay is defined as the difference in the number of days to reach the endpoint for a given treatment arm compared to vehicle control.[Oncotarget. 2015 Dec 29;6(42):44289-305.]

[1]. Lancet.2015 Feb 26;385 Suppl 1:S58
[2]. Sci Rep.2015 Aug 27;5:13545.

Background: DNA damage response (DDR) defects, particularly TP53 and biallelic ataxia telangiectasia mutated (ATM) aberrations, are associated with genomic instability, clonal evolution, and chemoresistance in chronic lymphocytic leukaemia (CLL). Therapies capable of providing long-term disease control in CLL patients with DDR defects are lacking. Using AZD6738, a novel ATR inhibitor, we investigated ATR pathway inhibition as a synthetically lethal strategy for targeting CLL cells with these defects. Methods: The effect of AZD6738 was assessed by western blotting and immunofluorescence of key DDR proteins. Cytotoxicity was assessed by CellTiter-Gloluminescence assay (Promega, Madison, WI, USA) and by propidium iodide exclusion. Primary CLL cells with biallelic TP53 or ATM inactivation were xenotransplanted into NOD/Shi-scid/IL-2Rγ mice. After treatment with AZD6738 or vehicle, tumour load was measured by flow cytometric analysis of infiltrated spleens, and subclonal composition by fluorescence in-situ hybridisation for 17p(TP53) or 11q(ATM) deletion. Findings: AZD6738 provided potent and specific inhibition of ATR signalling with compensatory activation of ATM/p53 pathway in cycling CLL cells in the presence of genotoxic stress. In p53 or ATM defective cells, AZD6738 treatment resulted in replication fork stalls and accumulation of unrepaired DNA damage, as evidenced by γH2AX and 53BP1 foci formation, which was carried through into mitosis, resulting in cell death by mitotic catastrophe. AZD6738 displayed selective cytotoxicity towards ATM or p53 deficient CLL cells, and was highly synergistic in combination with cytotoxic chemotherapy. This finding was confirmed in primary xenograft models of DDR-defective CLL, where treatment with AZD6738 resulted in decreased tumour load and selective reduction of CLL subclones with ATM or TP53 alterations. Interpretation: We have provided mechanistic insight and demonstrated in-vitro and in-vivo efficacy of a novel therapeutic approach that specifically targets p53-null or ATM-null CLL cells. Such an approach can potentially help to avert clonal evolution, a major cause of therapeutic resistance and disease relapse.[3]

---

Understanding the therapeutic effect of drug dose and scheduling is critical to inform the design and implementation of clinical trials. The increasing complexity of both mono, and particularly combination therapies presents a substantial challenge in the clinical stages of drug development for oncology. Using a systems pharmacology approach, we have extended an existing PK-PD model of tumor growth with a mechanistic model of the cell cycle, enabling simulation of mono and combination treatment with the ATR inhibitor AZD6738 and ionizing radiation. Using AZD6738, we have developed multi-parametric cell based assays measuring DNA damage and cell cycle transition, providing quantitative data suitable for model calibration. Our in vitro calibrated cell cycle model is predictive of tumor growth observed in in vivo mouse xenograft studies. The model is being used for phase I clinical trial designs for AZD6738, with the aim of improving patient care through quantitative dose and scheduling prediction.[4]

C20H24N6O2S

412.5086

412.17

C, 58.23; H, 5.86; N, 20.37; O, 7.76; S, 7.77

1352226-87-9

Ceralasertib;1352226-88-0; 1352280-98-8 (formate); 1352226-87-9 (S-isomer); 1352226-97-1 (racemic)

54761305

Typically exists as light yellow to yellow solids at room temperature

2.6

116Ų

2

7

4

29

724

2

C[C@@H]1COCCN1C2=NC(=NC(=C2)C3(CC3)[S@@](=N)(=O)C)C4=C5C=CNC5=NC=C4

OHUHVTCQTUDPIJ-MUWSIPGASA-N

InChI=1S/C20H24N6O2S/c1-13-12-28-10-9-26(13)17-11-16(20(5-6-20)29(2,21)27)24-19(25-17)15-4-8-23-18-14(15)3-7-22-18/h3-4,7-8,11,13,21H,5-6,9-10,12H2,1-2H3,(H,22,23)/t13-,29+/m1/s1

imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-lambda6-sulfane

(S)-Ceralasertib; 1352226-87-9; (S)-AZD6738; imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-lambda6-sulfane; BDBM60432; AZD6738; AZD 6738; SCHEMBL9979159;

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)

DMSO : ~100 mg/mL (~242.42 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.06 mM) (饱和度未知) 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。
例如，若需制备1 mL的工作液，可将100 μL 25.0 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。

---

配方 2 中的溶解度: ≥ 2.5 mg/mL (6.06 mM) (饱和度未知) 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), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 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。

---

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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。