AZD8055

mTOR (IC50 = 0.13 nM); mTOR (IC50 = 0.8 nM)

AZD8055 对所有 PI3K 同工型（α、β、γ、δ）和 PI3K 样激酶家族的其他成员（ATM 和 DNA-PK）均表现出低活性（约 1,000 倍）。 AZD8055 可防止 mTORC1（p70S6K 和 4E-BP1）、mTORC2 (Akt) 和下游蛋白的磷酸化。 AZD8055 可以抑制大量的帽依赖性翻译，因为它完全抑制 4E-BP1 上的雷帕霉素抗性 T37/46 磷酸化位点。 AZD8055 的 IC50 值分别为 53、50 和 20 nM，可有效抑制 U87MG、A549 和 H838 细胞的增殖。此外，AZD8055 在 H838 和 A549 细胞中诱导自噬和升高 LC3-II 水平。 [1] AML 母细胞增殖和细胞周期进程减少，白血病祖细胞的克隆生长受到抑制，AZD8055 会诱导白血病细胞中的 caspase 依赖性细胞凋亡，但不会诱导健康、未成熟的 CD34+ 细胞。 [2] AZD8055 的 IC50 为 24.7 nM，对儿科临床前测试计划 (PPTP) 细胞系表现出抑制活性，并导致 EFS 分布出现明显变化。 [3]

AZD8055 以 2.5/10 mg/kg 浓度抑制 U87MG 和 A549 异种移植物中的 pS6 和 pAkt，从而抑制肿瘤生长。在 10–20 mg/kg 剂量下，AZD8055 可显着抑制多种异种移植物中的肿瘤生长，包括 U87MG、BT474c、A549、Calu-3、LoVo、SW620、PC3 和 MES-SA。 [1] AZD8055 使肿瘤体积减少 40%，并且 Akt、S6K 和 SGK 蛋白激酶磷酸化也被消除。通过抑制 mTORC1 和 mTORC2 信号传导，给予 AZD8055（5 mg/kg，Bid）和 SAHA（100 mg/kg/d）可完全抑制小鼠 PTEN+/-LKB1+/hypo 异种移植物中的肿瘤生长。 [4]

为了鉴定 mTORC1 和 mTORC2 活性，使用 MDA-MB-468 细胞创建了基于细胞的高通量筛选测定法。将逐渐增加的量的 AZD8055 添加到细胞上两小时。将细胞固定、清洗，然后在孵育期结束时用 S473 pAkt 或 S235/236 磷酸化 S6 (pS6) 抗体进行探测。利用 Acumen 激光扫描细胞仪测量磷酸化水平。细胞检测 mTORC1 和 mTORC2 活性。将细胞暴露于浓度不断增加的 AZD8055 中 2 小时。孵育期结束时，将细胞固定、洗涤并用针对 S473 pAkt 或针对 S235/236 磷酸化 S6 (pS6) 的抗体进行探测。使用 Acumen 激光扫描细胞仪评估磷酸化水平。

对暴露于 AZD8055 72 至 96 小时的细胞中的细胞核（0.03 mg/mL Hoechst 33342）和酸性囊泡（1 g/mL 吖啶橙）进行染色。在ArrayScan II平台上，在450和536 nm处拍摄图像，并对酸性囊泡的比例和细胞数量进行计数。在与 AZD8055 一起孵育之前，将细胞暴露于 e64d/胃酶抑素 (10 g/mL) 30 至 90 分钟，以评估 LC3。在冰上裂解后，使用免疫印迹检查细胞。

U87MG, BT474c, A549, Calu-3, LoVo, SW620, PC3 and MES-SA U87-MG and A549 are established in pathogen-free, female nude mice (nu/nu:Alpk).
2.5-20 mg/kg
Oral gavage once or twice daily

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In vivo, AZD8055 induces a dose-dependent pharmacodynamic effect on phosphorylated S6 and phosphorylated AKT at plasma concentrations leading to tumor growth inhibition. Notably, AZD8055 results in significant growth inhibition and/or regression in xenografts, representing a broad range of human tumor types. AZD8055 is currently in phase I clinical trials.[1]

Tumor cells (106 for U87-MG, 5 × 106 for A549) were injected s.c. in a volume of 0.1 mL, and mice were randomized into control and treatment groups when tumor size reached 0.2 cm3. AZD8055 was formulated in 30% (w/v) captisol (pH 3.0). The control group received the vehicle only. Tumor volumes (measured by caliper), animal body weight, and tumor condition were recorded twice weekly for the duration of the study. The tumor volume was calculated (taking length to be the longest diameter across the tumor and width to be the corresponding perpendicular diameter) using the following formula: (length × width) × √(length × width) × (π/6).[1]

For pharmacodynamic studies, animals were randomized when tumor size reached 0.5 cm3. The treatment groups received a single dose of AZD8055 and the control group received vehicle only. Tumor samples and blood were collected at various times after drug administration. The expression of pAKT and pS6 was determined in xenograft tissue by immunoblotting as described above. Ki67 nuclear staining was carried out using formalin-fixed, paraffin-embedded A549 xenografts.[1]

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In vivo AZD8055 induced significant differences in EFS distribution compared to controls in 23 of 36 (64%) evaluable solid tumor xenografts, and 1 of 6 evaluable ALL xenografts. Intermediate activity for the time to event activity measure (EFS T/C >2) was observed in 5 of 32 (16%) solid tumor xenografts evaluable. The best response was stable disease. PD2 (progressive disease with growth delay) was observed in 20 of 36 (55.6%) evaluable solid tumor xenografts. AZD8055 significantly inhibited 4E-BP1, S6, and Akt phosphorylation following day 1 and day 4 dosing, but suppression of mTORC1 or mTORC2 signaling did not predict tumor sensitivity.[3]

In Vivo Tumor Growth Inhibition Studies[3]
CB17SC scid−/− female mice (Taconic Farms, Germantown NY), were used to propagate subcutaneously implanted kidney/rhabdoid tumors, sarcomas, neuroblastoma, and non-glioblastoma brain tumors, while BALB/c nu/nu mice were used for glioma models, as previously described. Human leukemia cells were propagated by intravenous inoculation in female non-obese diabetic (NOD)/scid−/− mice as described previously 24. Female mice were used irrespective of the patient gender from which the original tumor was derived. All mice were maintained under barrier conditions and experiments were conducted using protocols and conditions approved by the institutional animal care and use committee of the appropriate consortium member. Ten mice were used in each control or treatment group. Tumor volumes (cm3) [solid tumor xenografts] or percentages of human CD45-positive [hCD45] cells [ALL xenografts] were determined as previously described 25 and responses were determined using three activity measures as previously described 25. An in-depth description of the analysis methods is included in the supplemental response definitions.[3]

Drugs and Formulation[3]
AZD8055 was provided to the PPTP by Astrazeneca, through the Cancer Therapy Evaluation Program (NCI). AZD8055 was dissolved in 0.5% hydroxypropylmethylcellulose containing 0.1% Tween 80 in water, sonicated and stirred overnight. AZD8055 was administered P.O. daily for 28 days at 20 mg/kg per day.

[1]. Cancer Res . 2010 Jan 1;70(1):288-98.

[2]. Leukemia . 2012 Jun;26(6):1195-202.

[3]. Pediatr Blood Cancer . 2012 Feb;58(2):191-9.

AZD-8055 is a pyridopyrimidine that is pyrido[2,3-d]pyrimidine which is substituted at positions 2 and 4 by (3S)-3-methylmorpholin-4-yl groups and at position 5 by a 3-(hydroxymethyl)-4-methoxyphenyl group. It is an mTOR complex 1/2 (mTORC1/2) dual inhibitor [mTOR = mammalian target of rapamycin]. It has a role as a mTOR inhibitor, an apoptosis inducer and an antineoplastic agent. It is a member of benzyl alcohols, a tertiary amino compound, a pyridopyrimidine and a member of morpholines.
AZD8055 has been used in trials studying the treatment of Cancer, Lymphomas, Solid Tumors, MALIGNANT GLIOMA, and brainstem glioma, among others.
mTOR Kinase Inhibitor AZD8055 is an inhibitor of the mammalian target of rapamycin (mTOR) with potential antineoplastic activity. mTOR kinase inhibitor AZD8055 inhibits the serine/threonine kinase activity of mTOR, resulting in decreased expression of mRNAs necessary for cell cycle progression, which may induce cell cycle arrest and tumor cell apoptosis. mTOR phosphorylates transcription factors, such as S6K1 and 4E-BP1, which stimulate protein synthesis and regulate cell growth, proliferation, motility, and survival.

C25H31N5O4

465.5447

465.237

C, 64.50; H, 6.71; N, 15.04; O, 13.75

1009298-09-2

25262965

Yellow solid powder

1.2±0.1 g/cm3

694.3±65.0 °C at 760 mmHg

373.7±34.3 °C

0.0±2.3 mmHg at 25°C

1.609

0.27

93.07

1

9

5

34

659

2

O1C([H])([H])C([H])([H])N(C2C3C([H])=C([H])C(C4C([H])=C([H])C(=C(C([H])([H])O[H])C=4[H])OC([H])([H])[H])=NC=3N=C(N=2)N2C([H])([H])C([H])([H])OC([H])([H])[C@]2([H])C([H])([H])[H])[C@@]([H])(C([H])([H])[H])C1([H])[H]

KVLFRAWTRWDEDF-IRXDYDNUSA-N

InChI=1S/C25H31N5O4/c1-16-14-33-10-8-29(16)24-20-5-6-21(18-4-7-22(32-3)19(12-18)13-31)26-23(20)27-25(28-24)30-9-11-34-15-17(30)2/h4-7,12,16-17,31H,8-11,13-15H2,1-3H3/t16-,17-/m0/s1

[5-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-2-methoxyphenyl]methanol

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: ~50 mg/mL (~107.4 mM)
Water: <1 mg/mL
Ethanol: ~3 mg/mL (~6.4 mM)

配方 1 中的溶解度: ≥ 5 mg/mL (10.74 mM) (饱和度未知) 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。
例如，若需制备1 mL的工作液，可将100 μL 50.0 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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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.37 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。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.37 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。

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配方 4 中的溶解度: ≥ 2.5 mg/mL (5.37 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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 5 中的溶解度: ≥ 2.5 mg/mL (5.37 mM) (饱和度未知) in 5% DMSO + 95% (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。
*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 6 中的溶解度: 4% DMSO+30% PEG 300+ddH2O: 5mg/mL

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配方 7 中的溶解度: 50 mg/mL (107.40 mM) in 30 % SBE-β-CD (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.

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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；
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。