EGFR^L858R (IC50 = 12 nM); EGFR^L858R/T790M (IC50 = 1 nM)

体外活性：与野生型体外相比，AZD9291 对突变型 EGFR 细胞系的增殖具有明显更有效的抑制作用。激酶测定：Osimertinib（以前称为 AZD-9291 和 mereletinib）是一种口服的、不可逆的、突变选择性的第三代 EGFR 抑制剂，对于外显子 19 缺失 EGFR、L858R/T790M EGFR、IC50 分别为 12.92、11.44 和 493.8 nM。和 WT EGFR 分别在 LoVo 细胞中。它抑制激活性和耐药性 EGFR 突变，同时保留正常皮肤和肠道细胞中存在的正常形式 EGFR，从而减少当前可用药物遇到的副作用。细胞测定：AZD9291 在体外有效抑制 EGFRm+（例如 PC9；< 25 nM）和 EGFR m+/T790M（例如 H1975；< 25 nM）细胞系中的 EGFR 磷酸化，同时对野生型 EGFR 系（例如 LoVo）的活性要低得多；> 500 nM)。一致地，与野生型体外相比，AZD9291 在突变型 EGFR 细胞系中显示出更有效的增殖抑制作用。

AZD9291（5mg/kg po）可引起 EGFRm+ (PC9) 和 EGFRm+/T790M (H1975) 肿瘤模型的肿瘤深度消退，并在体内深度抑制 EGFR 磷酸化和关键下游信号通路（如 AKT 和 ERK）。

在康宁黑色透明底 384 孔板中，每孔在生长培养基中接种 10,000 个细胞，然后将板在 37°C、5% CO2 下孵育整晚。将化合物在 100% DMSO 中连续稀释，并使用 Echo 555 对细胞进行声学给药。吸出培养基并将板再孵育两小时后，向每孔中添加 40μL 裂解缓冲液。 Greiner black 高结合 384 孔板在包被捕获抗体后用 3% BSA 封闭。去除块后，将15μL裂解液添加至Greiner black高结合384孔板中，并将板孵育2小时。添加检测抗体(20μL)，并在抽吸和PBS洗涤后将板孵育两小时。抽吸和 PBS 洗涤后添加 20μL QuantaBlu 荧光过氧化物酶底物并孵育 1 小时。在每个板中添加 20μL QuantaBlu 终止液，并使用 Envision 读板器读取 352 nm（激发）和 460 nm（发射）的荧光。使用合适的软件程序导出每种化合物获得的数据并进行曲线拟合分析。通过计算产生 50% 效果所需的化合物浓度，从该数据获得 IC50 值。

在体外，AZD9291 在 EGFRm+（例如 PC9；< 25 nM）和 EGFR m+/T790M（例如 H1975；< 25 nM）细胞系中显示出 EGFR 磷酸化的显着抑制作用，但对野生型 EGFR 细胞系（例如，LoVo；> 500 nM）。在体外，与野生型相比，AZD9291 在突变型 EGFR 细胞系中持续表现出更强大的增殖抑制作用。

In Vivo Antitumor Efficacy Studies[5]
All in vivo efficacy studies were performed as reported previously by ourselves.

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Rat in Vivo Toxicology Studies[5]
The animals used were 10-week-old male RccHan:WIST rats obtained from Harlan, U.K. Animals (n = 3/compound) received a single oral dose of compound as a suspension in 0.5% w/v HPMC/0.1% w/v Tween in deionized water at a concentration of 20 mg/mL. Blood glucose levels were measured using an Accuchek Active meter. Serum insulin concentrations were determined using a commercial rat ELISA kit. Water and food were available ad libitum.

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In vivo studies[4]
NSCLC cell lines (PC9, H1975, or MGH134) were evaluated by IMPACT testing prior to their use in vivo. ~0.5–1 × 10^6 cells were suspended in a PBS and Matrigel solution (PBS: Matrigel = 1:1), and 100μl of cell suspension was subcutaneously injected into the flank of ~6–8 week-old female nude mice. Tumor size was measured three times weekly with calipers and tumor volume was calculated by the formula, V = L × W2 × 0.52 (L = longest diameter, W = shortest diameter). When tumor volume reached ~100–200 mm3, mice were randomized into treatment groups, with each group having 5–6 mice. AZD0156 was resuspended in Ora-Plus suspension, and osimertinib was dissolved in a 10% DMSO, 30% PEG400 and 60% H2O solution. All drugs were administered orally with 100μl drug suspension/dose per mouse. AZD0156 was administrated at 50mg/kg daily, and osimertinib was administrated at 5mg/kg daily. All mice were dosed Monday-Friday (5 days/week). Tumor size was monitored two to three times per week until the end point when tumors reached ~1,000 mm3 or tumors were ulcerated.

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5 mg/kg; p.o

EGFRm+ and EGFRm+/T790M transgenic mice

Absorption, Distribution and Excretion
The median time to Cmax was found to be 6 hours.
Osimertinib is primarily eliminated through excretion in the feces (68%), to a lesser extent through urine (14%), while only 2% is excreted unchanged.
The mean volume of distribution at steady state is 918 L.
Oral clearance is 14.3 L/hr.

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Metabolism / Metabolites
Osimertinib is metabolized to at least two pharmacologically active metabolites, AZ7550 and AZ5104, that circulate at approximately 10% of the concentration of the parent compound. Biochemical assays have shown that AZ7550 has similar potency and efficacy to osimertinib, while AZ5104 is more potent against mutant and wild-type EGFR. The main metabolic pathways are oxidation (predominantly by CYP3A) and dealkylation.

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Biological Half-Life
The population estimated mean half-life is 48 hours.

Hepatotoxicity
Elevations in serum aminotransferase levels are uncommon during osimertinib therapy occurring in 4% to 5% of patients and rising above 5 times the upper limit of the normal range in only 1% or less. In preregistration trials, there was a single incidence of clinically apparent liver injury attributed to osimertinib therapy, but the clinical features and relatedness to therapy were not defined. Since its approval and more widespread use, there have been no published cases of liver injury due to osimertinib.
Likelihood score: E* (unproven but suspected cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of osimertinib during breastfeeding. Because osimertinib is 95% bound to plasma proteins, the amount in milk is likely to be low. However, its half-life is about 48 hours and it might accumulate in the infant. The drug also has 2 active metabolites that have not been studied in breastmilk. The manufacturer recommends that breastfeeding be discontinued during osimertinib therapy and for 2 weeks after the final dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Plasma protein binding of osimertinib is 95%.

[1]. Patent. 2013, WO2013014448 A1.

[2]. Mol Cancer Ther (2013) 12 (11_Supplement): A109.

[3]. Sci Transl Med. 2022 Mar 30;14(638):eabc7480.

[4]. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discov. 2014 Sep;4(9):1046-61.
[5]. Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistancemutations that spares the wild type form of the receptor. J Med Chem. 2014 Oct 23;57(20):8249-67.

Pharmacodynamics
A pharmacokinetic/pharmacodynamic analysis suggested a concentration-dependent QTc interval prolongation of 14 msec (upper bound of two-sided 90% CI: 16 msec) at a dose of osimertinib 80 mg.

C28H33N7O2

499.61

499.269

C, 67.31; H, 6.66; N, 19.62; O, 6.40

1421373-65-0

Osimertinib mesylate;1421373-66-1;Osimertinib-d6;1638281-44-3;Osimertinib dimesylate;2070014-82-1;Osimertinib-13C,d3;2254100-49-5

71496458

Brown to yellow solid powder

1.2±0.1 g/cm3

1.618

3.3

87.55

2

7

10

37

752

0

O(C([H])([H])[H])C1=C(C([H])=C(C(=C1[H])N(C([H])([H])[H])C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H])N([H])C(C([H])=C([H])[H])=O)N([H])C1=NC([H])=C([H])C(C2=C([H])N(C([H])([H])[H])C3=C([H])C([H])=C([H])C([H])=C32)=N1

DUYJMQONPNNFPI-UHFFFAOYSA-N

InChI=1S/C28H33N7O2/c1-7-27(36)30-22-16-23(26(37-6)17-25(22)34(4)15-14-33(2)3)32-28-29-13-12-21(31-28)20-18-35(5)24-11-9-8-10-19(20)24/h7-13,16-18H,1,14-15H2,2-6H3,(H,30,36)(H,29,31,32)

N-[2-[2-(dimethylamino)ethyl-methylamino]-4-methoxy-5-[[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino]phenyl]prop-2-enamide

AZD-9291; AZD9291; AZD 9291; Mereletinib; AZD9291; AZD 9291; UNII-3C06JJ0Z2O; Osimertinib [USAN]; Osimertinib free base; Mereletinib; Trade name: Tagrisso

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: ≥ 2.5 mg/mL (5.00 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 25.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.00 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 3 中的溶解度: ≥ 2.08 mg/mL (4.16 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 20.8 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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 4 中的溶解度: 1% DMSO+30% PEG 300+dd H2O: 30mg/mL

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配方 5 中的溶解度: 5 mg/mL (10.01 mM) in 0.5%HPMC 1%Tween80 (这些助溶剂从左到右依次添加，逐一添加), 悬浮液； 超声助溶。 (<60°C).

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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。