EGFR

Firmonertinib 旨在阻断 T790M 获得性耐药突变和 EGFR 活性突变[1]。
本研究采用CRISPR方法构建4株EGFR S768I突变细胞系，检测EGFR对阿蒙尼替尼和阿氟替尼的敏感性，阳性对照为第1代(吉非替尼)、第2代(阿法替尼)、第3代(奥西替尼)药物。 结果:本研究结果表明，almonertinib和alflutinib可通过ERK或AKT通路，通过阻断细胞周期和抑制细胞凋亡，以时间依赖性的方式有效抑制罕见EGFR S768I突变的细胞活力和增殖。 结论:这些发现提示，阿莫那替尼和阿氟替尼可能是EGFR S768I突变的非小细胞肺癌患者的潜在治疗选择。[2]

从2016年12月27日至2017年8月21日，共有17名患者在4个队列中接受了至少一剂AST2818/alflutinib (20mg、40mg、80mg和160mg QD)。最大耐受剂量尚未达到。最常见的治疗相关ae是1级蛋白尿(25%，3/12)。其他ae包括疲劳、Q-T间隔延长等，均小于10%，为1级或2级。前12例患者的ORR为58.3%(7/12)，疾病控制率为91.7%(11/12)。在20mg组中已经观察到深刻和持续的肿瘤消退。以Cmax和AUC 0-24h测量的AST2818血浆暴露呈剂量正比增加。 结论 在第一代EGFR- tkis治疗失败后，AST2818在EGFR T790M突变型NSCLC中耐受性良好，具有良好的临床活性和持久的疾病控制。[1]

阿氟替尼在HLMs中的代谢[3]
在开始实验之前，将HLMs在冰上轻轻解冻。然后，将3µM阿氟替尼(0.5 mg蛋白/mL)加入到100 mM磷酸盐缓冲盐水(PBS;pH 7.4)至总容积为100 μL。37℃孵育3min后，加入1.0 mM NADPH启动反应。孵育1小时后，用相同体积的冰凉乙腈混合终止反应。所有孵育重复进行，然后用UPLC-UV/Q-TOF质谱分析[3]

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特异性CYP抑制剂对HLMs的影响[3]
采用HLMs研究CYP酶抑制剂对阿氟替尼代谢的影响。孵育混合物(100µL)由阿氟替尼(3µM)、HLMs (0.5 mg蛋白/mL)、NADPH (1 mM)、PBS (100 mM, pH 7.4)和选择性CYP抑制剂组成。化学抑制剂分别为:CYP1A/ 2c α-萘黄酮(2µM)、CYP2C8槲皮素(20µM)、CYP2C9磺胺苯唑(6µM)、CYP2B6/2C19噻氯匹定(24µM)、CYP2D6奎尼丁(8µM)、CYP2E1氯甲基唑(24µM)、CYP3A酮康唑(2µM)、ABT(1µM)。在加入底物之前，将这些抑制剂与HLMs在NADPH存在下预孵育10分钟。然后，在37°C下孵育60分钟开始反应。最后，加入100µL冰凉的乙腈终止反应。所有的孵育重复进行，在没有或存在抑制剂的情况下评估代谢物的形成[3]

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。 重组人CYP同工酶对阿氟替尼的代谢作用[3]
为了鉴定参与阿氟替尼代谢的特异性异构体，将3µM阿氟替尼与重组人CYP1A2、2A6、2B6、2C8、2C9、2C19、2D6、2E1、3A4或3A5 (25 pmol P450/mL)混合，总体积为100 μL。分别加入1 mM NADPH和100µL冰凉乙腈开始和终止反应。37℃孵育60 min。[3]

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阿氟替尼对人主要CYP酶的抑制作用[3]
所有cypp异构体特异性探针，如非那西丁、安非他酮、紫杉醇、甲磺丁酰胺、s -甲苯妥英和右美沙芬(分别用于CYP1A2、2B6、2C8、2C9、2C19和2D6)以及咪达唑仑和睾酮(用于CYP3A4)，均溶解于甲醇中。阿氟替尼作为100mm的原液溶解在DMSO中。总体积为100 μL，培养基为100 mM PBS，含有混合HLMs (0.5 mg/mL)、探针底物、NADPH (1 mM)和不同浓度的阿氟替尼(0、0.1、0.33、1、3.30、10、33.3和100 μM)。37℃预孵育5 min。在实验条件下，酶浓度与反应体系孵育时间均在线性范围内(补充表S1)。[3]

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阿氟替尼和AST5902对人CYP3A4酶的酶促作用[3]
为了评估酶的诱导作用，将7 × 105个肝细胞/mL接种于胶原包被的24孔板中，置于37℃加5% CO2的加湿培养箱中24 h。用人CYP3A4酶诱导剂利福平(10 μM)、阿氟替尼或AST5902(0.003、0.01、0.03、0.1、0.3、1、3或5 μM)或0.1% DMSO(对照组)处理肝细胞，每天1次，连续3天。处理后，按照制造商的方案用TRIzol进行RNA提取。采用PrimeScript RT试剂盒进行cDNA合成。采用SYBR green Premix Ex Taq试剂盒，在StepOnePlus实时PCR系统上进行实时PCR。CYP3A4的正向引物为5 ' -ATCACTAGCACATCATTTGGAG-3 '，反向引物为5 ' -GGAATGGAAAGGTTATTGAGAG-3 '。GAPDH正向引物为5′-AGAAGGCTGGGGCTCATTTG-3′，反向引物为5′-GAGGGGCCATCCACAGTCTTC-3′。以GAPDH为内标，采用比较阈值循环法定量cDNA水平。

细胞存活与生长试验[2]
采用MTT法测定H3255、H3255Cas9+S768I、H3255L858R+S768I、PC9、PC9Cas9+S768I、PC919Del+S768I细胞的活力，以确定不同TKIs的影响。将细胞消化成单细胞悬液，接种于96孔板，在RPMI 1640培养基中培养，每孔5000个细胞/100 μL。去除原始培养基后，每孔加入100μL不同的TKIs处理细胞。细胞培养24或48 h后，每孔中加入20 μL MTT, 37℃孵育4 h，丢弃上清，每孔中加入100 μL DMSO溶液。将96孔板在37℃避光条件下孵育15 min，之后使用微孔板仪在490 nm处测量光密度(OD)。[2]

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菌落形成试验[2]
将H3255、H3255Cas9+S768I、H3255L858R+S768I、PC9、PC9Cas9+S768I、PC919Del+S768I细胞接种于6孔板中，密度为每孔500个细胞，接种于2ml培养基中。18 h后，取出培养基，用不同浓度的TKIs孵育细胞。每2-3天更换一次含药培养基。在37℃下培养14天后，丢弃培养基。每孔用PBS缓冲液洗涤，4%多聚甲醛固定15分钟，室温下用吉姆萨染色20分钟。然后丢弃吉姆萨染色液，每孔用双蒸馏水冲洗三至四次。最后自然风干后计数菌落。[2]

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细胞增殖试验[2]
将H3255、H3255Cas9+S768I、H3255L858R+S768I、PC9、PC9Cas9+S768I和PC919Del+S768I细胞以每孔5000个细胞的密度接种于96孔板中。18 h后，更换不同的TKIs培养液，孵育48 h，丢弃培养液，用PBS缓冲液洗涤细胞。随后，每口井依次用4%多聚甲醛、3% BSA PBS和0.3% Triton X-100 PBS进行处理。每次处理后，用PBS缓冲液冲洗细胞三次。然后在每孔中加入50 μL的click反应液，标记增殖细胞。细胞在黑暗中孵育30分钟。去除培养基后，细胞洗涤三次。然后，加入50 μL的Hoechst染色剂标记所有细胞核，细胞在黑暗中孵育10分钟，丢弃培养基后，细胞清洗3次，立即荧光显微镜下拍照。

Patients with histologically diagnosed, EGFR T790M mutant stage IV NSCLC were considered eligible, and they should have documented disease progression on EGFR-TKIs. In a 3+3 dose-escalation design, AST2818 was orally administered every day on a 21-day cycle at doses ranging from 20mg to 240 mg (NCT02973763). AST2818 was then explored in a dose-expansion cohort at doses ranging from 40 to 240 mg every day. Plasma samples were collected to evaluate pharmacokinetics of AST2818. EGFR T790M mutation in tissue samples was detected by amplification refractory mutation system. The primary endpoint was to determine dose limiting toxicity and objective response rate (ORR). Adverse events (AEs) were evaluated by CTCAE 4.03, and efficacy was assessed per RECIST v1.1 every 6 weeks.[1]

[1]. Y. Shi, et al. P2.03-028 Third Generation EGFR Inhibitor AST2818 (Alflutinib) in NSCLC Patients with EGFR T790M Mutation: A phase1/2 Multi-Center Clinical Trial.

Purpose EGFR classical mutations respond well to EGFR tyrosine kinase inhibitors. However, it is uncertain whether currently available EGFR-TKIs are effective against rare EGFR mutations and compound mutations. Herein, the effectiveness of almonertinib and alflutinib, the third-generation tyrosine kinase inhibitors developed in China, on rare EGFR S768I mutations and compound mutations is identified. Methods In this study, using CRISPR method, four EGFR S768I mutation cell lines were constructed, and the sensitivity of EGFR to almonertinib and alflutinib was tested, with positive controls being the 1st (gefitinib), 2nd (afatinib), and 3rd (osimertinib) generation drugs. Results The present results indicate that almonertinib and alflutinib can effectively inhibit cell viability and proliferation in rare EGFR S768I mutations through the ERK or AKT pathways in a time-dependent manner, by blocking the cell cycle and inhibiting apoptosis. Conclusions These findings suggest that almonertinib and alflutinib may be potential therapeutic options for non-small cell lung cancer patients with the EGFR S768I mutation.[2]

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Alflutinib (AST2818) is a third-generation epidermal growth factor receptor (EGFR) inhibitor that inhibits both EGFR-sensitive mutations and T790M mutations. Previous study has shown that after multiple dosages, alflutinib exhibits nonlinear pharmacokinetics and displays a time- and dose-dependent increase in the apparent clearance, probably due to its self-induction of cytochrome P450 (CYP) enzyme. In this study, we investigated the CYP isozymes involved in the metabolism of alflutinib and evaluated the enzyme inhibition and induction potential of alflutinib and its metabolites. The data showed that alflutinib in human liver microsomes (HLMs) was metabolized mainly by CYP3A4, which could catalyze the formation of AST5902. Alflutinib did not inhibit CYP isozymes in HLMs but could induce CYP3A4 in human hepatocytes. Rifampin is a known strong CYP3A4 inducer and is recommended by the FDA as a positive control in the CYP3A4 induction assay. We found that the induction potential of alflutinib was comparable to that of rifampin. The Emax of CYP3A4 induction by alflutinib in three lots of human hepatocytes were 9.24-, 11.2-, and 10.4-fold, while the fold-induction of rifampin (10 μM) were 7.22-, 19.4- and 9.46-fold, respectively. The EC50 of alflutinib-induced CYP3A4 mRNA expression was 0.25 μM, which was similar to that of rifampin. In addition, AST5902 exhibited much weak CYP3A4 induction potential compared to alflutinib. Given the plasma exposure of alflutinib and AST5902, both are likely to affect the pharmacokinetics of CYP3A4 substrates. Considering that alflutinib is a CYP3A4 substrate and a potent CYP3A4 inducer, drug–drug interactions are expected during alflutinib treatment.[3]

C28H31F3N8O2

568.593355417252

568.2522

C, 59.15; H, 5.50; F, 10.02; N, 19.71; O, 5.63

1869057-83-9

Firmonertinib mesylate;2130958-55-1

118861389

Typically exists as solid at room temperature

4.4

100Ų

GHKOONMJXNWOIW-UHFFFAOYSA-N

1S/C28H31F3N8O2/c1-6-24(40)33-21-15-22(26(41-17-28(29,30)31)36-25(21)38(4)14-13-37(2)3)35-27-32-12-11-20(34-27)19-16-39(5)23-10-8-7-9-18(19)23/h6-12,15-16H,1,13-14,17H2,2-5H3,(H,33,40)(H,32,34,35)

2-Propenamide, N-(2-((2-(dimethylamino)ethyl)methylamino)-5-((4-(1-methyl-1H-indol-3-yl)-2-pyrimidinyl)amino)-6-(2,2,2-trifluoroethoxy)-3-pyridinyl)-

Alflutinib, ASK120067; AST-2818, ASK-120067; firmonertinib; furmonertinib; AST2818; AST-2818; A49A7A5YN4; N-[2-[2-(dimethylamino)ethyl-methylamino]-5-[[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino]-6-(2,2,2-trifluoroethoxy)pyridin-3-yl]prop-2-enamide; AST2818, AST 2818

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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));
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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