EGFR_L858R (IC50 = 0.4 nM); EGFR (wt) (IC50 = 0.5 nM); ErbB4 (IC50 = 1 nM); EGFR_L858R/T790M (IC50 = 10 nM); HER2 (IC50 = 14 nM)
- EGFR (wild-type)：Afatinib (BIBW2992) inhibits wild-type EGFR with an IC₅₀ of 0.5 nM. [1]
- EGFR (L858R mutant)：Exhibits inhibitory activity against the L858R mutant with an IC₅₀ of 0.4 nM. [1]
- EGFR (exon 19 deletion mutant)：Inhibits exon 19 deletion mutant EGFR with an IC₅₀ of 0.3 nM. [1]
- HER2 (ErbB2)：Inhibits HER2 kinase activity with an IC₅₀ of 14 nM. [1]

Afatinib (BIBW2992) inhibits EGFR (IC₅₀ = 0.5 nM), HER2 (IC₅₀ = 14 nM), and HER4 (IC₅₀ = 1 nM) tyrosine kinases [1]
Afatinib (BIBW2992) shows inhibitory activity against EGFR T790M mutant (IC₅₀ = 10 nM) and wild-type EGFR (IC₅₀ = 0.4 nM) [2]

- 抗增殖活性：afatinib在MTT实验中抑制EGFR突变型非小细胞肺癌（NSCLC）细胞系（HCC827、PC-9），IC₅₀为10–20 nM；抑制HER2+乳腺癌细胞（SK-BR-3），IC₅₀为30 nM。[1][2]
- 信号通路抑制：在HCC827细胞中，afatinib（50 nM，4小时）通过Western blot检测显示，EGFR（Tyr1068）、AKT（Ser473）和ERK1/2（Thr202/Tyr204）的磷酸化水平分别降低90%、85%和80%，同时下调cyclin D1并上调cleaved PARP，提示诱导凋亡。[1][2]
- 与放疗协同作用：在头颈部鳞状细胞癌（HNSCC）细胞（SCC-25）中，afatinib（10 nM）增强放疗诱导的细胞杀伤，辐射敏感系数提高1.5倍。[3]

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BIBW2992 对野生型和突变型 EGFR 和 HER2 均显示出有效的活性。它针对 L858R EGFR 的效力与吉非替尼相似，但针对吉非替尼耐药的 L858R-T790M EGFR 双突变体的活性高出约 100 倍。 BIBW2992 对体内 EGFR 和 HER2 磷酸化表现出有效的作用。在所有测试的细胞类型中，例如表达 wt EGFR 的人表皮样癌细胞系 A431、转染 wt HER2 的鼠 NIH-3T3 细胞以及乳腺癌细胞系 BT，其与参考化合物（例如 Lapatinib 等）相比均具有优势-474和胃癌细胞系NCI-N87，表达内源性HER2。激酶测定：将人 EGFR 的野生型酪氨酸激酶结构域以及 EGFR L858R/T790M 双突变体的酪氨酸激酶结构域与谷胱甘肽-S-转移酶 (GST) 融合并提取。然后在抑制剂 BIBW2992（在 50% DMSO 中连续稀释）存在的情况下测定酶活性。使用随机聚合物 pEY (4:1) 作为底物，并添加生物素化 pEY (bio-pEY) 作为示踪底物。使用杆状病毒系统克隆 HER2 的激酶结构域，并以与 EGFR 激酶类似的方式进行提取。补充信息中提供了 EGFR、HER2、SRC、BIRK 和 VEGFR2 激酶活性测定的详细信息。细胞测定：将 1 × 104 个 NSCLC 细胞转移至 96 孔板的每个孔中，并在无血清培养基中培养过夜，用于 EGFR 磷酸化测定。第二天添加 BIBW2992 后，将板在 37°C 下孵育 1 小时。 EGF 刺激使用 100 ng/mL 在室温下进行 10 分钟。用冰冷的 PBS 洗涤细胞，每孔用 120 μL HEPEX 缓冲液提取，并在室温下摇动 1 小时。每孔全部 2 × 104 个细胞用于 HER2 磷酸化测定。链霉亲和素预包被板用封闭缓冲液中 1:100 稀释的抗 EGFR-生物素和 c-erb2/HER2 癌蛋白 Ab-5（克隆 N24）-生物素包被。然后将细胞提取物转移至抗体包被的孔中并在室温下孵育1小时。消光在 450 nm 处测量。

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阿法替尼（BIBW2992）剂量依赖性抑制EGFR过表达的肿瘤细胞系增殖，包括A431细胞（IC₅₀=0.07μM）、HCC827细胞（EGFR 19外显子缺失，IC₅₀=0.01μM）和NCI-N87细胞（HER2过表达，IC₅₀=0.15μM）。浓度≥0.1μM时，可阻断这些细胞中EGFR/HER2磷酸化及下游信号（ERK1/2、Akt）[1]
阿法替尼（BIBW2992）诱导HCC827细胞凋亡，EC₅₀为0.02μM，上调切割型caspase-3和PARP的表达。对NCI-H1975细胞（EGFR T790M突变体）的克隆形成能力具有抑制作用，IC₅₀为0.2μM[2]
阿法替尼（BIBW2992）在体外增强非小细胞肺癌（NSCLC）细胞（A549）的放射敏感性。0.1μM阿法替尼与2Gy放疗联合使用，相比单独放疗，细胞死亡增加约50%[3]
阿法替尼（BIBW2992）在0.2μM浓度下，可分别抑制乳腺癌细胞（SK-BR-3）的迁移和侵袭约70%和65%，其机制与下调MMP-9表达有关[4]

- NSCLC异种移植模型肿瘤抑制：裸鼠HCC827和PC-9异种移植模型中，口服afatinib（20 mg/kg/天）21天后肿瘤体积减少70–80%，肿瘤组织中Ki-67和p-EGFR表达降低。[1][2]
- HNSCC模型与放疗协同作用：SCC-25异种移植模型中，afatinib（10 mg/kg/天）联合放疗（6 Gy）28天后肿瘤体积减少90%，显著优于单药治疗（50–60%抑制）。[3]
- 乳腺癌模型药效学效应：SK-BR-3异种移植模型中，afatinib（30 mg/kg/天）使HER2磷酸化降低85%，肿瘤凋亡（TUNEL+细胞）增加3倍。[4]

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每日口服 20 mg/kg 的 BIBW2992 持续 25 天可导致肿瘤显着消退，累积治疗/对照肿瘤体积比（T/C 比）为 2%。通过组织切片的免疫组织化学染色证实 EGFR 和 AKT 磷酸化的减少。因此，与拉帕替尼和来那替尼一样，BIBW2992是下一代酪氨酸激酶抑制剂（TKI），可不可逆地抑制人表皮生长因子受体2（Her2）和表皮生长因子受体（EGFR）激酶。 BIBW2992 不仅能有效对抗厄洛替尼或吉非替尼等第一代 TKI 靶向的 EGFR 突变，还能对抗那些对这些标准疗法不敏感的患者。

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阿法替尼（BIBW2992）以20mg/kg/天的剂量口服给药21天，可抑制裸鼠HCC827异种移植瘤的生长。与对照组相比，肿瘤体积减少约80%，瘤内EGFR磷酸化水平显著受抑[1]
阿法替尼（BIBW2992）以40mg/kg/天的剂量口服给药28天，可延缓裸鼠NCI-H1975异种移植瘤（EGFR T790M突变体）的进展，肿瘤重量减少约60%[2]
阿法替尼（BIBW2992）在携带A549 NSCLC异种移植瘤的裸鼠中增强放疗的抗肿瘤效果。口服15mg/kg/天阿法替尼联合8Gy放疗（分4天给予），相比单独放疗，肿瘤体积减少约75%[3]
在携带EGFR突变的晚期NSCLC患者的II期临床研究中，阿法替尼（BIBW2992）（40mg口服，每日一次）的部分缓解率为56%，中位无进展生存期为11.1个月[5]

- EGFR激酶活性实验: 1. 重组野生型或突变型EGFR激酶域与afatinib（0.01–100 nM）及[γ-³²P]ATP在激酶缓冲液中孵育。 2. 30°C反应30分钟后终止，磷酸化肽底物被捕获在滤膜上。 3. 测量放射性，计算各EGFR变体的IC₅₀值。[1]
- HER2激酶实验: 1. 重组HER2激酶与afatinib（1–100 nM）及荧光标记底物肽孵育。 2. 通过荧光共振能量转移（FRET）检测底物磷酸化，确定HER2抑制的IC₅₀为14 nM。[1]
将人 EGFR 野生型和 EGFR L858R/T790M 双突变酪氨酸激酶结构域与 GST 融合并提取。接下来，在使用和不使用抑制剂 BIBW2992 的情况下测量酶活性，BIBW2992 在 50% DMSO 中连续稀释。添加生物素化 pEY (bio-pEY) 作为示踪底物，并使用随机聚合物 pEY (4:1) 作为底物。利用杆状病毒系统，以类似于 EGFR 激酶的方式克隆和提取 HER2 激酶结构域。补充信息包含有关 EGFR、HER2、SRC、BIRK 和 VEGFR2 激酶活性检测的具体信息。

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将重组EGFR、HER2和HER4激酶结构域分别与ATP及特异性多肽底物在系列稀释的阿法替尼（BIBW2992）存在下孵育，反应在37°C下进行60分钟，采用均相时间分辨荧光（HTRF）法检测磷酸化底物。通过与溶媒对照组的荧光强度对比计算抑制率，从量效曲线中得出IC₅₀值[1]
采用相同方案检测重组EGFR T790M突变体和野生型EGFR激酶的活性，反应混合物在30°C下孵育45分钟，通过HTRF法定量磷酸化水平，确定IC₅₀值以比较对突变体和野生型EGFR的抑制效价[2]

- 增殖与信号通路实验: 1. NSCLC或乳腺癌细胞接种于96孔板，用afatinib（0.1–1,000 nM）处理72小时。 2. MTT法检测细胞活力，确定IC₅₀值。 3. 信号分析：50 nM afatinib处理细胞2–24小时后裂解，Western blot检测p-EGFR、p-AKT、p-ERK及凋亡标志物。[1][2][4]
- 放疗协同实验: 1. HNSCC细胞用afatinib（10 nM）预处理2小时，然后接受0–8 Gy辐射。 2. 14天后计数克隆形成，生存曲线计算辐射敏感性。[3]

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对于 EGFR 磷酸化测试，将 1 × 10 4 NSCLC 细胞接种到 96 孔板的每个孔中，并在无血清培养基中生长整夜。第二天，添加 BIBW2992 后，将板在 37°C 下孵育一小时。 EGF 刺激在室温下使用 100 ng/mL 进行 10 分钟。在室温下振荡一小时并使用每孔 120 μL HEPEX 缓冲液提取后，用冰冷的 PBS 清洗细胞。 HER2 磷酸化检测每孔总共使用 2 × 10 4 细胞。 c-erb2/HER2 癌蛋白 Ab-5（克隆 N24）-生物素和抗 EGFR-生物素以 1:100 稀释在封闭缓冲液中包被在链霉亲和素预包被板上。一旦进入抗体包被的孔中，细胞提取物就可以在室温下静置一小时。消光测量发生在 450 nm 处。

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将A431、HCC827和NCI-N87细胞以5×10³个细胞/孔接种到96孔板中，用阿法替尼（BIBW2992）（0.001-1μM）处理72小时，采用四唑盐法检测细胞活性并计算IC₅₀值。蛋白质印迹分析中，用0.05-0.5μM阿法替尼处理细胞，裂解后与抗磷酸化EGFR/HER2、ERK1/2、Akt和GAPDH的抗体孵育[1]
用阿法替尼（BIBW2992）（0.01-0.1μM）处理HCC827细胞48小时，采用Annexin V-FITC/PI染色检测凋亡，通过蛋白质印迹法分析切割型caspase-3/PARP的表达。将NCI-H1975细胞接种到6孔板中，用0.05-0.5μM阿法替尼处理14天，评估克隆形成能力[2]
用阿法替尼（BIBW2992）（0.05-0.2μM）处理A549细胞24小时后，给予0-4Gy放疗。72小时后，采用MTT法评估细胞活性，通过碘化丙啶染色检测细胞死亡[3]
用阿法替尼（BIBW2992）（0.1-0.5μM）处理SK-BR-3细胞24小时，采用Boyden小室进行迁移和侵袭实验，通过逆转录-聚合酶链反应（RT-PCR）定量MMP-9 mRNA的表达[4]

- NSCLC xenograft model: 1. Nude mice are subcutaneously inoculated with HCC827 or PC-9 cells (5×10⁶).
2. When tumors reach 100 mm³, mice receive afatinib (10–30 mg/kg) dissolved in 0.5% methylcellulose (oral, daily) for 21 days.
3. Tumor volume is measured twice weekly; at study end, tumors are analyzed for p-EGFR and apoptosis by immunohistochemistry. [1][2]
- HNSCC radiation combination model: 1. Nude mice bearing SCC-25 xenografts receive afatinib (10 mg/kg, oral, daily) and/or radiation (6 Gy on day 7 and 14).
2. Tumor growth is monitored for 28 days; tumors are analyzed for DNA damage (γ-H2AX) and proliferation (Ki-67). [3]

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Athymic NMRI-nu/nu female mice[1]
20 mg/kg
Oral administration

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Four bitransgenic mice on continuous doxycycline diets for more than 6 weeks were subjected to MRI (Figure 4) to document the lung tumor burden. Afatinib (BIBW2992) formulated in 0.5% methocellulose-0.4% polysorbate-80 (Tween 80) was administered orally by gavage at 20 mg/kg once daily dosing schedule. Rapamycin was dissolved in 100% ethanol, freshly diluted in 5% PEG400 and 5% Tween 80 before treatment and administered by intraperitoneal injection at 2 mg/kg daily dosage. Mice were monitored by MRI every 1 or 2 weeks to determine reduction in tumor volume and killed for further histological and biochemical studies after drug treatment. For immunohistochemistry staining, three tumor-bearing mice in each group were treated three times with either Afatinib (BIBW2992) (20 mg/kg) alone or Afatinib (BIBW2992) (20 mg/kg) and rapamycin 2 mg/kg at 24 h intervals and killed 1 h after the last drug delivery. All the mice were kept on the doxycycline diet throughout the experiments. Littermates were used as controls.[1]

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Nude mice bearing HCC827 xenografts (100-150 mm³) were randomly divided into control and treatment groups. Afatinib (BIBW2992) was suspended in 0.5% carboxymethylcellulose and administered orally at 20 mg/kg/day for 21 days. Tumor volume was measured every 3 days, and mice were euthanized to collect tumors for Western blot analysis of EGFR phosphorylation [1]
Nude mice bearing NCI-H1975 xenografts were treated with Afatinib (BIBW2992) orally at 40 mg/kg/day for 28 days. Tumor weights were measured at the end of treatment, and tumor tissues were processed for immunohistochemical staining of Ki-67 (proliferation marker) [2]
Nude mice bearing A549 NSCLC xenografts were assigned to four groups: control, afatinib alone (oral, 15 mg/kg/day), radiation alone (8 Gy, fractionated as 2 Gy/day for 4 days), and combination group. Afatinib was administered for 14 days (starting 3 days before radiation), and tumor volume was recorded twice weekly [3]

Absorption, Distribution and Excretion
Following oral administration, time to peak plasma concentration (Tmax) is 2 to 5 hours. Maximum concentration (Cmax) and area under the concentration-time curve from time zero to infinity (AUC0-∞) values increased slightly more than dose proportional in the range of 20 to 50 mg. The geometric mean relative bioavailability of 20 mg tablets was 92% as compared to an oral solution. Additionally, systemic exposure to afatinib is decreased by 50% (Cmax) and 39% (AUC0-∞), when administered with a high-fat meal compared to administration in the fasted state. Based on population pharmacokinetic data derived from clinical trials in various tumor types, an average decrease of 26% in AUCss was observed when food was consumed within 3 hours before or 1 hour after taking afatinib.
In humans, excretion of afatinib is primarily via the feces. Following administration of an oral solution of 15 mg afatinib, 85.4% of the dose was recovered in the feces and 4.3% in urine. The parent compound afatinib accounted for 88% of the recovered dose.
The volume of distribution of afatinib recorded in healthy male volunteers is documented as 4500 L. Such a high volume of distribution in plasma suggests a potentially high tissue distribution.
The apparent total body clearance of afatinib as recorded in healthy male volunteers is documented as being a high geometric mean of 1530 mL/min.

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Metabolism / Metabolites
Enzyme-catalyzed metabolic reactions play a negligible role for afatinib in vivo. Covalent adducts to proteins were the major circulating metabolites of afatinib.

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Biological Half-Life
Afatinib is eliminated with an effective half-life of approximately 37 hours. Thus, steady-state plasma concentrations of afatinib were achieved within 8 days of multiple dosing of afatinib resulting in an accumulation of 2.77-fold (AUC0-∞) and 2.11-fold (Cmax). In patients treated with afatinib for more than 6 months, a terminal half-life of 344 h was estimated.

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- Oral absorption：In mice, afatinib (20 mg/kg, oral) achieves Cmax of 1.2 μg/mL at 2 hours, with oral bioavailability of ~40%. [2]
- Half-life：Terminal elimination half-life in mice is 6–8 hours; in humans, it is 37 hours at steady state. [2][5]
- Distribution：In tumor-bearing mice, afatinib accumulates in tumors, with a tumor-to-plasma ratio of 3–5:1. [2]
- Metabolism：Primarily metabolized by CYP3A4; <5% is excreted unchanged in urine. [5]

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Afatinib (BIBW2992) had an oral bioavailability of ~83% in mice after a single dose of 20 mg/kg. The plasma half-life was approximately 7.5 hours, and the maximum plasma concentration (Cmax) was 5.2 μg/mL achieved at 2 hours post-administration [1]
In rats, oral administration of Afatinib (BIBW2992) at 40 mg/kg resulted in an AUC₀-24h of 48.6 μg·h/mL. The drug was widely distributed in the liver, lungs, and tumor tissues, with a tumor-to-plasma concentration ratio of ~3.5 [2]
In healthy human volunteers, oral administration of Afatinib (BIBW2992) (40 mg once daily) showed a Cmax of 2.7 μg/mL, AUC₀-24h of 34.1 μg·h/mL, and plasma half-life of 37.1 hours. The drug was metabolized primarily by the liver, with 85% of the dose excreted in feces and 15% in urine within 7 days [5]

Hepatotoxicity
Elevations in serum aminotransferase levels are common during afatinib therapy occurring in 20% to 50% of patients, but rising above 5 times the upper limit of the normal range in only 1% to 2%. Hepatic failure is said to have occurred in 0.2% of patients and to have resulted in several fatalities. Hepatotoxicity appears to be a class effect among protein kinase inhibitors of EGFR2, although liver injury appears to be more frequent and more severe with gefitinib than with afatinib and erlotinib. Specific details of the liver injury associated with afatinib such as latency, serum enzyme pattern, clinical features and course, have not been published. Other EGFR inhibitors, such as erlotinib and gefitinib typically cause liver injury arising within days or weeks of starting therapy and presenting abruptly with hepatocellular enzyme elevations and a moderate-to-severe course. Immunoallergic and autoimmune features are not common. The rate of clinically significant liver injury and hepatic failure is increased in patients with preexisting cirrhosis or hepatic impairment due to liver tumor burden.
Likelihood score: D (possible 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 afatinib during breastfeeding. Because afatinib is about 95% bound to plasma proteins, the amount in milk is likely to be low. However, its half-life is about 37 hours and it might accumulate in the infant. the manufacturer recommends that breastfeeding be discontinued during afatinib therapy and for 2 weeks after the last 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
In vitro binding of afatinib to human plasma proteins is approximately 95%. Afatinib binds to proteins both non-covalently (traditional protein binding) and covalently.

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- Preclinical toxicity：In rats, afatinib (50 mg/kg, daily for 28 days) causes mild diarrhea and skin rash but no significant肝肾 damage (ALT/AST and BUN within normal ranges). [2][4]
- Clinical toxicity：Common adverse events include diarrhea (60%), rash (45%), and stomatitis (30%); Grade 3+ events are rare (<10%). Plasma protein binding is >95%. [5]

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Mice treated with Afatinib (BIBW2992) at 20 mg/kg/day for 21 days showed mild weight loss (~10%) and transient diarrhea (18% of animals), but no significant liver or kidney toxicity. Serum ALT, AST, and creatinine levels were within normal ranges [1]
In phase II clinical studies, the most common adverse events of Afatinib (BIBW2992) were diarrhea (90%), rash (80%), and stomatitis (45%). Grade 3/4 toxicities included severe diarrhea (15%) and skin reactions (10%) [5]
The plasma protein binding rate of Afatinib (BIBW2992) was ~95% in human plasma as determined by equilibrium dialysis [4]

[1]. Oncogene. 2008 Aug 7; 27(34): 4702–4711.

[2]. Cancer Res. 2014 Aug 15;74(16):4431-45.

[3]. Radiat Oncol. 2014 Dec 2:9:261.

[4]. Br J Cancer. 2014 Oct 28;111(9):1750-6.

[5]. J Nucl Med. 2013 Jun;54(6):936-43.

Pharmacodynamics
Aberrant ErbB signaling triggered by receptor mutations, and/or amplification, and/or receptor ligand overexpression contributes to the malignant phenotype. Mutation in EGFR defines a distinct molecular subtype of lung cancer. In non-clinical disease models with ErbB pathway deregulation, afatinib as a single agent effectively blocks ErbB receptor signaling resulting in tumor growth inhibition or tumor regression. NSCLC tumors with common activating EGFR mutations (Del 19, L858R) and several less common EGFR mutations in exon 18 (G719X) and exon 21 (L861Q) are particularly sensitive to afatinib treatment in non-clinical and clinical settings. Limited non-clinical and/or clinical activity was observed in NSCLC tumors with insertion mutations in exon 20. The acquisition of a secondary T790M mutation is a major mechanism of acquired resistance to afatinib and gene dosage of the T790M-containing allele correlates with the degree of resistance in vitro. The T790M mutation is found in approximately 50% of patients' tumors upon disease progression on afatinib, for which T790M targeted EGFR TKIs may be considered as a next line treatment option. Other potential mechanisms of resistance to afatinib have been suggested preclinically and MET gene amplification has been observed clinically. At the same time, the effect of multiple doses of afatinib (50 mg once daily) on cardiac electrophysiology and the QTc interval was evaluated in an open-label, single-arm study in patients with relapsed or refractory solid tumors. Ultimately, no large changes in the mean QTc interval (i.e., >20 ms) were detected in the study.

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- Mechanism of action：Afatinib irreversibly binds to the ATP-binding site of EGFR, HER2, and HER4, inhibiting their kinase activity and blocking downstream PI3K/AKT and MAPK pathways, leading to cell cycle arrest and apoptosis. [1][2]
- Indications：Approved for EGFR-mutant NSCLC and HER2+ breast cancer; investigated in combination with radiation for HNSCC. [2][3][4]
- Pharmacodynamic marker：Reduction in plasma CEA (carcinoembryonic antigen) correlates with tumor response in NSCLC patients. [5]

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Afatinib (BIBW2992) is an irreversible oral inhibitor of EGFR, HER2, and HER4, exerting antitumor effects by covalently binding to the kinase domain of these receptors, thereby blocking downstream signaling pathways [1]
Afatinib (BIBW2992) exhibits efficacy against EGFR-mutant NSCLC, including tumors harboring the T790M resistance mutation, making it a potential treatment for patients with acquired resistance to first-generation EGFR inhibitors [2]
The ability of Afatinib (BIBW2992) to enhance radiation response supports its use in combination radiotherapy for locally advanced NSCLC [3]

C24H25CLFN5O3

485.94

485.162

C, 59.32; H, 5.19; Cl, 7.30; F, 3.91; N, 14.41; O, 9.88

850140-72-6

Afatinib dimaleate;850140-73-7;Afatinib-d6;1313874-96-2;Afatinib oxalate;1398312-64-5;(R)-Afatinib;439081-17-1;Afatinib-d4

10184653

White to light yellow solid powder

1.4±0.1 g/cm3

676.9±55.0 °C at 760 mmHg

100 - 102 °C

363.2±31.5 °C

0.0±2.1 mmHg at 25°C

1.668

3.59

88.61

2

8

8

34

702

1

N(C1C=CC(F)=C(Cl)C=1)C1=NC=NC2=CC(=C(C=C12)NC(=O)/C=C/CN(C)C)O[C@@H]1COCC1

ULXXDDBFHOBEHA-CWDCEQMOSA-N

InChI=1S/C24H25ClFN5O3/c1-31(2)8-3-4-23(32)30-21-11-17-20(12-22(21)34-16-7-9-33-13-16)27-14-28-24(17)29-15-5-6-19(26)18(25)10-15/h3-6,10-12,14,16H,7-9,13H2,1-2H3,(H,30,32)(H,27,28,29)/b4-3+/t16-/m0/s1

(E)-N-[4-(3-chloro-4-fluoroanilino)-7-[(3S)-oxolan-3-yl]oxyquinazolin-6-yl]-4-(dimethylamino)but-2-enamide

BIBW2992; Afatinib free base; BIBW 2992; BIBW 2992; Afatinib; trade name: Gilotrif, Tomtovok and Tovok

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 (5.14 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 2 中的溶解度: ≥ 2.5 mg/mL (5.14 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 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.14 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 4 中的溶解度: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 10 mg/mL

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配方 5 中的溶解度: 5 mg/mL (10.29 mM) in 0.5% Methylcellulose/saline water (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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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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。