EGFR L858R (IC50 = 0.4 nM); EGFR (IC50 = 0.5 nM); EGFR L858R/T790M (IC50 = 10 nM); HER2 (IC50 = 14 nM); HER3
- 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) Dimaleate inhibits EGFR (IC₅₀ = 0.5 nM), HER2 (IC₅₀ = 14 nM), and HER4 (IC₅₀ = 1 nM) tyrosine kinases [1]
Afatinib (BIBW2992) Dimaleate shows inhibitory activity against EGFR T790M mutant (IC₅₀ = 10 nM) and wild-type EGFR (IC₅₀ = 0.4 nM) [2]

BIBW2992 is more efficient at preventing lung cancer cell lines carrying wild-type (H1666) or L858R/T790M (NCI-H1975) EGFR from surviving than erlotinib, gefitinib, or lapatinib. Comparably effective against NSCLC lines expressing EGFR E746_A750del (HCC827) or HER2 776insV (NCI-H1781), BIBW2992 exhibits no activity against A549 cells, which express EGFR and HER2 in their wild-type forms.[1] Afatinib increases the apoptosis that topotecan and mitoxantrone induce in SP cells as well as the cytotoxicity of these substances to SP cells.[2]

---

- Antiproliferative activity：Afatinib inhibits proliferation of EGFR-mutant non-small cell lung cancer (NSCLC) cell lines (HCC827, PC-9) with IC₅₀ values of 10–20 nM, and HER2+ breast cancer cells (SK-BR-3) with an IC₅₀ of 30 nM in MTT assays. [1][2]
- Signal pathway inhibition：In HCC827 cells, afatinib (50 nM, 4 hours) reduces phosphorylation of EGFR (Tyr1068), AKT (Ser473), and ERK1/2 (Thr202/Tyr204) by 90%, 85%, and 80%, respectively, as measured by Western blot. It also downregulates cyclin D1 and upregulates cleaved PARP, indicating apoptosis induction. [1][2]
- Synergy with radiation：In head and neck squamous cell carcinoma (HNSCC) cells (SCC-25), afatinib (10 nM) enhances radiation-induced cell killing, increasing the radiation sensitivity factor by 1.5-fold. [3]

---

Afatinib (BIBW2992) Dimaleate dose-dependently inhibited the proliferation of EGFR-overexpressing tumor cell lines, including A431 (IC₅₀ = 0.07 μM), HCC827 (EGFR exon 19 deletion, IC₅₀ = 0.01 μM), and NCI-N87 (HER2-overexpressing, IC₅₀ = 0.15 μM). It blocked EGFR/HER2 phosphorylation and downstream signaling (ERK1/2, Akt) in these cells at concentrations ≥ 0.1 μM [1]
Afatinib (BIBW2992) Dimaleate induced apoptosis in HCC827 cells with an EC₅₀ of 0.02 μM, increasing cleaved caspase-3 and PARP levels. It also suppressed clonogenicity of NCI-H1975 cells (EGFR T790M mutant) with an IC₅₀ of 0.2 μM [2]
Afatinib (BIBW2992) Dimaleate enhanced the radiosensitivity of non-small cell lung cancer (NSCLC) cells (A549) in vitro. Combination of 0.1 μM afatinib with 2 Gy radiation increased cell death by ~50% compared to radiation alone [3]
Afatinib (BIBW2992) Dimaleate inhibited the migration and invasion of breast cancer cells (SK-BR-3) by ~70% and ~65% at 0.2 μM, respectively, by downregulating MMP-9 expression [4]

BIBW2992 (20 mg/kg, p.o.) causes a significant tumor regression with a cumulative treated/control tumor volume ratio (T/C ratio) of 2% in the MDA-MB-453 xenograft model. It also results in the downregulation of EGFR and AKT phosphorylation.[1] Applying BIBW 2992 (30 mg/kg, p.o.) to xenograft models A7, A431, FaDu, UT-SCC-14, and UT-SCC-15 significantly extends the time for tumor growth.[3] Afatinib (30 mg/kg, p.o.) causes a notable inhibition of tumor growth and a notable extension of overall survival in HER2-amplified xenograft models. [4] Afatinib (25 mg/kg, p.o.) causes a dramatic tumor volume regression within 4 days and nearly complete tumor resolution after 21 days of treatment in HER2-positive gastric cancer NCI-N87 xenograft.[5]

---

- Tumor growth inhibition in NSCLC xenografts：Oral afatinib (20 mg/kg, daily) reduces tumor volume by 70–80% in HCC827 and PC-9 xenografts in nude mice after 21 days, with decreased Ki-67 and p-EGFR expression in tumor tissues. [1][2]
- Synergy with radiation in HNSCC models：In SCC-25 xenografts, afatinib (10 mg/kg, daily) combined with radiation (6 Gy) reduces tumor volume by 90% after 28 days, significantly more than either treatment alone (50–60% inhibition). [3]
- Pharmacodynamic effects in breast cancer models：In SK-BR-3 xenografts, afatinib (30 mg/kg, daily) decreases HER2 phosphorylation by 85% and increases tumor apoptosis (TUNEL+ cells) by 3-fold. [4]

---

Afatinib (BIBW2992) Dimaleate inhibited tumor growth in nude mice bearing HCC827 xenografts when administered orally at 20 mg/kg/day for 21 days. Tumor volume was reduced by ~80% compared to the control group, and intratumoral EGFR phosphorylation was significantly suppressed [1]
Afatinib (BIBW2992) Dimaleate delayed tumor progression in nude mice bearing NCI-H1975 xenografts (EGFR T790M mutant) at an oral dose of 40 mg/kg/day for 28 days, resulting in a ~60% reduction in tumor weight [2]
Afatinib (BIBW2992) Dimaleate augmented the antitumor effect of radiation in nude mice bearing A549 NSCLC xenografts. Oral administration of 15 mg/kg/day afatinib plus 8 Gy radiation (fractionated over 4 days) reduced tumor volume by ~75% compared to radiation alone [3]
In a phase II clinical study of patients with advanced NSCLC harboring EGFR mutations, Afatinib (BIBW2992) Dimaleate (40 mg orally once daily) showed a partial response rate of 56% and a median progression-free survival of 11.1 months [5]

EGFR kinase: 10 μL of inhibitor in 50% Me2SO, 20 μL of substrate solution (200 mM HEPES pH 7.4, 50 mM Mg-acetate, 2.5 mg/mL poly (EY), 5 μg/mL bio-pEY), and 20 µL enzyme preparation were included in each 100 µL enzyme reaction. The addition of 50 µL of a 100 µM ATP solution prepared in 10 mM MgCl2 initiates the enzymatic reaction. After 30 minutes of assaying at room temperature, 50 µL of stop solution (250 mM EDTA in 20 mM HEPES pH 7.4) is added to end the assay. 100 µL are added to a microtiterplate coated with streptavidin, and after 60 minutes of room temperature incubation, the plate is cleaned with 200 µL of wash solution (50 mM Tris, 0.05% Tween20). The wells are filled with a 100 µL aliquot of PY20H Anti-Ptyr:HRP, a 250 ng/mL HRPO-labeled anti-PY antibody. Following a 60-minute incubation period, the plate is three times cleaned using a 200 µL wash solution. Following that, 100µL of TMB Peroxidase Solution (A:B=1:1) is used to develop the samples. After ten minutes, the reaction is stopped. After the plate is placed in an ELISA reader, the extinction at OD450nm is calculated. The enzyme HER2-IC: The assay of enzyme activity is conducted in 50% Me2SO with or without serial inhibitor dilutions. Similar components as described for the EGFR kinase assay are included in each 100 µL reaction, along with the addition of 1000 µM Na3VO4. The addition of 50µL of a 500 µM ATP solution prepared in 10 mM magnesium acetate initiates the enzymatic reaction. The enzyme is diluted to the point where the amount of enzyme and the amount of time it takes for phosphate to be incorporated into bio-pEY are linear. The mixture of 20 mM HEPES pH 7.4, 130 mM NaCl, 0.05% Triton X-100, 1 mM DTT, and 10% glycerol is used to dilute the enzyme preparation. After 30 minutes of assaying at room temperature, 50 µL of stop solution is added to end the procedure. Src kinase assays: 10 µL of inhibitor in 50% Me2SO, 20 µL of enzyme preparation, and 20 µL of substrate solution enhanced with 1000 µM Na3VO4 were included in each 100 µL reaction. The addition of 50 µL of a 1000 µM ATP solution prepared in 10 mM Mg-acetate initiates the enzymatic reaction. Assay for BIRK kinase: 50 µL of a 2 mM ATP solution prepared in 8 mM MnCl2 and 20 mM Mg-acetate is added to 250 mM Tris pH 7.4, 10 mM DTT, 2.5 mg/mL poly(EY), and 5 mg/mL bio-pEY as the substrate solution to initiate the enzymatic reaction. HGFR kinase and VEGF2 assays: The assay is completed by adding 10 µL of 5% H3PO4 after it has been running at room temperature for 20 minutes. The precipitate is then collected using a 96 well filter mate universal harvester and trapped onto GF/B filters. The filter plate is thoroughly cleaned, dried for one hour at 50°C, sealed, and the radioactivity is measured using scintillation counting with either a TopCount TM or a Microbeta b counter TM .

---

- EGFR kinase activity assay: 1. Recombinant wild-type or mutant EGFR kinase domains are incubated with afatinib (0.01–100 nM) and [γ-³²P]ATP in kinase buffer.
2. After 30 minutes at 30°C, reactions are stopped, and phosphorylated peptide substrates are captured on filters.
3. Radioactivity is measured, and IC₅₀ values are calculated for each EGFR variant. [1]
- HER2 kinase assay: 1. Recombinant HER2 kinase is incubated with afatinib (1–100 nM) and fluorescently labeled substrate peptide.
2. Kinase activity is measured via fluorescence resonance energy transfer (FRET) to detect substrate phosphorylation.
3. The IC₅₀ for HER2 inhibition is determined as 14 nM. [1]

---

Recombinant EGFR, HER2, and HER4 kinase domains were individually incubated with ATP and specific peptide substrates in the presence of serial dilutions of Afatinib (BIBW2992) Dimaleate. Reactions were carried out at 37°C for 60 minutes, and phosphorylated substrates were detected using a homogeneous time-resolved fluorescence (HTRF) assay. Inhibition rates were calculated by comparing fluorescence intensity with vehicle controls, and IC₅₀ values were derived from dose-response curves [1]
Recombinant EGFR T790M mutant and wild-type EGFR kinase domains were tested using the same protocol. The reaction mixture was incubated at 30°C for 45 minutes, and phosphorylation was quantified by HTRF. IC₅₀ values were determined to compare inhibitory potency against mutant and wild-type EGFR [2]

Proliferation and signaling assay: 1. NSCLC or breast cancer cells are seeded in 96-well plates and treated with afatinib (0.1–1,000 nM) for 72 hours.
2. Cell viability is measured by MTT assay to determine IC₅₀ values.
3. For signaling analysis, cells are treated with 50 nM afatinib for 2–24 hours, lysed, and p-EGFR, p-AKT, p-ERK, and apoptotic markers are detected by Western blot. [1][2][4]
- Radiation synergy assay: 1. HNSCC cells are pre-treated with afatinib (10 nM) for 2 hours, then irradiated with 0–8 Gy.
2. Clonogenic survival is assessed by counting colonies after 14 days; survival curves are used to calculate radiation sensitivity. [3]
The MTT assay is used to determine cytotoxicity. The drug concentration that causes 50% of cells to die is known as the IC 50 value. The Bliss method is used to calculate the IC50 as well as the fitted sigmoidal dose response curve.

---

A431, HCC827, and NCI-N87 cells were seeded in 96-well plates at 5×10³ cells/well and treated with Afatinib (BIBW2992) Dimaleate (0.001-1 μM) for 72 hours. Cell viability was measured using a tetrazolium-based assay to calculate IC₅₀ values. For Western blot analysis, cells were treated with 0.05-0.5 μM afatinib, lysed, and probed with antibodies against phosphorylated EGFR/HER2, ERK1/2, Akt, and GAPDH [1]
HCC827 cells were treated with Afatinib (BIBW2992) Dimaleate (0.01-0.1 μM) for 48 hours. Apoptosis was detected by Annexin V-FITC/PI staining, and cleaved caspase-3/PARP expression was analyzed by Western blot. NCI-H1975 cells were seeded in 6-well plates and treated with 0.05-0.5 μM afatinib for 14 days to assess clonogenicity [2]
A549 cells were treated with Afatinib (BIBW2992) Dimaleate (0.05-0.2 μM) for 24 hours, followed by radiation (0-4 Gy). After 72 hours, cell viability was assessed by MTT assay, and cell death was detected by propidium iodide staining [3]
SK-BR-3 cells were treated with Afatinib (BIBW2992) Dimaleate (0.1-0.5 μM) for 24 hours. Migration and invasion assays were performed using Boyden chambers, and MMP-9 mRNA expression was quantified by RT-PCR [4]

SCID mice harbouring ARK2 xenografts
25 mg/kg
p.o.

---

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]

---

- 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]

---

Nude mice bearing HCC827 xenografts (100-150 mm³) were randomly divided into control and treatment groups. Afatinib (BIBW2992) Dimaleate 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) Dimaleate 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]

Oral absorption: In mice, the peak plasma concentration (Cmax) of afatinib (20 mg/kg) was 1.2 μg/mL 2 hours after oral administration, and the oral bioavailability was approximately 40%. [2]
- Half-life: The terminal elimination half-life in mice was 6-8 hours; in humans, the steady-state half-life was 37 hours. [2][5]
- Distribution: In tumor-bearing mice, afatinib mainly accumulated in the tumor, with a tumor-to-plasma concentration ratio of 3-5:1. [2]
- Metabolism: Mainly metabolized by CYP3A4; <5% was excreted unchanged in the urine. [5]

---

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

Preclinical toxicity: In rats, afatinib (50 mg/kg once daily for 28 days) caused mild diarrhea and rash, but no significant liver or kidney damage was observed (ALT/AST and BUN were within the normal range). [2][4] - Clinical toxicity: Common adverse reactions included diarrhea (60%), rash (45%) and stomatitis (30%); Grade 3 or higher adverse reactions were rare (<10%). Plasma protein binding was >95%. [5] Mice administered afatinib (BIBW2992) dimaleate 20 mg/kg/day for 21 days showed mild weight loss (approximately 10%) and transient diarrhea (18% of animals), but no significant liver or kidney toxicity was observed. Serum ALT, AST, and creatinine levels were all within the normal range [1]. In the phase II clinical trial, the most common adverse reactions to afatinib (BIBW2992) dimaleate 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) dimaleate in human plasma was approximately 95% as determined by balanced 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.

Afatinib dimaleate is a maleate form of afatinib combined with two molar equivalents of maleic acid. It is used as a first-line treatment for patients with metastatic non-small cell lung cancer. It is a tyrosine kinase inhibitor and an antitumor drug. Its main component is afatinib. Afatinib dimaleate is the dimaleate form of afatinib, a highly bioavailable orally bioavailable aniline-2-quinazoline derivative that is an inhibitor of the epidermal growth factor receptor (ErbB; EGFR) family of receptor tyrosine kinases (RTKs) and possesses antitumor activity. After administration, afatinib selectively and irreversibly binds to and inhibits epidermal growth factor receptors 1 (ErbB1; EGFR), 2 (ErbB2; HER2), and 4 (ErbB4; HER4), as well as certain EGFR mutants, including those caused by EGFR exon 19 deletion mutations or exon 21 (L858R) mutations. This may lead to inhibition of tumor growth and angiogenesis in tumor cells that overexpress these receptor tyrosine kinases (RTKs). In addition, afatinib can inhibit EGFR T790M "gatekeeper" mutations resistant to first-generation EGFR inhibitors. EGFR, HER2, and HER4 are RTKs belonging to the EGFR superfamily. They play important roles in tumor cell proliferation and tumor angiogenesis and are overexpressed in various cancer cell types. A quinazoline and butenamide derivative, as a tyrosine kinase inhibitor of the epidermal growth factor receptor (ERBB receptor), is used to treat metastatic non-small cell lung cancer. See also: Afatinib (with the active moiety). Drug Indications Gelatinib monotherapy is indicated for the treatment of adult patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) with EGFR activating mutations who have not previously received epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy; and locally advanced or metastatic squamous cell carcinoma NSCLC that has progressed during or after platinum-based chemotherapy.

---

- Mechanism of action: Afatinib irreversibly binds to the ATP binding sites of EGFR, HER2 and HER4, inhibiting their kinase activity and blocking downstream PI3K/AKT and MAPK pathways, thereby leading to cell cycle arrest and apoptosis. [1][2]
- Indications: Approved for EGFR-mutant non-small cell lung cancer (NSCLC) and HER2-positive breast cancer; its efficacy in combination with radiotherapy for head and neck squamous cell carcinoma (HNSCC) is under investigation. [2][3][4]
- Pharmacodynamic markers: Decreased plasma carcinoembryonic antigen (CEA) levels are associated with tumor response in NSCLC patients. [5]

---

Afatinib (BIBW2992) dimaleate is an irreversible oral EGFR, HER2 and HER4 inhibitor that exerts its antitumor effect by covalently binding to the kinase domains of these receptors, thereby blocking downstream signaling pathways[1]
Afatinib (BIBW2992) dimaleate is effective against EGFR-mutant non-small cell lung cancer (NSCLC), including tumors carrying the T790M resistance mutation, making it a potential treatment option for patients resistant to first-generation EGFR inhibitors[2]
The ability of afatinib (BIBW2992) dimaleate to enhance the response to radiotherapy supports its use in combination with radiotherapy in locally advanced NSCLC[3]

C32H33CLFN5O11

717.18

717.184

C, 53.52; H, 4.63; Cl, 4.94; F, 2.65; N, 9.75; O, 24.51

850140-73-7

Afatinib;850140-72-6;Afatinib-d6 dimaleate;Afatinib oxalate;1398312-64-5

15606394

White to off-white solid powder

4.536

6

16

12

50

821

1

C(/C(=O)O)=C/C(=O)O.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

USNRYVNRPYXCSP-JUGPPOIOSA-N

InChI=1S/C24H25ClFN5O3.2C4H4O4/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;2*5-3(6)1-2-4(7)8/h3-6,10-12,14,16H,7-9,13H2,1-2H3,(H,30,32)(H,27,28,29);2*1-2H,(H,5,6)(H,7,8)/b4-3+;2*2-1-/t16-;;/m0../s1

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

Afatinib dimaleate; BIBW 2992; Afatinib (diMaleate); BIBW-2992; BIBW2992; UNII-V1T5K7RZ0B; Afatinib dimaleate [USAN]; 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

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 100 mg/mL (139.26 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

---

Solubility in Formulation 2: 5% DMSO+30% PEG 300+ddH2O: 28 mg/mL

---

 (Please use freshly prepared in vivo formulations for optimal results.)