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]

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]

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- 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]

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.

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- 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]

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.

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

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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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- 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]

- 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]

- 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]

[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 salt obtained by combining afatinib with two molar equivalents of maleic acid. Used for the first-line treatment of patients with metastatic non-small cell lung cancer. It has a role as a tyrosine kinase inhibitor and an antineoplastic agent. It contains an afatinib.
Afatinib Dimaleate is the dimaleate salt form of afatinib, an orally bioavailable anilino-quinazoline derivative and inhibitor of the receptor tyrosine kinase (RTK) epidermal growth factor receptor (ErbB; EGFR) family, with antineoplastic activity. Upon administration, afatinib selectively and irreversibly binds to and inhibits the epidermal growth factor receptors 1 (ErbB1; EGFR), 2 (ErbB2; HER2), and 4 (ErbB4; HER4), and certain EGFR mutants, including those caused by EGFR exon 19 deletion mutations or exon 21 (L858R) mutations. This may result in the inhibition of tumor growth and angiogenesis in tumor cells overexpressing these RTKs. Additionally, afatinib inhibits the EGFR T790M gatekeeper mutation which is resistant to treatment with first-generation EGFR inhibitors. EGFR, HER2 and HER4 are RTKs that belong to the EGFR superfamily; they play major roles in both tumor cell proliferation and tumor vascularization and are overexpressed in many cancer cell types.
A quinazoline and butenamide derivative that acts as a tyrosine kinase inhibitor of epidermal growth factor receptors (ERBB RECEPTORS) and is used in the treatment of metastatic NON-SMALL CELL LUNG CANCER.
See also: Afatinib (has active moiety).

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Drug Indication
Giotrif as monotherapy is indicated for the treatment ofEpidermal Growth Factor Receptor (EGFR) TKI-naïve adult patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) with activating EGFR mutation(s); locally advanced or metastatic NSCLC of squamous histology progressing on or after platinum-based chemotherapy.

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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]

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.

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

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