EGFR

E/Z-Afatinib is a strong, irreversible inhibitor of EGFR kinase, Her2/ErbB 2. Afatinib, a small molecule tyrosine kinase inhibitor (TKI) targeting EGFR, HER-2 and HER-4, reversed the chemoresistance mediated by ABCG2 in vitro, but had no effect on that mediated by multidrug resistance protein ABCB1 and ABCC1. [2]

Genetic alterations in the kinase domain of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) patients are associated with sensitivity to treatment with small molecule tyrosine kinase inhibitors. Although first-generation reversible, ATP-competitive inhibitors showed encouraging clinical responses in lung adenocarcinoma tumors harboring such EGFR mutations, almost all patients developed resistance to these inhibitors over time. Such resistance to first-generation EGFR inhibitors was frequently linked to an acquired T790M point mutation in the kinase domain of EGFR, or upregulation of signaling pathways downstream of HER3. Overcoming these mechanisms of resistance, as well as primary resistance to reversible EGFR inhibitors driven by a subset of EGFR mutations, will be necessary for development of an effective targeted therapy regimen. Here, we show that BIBW2992, an anilino-quinazoline designed to irreversibly bind EGFR and HER2, potently suppresses the kinase activity of wild-type and activated EGFR and HER2 mutants, including erlotinib-resistant isoforms. Consistent with this activity, BIBW2992 suppresses transformation in isogenic cell-based assays, inhibits survival of cancer cell lines and induces tumor regression in xenograft and transgenic lung cancer models, with superior activity over erlotinib. These findings encourage further testing of BIBW2992 in lung cancer patients harboring EGFR or HER2 oncogenes.[3]

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In addition, afatinib, in combination with topotecan, significantly inhibited the growth of ABCG2- overexpressing cell xenograft tumors in vivo. [2]

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 TopCountTM or a Microbeta b counterTM.

Esophageal squamous cell carcinoma (ESCC) is the eighth most common cancer worldwide. Epidermal growth factor receptors (EGFR) are often overexpressed in esophageal cancers, thus anti-EGFR inhibitors have been evaluated in ESCC. Afatinib was an irreversible inhibitor of these ErbB family receptors. This study characterized the preclinical activity of afatinib in five ESCC cell lines: HKESC-1, HKESC-2, KYSE510, SLMT-1 and EC-1. ESCC cell lines were sensitive to afatinib with IC50 concentrations at lower micro-molar range (at 72 hour incubation: HKESC-1 = 0.002 μM, HKESC-2 = 0.002 μM, KYSE510 = 1.090 μM, SLMT-1 = 1.161 μM and EC-1 = 0.109 μM) with a maximum growth inhibition over 95%. Afatinib can strongly induce G0/G1 cell cycle arrest in HKESC-2 and EC-1 in a dose- and time-dependent manner. The phosphorylation of ErbB family downstream effectors such as pAKT, pS6 and pMAPK were significantly inhibited in HKESC-2 and EC-1. Apoptosis was observed in both cell lines at 24 hours after exposure to afatinib, as determined by the presence of cleaved PARP. Afatinib could effectively inhibit HKESC-2 tumor growth in mice without obvious toxicity. Afatinib alone has shown excellent growth inhibitory effect on ESCC in both in vitro and in vivo models, however, no synergistic effect was observed when it was combined with chemotherapeutic agents such as 5-fluorouracil (5-FU) and cisplatin. In summary, afatinib can inhibit cell proliferation effectively by arresting the cells in G0/G1 phase, as well as inducing apoptosis in ESCC. These findings warrant further studies of afatinib as therapeutic agent in treating ESCC.[4]

H460/MX20 cells (3 × 106) were subcutaneously injected into the right flank of athymic nude mice (BALB/c-nu/nu, both sexes, 5 to 6 weeks old). When xenograft size reached 5 mm in diameter, mice were randomized into four groups (12 in each group), and then received various treatments: (a) saline (every 3 d × 6, intraperitoneally [IP]); (b) topotecan (every 3 d × 6, IP, 3 mg/kg); (c) Afatinib (BIBW2992) (every 3 d × 6, orally [PO], 20 mg/kg); (d) topotecan (every 3 d × 6, IP, 3 mg/kg) plus afatinib (every 3 d × 6, PO, 20 mg/kg) (afatinib was given 1 h before topotecan administration). Tumor size was measured with linear calipers every 3 days. Tumor volumes (V) were calculated using the formula: (length×width2/2). The mice were euthanized on day 30 and the xenografts were excised and weighed. [2]

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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.[3]

Absorption, Distribution and Excretion
Following oral administration, the time to peak concentration (Tmax) ranges from 2 to 5 hours. Within the dose range of 20 to 50 mg, the increases in maximum concentration (Cmax) and area under the concentration-time curve (AUC0-∞) are slightly greater than the dose-proportional increase. The geometric mean relative bioavailability of the 20 mg tablet is 92% compared to the oral solution. Furthermore, compared to fasting, systemic exposure to afatinib is reduced by 50% (Cmax) and 39% (AUC0-∞) when taken with a high-fat meal, respectively. Based on population pharmacokinetic data from clinical trials across various tumor types, a mean reduction in AUCss was observed when food was consumed within 3 hours before or 1 hour after afatinib administration. In humans, afatinib is primarily excreted via feces. After oral administration of 15 mg afatinib solution, 85.4% of the dose is recovered in feces, and 4.3% in urine. 88% of the recovered dose was the original drug, afatinib. The volume of distribution of afatinib was 4500 L in healthy male volunteers. Such a high plasma volume of distribution suggests potentially high tissue distribution. The geometric mean of apparent systemic clearance of afatinib was as high as 1530 mL/min in healthy male volunteers. Metabolites/Metabolites: Enzymatic metabolism has negligible effect on afatinib in vivo. Protein covalent adducts are the main circulating metabolites of afatinib. Biological Half-Life: The effective half-life of afatinib is approximately 37 hours. Therefore, after multiple doses, steady-state plasma concentrations of afatinib can be reached within 8 days, resulting in a 2.77-fold (AUC0-∞) and 2.11-fold (Cmax) increase in cumulative drug dose. In patients treated with afatinib for more than 6 months, the estimated terminal half-life is 344 hours.

Hepatotoxicity
Elevated serum transaminase levels are common during afatinib treatment, occurring in 20% to 50% of patients, but only 1% to 2% of patients have transaminase levels exceeding five times the upper limit of normal. Liver failure has been reported in 0.2% of patients, leading to several deaths. Hepatotoxicity appears to be a class effect of EGFR2 protein kinase inhibitors, although liver injury caused by gefitinib appears to be more common and severe than that caused by afatinib and erlotinib. Specific details regarding afatinib-related liver injury, such as latency, serum enzyme profiles, clinical features, and disease course, have not been published. Other EGFR inhibitors, such as erlotinib and gefitinib, typically cause liver injury within days or weeks of treatment initiation, manifested as elevated hepatocyte enzymes, with a moderate to severe course. Immune hypersensitivity and autoimmune features are uncommon. Patients with a history of cirrhosis or liver dysfunction due to hepatic tumor burden have an increased risk of clinically significant liver injury and liver failure. Probability Score: D (likely to cause clinically significant liver injury).

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Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information on the clinical use of afatinib during lactation. Because afatinib binds to plasma proteins at a rate of approximately 95%, its concentration in breast milk may be low. However, its half-life is approximately 37 hours, which may allow it to accumulate in the infant. The manufacturer recommends discontinuing breastfeeding during afatinib treatment and for two weeks after the last dose.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
Afatinib binds to human plasma proteins at a rate of approximately 95% in vitro. Afatinib binds to proteins via non-covalent binding (traditional protein binding) and covalent binding.

[1]. Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer. Cancer Sci. 2018 Apr;109(4):1166-1176.

[2]. Afatinib circumvents multidrug resistance via dually inhibiting ATP binding cassette subfamily G member 2 in vitro and in vivo. Oncotarget. 2014 Dec 15;5(23):11971-85.

[3]. BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene. 2008 Aug 7;27(34):4702-11.

[4]. Preclinical evaluation of afatinib (BIBW2992) in esophageal squamous cell carcinoma (ESCC). Am J Cancer Res. 2015 Nov 15;5(12):3588-99.

Pharmacodynamics
Abnormal ErbB signaling induced by receptor mutations, amplification, and/or receptor ligand overexpression leads to malignant phenotypes. EGFR mutations define a unique molecular subtype of lung cancer. In non-clinical disease models of ErbB pathway dysregulation, afatinib as monotherapy effectively blocks ErbB receptor signaling, thereby inhibiting tumor growth or causing tumor regression. In both non-clinical and clinical settings, non-small cell lung cancer (NSCLC) tumors carrying common EGFR activating mutations (Del 19, L858R) and some less common EGFR mutations (located in exon 18 (G719X) and exon 21 (L861Q)) are particularly sensitive to afatinib treatment. Limited non-clinical and/or clinical activity has been observed in NSCLC tumors carrying exon 20 insertion mutations. Acquired secondary T790M mutations are the main mechanism of acquired resistance to afatinib, and the gene dose of the T790M mutation allele is correlated with the degree of resistance in vitro. In patients who progress on afatinib treatment, approximately 50% of tumors have the T790M mutation. For these patients, an EGFR-TKI targeting T790M can be considered as a second-line treatment option. Preclinical studies have suggested other potential afatinib resistance mechanisms, and MET gene amplification has also been observed clinically. Meanwhile, an open-label, single-arm study evaluated the effects of repeated afatinib administration (50 mg once daily) on cardiac electrophysiology and QTc interval in patients with relapsed or refractory solid tumors. Ultimately, this study did not detect significant changes in the mean QTc interval (i.e., >20 ms).

C24H25CLFN5O3

485.94

485.162

439081-18-2

Afatinib oxalate;1398312-64-5

10184653

White to off-white 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

2

8

8

34

702

1

CN(C)C/C=C/C(=O)NC1=C(C=C2C(=C1)C(=NC=N2)NC3=CC(=C(C=C3)F)Cl)O[C@H]4CCOC4

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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