EGFR

Gefitinib dihydrochloride (0.01–0.1 μM, 72 h) enhances anchorage-dependent growth and proliferation, raises ERK signaling, and increases the receptor's phosphotyrosine loading [2]. Significant reductions in EGFRvIII phosphotyrosine loading, EGFRvIII-mediated proliferation, and anchorage-independent growth are observed with gefitinib dihydrochloride (1-2 μM, 72 hours) [2]. Through the STAT6-dependent signaling route, gefitinib diHClide (0.62 μM, 24-72 h) suppresses the M2-like polarization of RAW 264.7 cells generated by IL-13 [3]. M2-like macrophage-promoted invasion and migration are inhibited by gefitinib diHClide (0.62 μM, 72 h) [3]. In NSCLC cell lines (H3255 and HCC827 cells), gefitinib diHClide (0–10 μM, 72 hours) causes apoptosis (induction of BIM protein) [4]. In HCC827 and A549 cells, gefitinib diHClide (100 nM, 24 h) promotes cellular absorption of extracellular vesicles (EVs) and inhibits macropinocytosis [6]. The growth of cisplatin-resistant wtEGFR NSCLC cell lines, H358R and A549R, is more inhibited by gefitinib diHClide (1.5–60 μM, 48 hours) [7].

Gefitinib dihydrochloride (oral, 75 mg/kg/d, 21 days) suppresses M2-like polarization of macrophages in an LLC mouse metastatic model [3]. Gefitinib dihydrochloride (orally, 75 mg/kg during the first week, once daily for 5 days per week) abolishes phosphorylation of HER2 and HER3 and signaling through MAPK and Akt in lobular hyperplasia and cancer, Increases MAPK activity and cytokine production in splenocytes, lymph nodes [5]. Gefitinib dihydrochloride (150 mg/kg orally, daily) improves the anticancer activity of cisplatin in H358R xenografts [7].

Western Blot Analysis[2]
Cell Types: NR6wtEGFR, NR6W and NR6M
Tested Concentrations: 1, 10, 100 μM
Incubation Duration: 5 hrs (hours)
Experimental Results: Inhibition of EGFR tyrosine phosphorylation.

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Cell migration assay [3]
Cell Types: LLCs Cell
Tested Concentrations: 0.62 μM
Incubation Duration: 72 h
Experimental Results: Eliminated M2-like macrophages promoted the invasion and migration of LLCs.

Animal/Disease Models: LLC mouse metastasis model [3]
Doses: 75 mg/kg/d for 21 days.
Route of Administration: Oral administration
Experimental Results: Reduce the number of lung metastasis nodules, down-regulate the expression of M2 marker genes and the percentage of CD206+ and CD68+ macrophages in tumor tissue.

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Animal/Disease Models: BALB-NeuT transgenic mouse model [5]
Doses: 75 mg/kg in the first week, increasing by 15 mg/kg every other week, one time/day, for 5 days a week, followed by 2 days of no treatment, repeated for 8- 9 weeks.
Route of Administration: Oral administration
Experimental Results: Treated mice diminished tumor multiplicity from 9.6 to 0.58 (83%) and diminished the number and size of lobules and lobular nodules.

[1]. Wakeling AE, et al. ZD1839: an orally active inhibitor of epidermal growth factor signaling with potential for cancer therapy. Cancer Res. 2002 Oct 15;62(20):5749-54.
[2]. Pedersen MW, et al. Differential response to gefitinib of cells expressing normal EGFR and the mutant EGFRvIII. Br J Cancer. 2005 Oct 17;93(8):915-23.
[3]. Muhammad Tariq, et al. Gefitinib inhibits M2-like polarization of tumor-associated macrophages in Lewis lung cancer by targeting the STAT6 signaling pathway. Acta Pharmacol Sin. 2017 Nov;38(11):1501-1511.
[4]. Mark S Cragg, et al. Gefitinib-induced killing of NSCLC cell lines expressing mutant EGFR requires BIM and can be enhanced by BH3 mimetics. PLoS Med. 2007 Oct;4(10):1681-89; discussion 1690.
[5]. Marie P Piechocki, et al. Gefitinib prevents cancer progression in mice expressing the activated rat HER2/neu. Int J Cancer. 2008 Apr 15;122(8):1722-9.
[6]. Tomoya Takenaka, et al. Effects of gefitinib treatment on cellular uptake of extracellular vesicles in EGFR-mutant non-small cell lung cancer cells. Int J Pharm. 2019 Dec 15;572:118762.
[7]. Amin Li, et al. Gefitinib sensitization of cisplatin-resistant wild-type EGFR non-small cell lung cancer cells. J Cancer Res Clin Oncol. 2020 Jul;146(7):1737-1749.

Epidermal growth factor receptor (EGFR) has become a promising target for anticancer therapy due to its important role in tumor growth, metastasis, angiogenesis, and tumor resistance to chemotherapy and radiotherapy. We developed a low molecular weight EGFR tyrosine kinase inhibitor (EGFR-TKI) ZD1839 (Iressa(2)). ZD1839 is a substituted aniline quinazoline compound and a potent EGFR-TKI (IC50 = 0.033 μM) that selectively inhibits EGF-stimulated tumor cell growth (IC50 = 0.054 μM) and blocks EGFR autophosphorylation in EGF-stimulated tumor cells. In mouse models carrying multiple human xenograft tumors, once-daily oral administration of ZD1839 dose-dependently inhibited tumor growth. EGFR expression levels did not determine the sensitivity of xenograft tumors to ZD1839. Long-term (>3 months) ZD1839 treatment was well tolerated in mice carrying A431 xenografts. ZD1839 completely inhibited tumor growth and induced regression of existing tumors. No drug-resistant tumors were observed during ZD1839 treatment, but some tumors relapsed after discontinuation of the drug. These studies suggest that ZD1839 has potential value in the treatment of various human tumors and that once-daily oral administration may be a suitable treatment regimen. [1] Epidermal growth factor receptor (EGFR) is frequently amplified and/or mutated in various human tumors, and abnormal signaling of this receptor is thought to be associated with the malignant phenotype of these tumors. Gefitinib is a small molecule inhibitor that specifically binds to and inhibits EGFR tyrosine kinase and has been shown to inhibit the growth, proliferation, survival and invasion of various EGFR-overexpressing tumor cells. However, there is a lack of correlation between the clinical efficacy of gefitinib and EGFR levels and activity, suggesting that other molecular mechanisms, such as downstream signaling pathways and gene mutations, may play an important role in predicting clinical efficacy. Therefore, we investigated the effects of the specific EGFR inhibitor gefitinib on phosphorylation levels, signaling pathways, and growth in cells expressing the naturally occurring constitutively active EGFR variant EGFRvIII, low-level unconverted EGFR, and high-level converted EGFR. The results showed that gefitinib doses sufficient to inhibit EGFR phosphorylation, EGFR-mediated proliferation, and EGFR-mediated anchorage-independent growth were insufficient to inhibit these characteristics in EGFRvIII-expressing cells. Furthermore, data indicated that long-term exposure of EGFRvIII-expressing cells to low concentrations of gefitinib (0.01–0.1 μM) led to increased receptor phosphotyrosine levels, enhanced ERK signaling, and stimulation of cell proliferation and anchorage-independent growth, likely through the induction of EGFRvIII dimerization. Conversely, higher concentrations of gefitinib (1–2 μM) significantly reduced EGFRvIII phosphotyrosine levels, EGFRvIII-mediated cell proliferation, and anchorage-independent growth. Further investigation is needed to explore the clinical implications of these important findings. [2] M2-like polarized tumor-associated macrophages (TAMs) play a key role in promoting cancer cell growth, invasion, metastasis, and angiogenesis. Identifying M2-like TAMs during tumor progression is an attractive strategy for cancer treatment. This study investigated the role of macrophage polarization and the antitumor effects of gefitinib in Lewis lung cancer (LLC) in vitro and in vivo. Gefitinib at concentrations below 2.5 μmol/L did not significantly inhibit the growth of LLC and RAW 264.7 cell lines, as well as bone marrow-derived macrophages (BMDM). However, low concentrations of gefitinib (0.62 μmol/L) significantly inhibited IL-13-induced macrophage M2-like polarization, manifested by decreased expression of M2 surface markers CD206 and CD163, downregulation of specific M2 marker genes (Mrc1, Ym1, Fizz1, Arg1, IL-10, and CCL2), and suppression of M2-like macrophage-mediated LLC cell invasion and migration. In RAW 264.7 cells, gefitinib inhibited IL-13-induced STAT6 phosphorylation, a key signaling pathway in macrophage M2-like polarization. In an LLC mouse metastasis model, oral administration of gefitinib (75 mg·kg-1·d-1, for 21 consecutive days) significantly reduced the number of lung metastatic nodules, downregulated the expression of M2 marker genes in tumor tissue, and the proportion of CD206+ and CD68+ macrophages. These results indicate that gefitinib can effectively inhibit M2-like polarization both in vitro and in vivo, revealing a new potential mechanism of gefitinib's chemopreventive effect. [3]

C22H24N4O3FCL.2[HCL]

519.82424

518.105

184475-56-7

Gefitinib;184475-35-2

19077503

Typically exists as solid at room temperature

628.2ºC at 760 mmHg

333.7ºC

4.9E-16mmHg at 25°C

5.89

3

8

8

33

545

0

OHAYARNLYSPHOJ-UHFFFAOYSA-N

InChI=1S/C22H24ClFN4O3.2ClH/c1-29-20-13-19-16(12-21(20)31-8-2-5-28-6-9-30-10-7-28)22(26-14-25-19)27-15-3-4-18(24)17(23)11-15;;/h3-4,11-14H,2,5-10H2,1H3,(H,25,26,27);2*1H

N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine;dihydrochloride

184475-56-7; Gefitinib (Dihydrochloride); 4-(3-Chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxy]quinazoline dihydrochloride; Gefitinib 2hydrochloride salt; gefitinib dihydrochloride; N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine;dihydrochloride; ZD 1839 Dihydrochloride;ZD-1839 Dihydrochloride;ZD1839 Dihydrochloride; SCHEMBL8208642;

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

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.)