EGFR tyrosine kinase
Gefitinib hydrochloride (Gefitinib) targets epidermal growth factor receptor (EGFR) tyrosine kinase (IC₅₀ = 0.033 μM for EGFR kinase activity; IC₅₀ = 0.054 μM for inhibiting EGF-stimulated tumor cell growth) [1]
Gefitinib hydrochloride (Gefitinib) targets human epidermal growth factor receptor 2 (HER2) and inhibits HER2-driven signaling in HER2-overexpressing tumor cells [3]
Gefitinib hydrochloride (Gefitinib) targets wild-type EGFR and mutant EGFRvIII: it inhibits wild-type EGFR-mediated phosphorylation, proliferation and anchorage-independent growth at low concentrations; for EGFRvIII-expressing cells, low concentrations (0.01-0.1 μM) increase EGFRvIII phosphotyrosine load and downstream ERK signaling, while high concentrations (1-2 μM) inhibit EGFRvIII-mediated activities [2]

After long-term exposure of EGFRvIII-expressing cells, gefitinib (0.01-0.1 mM) increases the phosphotyrosine load of the receptor, increases signaling to ERK, and stimulates proliferation and anchorage-independent growth. This effect is likely caused by the induction of EGFRvIII dimerization. Conversely, EGFRvIII phosphotyrosine load, EGFRvIII-mediated proliferation, and anchorage-independent growth are all markedly reduced by gefitinib (1-2 mM)[1]. With an IC50 of 20 nM, gefitinib (ZD1839) prevents these EGF-driven untransformed cells from growing monolayer[2]. With an IC50 of 2 μM, gefitinib inhibits the growth of GLC82 and CALU-3 cells[3].

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Epidermal growth factor receptor (EGFR) is frequently amplified and/or mutated in a number of human tumours and abnormal signalling from this receptor is believed to contribute to the malignant phenotype seen in these tumours. Gefitinib is a small molecule inhibitor that specifically binds and inhibits the EGFR tyrosine kinase and has been shown to inhibit the growth, proliferation, survival and invasion of a range of tumour cells overexpressing EGFR. However, clinical response to gefitinib has failed to correlate with EGFR levels and activity, indicating that other molecular mechanisms such as downstream signalling and mutations could be of importance in predicting clinical response. We therefore investigated the effect of the specific EGFR inhibitor gefitinib on the phosphorylation level, signalling and growth of cells expressing the naturally occurring constitutively active EGFR variant EGFRvIII, a low nontransforming level of EGFR and a high transforming level of EGFR. Results show that levels of gefitinib sufficient to suppress EGFR phosphorylations, EGFR-mediated proliferation and EGFR-mediated anchorage-independent growth are not sufficient to inhibit these features in cells expressing EGFRvIII. Furthermore, the data indicate that long-term exposure of EGFRvIII-expressing cells to low concentrations of gefitinib (0.01-0.1 microM) result in increased phosphotyrosine load of the receptor, increased signalling to ERK and stimulation of proliferation and anchorage-independent growth, presumably by inducing EGFRvIII dimerisation. Higher concentrations of gefitinib (1-2 microM), on the other hand, significantly decreased EGFRvIII phosphotyrosine load, EGFRvIII-mediated proliferation and anchorage-independent growth. Further studies are needed to investigate the implications of these important findings in the clinical setting. [2]

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The epidermal growth factor receptor (EGFR) is commonly overexpressed in many human tumors and provides a new target for anticancer drug development. ZD1839 ("Iressa"), a quinazoline tyrosine kinase inhibitor selective for the EGFR, has shown good activity in preclinical studies and in the early phase of clinical trials. However, because it remains unclear which tumor types are the best targets for treatment with this agent, the molecular characteristics that correlate with tumor sensitivity to ZD1839 have been studied. In a panel of human breast cancer and other epithelial tumor cell lines, HER2-overexpressing tumors were particularly sensitive to ZD1839. Growth inhibition of these tumor cell lines was associated with the dephosphorylation of EGFR, HER2, and HER3, accompanied by the loss of association of HER3 with phosphatidylinositol 3-kinase, and down-regulation of Akt activity. These studies suggest that HER2-overexpressing tumors are particularly susceptible to the inhibition of HER family tyrosine kinase signaling and suggest novel strategies to treat these particularly aggressive tumors.[3]

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1. Gefitinib hydrochloride (Gefitinib) potently inhibits EGFR tyrosine kinase activity (IC₅₀ = 0.033 μM) and selectively suppresses EGF-stimulated tumor cell proliferation (IC₅₀ = 0.054 μM); it also blocks EGF-induced EGFR autophosphorylation in tumor cells, confirming its target-specific inhibition of EGFR signaling [1]
2. In cells expressing wild-type EGFR (NR6wtEGFR, low EGFR; NR6W, high EGFR), Gefitinib hydrochloride (Gefitinib) inhibits EGF-induced EGFR phosphorylation (at Tyr845, Tyr992, Tyr1045, Tyr1068, Tyr1086, Tyr1148, Tyr1173), downstream signaling (p-ERK, p-PLC-γ, p-AKT, p-STAT3) and cell proliferation at low concentrations; in EGFRvIII-expressing cells (NR6M), low concentrations (0.01-0.1 μM) of the compound increase EGFRvIII phosphotyrosine load, enhance ERK signaling, and stimulate cell proliferationchorage-independent growth (via inducing EGFRvIII dimerization), while high concentrations (1-2 μM) significantly reduce EGFRvIII phosphorylation and inhibit its mediated proliferation and anchorage-independent growth [2]
3. Gefitinib hydrochloride (Gefitinib) suppresses the growth of HER2-overexpressing breast cancer and epithelial tumor cell lines; this growth inhibition is associated with dephosphorylation of EGFR, HER2, and HER3, reduced association of HER3 with phosphatidylinositol 3-kinase (PI3K), and down-regulation of Akt activity [3]
4. In LKB1 wild-type non-small cell lung cancer (NSCLC) cell lines, combination of Gefitinib hydrochloride (Gefitinib) with metformin exerts synergistic antiproliferative and proapoptotic effects: metformin alone activates mitogen-activated protein kinase (MAPK) via increased C-RAF/B-RAF heterodimerization, while Gefitinib hydrochloride (Gefitinib) inhibits EGFR phosphorylation and downstream signaling, abrogating metformin-induced MAPK activation and enhancing apoptotic effects [4]
5. Gefitinib hydrochloride (Gefitinib) inhibits proliferation and EGFR expression in lung fibroblasts isolated from irradiated rats, which contributes to the attenuation of radiation-induced lung fibrosis; however, it increases infiltration of inflammatory cells and production of pro-inflammatory cytokines (IL-6, IL-1β) in irradiated rat lungs [5]

When metformin and gefitinib (150 mg/kg, po) are given to nude mice containing H1299 or CALU-3 GEF-R cells that are cultured subcutaneously as tumor xenografts, the tumor growth is significantly reduced[3]. While gefitinib therapy attenuates fibrotic lung remodeling due to the reduction of lung fibroblast proliferation, it increases lung inflammation in irradiated rats, including inflammatory cell infiltration and pro-inflammatory cytokine expression[4].

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In studies with mice bearing a range of human tumor-derived xenografts, ZD1839 given p.o. once a day inhibited tumor growth in a dose-dependent manner. The level of expression of EGFR did not determine xenograft tumor sensitivity to ZD1839. Long-term ZD1839 (>3 months) treatment of mice bearing A431 xenografts was well tolerated, and ZD1839 completely inhibited tumor growth and induced regression of established tumors. No drug-resistant tumors appeared during ZD1839 treatment, but some tumors regrew after drug withdrawal. These studies indicate the potential utility of ZD1839 in the treatment of many human tumors and indicate that continuous once-a-day p.o. dosing might be a suitable therapeutic regimen.[1]

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Gefitinib treatment increased the infiltration of inflammatory cells, which produced more pro-inflammatory cytokines (IL-6, IL-1β), in the lungs of the irradiated rats on days 15 and 57, while gefitinib treatment reduced collagen content of the lungs in irradiated rats and decreased proliferation and EGFR expression in the lung fibroblasts from irradiated rats on day 57. Conclusions: In irradiated rats, gefitinib treatment augmented lung inflammation, including inflammatory cell infiltration and pro-inflammatory cytokine expression, while gefitinib treatment attenuated fibrotic lung remodeling due to the inhibition of lung fibroblast proliferation [5].

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1. In nude mice bearing various human tumor-derived xenografts (including A431), oral administration of Gefitinib hydrochloride (Gefitinib) once daily inhibits tumor growth in a dose-dependent manner (doses tested: 3.125-200 mg/kg); long-term treatment (>3 months) is well-tolerated, completely inhibits A431 xenograft growth, and induces regression of established tumors; no drug-resistant tumors emerge during treatment, but some tumors regrow after drug withdrawal [1]
2. In mouse xenograft models of NSCLC, combination of Gefitinib hydrochloride (Gefitinib) with metformin shows stronger tumor growth inhibition compared to either agent alone, confirming the in vivo synergistic antitumor effect of the combination in LKB1 wild-type NSCLC [4]
3. In Wistar rats with radiation-induced lung injury (single 20 Gy X-ray irradiation to the thorax), oral administration of Gefitinib hydrochloride (Gefitinib) (50 mg/kg/day) from day 1 to 14: on day 15, it increases inflammatory cell infiltration and pro-inflammatory cytokine (IL-6, IL-1β) levels in the lungs; on day 57, it reduces lung collagen content and inhibits lung fibroblast proliferation, attenuating fibrotic lung remodeling [5]

1. EGFR tyrosine kinase activity assay: Recombinant EGFR tyrosine kinase domain is incubated with serial concentrations of Gefitinib hydrochloride (Gefitinib) in the presence of ATP and a specific peptide substrate for EGFR. After incubation at appropriate temperature for a set period, the phosphorylation level of the peptide substrate is detected using a suitable method (e.g., immunoblotting or kinase activity detection kits), and the inhibition rate is calculated to determine the IC₅₀ value of Gefitinib hydrochloride (Gefitinib) for EGFR kinase activity [1]
2. EGFR/EGFRvIII phosphorylation assay: Cells expressing wild-type EGFR (NR6wtEGFR, NR6W) or mutant EGFRvIII (NR6M) are treated with different concentrations of Gefitinib hydrochloride (Gefitinib); for EGF-stimulated cells, EGF (10 nM) is added after drug pretreatment. Whole-cell lysates are prepared, and phosphorylated EGFR/EGFRvIII at specific tyrosine residues (Tyr845, Tyr992, Tyr1045, Tyr1068, Tyr1086, Tyr1148, Tyr1173) is detected by immunoblotting with phospho-specific antibodies; total EGFR/EGFRvIII levels are measured as internal controls, and band intensities are quantified to assess the effect of the compound on receptor phosphorylation [2]
3. HER family phosphorylation and PI3K association assay: HER2-overexpressing tumor cells are treated with Gefitinib hydrochloride (Gefitinib), and cell lysates are prepared. Phosphorylated EGFR, HER2, and HER3 are detected by immunoblotting; co-immunoprecipitation (co-IP) is performed to measure the association between HER3 and PI3K. The changes in phosphorylation levels and protein-protein interaction are analyzed to evaluate the inhibition of HER family signaling by the compound [3]

Purpose: EGF receptor (EGFR) tyrosine kinase inhibitors (TKI) have been found to be effective against lung cancer, but clinical resistance to these agents has developed as their usage has increased. Metformin is a widely used antidiabetic drug and also displays significant growth-inhibitory and proapoptotic effects in several cancer models, alone or in combination with chemotherapeutic drugs.

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Experimental design: The effects of gefitinib, a selective EGFR-TKI, and metformin on a panel of non-small cell lung cancer (NSCLC) cell lines were assessed by using MTT, bromide assay, flow cytometry, anchorage-independent growth, coimmunoprecipitation, and Western blot analysis.

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Results: The combination of metformin with gefitinib induced a strong antiproliferative and proapoptotic effect in NSCLC cell lines that harbored wild-type LKB1 gene. Treatment with metformin as single agent, however, induced an activation and phosphorylation of mitogen-activated protein kinase (MAPK) through an increased C-RAF/B-RAF heterodimerization. The inhibition of EGFR phosphorylation and of downstream signaling by adding gefitinib to metformin treatment abrogated this phenomenon and induced a strong apoptotic effect in vitro and in vivo.

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Conclusions: Metformin and gefitinib are synergistic in LKB1 wild-type NSCLC cells. However, further studies are required to investigate better the effect of metformin action on the RAS/RAF/MAPK pathway and the best context in which to use metformin in combination with molecular targeted agents.[4]

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1. Cell proliferation assay (EGFR-dependent cells): Tumor cells sensitive to EGF are seeded in 96-well plates and cultured overnight. Serial dilutions of Gefitinib hydrochloride (Gefitinib) are added, and cells are incubated for 72 hours under standard culture conditions. Cell viability is measured using a colorimetric reagent (e.g., MTT or bromide assay), and the EC₅₀/IC₅₀ values for inhibiting cell proliferation are calculated from dose-response curves [1]
2. Anchorage-independent growth assay: NR6 (control), NR6wtEGFR, NR6W (high EGFR), and NR6M (EGFRvIII) cells are treated with 0-2 μM Gefitinib hydrochloride (Gefitinib) and seeded in soft agar. NR6wtEGFR and NR6W cells are stimulated with 10 nM and 0.1 nM EGF, respectively. After 18 days of culture, colonies are counted; the number of colonies is plotted as a percentage of untreated cells to assess the effect of the compound on anchorage-independent growth [2]
3. Western blot for downstream signaling: Cells (e.g., NSCLC cells, HER2-overexpressing cells) are treated with Gefitinib hydrochloride (Gefitinib) (with or without metformin pretreatment for combination studies). Cell lysates are separated by SDS-PAGE, transferred to membranes, and probed with antibodies against phosphorylated and total downstream signaling molecules (ERK, AKT, PLC-γ, STAT3). Band intensities are quantified to analyze the effect of the compound on signaling pathway activation [3,4]
4. Apoptosis assay (combination treatment): LKB1 wild-type NSCLC cells are treated with Gefitinib hydrochloride (Gefitinib) alone, metformin alone, or their combination. Apoptosis is detected by flow cytometry (using Annexin V/PI staining) or immunoblotting (detecting cleaved caspase-3 or PARP); the percentage of apoptotic cells or levels of apoptotic markers are compared between groups to evaluate synergistic proapoptotic effects [4]
5. Lung fibroblast proliferation assay: Lung fibroblasts are isolated from irradiated rats and treated with Gefitinib hydrochloride (Gefitinib). Cell proliferation is measured using a cell counting kit or BrdU incorporation assay; EGFR expression in fibroblasts is detected by immunoblotting to assess the effect of the compound on fibroblast function [5]

Dissolved in 0.5% polysorbate; 100 mg/kg; Oral gavage
Nude mice (cba nu/nu) are intra-dermal injected with LoVo cells. Background: Gefitinib, an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, has been reported to be associated with interstitial lung disorders, and their high incidence and mortality have become a matter of great concern, especially in Japan. In this study, we investigated the effect of gefitinib on different phases of radiation-induced lung disorders in an experimental model. Methods: The thoraxes of Wistar rats were irradiated on day 1 with a single X-ray dose of 20 Gy, and gefitinib (50 mg/kg/day) was orally administered from day 1 to 14. The rat lungs were harvested on days 15 and 57 and the bronchoalveolar lavage (BAL) was performed.[5]

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1. Tumor xenograft model (efficacy study): Female nude mice are subcutaneously implanted with human tumor cells (e.g., A431, HER2-overexpressing breast cancer cells, NSCLC cells). When tumors reach a mean volume of ~100-150 mm³, mice are randomized into treatment and control groups. Gefitinib hydrochloride (Gefitinib) is dissolved in a suitable vehicle (e.g., aqueous solution or appropriate organic solvent mixture) and administered orally by gavage once daily at doses of 3.125-200 mg/kg. Tumor volume and body weight are measured twice weekly for 2-4 weeks (or >3 months for long-term studies); tumor growth inhibition (TGI) is calculated relative to the vehicle control [1]
2. Radiation-induced lung injury model: Male Wistar rats receive a single 20 Gy X-ray irradiation to the thorax on day 1. Gefitinib hydrochloride (Gefitinib) is administered orally at 50 mg/kg/day from day 1 to 14. Rats are euthanized on day 15 and 57; bronchoalveolar lavage (BAL) is performed to collect inflammatory cells, and lung tissues are harvested for histopathological analysis (inflammatory cell infiltration, collagen content) and detection of pro-inflammatory cytokines (IL-6, IL-1β) [5]
3. Combination therapy xenograft model: Nude mice bearing LKB1 wild-type NSCLC xenografts are randomized into four groups: vehicle control, Gefitinib hydrochloride (Gefitinib) alone (oral gavage, dose based on previous efficacy studies), metformin alone (oral administration), and combination of both agents. Drug administration is continued for a set period; tumor burden is measured regularly, and tumor growth curves are generated to assess synergistic effects [4]

Gefitinib hydrochloride (gefitinib) has shown good oral bioavailability in preclinical studies, supporting its once-daily oral dosing regimen; however, detailed data on its half-life, absorption, distribution, metabolism or excretion (e.g., clearance, volume of distribution) are not provided in the relevant literature [1].

1. In nude mice, long-term (>3 months) administration of effective tumor-inhibiting doses of gefitinib hydrochloride (gefitinib) was well tolerated; no significant treatment-related toxicities (e.g., weight loss, organ damage) were observed.[1] 2. In Wistar rats treated with gefitinib hydrochloride (gefitinib) (50 mg/kg/day, orally, 1–14 days) after chest irradiation: it exacerbated radiation-induced pneumonia (increased inflammatory cell infiltration and pro-inflammatory cytokines IL-6/IL-1β in the lungs on day 15), but reduced radiation-induced pulmonary fibrosis (decreased lung collagen content and inhibited lung fibroblast proliferation on day 57).[5]

[1]. ZD1839 (Iressa): an orally active inhibitor of epidermal growth factor signaling with potential for cancer therapy. Cancer Res. 2002 Oct 15;62(20):5749-54.

[2]. Differential response to gefitinib of cells expressing normal EGFR and the mutant EGFRvIII. Br J Cancer. 2005 Oct 17;93(8):915-23.

[3]. The tyrosine kinase inhibitor ZD1839 ("Iressa") inhibits HER2-driven signaling and suppresses the growth of HER2-overexpressing tumor cells. Cancer Res. 2001 Oct 1;61(19):7184-8.

[4]. Synergistic effects of metformin treatment in combination with gefitinib, a selective EGFR tyrosine kinase inhibitor, in LKB1 wild-type NSCLC cell lines. Clin Cancer Res. 2013 Jul 1;19(13):3508-19.

[5]. Epidermal growth factor receptor-tyrosine kinase inhibitor (gefitinib) augments pneumonitis, but attenuates lung fibrosis in response to radiation injury in rats. J Med Invest. 2012;59(1-2):174-85.

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 xenograft tumors. ZD1839 completely inhibited tumor growth and induced regression of established tumors. No drug-resistant tumors developed during ZD1839 treatment, but some tumors relapsed after discontinuation of the drug. These studies suggest that ZD1839 has potential applications in treating various human tumors and indicate that once-daily oral administration may be a suitable treatment regimen. [1]

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1. Gefitinib hydrochloride (gefitinib) is a low molecular weight, orally effective aniline quinazoline derivative and the first selective EGFR tyrosine kinase inhibitor (EGFR-TKI) for cancer treatment; it exerts its antitumor effect by inhibiting EGFR-mediated signal transduction, which is crucial for tumor growth, metastasis, angiogenesis, and resistance to chemotherapy/radiotherapy [1]
2. The sensitivity of tumor cells to gefitinib hydrochloride (gefitinib) is not entirely determined by the level of EGFR expression; factors such as EGFR mutations (e.g., EGFRvIII) and HER2 overexpression also affect efficacy: tumors with HER2 overexpression are particularly sensitive, while cells expressing EGFRvIII show a biphasic response (low-dose stimulation, high-dose inhibition) [2,3]
3. The synergistic effect of gefitinib and metformin is specifically targeted at the LKB1 wild-type non-small cell lung cancer (NSCLC) cell line, because metformin alone can activate the MAPK pathway in these cells, while gefitinib can block this non-targeted activation, thereby enhancing the anti-tumor efficacy [4]. Gefitinib has a dual effect on radiation-induced lung injury: it exacerbates the inflammatory phase (pneumonia) but can also alleviate the fibrotic phase, suggesting that there may be risks and benefits when used in combination with thoracic radiotherapy [5].

C22H24N4O3FCL.HCL

483.3633

482.128

184475-55-6

Gefitinib;184475-35-2

19077490

Typically exists as white to off-white solids at room temperature

607.7ºC at 760 mmHg

321.3ºC

4.9E-15mmHg at 25°C

5.088

2

8

8

32

545

0

ClC1=C(C=CC(NC2=NC=NC3=C2C=C(C(OC)=C3)OCCCN4CCOCC4)=C1)F.[H]Cl

QUINXWLATMJDQF-UHFFFAOYSA-N

InChI=1S/C22H24ClFN4O3.ClH/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);1H

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

ZD1839 HCl; ZD 1839 HCl; ZD-1839 HCl; Gefitinib HCl; trade name: Iressa.Gefitinib hydrochloride; 184475-55-6; gefitinib hcl; Gefitinib (hydrochloride); ZD-1839 hydrochloride; Gefitinib hydrochloride salt; N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine;hydrochloride; 4-Quinazolinamine,N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-,monohydrochloride;

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)