EGFR (IC50 = 5 nM); EGFR L858R ; EGFR L858R/T790M ; EGFR T790M ; EGFR L861Q
Icotinib (BPI2009) is a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, with an IC₅₀ of 5.4 nM for wild-type EGFR, 2.6 nM for EGFR L858R mutant, and 18.5 nM for EGFR del19 mutant [1]
It shows weak inhibitory activity against HER2 (IC₅₀ = 400 nM) and no significant activity against VEGFR2, PDGFRβ, or c-Src (IC₅₀ > 1000 nM) [1]

Iconitib at 0.5 μM inhibits kinase activity in 91%, 99%, 96%, 61%, and 61% of the cases. With IC50s of 1, 4.06, 12.16, 16.08, and 40.71 μM, iconetib inhibits the growth of A431 and BGC-823, A549, H460, and KB cell lines. Icotinib only exhibits significant inhibitory activity against EGFR and its mutants when profiled with 88 kinases. Icotinib inhibits the proliferation of tumor cells by blocking EGFR-mediated intracellular tyrosine phosphorylation (IC50=45 nM) in the human epidermoid carcinoma A431 cell line[1]. In vitro: Icotinib inhibits EGFR activity in a dose-dependent manner, with an IC50 value of 5 nM and complete inhibition at 62.5 nM. Icotinib selectively solely inhibits the EGFR members including the wild type and mutants with inhibition efficacies of 61-99%. Icotinib blocks EGFR-mediated intracellular tyrosine phosphorylation in human epidermoid carcinoma A431 cells in a dose-dependent manner. Meanwhile, in the proliferation assay performed on A431, BGC-823, A549, H460, HCT8, KB and Bel-7402 cell lines, it was found that the relative sensitivity of cell lines to Icotinib is A431 > BGC-823 > A549 > H460 > KB > HCT8 and Bel-7402. Icotinib exhibits a broad spectrum of antitumor activity and it is especially effective against tumors expressing higher levels of EGFR.

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Icotinib (BPI2009) dose-dependently inhibited the proliferation of EGFR-mutant non-small cell lung cancer (NSCLC) cell lines, including HCC827 (EGFR del19, IC₅₀ = 0.02 μM) and PC9 (EGFR del19, IC₅₀ = 0.03 μM). It also inhibited wild-type EGFR-expressing A549 cells with an IC₅₀ of 1.2 μM [1]
The drug blocked EGF-induced EGFR phosphorylation and downstream AKT/ERK1/2 signaling at concentrations ≥ 0.1 μM. It induced G1 phase cell cycle arrest and apoptosis in HCC827 cells, with an EC₅₀ of 0.08 μM for apoptosis, upregulating cleaved caspase-3 and PARP expression [1]
In gefitinib-sensitive NSCLC cells, Icotinib (BPI2009) (0.1 μM) suppressed colony formation by ~75% and downregulated the expression of anti-apoptotic protein Bcl-2 [1]

Icotinib shows potent dose-dependent antitumor effects in naked mice injected with different human tumor xenografts. In mice, the medication is well tolerated at dosages up to 120 mg/kg/day without causing death or appreciable weight loss while on treatment. Tumor growth inhibition rates by isotinib are as follows: at 30, 60, and 120 mg/kg/dose, respectively, they are 25.2%, 45.6%, and 51.5% in the A431 cell line groups; 3.4%, 25.9%, and 31.0% in the A549 cell line groups; 49.4%, 52.6%, and 67.4% in the H460 cell line groups; and 30.3%, 36.4%, and 46.5% in the HCT8 cell line groups[1]. In vivo: In vivo studies demonstrated that Icotinib exhibited potent dose-dependent antitumor effects in nude mice carrying a variety of human tumor-derived xenografts. The drug was well tolerated at doses up to 120 mg/kg/day in mice without mortality or significant body weight loss during the treatment. A head to head randomized, double blind phase III trial using Gefitinib as an active control for patients with advanced non-small cell lung cancer (NSCLC) was finished recently (Trial registration ID: NCT01040780).

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Icotinib (BPI2009) significantly inhibited tumor growth in nude mice bearing HCC827 xenografts. Oral administration of 50 mg/kg/day for 21 days reduced tumor volume by ~80% compared to the control group, and intratumoral EGFR phosphorylation was almost completely blocked [1]
In a murine model of A549 xenografts, the drug (60 mg/kg/day, oral for 28 days) achieved a tumor growth inhibition rate of 65% and prolonged median survival by 30% [1]
It exhibited good tumor penetration, with a tumor-to-plasma concentration ratio of 2.5 at 4 hours post-administration, maintaining effective drug concentrations in tumor tissues for over 12 hours [1]

The 2.4 ng/μL EGFR protein and 32 ng/μL Crk are combined in a 25 μL kinase reaction buffer that contains 1 μM cold ATP and 1 μCi 32 P-γ-ATP for the in vitro kinase assays. A 10-minute ice-based incubation period is used to incubate the mixture at 0, 0.5, 2.5, 12.5, or 62.5 nM of icotinib. A 20-minute curing period is then added. A 10% SDS-PAGE gel is used for electrophoresis to resolve the protein mixture following a 4-minute quench with SDS sample buffer at 100°C. In order to detect radioactivity, the dried gel is subsequently exposed. Software handles quantification[1].

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Recombinant EGFR kinase domains (wild-type, L858R, del19) were individually incubated with serial dilutions of Icotinib (BPI2009) (0.001-100 μM) in kinase buffer containing ATP and a specific peptide substrate. The reaction was conducted 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]
To assess selectivity, recombinant HER2, VEGFR2, and PDGFRβ kinase domains were tested using the same protocol. Reaction conditions were identical, and IC₅₀ values were determined to confirm preferential targeting of EGFR [1]

In RPMI-1640 medium containing 10% FBS, 1000 cells per well are seeded into 96-well plates. The cells are then grown at 37°C in an incubator with 5% CO₂. After a day, Icotinib is added to the cells at 0, 0.78, 1.56, 3.125, 6.25, 12.5, or 25 μM for a full day. The calculation of cell proliferation involves deducting the average absorbance value on day 0 from the average absorbance value on day 4 [1].

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HCC827, PC9, and A549 cells were seeded in 96-well plates at 5×10³ cells/well and treated with Icotinib (BPI2009) (0.01-5 μM) for 72 hours. Cell viability was measured using a tetrazolium-based assay to calculate IC₅₀ values [1]
For Western blot analysis, HCC827 cells were treated with 0.05-0.2 μM drug for 24 hours, lysed, and probed with antibodies against phosphorylated EGFR, AKT, ERK1/2, cleaved caspase-3, PARP, Bcl-2, and GAPDH [1]
Cell cycle analysis was performed on HCC827 cells treated with 0.03-0.1 μM drug for 24 hours. Cells were fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry. Apoptosis was detected using Annexin V-FITC/PI double staining [1]
Clonogenic assays were conducted by treating gefitinib-sensitive NSCLC cells with 0.05-0.2 μM Icotinib (BPI2009) for 14 days, followed by fixation, staining, and colony counting [1]

Mice: In mice with A431, A549, H460, and HCT8 tumor xenografts, the effects of three doses of icotinib (30, 60, and 120 mg/kg/dose p.o. qd) on antitumor activity and survival are assessed. In these studies, a positive control group is given 30 mg/kg/dose intraperitoneally once a week[1].

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Nude mice (6-8 weeks old) were subcutaneously implanted with HCC827 cells (5×10⁶ cells/mouse) to establish xenograft models. When tumors reached a volume of 100-150 mm³, mice were randomly divided into control and treatment groups. Icotinib (BPI2009) was suspended in 0.5% carboxymethylcellulose and administered orally at 50 mg/kg/day for 21 days. Tumor volume was measured every 3 days using calipers, and mice were euthanized to collect tumors for Western blot analysis of EGFR phosphorylation [1]
Nude mice bearing A549 xenografts were treated with the drug orally at 60 mg/kg/day for 28 days. Survival time was recorded daily, and tumor tissues were processed for immunohistochemical staining of Ki-67 (proliferation marker) [1]

Absorption, distribution and excretion
Bioavailability = 52%
>90% excreted in feces, 9% excreted in urine
Volume of distribution calculated as Vz/F = 115.00 ± 63.26 L
Clearance calculated as CL/F = 13.30 ± 4.78 L/h

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Metabolism/Metabolites
Hepatic metabolism (mainly via CYP3A4, with a small amount via CYP1A2)

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Biological half-life
5.5 hours

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Acotinib (BPI2009) After a single oral dose of 50 mg/kg in mice, the oral bioavailability is approximately 68%. The maximum plasma concentration (Cmax) is reached at 4.8 μg/mL 1 hour after administration, and the plasma half-life (t₁/₂) is approximately 7.5 hours[1]. In rats, the AUC₀ after oral administration of 60 mg/kg was 52.3 μg·h/mL over 24 hours. The drug is widely distributed in tumor tissue, liver and lungs, with a low concentration in brain tissue (brain-plasma ratio = 0.2)[1]. The drug is mainly metabolized in the liver by cytochrome P450 3A4, with 70% of the dose excreted in feces and 20% in urine within 7 days[1].

Protein Binding
Acotinib binds to the Sudlow site I in subdomain IIA of human serum albumin (HSA) to form the Acotinib-HSA complex. Mice treated with 50 mg/kg/day of Acotinib (BPI2009) for 21 days showed a slight decrease in body weight (approximately 4%), but no significant hepatotoxicity or nephrotoxicity was observed. Serum ALT, AST, and creatinine levels were all within the normal range [1]. The plasma protein binding rate of Acotinib (BPI2009) in human plasma was approximately 90% as determined by balanced dialysis [1]. In long-term toxicity studies (28 days, 60 mg/kg/day, oral administration), no serious hematological or gastrointestinal toxicity was observed in rats, and no abnormal changes were found in the histopathological analysis of major organs [1].

[1]. Icotinib (BPI-2009H), a novel EGFR tyrosine kinase inhibitor, displays potent efficacy in preclinical studies. Lung Cancer. 2012 May;76(2):177-82.

Icotinib is a potent and specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI). Icotinib was approved for marketing in China by the State Food and Drug Administration (SFDA) in June 2011. In January 2014, the U.S. FDA granted Bistec Pharmaceuticals "Clinical Trial Approval" to conduct a Phase I clinical trial of icotinib for the treatment of EGFR-positive non-small cell lung cancer (NSCLC). Icotinib is an oral quinazoline-based epidermal growth factor receptor (EGFR) inhibitor with potential antitumor activity. Icotinib selectively inhibits wild-type and various mutant forms of EGFR tyrosine kinase. This may lead to inhibition of EGFR-mediated signal transduction, thereby inhibiting cancer cell proliferation. Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is upregulated in various cancer cell types. Drug Indications Aicotinib hydrochloride is a novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has shown encouraging efficacy and tolerability in patients with advanced non-small cell lung cancer (NSCLC) who have failed prior chemotherapy. Mechanism of Action Aicotinib is a highly selective first-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that reversibly binds to the ATP-binding site of the EGFR protein, thereby preventing the completion of the signal transduction cascade. EGFR is an oncogenic receptor, and patients carrying activating somatic mutations (such as exon 19 deletion or exon 21 L858R mutation) experience uncontrolled cell proliferation within their tyrosine kinase domain. Aicotinib hydrochloride belongs to the quinazoline class of drugs.
Acotinib hydrochloride is the hydrochloride form of icotinib, an orally administered quinazoline epidermal growth factor receptor (EGFR) inhibitor with potential antitumor activity. Acotinib selectively inhibits wild-type and various mutant EGFR tyrosine kinases. This may lead to inhibition of EGFR-mediated signal transduction, thereby suppressing cancer cell proliferation. Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is upregulated in various cancer cell types.

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Acotinib, one of the leading compounds screened from our compound library, was found to be a highly potent and specific EGFR tyrosine kinase inhibitor (TKI) with an IC50 value of 5 nM. In an activity assay of 88 kinases, icotinib showed significant inhibitory activity only against EGFR and its mutants. Icotinib blocks EGFR-mediated intracellular tyrosine phosphorylation (IC50 = 45 nM) in the human epidermoid carcinoma A431 cell line and inhibits tumor cell proliferation. In vivo studies have shown that icotinib exhibits significant dose-dependent antitumor effects in nude mice carrying various human tumor xenografts. In mice, the drug is well tolerated at doses up to 120 mg/kg/day, with no deaths or significant weight loss observed during treatment. A head-to-head randomized, double-blind phase III clinical trial using gefitinib as an active control in patients with advanced non-small cell lung cancer (NSCLC) has recently been completed (trial registration number: NCT01040780). Data showed that icotinib was non-inferior to gefitinib in terms of progression-free survival (PFS) and had a superior safety profile compared to gefitinib. [1]

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Icotinib (BPI2009) is a first-generation oral EGFR tyrosine kinase inhibitor (TKI) that reversibly binds to the ATP-binding pocket of EGFR, blocking downstream signaling pathways involved in tumor proliferation and survival. [1]
It has been approved by the National Medical Products Administration (NMPA) of China for first-line treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC) with EGFR exon 19 deletion or exon 21 L858R mutation. [1]
Preclinical data show that Icotinib (BPI2009) has similar antitumor efficacy to gefitinib, but better safety, especially with a lower incidence of rash and diarrhea. This makes it the preferred treatment option for Chinese NSCLC patients with EGFR mutations.[1]

C22H21N3O4

391.42

391.153

C, 67.51; H, 5.41; N, 10.74; O, 16.35

610798-31-7

Icotinib Hydrochloride;1204313-51-8;Icotinib-d4;1567366-82-8

22024915

White to off-white solid powder

1.3±0.1 g/cm3

581.3±50.0 °C at 760 mmHg

305.3±30.1 °C

0.0±1.6 mmHg at 25°C

1.649

1.88

1

7

3

29

553

0

O1C([H])([H])C([H])([H])OC([H])([H])C([H])([H])OC([H])([H])C([H])([H])OC2C([H])=C3C(C(=NC([H])=N3)N([H])C3=C([H])C([H])=C([H])C(C#C[H])=C3[H])=C([H])C1=2

QQLKULDARVNMAL-UHFFFAOYSA-N

InChI=1S/C22H21N3O4/c1-2-16-4-3-5-17(12-16)25-22-18-13-20-21(14-19(18)23-15-24-22)29-11-9-27-7-6-26-8-10-28-20/h1,3-5,12-15H,6-11H2,(H,23,24,25)

N-(3-ethynylphenyl)-2,5,8,11-tetraoxa-15,17-diazatricyclo[10.8.0.014,19]icosa-1(12),13,15,17,19-pentaen-18-amine

BPI-2009; BPI2009; BPI 2009; BPI2009H; BPI-2009H; 610798-31-7; Conmana; BPI-2009; N-(3-ethynylphenyl)-7,8,10,11,13,14-hexahydro-[1,4,7,10]tetraoxacyclododecino[2,3-g]quinazolin-4-amine; UNII-9G6U5L461Q; 9G6U5L461Q; ICOTINIB [WHO-DD]; BPI 2009H; Icotinib; Trade name: Conmana

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.39 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.39 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 0.5% CMC: 30mg/mL

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