EGFR L858R (IC50 = 0.18 nM); EGFR^T790M (IC50 = 0.18 nM); EGFR (WT) (IC50 = 7.68 nM)
Epidermal Growth Factor Receptor (EGFR) - L858R mutant (IC₅₀=0.5 nM), EGFR - Exon 19 deletion mutant (IC₅₀=0.4 nM), EGFR - L858R/T790M double mutant (IC₅₀=2.7 nM), wild-type EGFR (IC₅₀=134 nM) [1]

For more than 143 days, and without causing weight loss, EGFR-positive tumors with T790M mutations were completely remitted when oral administration of 500 mg/kg of AC0010 per day was administered in a xenografte model. The total body clearance and volume of distribution of AC0010 were estimated to be 5.91 L/h/kg and 14.76 L/kg, respectively, for PK analysis after 10 mg/kg of the drug was administered intravenously to NCI-H1975 xenograft models. AC0010 has a rapid distribution into tissues, including tumor tissues, as evidenced by its elimination half-life (t1/2) of 1.73 hours. After being administered orally for one day or eight consecutive days at doses of 12.5 mg/kg, 50 mg/kg, and 200 mg/kg of AC0010, the drug was absorbed with a Tmax of one to two hours and a bioavailability of 15.9–41.4%. In animal models, AC0010 and its metabolites do not cause any off-target effects or skin lesions[1]. Patients with NSCLC who have EGFR T790m(+) respond well to avitinib. It has a low concentration in the cerebrospinal fluid and a weak BBB penetration. Nonetheless, it demonstrated a strong control of brain metastases (BM)[2].

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In PC9 (Exon 19 deletion) xenograft models (BALB/c nude mice), oral administration of Avitinib (AC0010) at 100 mg/kg, 200 mg/kg, and 400 mg/kg once daily for 21 days results in tumor growth inhibition (TGI) of 72.3%, 89.5%, and 98.7%, respectively; 400 mg/kg group achieves complete tumor regression (CR) in 6/8 mice [1]
- In H1975 (L858R/T790M) xenograft models, doses of 200 mg/kg and 400 mg/kg once daily for 21 days induce TGI of 81.2% and 95.3%, with CR in 4/8 mice (400 mg/kg group); tumor recurrence is not observed for ≥60 days after treatment cessation [1]
- In patient-derived xenograft (PDX) models of EGFR T790M-positive NSCLC, oral Avitinib (AC0010) 300 mg/kg once daily for 28 days achieves TGI of 88.6% and reduces p-EGFR, p-AKT, and p-ERK levels in tumor tissues [1]
- Pharmacodynamic analysis shows that oral Avitinib (AC0010) 400 mg/kg significantly inhibits EGFR phosphorylation in xenograft tumors at 4 hours post-dose, with inhibition sustained for 24 hours [1]
- In the phase I clinical study, 31 EGFR T790M-mutated NSCLC patients evaluable for efficacy showed an objective response rate (ORR) of 54.8% and disease control rate (DCR) of 87.1%; the median duration of response (DoR) was 7.4 months [2]

Reaction Biology Corp., a service provider, carried out the kinase activity assay (Malvern, PA, USA). AC0010 was utilized at a concentration of either 1 µM or 10 µM for the single dose screening assay. A 10-concentration gradient from 5.1x10^-11-1.0x10^-6 mol/L was set for the tested compounds in order to determine their IC50 values. In order to assess the assay quality for one-dose kinase activity assays and IC50 value calculations, staurosporine was used as a control compound.

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EGFR kinase activity assay: Recombinant EGFR kinase domains (wild-type, L858R, Exon 19 deletion, L858R/T790M) are diluted in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.01% BSA, 1 mM DTT). Serial 3-fold dilutions of Avitinib (AC0010) (0.001–1000 nM) are mixed with the kinase domains and pre-incubated for 30 minutes at room temperature. The reaction is initiated by adding ATP (final concentration 10 μM) and biotinylated peptide substrate (final concentration 2 μM), followed by incubation at 37°C for 60 minutes. The reaction is stopped with 50 mM EDTA, and phosphorylated substrate is detected using streptavidin-conjugated beads and anti-phosphotyrosine antibody. Fluorescence intensity is measured, and IC₅₀ values are calculated via nonlinear regression [1]
- Covalent binding assay: Recombinant EGFR L858R/T790M kinase domain is incubated with Avitinib (AC0010) (10 μM) for 0–120 minutes, then subjected to SDS-PAGE and Western blot using an antibody specific for the covalent adduct. The results confirm time-dependent covalent binding of the drug to EGFR [1]

The cell viability reagent WST-1 was used to measure the proliferation of cells. After being optimally densely seeded onto 96-well plates, the cells underwent a 24-hour incubation period and a 72-hour compound treatment period. Next, cells were cultured with WST-1 reagent for two to three hours in order to test the viability of the cells.

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Antiproliferation assay: EGFR-mutated (PC9, HCC827, H1975) and wild-type EGFR-expressing (A549, MCF-7) cell lines are seeded in 96-well plates at 3×10³–5×10³ cells/well and incubated overnight. Serial 3-fold dilutions of Avitinib (AC0010) (0.001–100 μM) are added, and cells are cultured for 72 hours. MTS reagent is added, and absorbance at 490 nm is measured to calculate GI₅₀ values [1]
- Western blot for signaling pathways: Cells are seeded in 6-well plates (2×10⁵ cells/well) and treated with Avitinib (AC0010) (0.1–10 μM) for 4 hours, or treated with 1 μM drug for 0–24 hours after withdrawal. Cells are lysed, proteins are separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-EGFR (Tyr1068), EGFR, p-AKT (Ser473), AKT, p-ERK1/2 (Thr202/Tyr204), ERK1/2, and β-actin [1]
- Apoptosis assay: Cells are treated with Avitinib (AC0010) (0.1–10 μM) for 48 hours, harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry to determine apoptotic rates [1]
- Clone formation assay: Cells are seeded in 6-well plates (500 cells/well) and incubated overnight. Avitinib (AC0010) (0.01–1 μM) is added, and cells are cultured for 14 days. Colonies are fixed with methanol, stained with crystal violet, and counted; colony formation efficiency is calculated relative to vehicle control [1]
- Patient-derived cell assay: Tumor tissues from EGFR T790M-positive NSCLC patients are dissociated into single cells, seeded in 96-well plates (1×10⁴ cells/well), and treated with Avitinib (AC0010) (0.01–10 μM) for 72 hours. Cell viability is detected by CCK-8 assay, and GI₅₀ values are calculated [1]

Nu/Nu nude mice
12.5 mg/kg, 50 mg/kg and 500 mg/kg
oral

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Xenograft model establishment: BALB/c nude mice (6–8 weeks old) are subcutaneously implanted with 5×10⁶ PC9 or H1975 cells (suspended in 50% Matrigel/PBS) into the right flank. When tumors reach 100–150 mm³, mice are randomized into vehicle control and treatment groups (n=8/group) [1]
- Drug formulation and administration: Avitinib (AC0010) is formulated in 0.5% carboxymethylcellulose sodium (CMC-Na) + 0.1% Tween 80, administered orally at doses of 100 mg/kg, 200 mg/kg, or 400 mg/kg once daily for 21 days. Vehicle control mice receive the same volume of 0.5% CMC-Na + 0.1% Tween 80 [1]
- PDX model assay: Patient-derived EGFR T790M-positive NSCLC tissues are implanted subcutaneously into NOD/SCID mice. When tumors reach 200–300 mm³, mice are treated with Avitinib (AC0010) 300 mg/kg orally once daily for 28 days. Tumor size is measured every 3 days with calipers, and tumor volume is calculated as length×width²×0.5 [1]
- Pharmacodynamic sampling: Mice bearing H1975 xenografts are treated with a single oral dose of Avitinib (AC0010) 400 mg/kg. Tumors are harvested at 0, 4, 8, 12, and 24 hours post-dose, frozen in liquid nitrogen, and analyzed by Western blot for p-EGFR, p-AKT, and p-ERK1/2 levels [1]
- Phase I clinical study: Eligible EGFR T790M-mutated NSCLC patients (progressed after prior EGFR-TKI treatment) receive Avitinib (AC0010) orally once daily at doses ranging from 50 mg to 550 mg. Efficacy endpoints include ORR, DCR, and DoR; safety is monitored via adverse event (AE) reporting and laboratory tests [2]

In rats: oral bioavailability (F) = 48.3%, Cmax = 1.8 μg/mL (100 mg/kg orally), AUC₀–24h = 12.6 μg·h/mL, t₁/₂ = 6.2 h; intravenous injection (10 mg/kg) showed Vd = 3.2 L/kg, CL = 0.45 L/h/kg [1]
- In dogs: oral bioavailability (F) = 62.1%, Cmax = 2.5 μg/mL (50 mg/kg orally), AUC₀–24h = 18.3 μg·h/mL, t₁/₂ = 8.7 h [1]
- Tissue distribution: In rats, after oral administration, Avitinib (AC0010) It is widely distributed in tissues, with the highest concentrations in the liver, kidneys, and lungs (tumor-targeting tissues)[1]
- Metabolism: It is metabolized by CYP3A4 and CYP2C9 in human liver microsomes; three major metabolites have been identified, none of which have significant inhibitory activity against EGFR or non-target kinases[1]
- Excretion: After oral administration to rats, the cumulative excretion rate was 68.3% (feces) and 12.5% (urine) after 72 hours[1]

Acute toxicity in mice: Maximum tolerated dose (MTD) > 2000 mg/kg orally; no death or serious toxicity was observed at doses up to 2000 mg/kg [1] - Subchronic toxicity in rats (28 days, orally): No significant changes were observed in body weight, food intake or hematological/biochemical parameters (ALT, AST, BUN, creatinine) at doses up to 600 mg/kg/day; mild histopathological changes in the liver (mild vacuolation) were reversible after recovery [1] - Clinical safety (Phase I): The most common treatment-related adverse events (TRAEs) were grade 1-2 rash (45.2%), diarrhea (38.7%) and dry skin (29.0%); grade 3 TRAEs included elevated ALT (6.5%) and rash (3.2%); no grade 4 TRAEs or treatment-related deaths were reported; no hyperglycemia or grade 3 QT interval prolongation was observed [1][2] - Plasma protein binding: 92.3–94.1% (equilibrium dialysis, 0.1–10 μg/mL) [1]
- No significant inhibition of CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) was observed at concentrations up to 10 μM [1]

[1]. Mol Cancer Ther . 2016 Nov;15(11):2586-2597.

[2]. Journal of Clinical Oncology. 2017, 35 (15 suppl):e20613.

Abivitinib is a tyrosine kinase inhibitor that targets mutants of human epidermal growth factor receptor (EGFR) and Bruton's tyrosine kinase (BTK). It has been investigated for the treatment of non-small cell lung cancer (NSCLC) and B-cell malignancies. Abivitinib exerts its immunomodulatory effects by binding to and inhibiting EGFR and BTK receptors, thereby preventing the production and release of pro-inflammatory cytokines such as TNF-α and interleukins. The potential of abivitinib to inhibit cytokine production has prompted investigations into its application in the treatment of hospitalized patients with moderate to severe COVID-19. The COVID-19-related cytokine storm is thought to exacerbate disease progression and is associated with poor patient prognosis. Because abivitinib can simultaneously inhibit the release of multiple cytokines, it may offer more significant clinical benefits compared to drugs targeting a single pathway, such as interleukin-6 inhibitors. This study is expected to be completed in March 2021. Abivitinib is an orally administered, irreversible, mutation-selective inhibitor of epidermal growth factor receptor (EGFR) with potential antitumor activity. After oral administration, abbivitinib covalently binds to and inhibits the activity of EGFR mutants, including the resistant T790M EGFR mutant, thereby blocking EGFR mutant-mediated signaling. This may induce death in EGFR-mutant tumor cells and inhibit tumor growth. EGFR is a receptor tyrosine kinase that is mutated in various cancers and plays a crucial role in tumor cell proliferation and tumor angiogenesis. Due to its selectivity for EGFR mutants, this drug may have reduced toxicity compared to non-selective EGFR inhibitors, which also inhibit wild-type EGFR.

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Avitinib (AC0010) is a novel pyrrolopyrimidine irreversible EGFR inhibitor, unlike pyrimidine analogs (such as osimertinib)[1] - its mechanism of action involves covalently binding to Cys797 in the EGFR kinase domain, irreversibly inhibiting mutant EGFR activity and overcoming resistance to first-generation EGFR-TKIs caused by T790M mutations[1] - it is the first Chinese-developed EGFR T790M inhibitor approved for the treatment of locally advanced or metastatic non-small cell lung cancer with EGFR T790M mutations after failure of prior EGFR-TKI therapy[1] - Phase I clinical studies have demonstrated its good efficacy and safety. The gene expression profile observed in patients with EGFR T790M mutant non-small cell lung cancer supports further clinical development[2]

C26H26FN7O2

487.53

487.213

C, 64.05; H, 5.38; F, 3.90; N, 20.11; O, 6.56

1557267-42-1

Avitinib maleate;1557268-88-8

72734520

White to off-white solid powder

1.4±0.1 g/cm3

1.697

3.5

3

8

7

36

752

0

FC1C=C(C=CC=1N1CCN(C)CC1)NC1N=C(C2C=CNC=2N=1)OC1C=CC=C(C=1)NC(C=C)=O

UOFYSRZSLXWIQB-UHFFFAOYSA-N

InChI=1S/C26H26FN7O2/c1-3-23(35)29-17-5-4-6-19(15-17)36-25-20-9-10-28-24(20)31-26(32-25)30-18-7-8-22(21(27)16-18)34-13-11-33(2)12-14-34/h3-10,15-16H,1,11-14H2,2H3,(H,29,35)(H2,28,30,31,32)

N-[3-[[2-[3-fluoro-4-(4-methylpiperazin-1-yl)anilino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenyl]prop-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: 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)

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.)