EGFR

Firmonertinib is intended to block both the T790M acquired resistance mutation and EGFR active mutations [1].
In this study, using CRISPR method, four EGFR S768I mutation cell lines were constructed, and the sensitivity of EGFR to almonertinib and alflutinib was tested, with positive controls being the 1st (gefitinib), 2nd (afatinib), and 3rd (osimertinib) generation drugs. Results: The present results indicate that almonertinib and alflutinib can effectively inhibit cell viability and proliferation in rare EGFR S768I mutations through the ERK or AKT pathways in a time-dependent manner, by blocking the cell cycle and inhibiting apoptosis. Conclusions: These findings suggest that almonertinib and alflutinib may be potential therapeutic options for non-small cell lung cancer patients with the EGFR S768I mutation.[2]

From December 27, 2016 to August 21, 2017, 17 patients received at least one dose of AST2818 across four cohorts (20mg, 40mg, 80mg and 160 mg QD). Maximum tolerated dose has not been reached. The most common treatment-related AEs were grade 1 proteinuria (25%, 3/12). Other AEs included fatigue and prolonged Q-T interval, etc, all less than 10% and grade 1 or 2. The first 12 patients had been evaluated with an ORR of 58.3% (7/12) and a disease control rate of 91.7% (11/12). Profound and sustained tumor regression had already been observed at 20mg cohort. AST2818 plasma exposure, measured as Cmax and AUC 0-24h showed a dose-proportional increase. Conclusion AST2818 was well tolerated and had promising clinical activity with durable disease control in EGFR T790M mutant NSCLC after first-generation EGFR-TKIs treatment failure.[1]

Metabolism of alflutinib in HLMs[3]
Before starting the experiments, the HLMs were thawed gently on ice. Then, 3 µM alflutinib was added to the HLMs (0.5 mg protein/mL) in 100 mM phosphate-buffered saline (PBS; pH 7.4) to a total volume of 100 μL. After incubating at 37 °C for 3 min, the reactions were initiated by the addition of 1.0 mM NADPH. Following 1 h of incubation, the reactions were terminated by mixing with ice-cold acetonitrile at the same volume. All incubations were performed in duplicate and then analyzed by UPLC-UV/Q-TOF MS.[3]

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Incubation of HLMs with specific CYP inhibitors[3]
HLMs were used to study the effects of CYP enzyme inhibitors on the metabolism of alflutinib. The incubation mixture (100 µL) consisted of alflutinib (3 µM), HLMs (0.5 mg protein/mL), NADPH (1 mM), PBS (100 mM, pH 7.4) and a selective CYP inhibitor. The chemical inhibitors were as follows: α-naphthoflavone (2 µM) for CYP1A/2 C, quercetin (20 µM) for CYP2C8, sulfaphenazole (6 µM) for CYP2C9, ticlopidine (24 µM) for CYP2B6/2C19, quinidine (8 µM) for CYP2D6, chlormethiazole (24 µM) for CYP2E1, ketoconazole (2 µM) for CYP3A and ABT (1 mM) for all CYP enzymes. These inhibitors were preincubated with HLMs in the presence of NADPH for 10 min before adding the substrate. After that, the reactions were initiated by incubation at 37 °C for 60 min. Finally, the reactions were terminated by the addition of 100 µL of ice-cold acetonitrile. All incubations were performed in duplicate, and the formation of metabolites was evaluated in the absence or presence of inhibitors.[3]

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The metabolism of alflutinib through recombinant human CYP isoenzyme[3]
To identify the specific isoform that participates in the metabolism of alflutinib, 3 µM alflutinib was mixed with recombinant human CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, or 3A5 (25 pmol P450/mL) in a total volume of 100 μL. The reactions were initiated and terminated by the addition of 1 mM NADPH and 100 µL of ice-cold acetonitrile, respectively. The incubation was carried at 37 °C for 60 min. [3]

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Enzyme inhibition of alflutinib on major human CYP enzymes[3]
All CYP isoform-specific probes, such as phenacetin, bupropion, paclitaxel, tolbutamide, S-mephenytoin and dextromethorphan (for CYP1A2, 2B6, 2C8, 2C9, 2C19, and 2D6, respectively), as well as midazolam and testosterone (for CYP3A4), were dissolved in methanol. Alflutinib was dissolved in DMSO as a 100 mM stock solution. The total volume was 100 μL, and the medium was 100 mM PBS containing mixed HLMs (0.5 mg/mL), probe substrates, NADPH (1 mM) and different concentrations of alflutinib (0, 0.1, 0.33, 1, 3.30, 10, 33.3, and 100 μM). The reactions were preincubated at 37 °C for 5 min. Under the experimental conditions, the enzyme concentration and incubation time of the reaction system were all within a linear range (Supplementary Table S1).[3]

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Enzyme induction of alflutinib and AST5902 on the human CYP3A4 enzyme[3]
For the assessment of enzyme induction, 7 × 105 hepatocytes/mL were seeded into a collagen-coated 24-well plate and placed in a 37 °C humidified incubator with 5% CO2 for 24 h. The hepatocytes were treated with the human CYP3A4 enzyme inducer rifampin (10 μM), alflutinib or AST5902 (0.003, 0.01, 0.03, 0.1, 0.3, 1, 3 or 5 μM) or 0.1% DMSO (control group) once daily for three consecutive days. After treatment, RNA extraction was performed with TRIzol according to the manufacturer’s protocol. cDNA synthesis was carried out using the PrimeScript RT reagent kit. Real-time PCR was conducted on a StepOnePlus real-time PCR system sing the SYBR green Premix Ex Taq kit. The forward primer and reverse primer for CYP3A4 were 5′-ATCACTAGCACATCATTTGGAG-3′ and 5′-GGAATGGAAAGGTTATTGAGAG-3′, respectively. For GAPDH, the forward and reverse primers were 5′-AGAAGGCTGGGGCTCATTTG-3′ and 5′-GAGGGGCCATCCACAGTCTTC-3′, respectively. The levels of cDNA were quantitated by the comparative threshold cycle method using GAPDH as an internal standard.

Cell survival and growth assay[2]
The MTT assay was used to assess the viability of H3255, H3255Cas9+S768I, H3255L858R+S768I, PC9, PC9Cas9+S768I, and PC919Del+S768I cells, in order to determine the effect of different TKIs. The cells were digested into a single-cell suspension, inoculated into 96-well plates, and cultured in RPMI 1640 medium with 5000 cells/100 μL per well. After removing the original medium, 100μL of different TKIs were added to each well to treat the cells. Following 24 or 48 h of cell culture, 20 μL of MTT was added to each well and the cells were incubated at 37 °C for 4 h. The supernatant was then discarded, and 100 μL of DMSO solution was added to each well. The 96-well plate was incubated at 37 °C in light-proof conditions for 15 min, after which the optical density (OD) was measured at 490 nm using a microplate reader.[2]

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Colony formation assay[2]
Cells including H3255, H3255Cas9+S768I, H3255L858R+S768I, PC9, PC9Cas9+S768I, and PC919Del+S768I were inoculated into 6-well plates at a density of 500 cells per well in 2 ml of medium. After 18 h, the medium was removed, and the cells were incubated with varying concentrations of TKIs. The drug-containing medium was changed every 2–3 days. Following a 14-day incubation period at 37 °C, the culture medium was discarded. Each well was washed with PBS buffer, fixed with 4% paraformaldehyde for 15 min, and stained with Giemsa for 20 min at room temperature. The Giemsa staining solution was then discarded, and each well was rinsed with double-distilled water three to four times. Finally, the colonies were counted after natural air-drying.[2]

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Cell proliferation assay[2]
Cells including H3255, H3255Cas9+S768I, H3255L858R+S768I, PC9, PC9Cas9+S768I, and PC919Del+S768I were seeded in 96-well plates at a density of 5,000 cells per well. After 18 h, the medium was replaced with different TKIs and incubated for 48 h. The culture medium was then discarded, and the cells were washed with PBS buffer. Subsequently, each well was treated with 4% paraformaldehyde, 3% BSA PBS, and 0.3% Triton X-100 PBS in sequence. Following each treatment, the cells were washed three times with PBS buffer. Then, 50 μL of the click reaction solution was added to each well to label the proliferating cells. The cells were incubated in the dark for 30 min. After removing the medium, the cells were washed three times. Next, 50 μL of Hoechst stain was added to label all cell nuclei, and the cells were incubated in the dark for 10 min. After discarding the medium, the cells were washed three times and then immediately photographed using a fluorescence microscope.

Patients with histologically diagnosed, EGFR T790M mutant stage IV NSCLC were considered eligible, and they should have documented disease progression on EGFR-TKIs. In a 3+3 dose-escalation design, AST2818 was orally administered every day on a 21-day cycle at doses ranging from 20mg to 240 mg (NCT02973763). AST2818 was then explored in a dose-expansion cohort at doses ranging from 40 to 240 mg every day. Plasma samples were collected to evaluate pharmacokinetics of AST2818. EGFR T790M mutation in tissue samples was detected by amplification refractory mutation system. The primary endpoint was to determine dose limiting toxicity and objective response rate (ORR). Adverse events (AEs) were evaluated by CTCAE 4.03, and efficacy was assessed per RECIST v1.1 every 6 weeks.[1]

[1]. Y. Shi, et al. P2.03-028 Third Generation EGFR Inhibitor AST2818 (Alflutinib) in NSCLC Patients with EGFR T790M Mutation: A phase1/2 Multi-Center Clinical Trial.

Alflutinib is under investigation in clinical trial NCT03452592 (Efficacy and Safety of Alflutinib in Locally Advanced or Metastatic Non-small Cell Lung Cancer Patients With T790M).
Firmonertinib is an orally available selective inhibitor of the epidermal growth factor receptor (EGFR) mutant form T790M, with potential antineoplastic activity. Upon administration, firmonertinib specifically binds to and inhibits the tyrosine kinase activity of EGFR T790M, a secondarily acquired resistance mutation. This prevents EGFR T790M-mediated signaling and leads to cell death in EGFR T790M-expressing tumor cells. EGFR, a receptor tyrosine kinase that is mutated in many tumor cell types, plays a key role in tumor cell proliferation and tumor vascularization. Compared to some other EGFR inhibitors, alflutinib may have therapeutic benefits in tumors with T790M-mediated drug resistance.

C28H31F3N8O2

568.593355417252

568.252

C, 59.15; H, 5.50; F, 10.02; N, 19.71; O, 5.63

1869057-83-9

Firmonertinib mesylate;2130958-55-1

118861389

White to off-white solid powder

4.4

2

11

11

41

865

0

GHKOONMJXNWOIW-UHFFFAOYSA-N

1S/C28H31F3N8O2/c1-6-24(40)33-21-15-22(26(41-17-28(29,30)31)36-25(21)38(4)14-13-37(2)3)35-27-32-12-11-20(34-27)19-16-39(5)23-10-8-7-9-18(19)23/h6-12,15-16H,1,13-14,17H2,2-5H3,(H,33,40)(H,32,34,35)

2-Propenamide, N-(2-((2-(dimethylamino)ethyl)methylamino)-5-((4-(1-methyl-1H-indol-3-yl)-2-pyrimidinyl)amino)-6-(2,2,2-trifluoroethoxy)-3-pyridinyl)-

Alflutinib, ASK120067; AST-2818, ASK-120067; firmonertinib; furmonertinib; AST2818; AST-2818; A49A7A5YN4; N-[2-[2-(dimethylamino)ethyl-methylamino]-5-[[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino]-6-(2,2,2-trifluoroethoxy)pyridin-3-yl]prop-2-enamide; AST2818, AST 2818

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.)