生物活性

EGF816 is a third-generation covalent EGFR inhibitor that has potent inhibitory activity against activating and resistant T790M mutants with low IC50 in various cellular assays.

Cellularpotency of EGF816[1]

Enzyme activities[1]

Biologicalactivities of EGF816[2]

体外研究

体内研究

激酶实验

Receptor occupancy determination[2]

Cells were labeled with ¹⁴C-EGF816for 3 hours in an incubator, washed with PBS, and lysed with 1% Triton X-100buffer supplemented with protease inhibitors. Anti-EGFR sepharose was used forovernight immunoprecipitation. Beads were washed with lysis buffer and preparedfor NuPAGE. Phosphor-imaging of dried gels was carried out with a TyphoonPhosphor Imager and EC50 values determined by graphical analysis.

细胞实验

Cellular target modulation assays[2]

H1975, H3255, HCC827, A431, and HaCaT cellswere maintained in RPMI media supplemented with antibiotics and 10% FBS. HEKncells were maintained in EpiLife media supplemented with growth supplement. Allcells were maintained in a 37^oC, 5% CO₂ humidifiedincubator. In 384-well plates, H1975, H3255, HCC827, A431, or HaCaT were seededwith RPMI media supplemented with antibiotics and 5% FBS, whereas HEKn cellswere seeded in EpiLife media supplemented with 5% FBS. After an overnightincubation, serial diluted compounds were transferred to cells and incubatedfor 3 hours. HaCaT and HEKn cells were stimulated with 10ng/mL EGF (50ng/mL EGFfor A431) for 5 minutes. Cells were lysed in 1% Triton X-100 buffer containingprotease and phosphatase inhibitors. Lysates were analyzed by sandwich ELISAutilizing goat anti-EGFR capture antibody, anti-phospho-EGFR (Y1173), andanti-rabbit HRP. Signal was measured by chemiluminescent detection.

Cellularproliferation assays

Cells were seeded 500cells/well in solidwhite 384-well plates in maintenance media. Serial diluted compounds weretransferred to cells. After 3 days, cell viability was measured by CellTiter-Gloaccording to the manufacturer's instructions. BaF3 cell viability was measured2 days after compound treatment using Bright-Glo Luciferase Assay System. Luminescentreadout was normalized to 0.1% DMSO-treated cells and empty wells.

动物实验

In Vivo Efficacy Studies[1]

Foxn1 nude mice bearing the H1975 tumorswere randomized and used for efficacy studies. Compounds were formulated in 0.5%MC, 0.5% Tween80 suspension formulation and administered by oral gavage at adosing volume of 10 μL/g of the animal body weight. Animals in each groupreceived one oral dose of either vehicle (n = 6) or the different testcompounds (n = 6 in each dose group). Plasma samples were collected for PKmeasurements at 30 min, 3 h, 7 h, and 24 h after last dose on day 5. Bodyweight was monitored daily, and the % change in body weight was calculated as[(BW_current − BW_initial)/ (BW_initial)] × 100.Data are presented as percent body weight change from the day of treatmentinitiation. Tumor sizes were assessed three times during the efficacy study for5 days. Tumor sizes were determined by using caliper measurements. Tumorvolumes were calculated with the formula (length × width × width)/2.

Percent treatment/control (T/C) values fortumor were calculated using the following formula: % T/C = 100 × ΔT/ΔC if ΔT> 0; % regression = 100 × ΔT/Tinitial if ΔT < 0; where T is the meantumor volume of the drug-treated group on the final day of the study; ΔT is meantumor volume of the drug-treated group on the final day of the study minus meantumor volume of the drug-treated group on initial day of dosing; Tinitial isthe mean tumor volume of the drug-treated group on initial day of dosing; C isthe mean tumor volume of the control group on the final day of the study; andΔC is mean tumor volume of the control group on the final day of the studyminus mean tumor volume of the control group on initial day of dosing.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Lelais G, Epple R, Marsilje TH, et al. Discovery of (R,E)-N-(7-Chloro-1-(1-[4-(dimethylamino)but-2-enoyl]azepan-3-yl)-1H-benzo[d]imid azol-2-yl)-2-methylisonicotinamide (EGF816), a Novel, Potent, and WT Sparing Covalent Inhibitor of Oncogenic (L858R, ex19del) and Resistant (T790M) EGFR Mutants for the Treatment of EGFR Mutant Non-Small-Cell Lung Cancers. J Med Chem. 2016;59(14):6671-6689.
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体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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