1、Product Data SheetNeratinibCat. No.: HY-32721CAS No.: 698387-09-6分式: CHClNO分量: 557.04作靶點(diǎn): EGFR作通路: JAK/STAT Signaling; Protein Tyrosine Kinase/RTK儲(chǔ)存式: Powder -20C 3 years4C 2 yearsIn solvent -80C 6 months-20C 1 month溶解性數(shù)據(jù)體外實(shí)驗(yàn) DMSO : 20 mg/mL (35.90 mM; Need ultrasonic)H2O : 230-fold potencycompared

2、with non-transfected 3T3 cells as well as MDA-MB-435 and SW620 which are EGFR- and HER2-negative.Neratinib also inhibits the proliferation of EGFR-dependent A431 cells with an IC50 of 81 nM. Neratinib reduces HER2receptor autophosphorylation in BT474 cells with an IC50 of 5 nM, and EGF-dependent pho

3、sphorylation of EGFR inA431 cells with IC50 of 3 nM. Blocking of HER-2 by Neratinib results in inhibition of downstream MAPK and Aktpathways with IC50 of 2 nM. Neratinib inhibits the cyclin D1 expression and the phosphorylation of the Rb-susceptibility gene production in BT474 cells with IC50 of 9 n

4、M, leading to G1-S arrest and ultimately decreased cellproliferation1.體內(nèi)研究 Orally treated neratinib significantly inhibits the growth of 3T3/neu xenografts, with inhibition of 34%, 53%, 98%, and98% at dose of 10, 20, 40, and 80 mg/kg/day, respectively. Consistent with the inhibition of HER-2 phospho

5、rylation by84% within 1 hour of administration at 40 mg/kg/day, Neratinib inhibits the growth of BT474 xenografts by 70-82%,67%, and 93% at dose of 5, 10, and 40 mg/kg/day, respectively. Neratinib is also effective against SK-OV-3 xenograftswith inhibition of 31% and 85% at 5 and 60 mg/kg/day, respe

6、ctively. Neratinib is less potent against EGFR-dependentA431 xenografts than HER-2-dependent tumors, with 32% and 44% inhibition at 5 and 20 mg/kg/day, respectively.Neratinib displays little activity against MCF-7 and MX-1 xenografts expressing low levels of HER-2 and EGFR, withonly 28% inhibition a

7、t 80 mg/kg/day, suggesting that Neratinib has selective activity for cells expressing HER-2 orEGFR1.PROTOCOLKinase Assay 1 Neratinib is prepared as 10 mg/mL stocks in DMSO and diluted in 25 mM HEPES (pH 7.5; 0.002 ng/mL-20 g/mL).Purified recombinant COOH-terminal fragments of HER2 (amino acids 676-1

8、255) or epidermal growth factor receptor(EGFR) (amino acids 645-1186) diluted in 100 mM HEPES (pH 7.5) and 50% glycerol is incubated with increasingconcentrations of Neratinib in 4 mM HEPES (pH 7.5), 0.4 mM MnCl2, 20 M sodium vanadate, and 0.2 mM DTT for 15minutes at room temperature in 96-well ELIS

9、A plates. The kinase reaction is initiated by the addition of 40 M ATPand 20 mM MgCl2 and allowed to proceed for 1 hour at room temperature. Plates are washed, and phosphorylationis detected using Europium-labeled anti-phospho-tyrosine antibodies (15 ng/well). After washing and enhancementsteps, sig

10、nal is detected using a Victor2 fluorescence reader (excitation wavelength 340 nm, emission wavelength 615nm). The concentration of Neratinib that inhibits receptor phosphorylation by 50% (IC50) is calculated from inhibitioncurves.Page 2 of 3 www.MedChemEMCE has not independently confirmed the accur

11、acy of these methods. They are for reference only.Cell Assay 1 Cells are exposed to various concentrations of Neratinib for 2, or 6 days. Cell proliferation is determined usingsulforhodamine B, a protein binding dye. Briefly, cells are fixed with 10% trichloroacetic acid and washed extensivelywith w

12、ater. Cells are then stained with 0.1% sulforhodamine B and washed in 5% acetic acid. Protein-associated dye issolubilized in 10 mM Tris, and absorbance is measured at 450 nM. The concentration of Neratinib that inhibits cellproliferation by 50% (IC50) is determined from inhibition curves.MCE has no

13、t independently confirmed the accuracy of these methods. They are for reference only.Animal Tumor cells (maintained in tissue culture) or tumor fragments are implanted s.c. in the flanks of female athymicAdministration 1 (nude) mice. For estrogen-dependent cell lines (BT474, MCF-7, and SK-OV-3), ani

14、mals are implanted with hormonepellets 1 week before implantation of tumors. Additionally, SK-OV-3 cells are suspended in Matrigel basementmembrane matrix for implantation. Treatment is initiated after tumors hads reached a size of 90-200 mg, followingrandom assignment of the animals to different tr

15、eatment groups (staging, day 0). For 3T3/neu xenografts, treatmentis initiated the day after tumor implantation (day 0). HKI-272 is formulated in 0.5% methocellulose-0.4% polysorbate-80 (Tween 80) and administered daily, p.o., by gavage. Tumor mass (length width2)/2 is determined every 7 days.Tumor

16、outgrowth in all xenograft studies, except 3T3/neu, is expressed as relative tumor growth: the ratio of themean tumor mass to the mean tumor mass on day 0. Inhibition of tumor growth is calculated relative to vehicle-treated controls. Statistical significance of inhibition is demonstrated using one-

17、tailed Students t test (equal variance)after log transformation of the data.MCE has not independently confirmed the accuracy of these methods. They are for reference only.戶使本產(chǎn)品發(fā)表的科研獻(xiàn) Sci Transl Med. 2018 Jul 18;10(450). pii: eaaq1093. Sci Transl Med. 2018 Jun 20;10(446). pii: eaao2565. Cell Syst. 20

18、19 Jul 24;9(1):35-48.e5. Mol Cancer Ther. 2018 Mar;17(3):603-613. Cancer Sci. 2018 Apr;109(4):1166-1176.See more customer validations on HYPERLINK www.MedChemE www.MedChemEREFERENCES1. Rabindran SK, et al. Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase. Cancer Res, 2004, 64(11), 3958-3965.2. Yoshioka T, et al. Antitumor activity of pan-HER inhibitors in HER2-positi