Neratinib (HKI-272)

 

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Biological Activity

Description	Neratinib (HKI-272) is a highly selective HER2 and EGFR inhibitor with IC50 of 59 nM and 92 nM; weakly inhibits KDR and Src, no significant inhibition to Akt, CDK1/2/4, IKK-2, MK-2, PDK1, c-Raf and c-Met. Phase 3.
Targets	HER2	EGFR
IC50	59 nM	92 nM [1]
In vitro	Neratinib weakly inhibits tyrosine kinases KDR and Src with IC50 of 0.8 μM and 1.4 μM, respectively, being 14- and 24-fold less active compared with HER2. Neratinib displays no activity against other serine-threonine kinases such as Akt, cyclin D1/cdk4, cyclin E/cdk2, cyclin B1/cdk1, IKK-2, MK-2, PDK1, c-Raf, and Tpl-2, as well as the tyrosine kinase c-Met. Neratinib selectively inhibits the proliferation of 3T3 cells transfected with the HER2 (3T3/neu), as well as two other HER-2-overexpressing SK-Br-3 and BT474 cells with IC50 values of 2-3 nM, displaying >230-fold potency compared 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 HER2 receptor autophosphorylation in BT474 cells with an IC50 of 5 nM, and EGF-dependent phosphorylation of EGFR in A431 cells with IC50 of 3 nM. Blocking of HER-2 by Neratinib results in inhibition of downstream MAPK and Akt pathways with IC50 of 2 nM, more potently than Trastuzumab. Neratinib inhibits the cyclin D1 expression and the phosphorylation of the Rb-susceptibility gene production in BT474 cells with IC50 of 9 nM, leading to G1-S arrest and ultimately decreased cell proliferation.[1]
In vivo	Oral administration of Neratinib significantly inhibits the growth of 3T3/neu xenografts, with inhibition of 34%, 53%, 98%, and 98% at dose of 10, 20, 40, and 80 mg/kg/day, respectively. Consistent with the inhibition of HER-2 phosphorylation by 84% 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 xenografts with inhibition of 31% and 85% at 5 and 60 mg/kg/day, respectively. Neratinib is less potent against EGFR-dependent A431 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, with only 28% inhibition at 80 mg/kg/day, suggesting that Neratinib has selective activity for cells expressing HER-2 or EGFR. [1]
Features

Protocol(Only for Reference)

Kinase Assay: ^[1]

Cell-free autophosphorylation assay using time-resolved fluorometry

Neratinib is prepared as 10 mg/mL stocks in DMSO and diluted in 25 mM HEPES (pH 7.5; 0.002 ng/mL-20 μg/mL). Purified recombinant COOH-terminal fragments of HER2 (amino acids 676-1255) or epidermal growth factor receptor (EGFR) (amino acids 645-1186) [diluted in 100 mM HEPES (pH 7.5) and 50% glycerol] is incubated with increasing concentrations of Neratinib in 4 mM HEPES (pH 7.5), 0.4 mM MnCl2, 20 μM sodium vanadate, and 0.2 mM DTT for 15 minutes at room temperature in 96-well ELISA plates. The kinase reaction is initiated by the addition of 40 μM ATP and 20 mM MgCl2 and allowed to proceed for 1 hour at room temperature. Plates are washed, and phosphorylation is detected using Europium-labeled anti-phospho-tyrosine antibodies (15 ng/well). After washing and enhancement steps, signal is detected using a Victor2 fluorescence reader (excitation wavelength 340 nm, emission wavelength 615 nm). The concentration of Neratinib that inhibits receptor phosphorylation by 50% (IC50) is calculated from inhibition curves.

Cell Assay: ^[1]

Cell lines	3T3, 3T3/neu, A431, BT474, SK-Br-3, MDA-MB-435, and SW480
Concentrations	Dissolved in DMSO, final concentrations 0.5 ng/mL-5 μg/mL
Incubation Time	2 or 6 days
Method	Cells are exposed to various concentrations of Neratinib for 2, or 6 days. Cell proliferation is determined using sulforhodamine B, a protein binding dye. Briefly, cells are fixed with 10% trichloroacetic acid and washed extensively with water. Cells are then stained with 0.1% sulforhodamine B and washed in 5% acetic acid. Protein-associated dye is solubilized in 10 mM Tris, and absorbance is measured at 450 nM. The concentration of Neratinib that inhibits cell proliferation by 50% (IC50) is determined from inhibition curves.

Animal Study: ^[1]

Animal Models	Female athymic (nude) mice implanted s.c. with 3T3/neu, BT474, MCF-7, or SK-OV-3 cells
Dosages	~80 mg/kg/day
Administration	Gavage
Solubility	30% PEG400/0.5% Tween80/5% propylene glycol 5 mg/mL

1

References

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

NCT Number	Recruitment	Conditions	Sponsor /Collaborators	Start Date	Phases
NCT01111825	Recruiting	Breast Cancer	Puma Biotechnology, Inc.	2010-04	Phase 1|Phase 2
NCT01128842	Completed	Advanced Malignant Solid Tumors	Puma Biotechnology, Inc.	2010-04	Phase 1
NCT01142063	Completed	Healthy	Puma Biotechnology, Inc.	2010-06	Phase 1
NCT01494662	Recruiting	Breast Cancer	Dana-Farber Cancer Institute|Translational Breast Cancer Research Consortium	2012-02	Phase 2
NCT00932464	Withdrawn	Healthy	Puma Biotechnology, Inc.	2012-08	Phase 1

Chemical Information

Download Neratinib (HKI-272) SDF

Chemical Name	(E)-N-(4-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)-3-cyano-7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2-enamide

Customer Reviews (3)

Click to enlarge	Rating
	Source	Cancer Discovery, 2012, 2, 458-471. Neratinib (HKI-272) purchased from Selleck
	Method	Western Blot
	Cell Lines	SKMG3
	Concentrations	200-1800 nM
	Incubation Time	3 day
	Results	Immunoblots of whole-cell lysates from SKMG3 cells treated with either inhibitor showed that CI-1033 inhibited EGFR phosphorylation less effectively than HKI-272 (Fig. C).

Product Citations (4)

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  Differential sensitivity of glioma- versus lung cancer-specific EGFR mutations to EGFR kinase inhibitors. [Vivanco I, et al. Cancer Discov 2012;2(5):458-71]

  
  PubMed: 22588883


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  Non-Covalent Wild-Type-Sparing Inhibitors of EGFR T790M. [Lee HJ, et al. Cancer Discov 2013;3(2):168-81]

  
  PubMed: 23229345


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  Activating HER2 mutations in HER2 gene amplification negative breast cancer. [Bose R, et al. Cancer Discov 2013;3(2):224-37]

  
  PubMed: 23220880