Autophosphorylation assay

Neratinib was 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] was 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 mins at room temperature in 96-well ELISA plates. The kinase reaction was initiated by the addition of 40 μM ATP and 20 mM MgCl2 and allowed to proceed for 1 hr at room temperature. Plates were washed, and phosphorylation was detected using Europium-labeled anti-phospho-tyrosine antibodies (15 ng/well). After washing and enhancement steps, signal was detected using a Victor2 fluorescence reader (excitation wavelength 340 nm, emission wavelength 615 nm). The IC50 values was calculated from inhibition curves.

Cell lines

3T3, 3T3/neu, A431, BT474, SK-Br-3, MDA-MB-435 and SW480 cells

Preparation method

The solubility of this compound in DMSO is limited. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 ℃ for several months.

Reacting condition

0.5 ng/mL ~ 5 μg/mL; 2 or 6 days

Applications

Neratinib selectively inhibited the proliferation of HER2-overexpressing 3T3/neu, SK-Br-3 and BT474 cells, with the IC50 values of 2 ~ 3 nM, displaying >230-fold potency in HER2-overexpressing cells than in non-transfected 3T3 cells as well as EGFR- and HER2-negative MDA-MB-435 and SW620 cells. Neratinib also blocked the proliferation of EGFR-positive A431 cells, with an IC50 value of 81 nM.

Animal models

Nude mice bearing 3T3/neu and BT474 cells

Dosage form

5, 10, 20, 40 and 80 mg/kg/day; p.o.

Applications

In nude mice bearing 3T3/neu xenografts, Neratinib significantly inhibited tumor growth by 34%, 53%, 98% and 98% at the doses of 10, 20, 40 and 80 mg/kg/day, respectively. Neratinib also exhibited inhibitory effects on the growth of BT474 xenografts by 70 ~ 82%, 67% and 93% at corresponding doses of 5, 10 and 40 mg/kg/day.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

IC50: 59 nM (Her-2); 92 nM (EGFR)
HER-2 belongs to the ErbB family of receptor tyrosine kinases, which has been implicated in a variety of cancers. Overexpression of HER-2 is seen in 25–30% of breast cancer patients and predicts a poor outcome in patients with primary disease. Neratinib (HKI-272) is a tyrosine kinase inhibitor under investigation for the treatment breast cancer and other solid tumours.
In vitro: Neratinib (HKI-272) is a potent inhibitor of HER-2 and is highly active against HER-2-overexpressing human breast cancer cell lines in vitro. It also inhibits the epidermal growth factor receptor (EGFR) kinase and the proliferation of EGFR-dependent cells. Neratinib reduces HER-2 receptor autophosphorylation in cells at doses consistent with inhibition of cell proliferation and functions as an irreversible binding inhibitor, most likely by targeting a cysteine residue in the ATP-binding pocket of the receptor. In agreement with the predicted effects of HER-2 inactivation, Neratinib treatment of cells results in inhibition of downstream signal transduction events and cell cycle regulatory pathways. This leads to arrest at the G1-S (Gap 1/DNA synthesis)-phase transition of the cell division cycle, ultimately resulting in decreased cell proliferation [1].
In vivo: In vivo, Neratinib is active in HER-2- and EGFR-dependent tumor xenograft models when dosed orally on a once daily schedule. On the basis of its favorable preclinical pharmacological profile, Neratinib has been selected as a candidate for additional development as an antitumor agent in breast and other HER-2-dependent cancers [1].
Clinical trial: Neratinib is in development for the treatment of early- and late-stage HER2-positive breast cancer. Neratanib is being developed by Puma Biotechnology. It will be included in the forthcoming I-SPY2 breast cancer trial (http://en.wikipedia.org/wiki/Neratinib).
Reference:
[1] Rabindran SK, Discafani CM, Rosfjord EC, Baxter M, Floyd MB, Golas J, Hallett WA, Johnson BD, Nilakantan R, Overbeek E, Reich MF, Shen R, Shi X, Tsou HR, Wang YF, Wissner A. Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase. Cancer Res. 2004;64(11):3958-65.