Kinase experiment:

The catalytic domains of ERBB1, ERBB2, and ERBB4 tagged with glutathione S-transferase are expressed in insect cells and purified. ELISA-based enzyme assays and IC50 determinations for ERBB1, ERBB2, and ERBB4 are performed. Enzyme assays and IC50 determinations for all other kinases used in this study are performed[1].

Cell experiment:

Cells are seeded in duplicate at 5×103 to 5×104 cells per well in 24-well plates, and growth inhibition data is calculated. Briefly, day after plating, Dacomitinib is added at 10 μM and 2-fold dilutions over 12 concentrations are carried out to generate a dose-response curve. Control wells without the drug are also seeded. The cells are counted on day 1 when the drug is added, as well as after 6 days when the experiment ended. After the trypsinization cells are placed in an Isotone solution and immediately counted using a Coulter Z1 particle counter. The suspension cultures are counted using a Coulter Vi-Cell counter[2].

Animal experiment:

Mice[1] Nude mice (nu/nu; 6-8 weeks old) are used for in vivo studies. Mice are anesthetized using a 2% isoflurane (Baxter) inhalation oxygen mixture. A suspension of 5×106 HCC827-GFP or HCC827-Del/T790M lung cancer cells (in 0.2 mL of PBS) are inoculated s.c. into the lower-right quadrant of the flank of each mouse. Five mice are inoculated with either HCC827-GFP or HCC827-Del/T790M cells in the Gefitinib treatment group. Tumors are measured twice weekly using calipers, and volume is calculated using the following formula: length×width2×0.52. Mice are monitored daily for body weight and general condition. Mice are randomized to treatment when the mean tumor volume is 400 to 500 mm3. Gefitinib is administered at 150 mg/kg/d by daily oral gavage. Dacomitinib is administered at 10 mg/kg/d by daily oral gavage. The experiment is terminated when the mean size of the control tumors reached 2000 mm3.

IC50: 6 nM (EGFR); 45.7 nM (ERBB2); 73.7 nM (ERBB4)

The epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) is the cell-surface receptor for members of the epidermal growth factor family (EGF-family) of extracellular protein ligands. The epidermal growth factor receptor is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4). Mutations affecting EGFR expression or activity could result in cancer. Dacomitinib (PF-00299804) is an irreversible small molecule pan-HER inhibitor.

In vitro: Dacomitinib reduced the phosphorylation of HER2, EGFR, HER4, AKT, andERKin the majority of sensitive lines. Dacomitinib exerted its antiproliferative effect through a combined G0–G1 arrest and an induction of apoptosis. Dacomitinib inhibited growth in several HER2-amplified lines with de novo and acquired resistance to trastuzumab. Dacomitinib maintained a high activity in lines with acquired resistance to lapatinib. This study identifies HER2-amplified breast cancer lines as most sensitive to the antiproliferative effect of dacomitinib and provides a strong rationale for its clinical testing in HER2-amplified breast cancers resistant to trastuzumab and lapatinib [1].

In vivo: To evaluate the in vivo efficacy of PF00299804, the authors generated xenografts in nu/nu mice using HCC827 GFP and HCC827 Del/T790M cells and treated the mice with PF00299804. PF00299804 effectively inhibited the growth of HCC827 GFP xenografts. PF00299804 treatment was substantially more effective at inhibiting growth of this xenograft model than gefinitib. Thus, these preclinical models suggest that PF00299804 may be quite effective against lung cancers that become resistant to gefitinib or erlotinib via acquisition of a T790M mutation in EGFR [2].

Clinical trial: Dacomitinib (PF-00299804) is an experimental drug candidate under development by Pfizer for the treatment of non-small-cell lung carcinoma. It is a selective and irreversible inhibitor of EGFR. Dacomitinib has advanced to several Phase III clinical trials. The results of the first trials were disappointing, with a failure to meet the study goals. Additional Phase III trials are ongoing (http://en.wikipedia.org/wiki/Dacomitinib).

Reference:
[1] Kalous O, Conklin D, Desai AJ, O'Brien NA, Ginther C, Anderson L, Cohen DJ, Britten CD, Taylor I, Christensen JG, Slamon DJ, Finn RS.  Dacomitinib (PF-00299804), an irreversible Pan-HER inhibitor, inhibits proliferation of HER2-amplified breast cancer cell lines resistant to trastuzumab and lapatinib. Mol Cancer Ther. 2012;11(9):1978-87.
[2] Engelman JA, Zejnullahu K, Gale CM, Lifshits E, Gonzales AJ, Shimamura T, Zhao F, Vincent PW, Naumov GN, Bradner JE, Althaus IW, Gandhi L, Shapiro GI, Nelson JM, Heymach JV, Meyerson M, Wong KK, Jfinne PA.  PF00299804, an irreversible pan-ERBB inhibitor, is effective in lung cancer models with EGFR and ERBB2 mutations that are resistant to gefitinib. Cancer Res. 2007;67(24):11924-32.