AV-412

Kinase experiment:

Recombinant intracellular kinase domains of EGFR, EGFRL858R, EGFRT790M, EGFRL858R/T790M, and purified EGFR from A431 cell membranes are used. Kinase reactions are carried out in 8 mM MOPS (pH 7.0), 0.2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM MnCl2, 10 mM Mg acetate, 0.1 mg/mL poly(Glu, Tyr) 4:1, [γ33P-ATP], and 5–10 mU of enzyme, except that 250 µM of the GGMEDIYFEFMGGKKK peptide substrate is used for EGFRT790M. Phosphorylation is initiated by the addition of ATP and is allowed to proceed for 40 min at room temperature. The reaction is stopped by the addition of 3% phosphoric acid, then aliquots of the reaction mixture are spotted onto a filtermat. After rinsing to remove peptides bound non-specifically, the filter is scintillation counted[1].

Cell experiment:

To test the effects of AV-412 on growth factor-dependent cell proliferation, A431 and A7r5 cells are cultured for 24 h at 37°C in the presence of 1 ng/mL epidermal growth factor and 50 ng/mL platelet-derived growth factor, respectively. The 3H-thymidine incorporation during this period is measured[1].

Animal experiment:

Mice: For studies examining the dosing schedule in relation to efficacy against TE-8 tumors, AV-412 is administered either once daily, every other day, or once per week for 2 weeks. Mice are killed 1 day after the final treatment, and the tumors are dissected and weighed. For evaluation of tumor phosphorylation, tumor-bearing mice are given a single administration of AV-412 and tumors are dissected 4 h later[1].

References:

[1]. Suzuki T, et al. Pharmacological characterization of MP-412 (AV-412), a dual epidermal growth factor receptorand ErbB2 tyrosine kinase inhibitor. Cancer Sci. 2007 Dec;98(12):1977-84.