NVP-AEW541  (Synonyms: AEW541)

The activities of protein kinases are assayed in the presence or absence of inhibitors by measuring the incorporation of ³³P from [γ³³P]ATP (1000 Ci/mmol) into appropriate substrates. The protein kinase assays are carried out in 96-well plates at RT under conditions described in details below andterminated by the addition of 20 μL of 125 mM EDTA. Subsequently, 30 μL (c-Abl, c-Src, IGF-1R) or 40 μL (all other kinases) of the reaction mixture are transferred onto Immobilon-PVDF pre-soaked for 5 min with methanol, rinsed with water, then soaked for 5 min with 0.5 % H₃PO₄ and mounted on vacuum manifold. After spotting all samples, vacuum is connected and each well rinsed with 200 μL 0.5 % H₃PO₄. Membranes are removed and washed 4× on a shaker with 1% H₃PO₄, once with ethanol. After drying, mounting in Packard TopCount 96- well frame, and adding of 10 μL/well of Microscint, membranes are counted. IC₅₀ values are calculated by linear regression analysis of the percentage inhibition of each compound in duplicate, at four concentrations (usually 0.01, 0.1, 1, and 10 μM).One unit of protein kinase activity is defined as 1 nmole of ³³P transferred from [γ³³P]ATP to the substrate protein per minute per mg of protein at 37C^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Between 3000 and 6000 cells/well are seeded in 96-well plates with a total media volume of 100 μL/well. Increasing concentrations of the compound are added 24 hr thereafter in quadruplicate. 72 hr later, cells are fixed by addition of 25 μL/well Glutaraldehyde (20%) and incubation for 10 min at RT. Cells are then washed 2× with 200μL/well H2O and 100μL Methylene Blue (0.05%) is added. After incubation for 10 min at RT, cells are washed 3× with 200 μL/well H₂O. 200 μL/well HCl (3%) is added, and following incubation for 30 min at RT on a plate shaker, absorbance is measured at 650 nm^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
Female Harlan athymic nude mice are used NWT-21 cells are grown in DMEM (high glucose, 4.5 g/L), 10% FCS, 1% L-glutamine, and 1% Na-pyruvate. 5×10⁶ cells/animal are initially injected s.c. into the right flank of five mice. For the in vivo efficacy experiment, tumors of 500 to 800 mm³ are excised and nonnecrotic areas are cut to fragments of 3×3×3 mm. Tumor fragments are washed in sterile PBS and one tumor fragment per animal is trans- planted s.c. into the right flank. Tumor volumes (length×width ×height×π/6) and body weights are determined three times weekly. At the first day of treatment (day 0), the therapy group (NVP-AEW541) and the control group (vehicle only) are selected by stratification (8 animals per group, average tumor volume of about 95 mm³ per group). Animals are treated p.o. twice daily, 7 days/week either with NVP-AEW541 (20, 30, or 50 mg/kg; 10 mL/kg dissolved in 25 mM L(+)-tartaric acid, therapy group) or with 25 mM L(+)-tartaric acid (control group). Antitumor activity is expressed as T/C%(mean increase of tumor volumes of treated animals divided by the mean increase of tumor volumes of control animals multiplied by 100). The experiment is terminated when the mean tumor volume is about 1500 mm³.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. García-Echeverría C, et al. In vivo antitumor activity of NVP-AEW541-A novel, potent, and selective inhibitor of the IGF-IR kinase. Cancer Cell. 2004 Mar;5(3):231-9.  [Content Brief]

  [2]. Tanno B, et al. Down-regulation of IGF-1 receptor activity by NVP-AEW541 has an antitumor effect on neuroblastoma cells in vitro and in vivo. Clin Cancer Res. 2006, 12(22), 6772-6780.  [Content Brief]