In vitro activity: NVP-ADW742 exhibits a 6-fold greater selectivity for IGF-1R versus InsR with IC50 of 2.8 μM; minimal inhibitory activity against c-Kit, HER1, PDGFR, VEGFR2, or Bcr-Abl p210 with IC50 greater than 5 μM. NVP-ADW742 significantly inhibits the serum-stimulated cell proliferation in a variety of tumor cell lines in dose-dependent manner, with IC50 values of 0.1-0.5 μM for the multiple myeloma (MM) cell lines, and the antitumor effects on MM cells can not be overcome by the co-culture with BMSCs. NVP-ADW742 also abrogates the responsiveness of tumor cells to IL-6 in the presence of serum. In addition, NVP-ADW742 is active against MM cell lines with resistance to conventional (cytotoxic chemotherapy, dexamethasone) or investigational (thalidomide, CC-5013, TRAIL/Apo2L, PS-341) anticancer agents, as well as primary tumor cells from MM patients with multi-drug-resistant disease. NVP-ADW742 decreases the production of VEGF by tumor cells and bone marrow stromal cells, and suppresses the IGF-1-induced secretion of VEGF by various tumor types such as thyroid cancer cells or MM cells. IGF-1R inhibition by NVP-ADW742 (0.75 μM) sensitizes MM cells or prostate cancer cells to other anticancer agents such as doxorubicin, melphalan, dexamethasone, TRAIL/Apo2L, or PS-341.

Kinase Assay: The IC50 value for the effect of NVP-ADW742 on the autophosphorylation of IGF-1R is determined at the cellular level in the presence of increasing concentrations of NVP-ADW742, using 96-well “Capture ELISAs” assays. Briefly, NWT-21 cells are seeded into 96-well tissue culture plates in complete growth medium and grown to 70-80% confluency, and are then starved for 24 hours in 0.5% FCS medium. Subsequently, cells are incubated for 90 minutes in the presence of NVP-ADW742 followed by the stimulation with of IGF-I (10 ng/mL) for 10 minutes at 37 °C. Subsequently, the cells are washed twice with ice-cold PBS and lysed at 4 °C with 50 μL/well RIPA-buffer (50 mM Tris-HCl, pH 7.2, 120 mM NaCl, 1 mM EDTA, 6 mM EGTA, 1% NP-40, 20 mM NaF, 1 mM benzamidine, 15 mM sodium pyrophosphate, 1 mM PMSF and 0.5 mM Na3VO4). The lysates from each experiment are then transferred to black ELISA plates precoated with capture antibodies specific for IGF-1R. After capture by the antibodies, lysates are mixed with 40 μL of an alkaline phosphatase (AP) labelled anti-phosphotyrosine Ab (PY20(AP) diluted to 0.2 μg/mL in RIPA buffer, and incubated overnight at 4 °C. After washing (PBST) and incubation for 45 minutes at RT with the luminescent AP-substrate CDPStar RTU with Emerald II (90 μL/well), luminescence is measured using a Packard Top Count Scintillation Counter.

Cell Assay: Cells (MM-1S, MM-1R, RPMI-8226/S, OPM-1, OCI-My5, SKMM2, KMS-12-BM, XG-1, L363, S6B45 cells and et al.) are exposed to various concentrations of NVP-ADW742 for 48 hours in the presence or absence of serum. Cell survival is examined using MTT assay.

Cancer Cell. 2004 Mar;5(3):221-30.