Cell lines are plated in 96- or 384-well plates at optimum initial seeding densities to ensure that cells do not reach confluency by the end of the assay. The cells are treated with DMSO control or AMG 232 at various concentrations for 72 hours. CellTiter-Glo Luminescent Cell Viability or ATPlite 1step Luminescent assay kits are used to determine the numbers of viable cells. Luminescence is measured with an EnVision Multilabel Reader for each cell line at time zero (V₀) before the addition of compounds, as well as after 72 hours of compound treatment. Growth inhibition (GI) is calculated on a 200-point scale according to the following equations, where V₇₂ is luminescence of DMSO control at 72 hours and TV₇₂ is luminescence of the compound-treated sample: if T₇₂ > V₀, then GI=100 × (1 − ((T₇₂ − V₀)/(V₇₂ − V₀))); if T₇₂ < V₀, then GI=100 × (1 − ((T₇₂ − V₀)/V₀)). GI values of 0, 100, and 200 represent uninhibited cell growth (i.e., DMSO control), cell stasis, and complete cell killing, respectively. Dose-response curves are generated using XLFit software to calculate IC₅₀ values for AMG 232 in each cell line tested.

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

SJSA-1 cells (5×10⁶ cells with Matrigel at a ratio of 2:1), NCI-H460 cells (5 × 10⁶ cells with Matrigel at a ratio of 2:1), A375sq2 (5 × 10⁶ cells with Matrigel at a ratio of 2:1), or HCT116 (2 × 10⁶ cells) are injected subcutaneously in the flank of female athymic nude mice (n=10/group). Treatment begin when tumors are established and approximately 200 mm³. AMG 232 is administered once per day by oral gavage. Cisplatin, carboplatin, doxorubicin, and CPT-11 are dosed once per week by i.p or i.v. (doxorubicin) injection. Combination treatment studies are performed in a blinded manner. Tumor dimensions are assessed twice weekly with a Pro-Max electronic digital caliper and tumor volume is calculated using the formula: length×width×height and expressed as mm³. Data are expressed as mean±SEM. Body weight is recorded twice weekly to assess tolerability. Analysis of p21 mRNA at the end of the xenograft studies is performed as described for the p21 pharmacodynamic assay.

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

• [1]. Canon J, et al. The MDM2 Inhibitor AMG 232 Demonstrates Robust Antitumor Efficacy and Potentiates the Activity of p53-Inducing Cytotoxic Agents. Mol Cancer Ther. 2015 Mar;14(3):649-58.

  [2]. Ye Q, et al. Pharmacokinetics and metabolism of AMG 232, a novel orally bioavailable inhibitor of the MDM2-p53 interaction, in rats, dogs and monkeys: in vitro-in vivo correlation. Xenobiotica. 2015;45(8):681-92.

  [3]. Rew Y, et al. Discovery of a small molecule MDM2 inhibitor (AMG 232) for treating cancer. J Med Chem. 2014 Aug 14;57(15):6332-41.

568.55

C₂₈H₃₅Cl₂NO₅S

1352066-68-2

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Room temperature in continental US; may vary elsewhere