Cells are plated (3×10⁴ viable cells/well) in 96-well clear bottom white plates and the plates are incubated for ~24 hours at 37°C (5% CO₂/95% air) in a humidified incubator. The cells (six replicate wells) are then treated with various concentrations (0.5, 1, 2.5, 5, 10, 25, or 50 μM) of LCL161, or vehicle control (0.1% DMSO, final concentration) for 24 hours in Puracyp dosing media. After the incubation period, the cells are washed with PBS, lysed and the luciferase substrate is added according to the vendor instructions. An aliquot of each well is transferred to the identical wells of black 96-well plates. The luminescence of each well is measured with a TopCount NXT Microplate Scintillation and Luminescence Counter. Cell viability is measured in separate plates treated identically to the PXR-reporter gene assay plates by measurement of ATP content of the cells using the CellTiter-Glo^? Luminescent Cell Viability Assay kit. Cell viability is >80% for all treatments^[3].

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

Mice^[2]
Male NCr athymic nude mice (5-7 weeks of age) are used. Each mouse is inoculated s.c. in the dorsal flank with 1×10⁶ Huh-7 cells suspended in 0.1 mL of serum-free medium containing 50% Matrigel. When tumors reach 200-300 mm³, mice receives LCL161 (50 mg/kg) or SC-2001 (10 mg/kg) p.o., or a combination of LCL161 and SC-2001, once daily. Controls receive vehicle. Tumors are measured weekly using calipers and their volumes calculated using the following standard formula: width²×length×0.52. LCL161 is a first-in-class oral Smac mimetic shown to induce degradation of cIAP1 and cleavage of caspase 3 in mouse xenograft models .
Rats^[4]
LCL161 is administered orally, once weekly in 21-day cycles, at a starting dose of 10 mg (calculated by using one tenth of the dose that caused severe toxicity in 10% of rats and converted to a human-equivalent dose). In the MDA-MB-231 triple-negative breast cancer xenograft model, once-weekly and twice-daily LCL161 dosing are similarly efficacious. Once weekly is better tolerated, with reduced weight loss.

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

• [1]. Maria Ahn, et al. Potent, Dual cIAP1/XIAP Antagonists Induce Apoptosis in a Melanoma Stem Cell Population.

  [2]. Chen KF, et al. Inhibition of Bcl-2 improves effect of LCL161, a SMAC mimetic, in hepatocellular carcinoma cells. Biochem Pharmacol. 2012 Aug 1;84(3):268-77.  [Content Brief]

  [3]. Dhuria S, et al. Time-dependent inhibition and induction of human cytochrome P4503A4/5 by an oral IAP antagonist, LCL161, in vitro and in vivo in healthy subjects. J Clin Pharmacol. 2013 Jun;53(6):642-53.  [Content Brief]

  [4]. Infante JR, et al. Phase I dose-escalation study of LCL161, an oral inhibitor of apoptosis proteins inhibitor, in patients with advanced solid tumors. J Clin Oncol. 2014 Oct 1;32(28):3103-10.  [Content Brief]