Combination of low doses of NPI-0052 and lenalidomide inhibits human plasmacytoma growth in CB-17 SCID mice. (A) Average and SD of tumor volume (mm3) from group of mice (n = 5/group) versus time (days) when tumor was measured. Mice were treated with vehicle, NPI-0052 (orally), lenalidomide (orally), or NPI-0052 plus lenalidomide (orally) at the indicated doses for 24 days on a twice-weekly schedule for NPI-0052 and 4 consecutive days weekly for lenalidomide. A significant delay in tumor growth in NPI-0052 plus lenalidomide–treated mice was noted compared with vehicle-treated control mice (P = .002). Bars represent mean ± SD. (B) Micrographs show apoptotic cells in tumors sectioned on day 24 (endpoint) from untreated or NPI-0052 (0.15 mg/kg) plus lenalidomide (2.5 mg/kg)- treated mice as identified by caspase-3 cleavage (dark brown cells). Photographs are representative of similar observations in 2 different mice receiving treatment. Images were obtained with a Zeiss Axioimager M1 microscope (63×/1.4 Plan-Apochromat objective), axioCam HRc camera, a Axiovision Version 4.6 software, and permount imaging solution. (C) Kaplan-Meier plots showing survival for mice treated with NPI-0052, lenalidomide, or NPI-0052 plus lenalidomide at the indicated concentrations. NPI-0052 plus lenalidomide–treated mice show significantly increased survival (P < .002) compared with the untreated group. The mean overall survival (OS) was 48 days (95% confidence interval, 35-60) in the untreated or single agent–treated cohorts versus 135 days (95% confidence interval, 120-150) in groups treated with combination of NPI-0052 and lenalidomide (0.25 or 0.5 mg/kg). Overall, a 67% increase in survival was observed in mice receiving combined low dose of NPI-0052 (0.15 mg/kg plus lenalidomide (0.5 mg/mg) versus mice receiving either agent alone at these doses. A statistically significant prolongation in mean OS compared with control mice was observed in animals treated with 0.25 mg/kg (P < .002) and 0.5 mg/kg (P > .007). (D) Mice in vehicle-treated controls, NPI-0052–, lenalidomide-, or NPI-0052 plus lenalidomide–treated group were weighed every week. The average changes in body weight are shown. (E) Mice were treated with vehicle, NPI-0052, lenalidomide, or NPI-0052 plus lenalidomide (as in panel A) for 24 days; blood samples were obtained and subjected to analysis for serum bilirubin, hemoglobin, and creatine levels using Quantichrom Creatinine, Bilirubin, and Hemoglobin Assay kit (BioAssay Systems).
