The cytotoxicity of INNO-206 or doxorubicin was assessed in a concentration- and pH-dependent fashion in the 3 multiple myeloma cell lines RPMI8226, U266, and MM1S. First, drugs were prepared in pH 5 or 7 for 45 minutes before their addition to the cell culture. To compare equivalent concentrations of doxorubicin-bound INNO-206 to free doxorubicin, the INNO-206 concentrations were divided by 1.346 as this gives the amount of free doxorubicin contained within the INNO-206 compound. Cells were then exposed to increasing concentrations of INNO-206 from 0.27 to 2.16 μmol/L (free doxorubicin equivalent doses of 0.2–1.6 μmol/L) or doxorubicin (0.2–1.6 μmol/L) for 48 hours, and cell viability was determined with the MTS assay. A concentration- and pH-dependent decrease in viable RPMI8226 cells was observed after exposure to INNO-206 or doxorubicin (Fig. 1A). At pH 5, viable cells were essentially eliminated in cells cultured with INNO-206 at concentrations ≥0.54 μmol/L and doxorubicin was also effective but less so than INNO-206 (Fig. 1A). A similar concentration and pH-dependent inhibition of cell growth, as those observed earlier, was observed in the MM1S cell line after exposure to INNO-206 or doxorubicin (Fig. 1B). As the concentration was increased and pH was decreased, from pH 7 to 5, the percentage of viable MM1S cells within the INNO-206 group dramatically decreased, in contrast to what occurred with doxorubicin. In fact, the anti–multiple myeloma effects of doxorubicin at 0.4 and 0.8 μmol/L were less at pH 5 than 7. The diminishing anti–multiple myeloma effects of doxorubicin in an acidic environment were also observed in the U266 cell line (Fig. 1C), in contrast to INNO-206 where increased anti–multiple myeloma effects were observed at the lower pH. Because the data above was generated from drugs incubated at physiologic pH and at pH 5, the effect of an acidic pH alone on multiple myeloma cell lines was also tested. Exposure of multiple myeloma cells to pH 5 only resulted in a minimal reduction in viable cells compared to those cultured at pH 7. A representative example from all 3 cell lines tested is shown in Fig. 1D.

Reference: Clin Cancer Res. 2012 Jul 15;18(14):3856-67.

Mice bearing the LAGκ-1A tumor receiving INNO-206 once weekly via i.v. injection at 10.8 mg/kg (equivalent to 8.0 mg/kg of doxorubicin) showed significantly smaller tumor volumes and IgG levels on days 28 (tumor volumes: P = 0.0152; hIgG: P = 0.0019), 35 (tumor volumes: P = 0.0051; hIgG: P = 0.0006) and 42 (tumor volumes: P = 0.0036; hIgG: P = 0.0113) compared with vehicle—treated mice (Fig. 3A and B). This INNO-206 treatment regimen was well tolerated with 90% of mice surviving until the termination of the study (day 42).

Reference: Clin Cancer Res. 2012 Jul 15;18(14):3856-67.