Stage 2 combination testing of rapamycin with cytotoxic agents by the Pediatric Preclinical Testing Program

Abstract

Rapamycin demonstrated broad-spectrum tumor growth inhibition activity against the in vivo panels of childhood tumors used in the Pediatric Preclinical Testing Program (PPTP). Here we have evaluated rapamycin combined with agents used frequently in the treatment of childhood malignancies. Rapamycin was tested in vitro against 23 cell lines alone or in combination with melphalan, cisplatin, vincristine, or dexamethasone (leukemic models only). In vivo, the impact of combining rapamycin with a cytotoxic agent was evaluated using two measures: 1) the therapeutic enhancement measure, and 2) a linear regression model for time-to-event to formally evaluate for sub- and supraadditivity for the combination compared to the agents used alone. Combining rapamycin with cytotoxic agents in vitro gave predominantly subadditive or additive effects, except for dexamethasone in leukemia models for which supra-additive activity was observed. In vivo testing demonstrated that therapeutic enhancement was common for rapamycin in combination with cyclophosphamide and occurred for 4 of 11 evaluable xenografts for the rapamycin and vincristine combination. The combinations of rapamycin with either cyclophosphamide or vincristine were significantly more effective than the respective standard agents used alone at their maximum tolerated doses (MTD) for most evaluable xenografts. The combination of rapamycin and cisplatin produced excessive toxicity requiring cisplatin dose reductions, and therapeutic enhancement was not observed for this combination. Addition of rapamycin to either cyclophosphamide or vincristine at their respective MTDs appears promising, as these combinations are relatively well tolerated and as many of the pediatric preclinical models evaluated demonstrated therapeutic enhancement for these combinations.

Figure 1. Concentration response curves and EC₅₀ values with and withou rapamycin

The CHLA-258 cell line shows sub-additivity for melphalan (EC₅₀ ratio ± rapamycin significantly > 1) (Panel A), the NALM-6 cell line shows additivity for cisplatin (EC₅₀ ratio ± rapamycin ~ 1) (Panel B), and the NALM-6 cell line shows supra-additivity for dexamethasone (EC₅₀ ratio ± rapamycin significantly < 1) (Panel C). Closed squares are the combination of rapamycin plus the standard agent, while open squares are the standard agent alone. EC₅₀ values (Molar) are highlighted by arrows.

Figure 2. Rapamycin enhances the therapeutic activity of cyclophosphamide

Tumor bearing mice were treated with cyclophosphamide at the MTD (150 mg/kg q 7d × 6), rapamycin (5 mg/kg d×5 for 6 consecutive weeks) or the combination of cyclophosphamide and rapamycin. Tumor diameters were measured weekly. Panel 1 shows the Kaplan-Meier curves for EFS, control (black), rapamycin (green), cyclophosphamide (blue), or rapamycin + cyclophosphamide (red). Panel 2 shows median relative tumor volumes, control (black), rapamycin (green), cyclophosphamide (blue), or rapamycin + cyclophosphamide (red). Individual tumor growth curves are shown in panel 3, control (light gray), rapamycin (dark lines), and panel 4 cyclophosphamide (light gray), cyclophosphamide + rapamycin (dark lines). A. D456 glioblastoma; B. KT-14 rhabdoid tumor of kidney; C. Rh30 rhabdomyosarcoma.

Figure 3. Rapamycin enhances the therapeutic activity of vincristine

Tumor bearing mice were treated with vincristine at the MTD (1 mg/kg q 7d ×6) or 0.5MTD, rapamycin (5 mg/kg d×5 for 6 consecutive weeks) or the combination of vincristine and rapamycin. Tumor diameters were measured weekly. Panel 1 shows the Kaplan-Meier curves for EFS, control (black), rapamycin (green), vincristine (blue), or rapamycin + vincristine (red). Panel 2 shows median relative tumor volumes, control (black), rapamycin (green), vincristine (blue), or rapamycin + vincristine (red). Individual tumor growth curves are shown in panel 3, control (light gray), rapamycin (dark lines), and panel 4 vincristine (light gray), vincristine + rapamycin (dark lines). A. Rh18 rhabdomyosarcoma (0.5MTD); B. D456 glioblastoma (MTD); C. SK-NEP-1 Ewing sarcoma (MTD).