Synergistic anticancer activity of LB-1.2 combined with TMZ and Dox. 5 × 106 U87MG cells in log-phase growth were inoculated s.c. into each flank of SCID mice. After the xenografts reached 0.5 ± 0.1 cm (day zero), animals were randomized to 4 groups of 5 animals each. Animals were treated i.p. with vehicle alone day 1–12 (50% DMSO/H2O); LB-1.2 alone at 1.5 mg/kg on days 1–3, 5–7, and 9–11; TMZ alone at 80 mg/kg on days 4, 8, 12; or both drugs at the same doses and schedules as when used alone. For study of the effects of LB-1.2 on DOX anticancer efficacy, each of 10 animals with a single s.c. xenograft received i.p. either LB-1.2 at 1.5 mg/kg days 1, 4, and 7; DOX at 2.0 mg/kg days 2, 5, and 8; or both drugs at the same doses and schedules as when used alone. If xenografts reached a volume of 1,800 mm3, animals were killed. (A) Tumor volume of U87 xenografts. Control animals had rapid growth of all xenografts and were killed at 3 weeks; LB-1.2-treated animals had modest delay and were killed by week 4–5. TMZ treated animals had complete regression of all xenografts by week 5, but with recurrence and rapid growth requiring killing of all 5 animals between week 7 and 9. Each point is the mean of all xenografts ± 1 SD. LB-1.2 plus TMZ treated animals had complete regression of all xenografts by week 5 with recurrence of 1/2 xenografts in each of 3 mice at weeks 7, 11, and 13, requiring killing of 1 at week 11 and 2 at week 15. Two mice treated with the combination had no recurrence of either xenograft for 7 months. Average tumor volume is shown only through week 9 because 1/2 xenografts recurred in 3 mice with the treatment of 2-drug combination, and the mice were killed at weeks 11 and 15, which prevented accurate estimates of second xenografts in the combination group beyond week 9. (B) Survival curve combining the data from the study depicted in A with data from a second identical study; a total of 10 animals with 2 xenografts each per treatment. The animal survival was defined as no recurrence of either of the xenografts. Kaplan-Meier analysis revealed that survival following LB-1.2 plus TMZ was significantly greater than with LB1.2 alone and TMZ alone (logrank, P < 0.001). (C) Regression of SH-SY5Y xenografts. As in A, except that xenografts of SH-SY5Y cells were implanted in 1 flank only, and animals were treated with 1 additional cycle of drugs, i.e., LB-1.2 on days 13–15, TMZ on day 16, and LB-1.2 pus TMZ on the same schedules. Control animals had growth of all xenografts requiring killing by week 3. TMZ alone delayed growth but approached the maximum by week 7. LB-1.2 alone was more inhibitory with no growth until week 3 with progression thereafter, reaching about half the size of the TMZ alone by week 7. LB-1.2 plus TMZ completely inhibited xenograft growth but a residual tumor mass was still present at week 7. All xenografts in treatment arms ulcerated by week 7, necessitating killing per veterinarian recommendation. (D) Regression of GBM xenografts. The 2-drug combination caused regression of all xenografts, whereas LB-1.2 and DOX alone only slowed progression. One animal receiving the combination of drugs died on day 14, and all animals were then killed. (E) Histologic features of SH-SY5Y s.c. xenografts stained with H&E 24 h after i.p. vehicle alone (Upper Left), LB-1.2 at 1.5 mg/kg (Upper Right), TMZ at 80 mg/kg (Lower Left), and both drugs (Lower Right).