Anti-tumor activity of liposome encapsulated fluoroorotic acid as a single agent and in combination with liposome irinotecan

Abstract

To test the hypothesis that co-delivery of synergistic drug combinations in the same liposome provides a better anti-tumor effect than the drugs administered in separate liposomes, fluoroorotic acid (FOA) alone and in combination with irinotecan (IRN) were encapsulated in liposomes and evaluated for their anti-tumor activity in the C26 colon carcinoma mouse model. A new chaotropic loading strategy was devised wherein FOA was dissolved in 7 M urea to increase its solubility. This enabled the passive loading of FOA into liposomes at a high concentration. IRN was remote loaded into liposomes that contained the ammonium salt of the multi-valent 1,2,3,4-butanetetracarboxylic acid with a greater than 90% efficiency and at a drug to lipid ratio of 0.2:1. When the two molecules were loaded into the same liposome, FOA was used to remote load IRN. Modulation of the drug/lipid ratio, temperature, and loading time allowed for consistent co-encapsulation of FOA+IRN at various molar ratios. The anti-tumor activity of L-FOA, L-IRN, L-FOA-IRN (5:1), and the L-FOA+L-IRN mixture (5:1) were examined in the C26 mouse model. The maximum tolerated dose of L-FOA was 10 mg/kg given weekly as compared to 100 mg/kg of the non-encapsulated FOA. Delivering two drugs in the same liposome provided a statistically better anti-tumor effect than delivering the drugs in separate liposomes at the same drug ratio. However, the synergistic activity of the 5:1 ratio of free drugs measured on C26 cells in vitro was not observed in the C26 tumor mouse model. These findings point out the challenges to the design of synergistic treatment protocols based upon results from in vitro cytotoxicity studies. L-FOA at 10 mg/kg as a single agent provided the best anti-tumor efficacy which supports previous suggestions that L-FOA has useful properties as a liposome dependent drug.

Fig. 1

Maximum tolerated dose study of FOA 100 mg/kg and L-FOA 10 mg/kg in Balb/c mice. A. For single dose, FOA and L-FOA administered i.v. on Day 0. For multiple dose, FOA and L-FOA administered i.v. on Day 0 and four days apart (as indicated by arrows). B. FOA 100 mg/kg and L-FOA 10 mg/kg administered i.v. on a 3xq7d schedule starting on Day 0.

Fig. 2

Effect of IRN drug to lipid ratio on FOA and IRN co-encapsulation at 5:1 molar ratio. Loading temperature was 50 °C and loading time was 30 min. A. Effect of initial IRN drug/lipid ratio on the final encapsulated IRN concentration ([IRN]_f). B. Effect of initial IRN drug/lipid ratio on the final encapsulated FOA concentration ([FOA]_f). C. Effect of initial IRN drug/lipid ratio on the final ratio of FOA and IRN in the liposomes ([FOA]_f/[IRN]_f).

Fig. 3

Schematic diagram of proposed mechanism of co-encapsulation of FOA + IRN in liposomes.

Figure 4

FOA and IRN release profile from L-FOA-IRN, L-IRN, and L-FOA. A. In vitro leakage from liposomes during 96 hr incubation in 33% serum at 37°C. B. Ratio of IRN and FOA released from the co-encapsulated and the individual liposomes over time. *Statistical significance (P < 0.05) between the ratio from co-encapsulated and separate liposomes as measured by Student’s t-test.

Fig. 5

L-FOA-IRN combination therapy in C26 tumor-bearing mice. Balb/c mice (n=8) were treated with i.v. injections on Days 8 and 15 (as indicated by arrows). A. Anti-tumor activity. Error bars represent SEM. B. Effect of combination therapy on weight of C26 tumor-bearing mice. C and D. Survival curves. The treatment group s are PBS, L-FOA (10 mg/kg; 57.4 μmol/kg), L-IRN (50 mg/kg; 73.8 μmol/kg), L-FOA-IRN 5:1 (5 mg/kg or 28.7 μmol/kg FOA; 3.9 mg/kg or 5.7 μmol/kg IRN), L-FOA + L-IRN 5:1 (5 mg/kg or 28.7 μmol/kg FOA; 3.9 mg/kg or 5.7 μmol/kg IRN), and L-FOA + L-IRN 5:1 double strength (10 mg/kg or 57.4 μmol/kg FOA; 7.8 mg/kg or 11.5 μmol/kg IRN).