Fig 1.
Synthesis and characterization of the prodrugs AST-001, AST-002, and AST-003.
(a), AST-001, AST-002 and AST-003 were synthesized as described in the Methods and Materials. (b), The VEGFR2 biochemical assay was performed as described before using the recombinant VEGFR2 protein [21]. (c) and (d), AST-002 and AST-003 were rapidly hydrolyzed to Sunitinib and AST-004 in serum (Y axis, the percentage of AST-002 or AST-003 still in the solution, 100% at time 0; X axis, incubation time). The in vitro plasma stability assays were performed as described in the Materials and Methods. The data represent the average of duplicate experiments.
Fig 2.
AST-002, AST-003, and Sunitinib induced the dephosphorylation of STA3 and AKT as well as cellular changes.
786-O cells (a) and A549 cells (b) were treated with different compounds at the indicated concentrations. After a 2-h treatment, the cells were collected for a western blot analysis to detect the levels of phospho-STAT3 and phospho-AKT. Cytotoxic assays were performed in 786-O cells (c) and A549 cells (d) as described in the Materials and Methods. Error bars are the standard deviations of three replicates. (e) The morphology of 786-O cells treated with the compounds for 4 h at 20×magnification. A549 and 786-O cells (f) and HUVEC cells (g) were treated with different compounds for 8 h, and the compounds were then removed. The cells were incubated in complete medium without compounds for another 16hrs. Cytotoxicity assays were then performed. All data are the means of 4 replicates, with error bars representing the standard deviation (** P<0.01 vs. Sunitinib).
Fig 3.
AST-003 is better tolerated in mice.
The MTD experiments were performed as described in the Methods and Materials. AST-003 or Sunitinib was orally administered once daily at a dose of 50 mg/kg (a), 100 mg/kg (b), and 200 mg/kg (c). The body weights of mice treated with AST-003 (circle), Sunitinib (square) and vehicle (triangle) were measured. All data are the means of 5 mice, with error bars representing the standard deviation. (Statistical analysis: (b) AST-003 vs. Sunitinib, *** P<0.001; Sunitinib vs. Vehicle, *** P<0.001; (c) AST-003 vs. Vehicle, * P<0.05; AST-003 vs. Sunitinib, *** P<0.001; Sunitinib vs. Vehicle, *** P<0.001) At the end of the MTD assays, the biochemical activities of AST, ALT, and CHE (d), Glucose(Glu), Urea acid, blood urea nitrogen (BUN) (e), LDH, CK, and CKMB (f) were measured (* P<0.05; ** P<0.01). (g) AST-003 was orally administered at a dose of 40 mg/kg. The concentrations of AST-004 and Sunitinib were then measured. All data are the means of replicates from 5 mice, with error bars representing the standard deviation. (h) AST-003 and Sunitinib were orally administered at a dose of 40 mg/kg. The concentrations of AST-004 and Sunitinib were then measured 8 h after administration. All data are the means of replicates from 5 mice, with error bars representing the standard deviation.
Fig 4.
AST-003 more effectively inhibits tumors than Sunitinib.
Mice transplanted with A549 cells were treated i.p. with AST-003 (circle), Sunitinib (square) or vehicle (triangle) at doses of 1.5 mg/kg (a), 7.5 mg/kg (b), and 30 mg/kg (c) (* AST-003 vs. Sunitinib, p<0.05). In a separate experiment, these drugs were orally administered at doses of 5 mg/kg (d), 15 mg/kg (e), and 45 mg/kg (f) (* AST-003 vs. Sunitinib, p<0.05). (g) The concentrations of Sunitinib in different tissues were measured after two weeks of compound treatment. All data are the means of replicates from 5 mice, with error bars representing the standard deviation. (h) Mice with established A549 xenografts were orally treated with at 15 mg/kg Sunitinib, AST-003, or vehicle once daily for two days. Tumor samples were retrieved and fixed using the Zinc method. CD31 staining was then performed. Representative figures from 6 independent fields are shown here.
Fig 5.
The proposed mechanism of AST-003.
In mice treated with AST-003, the total Sunitinib (Sunitinib plus hydroxyl-methyl-Sunitinib) inhibits angiogenesis and cancer cell growth better than Sunitinib treatment alone because hydroxyl-methyl-Sunitinib kills vascular endothelial cells and cancer cells at a faster rate than Sunitinib. However, as hydroxyl-methyl-Sunitinib is less stable than Sunitinib, the lower sustained tissue concentrations of Sunitinib associated with AST-003 treatment are responsible for the observed reduced toxicity. Thus, AST-003 exhibits higher efficacy and lower systemic toxicity than Sunitinib.