Safety and Preclinical Efficacy of Aerosol Pioglitazone on Lung Adenoma Prevention in A/J Mice

Abstract

Pioglitazone is a PPARγ agonist commonly prescribed for the clinical treatment of diabetes. We sought to expand its use to lung cancer prevention in a benzo[a]pyrene (B[a]P) mouse model with direct lung delivery via inhalation. Initially, we conducted inhalational toxicity experiments with 0, 15, 50, 150, and 450 μg/kg body weight/day pioglitazone in 40 A/J mice. We examined the animals for any physical toxicity and bronchoalveolar lavage fluids for inflammatory and cytotoxicity markers. Doses up to and including 450 μg/kg bw/d failed to demonstrate toxicity with aerosol pioglitazone. For chemoprevention experiments, A/J mice were randomized to treatment groups of inhaled doses of 0, 50, 150, or 450 μg/kg bw/d pioglitazone 1 or 8 weeks after the last dose of B[a]P. For the early treatment group, we found up to 32% decrease in lung adenoma formation with 450 μg/kg bw/d pioglitazone. We repeated the treatments in a second late-stage experiment and found up to 44% decreases in lung adenoma formation in doses of pioglitazone of 150 and 450 μg/kg bw/day. Both the early- and the late-stage experiments demonstrated biologically relevant and statistically significant decreases in adenoma formation. We conclude that aerosol pioglitazone is well-tolerated in the A/J mouse model and a promising chemoprevention agent for the lower respiratory tract. Cancer Prev Res; 10(2); 124-32. ©2016 AACR.

Figure 1

Experimental schema and timeline. Flowchart of procedures, treatments, and analyses.

Figure 2

Evaluation of toxicity by body weight measurement. Animals were weighed before and after treatment for seven consecutive days with solvent, 15, 50, 150, and 450 µg/kg bw/day pioglitazone via aerosol. No significant differences were seen via one-way ANOVA analysis between any of the groups and no significant changes were seen in growth rate between any groups. Dunnett’s post test also showed no difference between any groups and the control. No animals died or had to be euthanized during the exposure period and no changes in coat or behavior were observed.

Figure 3

Evaluation of toxicity by bronchoalveolar lavage. Animals were treated via aerosol for seven consecutive days with solvent, 15, 50, 150, and 450 µg/kg bw/day pioglitazone. Bronchoalveolar lavage (BAL) was performed at sacrifice and demonstrates A) equivalent recovery of ~400 µL of fluid from all of the tracheobronchial trees, B) no statistical difference in the number of cells/mL in the BAL fluid, C) ~75% trypan blue exclusion at all pioglitazone concentrations, LDH concentrations were similar in all experimental groups of both airway fluids (D) and cytologic lysates (E), and total protein is unchanged across groups (F).

Figure 4

Effect of aerosolized pioglitazone on lung adenoma formation. After 15 (early stage intervention) or eight (late stage intervention) weeks of treatment, the animals were sacrificed and adenoma counts performed. (A) Animals did not experience statistically significant weight changes across experimental groups over the course of the experiment (B) or at sacrifice. (C) In the early treatment groups, adenoma formation reduced by 14% in the 150 µg/kg bw/day aerosol delivery group. At 450 µg/kg bw/day, there was a reduction of 32% compared to control animals. (* = p<0.05, Student T-test) However, one-way ANOVA analysis found no statistically significant difference over the three groups (F(2,66)=2.418,p=0.0969). Kruskal-Wallis non-parametric rank test yielded p=0.0580. (D) Neither 50 nor 450 µg/kg bw/day was statistically different from the control group in the late stage. 150 µg/kg bw/day decreased lung adenoma formation 44% versus solvent control (* = p<0.05, Student t-test and Dunnett’s post test) and means of all groups were statistically different via one-way ANOVA analysis (F(3,59)=4.716,p=0.0014).

Figure 5

Effect of pioglitazone on human bronchial epithelium cell proliferation. (A) Beas-2B cells were plated at 5×10³ cells/well and treated for 24, 48, and 72 hours with increasing concentrations of pioglitazone. Cell proliferation was determined via MTT assay at each time point. (*= p ≤ 0.01 vs. DMSO, **= p ≤ 0.001 vs. DMSO) (B) Beas-2B cells were treated for 24, 48, and 72 hours with 10 µM pioglitazone or 10 µM T0070907, an inhibitor of PPAR γ ligand binding. At 72 hours, pioglitazone alone produced significantly decreased cell proliferation versus pioglitazone plus the ligand binding inhibitor. (**= p ≤ 0.001)