Bleomycin Induces Drug Efflux in Lungs. A Pitfall for Pharmacological Studies of Pulmonary Fibrosis

Abstract

ATP-binding cassette (ABC) transporters are evolutionarily conserved membrane proteins that pump a variety of endogenous substrates across cell membranes. Certain subfamilies are known to interact with pharmaceutical compounds, potentially influencing drug delivery and treatment efficacy. However, the role of drug resistance-associated ABC transporters has not been examined in idiopathic pulmonary fibrosis (IPF) or its animal model: the bleomycin (BLM)-induced murine model. Here, we investigate the expression of two ABC transporters, P-gp (permeability glycoprotein) and BCRP (breast cancer resistance protein), in human IPF lung tissue and two different BLM-induced mouse models of pulmonary fibrosis. We obtained human IPF specimens from patients during lung transplantation and administered BLM to male C57BL/6J mice either by oropharyngeal aspiration (1 U/kg) or subcutaneous osmotic infusion (100 U/kg over 7 d). We report that P-gp and BCRP expression in lungs of patients with IPF was comparable to controls. However, murine lungs expressed increased levels of P-gp and BCRP after oropharyngeal and subcutaneous BLM administration. We localized this upregulation to multiple pulmonary cell types, including alveolar fibroblasts, endothelial cells, and type 2 epithelial cells. Functionally, this effect reduced murine lung exposure to nintedanib, a U.S. Food and Drug Administration-approved IPF therapy known to be a P-gp substrate. The study reveals a discrepancy between IPF pathophysiology and the common animal model of lung fibrosis. BLM-induced drug efflux in the murine lungs may present an uncontrolled confounding variable in the preclinical study of IPF drug candidates, and these findings will facilitate disease model validation and enhance new drug discoveries that will ultimately improve patient outcomes.

Keywords: ATP-binding cassette transporters; animal models; bleomycin; drug resistance; idiopathic pulmonary fibrosis.

Figure 1.

Both oropharyngeal bleomycin (OP-bleo) and subcutaneous bleomycin (SC-bleo) induce comparable pulmonary fibrosis with upregulating P-gp (permeability glycoprotein) and BCRP (breast cancer resistance protein) gene expression. (A) Percent initial body weight. Initial average body weight was 26 ± 2 g for each group. (B) Wet weight of the left lung was recorded after OP-bleo or SC-bleo. (C) Hydroxyproline (Hyp) content of the left lung was measured via liquid chromatography–tandem mass spectrometry (LC-MS/MS) after OP-bleo or SC-bleo. (D) Gene expression profiling of fibrosis markers. (E) Pressure–volume curve, (F) tissue elasticity, (G) tissue damping, as a measure of lung function obtained by flexiVent. Gene expression of drug efflux–associated ATP-binding cassette (ABC) transporters after (H) OP-bleo or (I) SC-bleo. n = 5 mice per group for control and 6 mice per OP-bleo group, and 5 mice per SC-bleo group. *P < 0.05 indicates significant difference from control group. PVP = pressure–volume curve.

Figure 2.

P-gp and BCRP protein levels are elevated in the bleo models but not in human idiopathic pulmonary fibrosis (IPF). Mouse and human lung sections were stained with Masson’s trichrome staining. Fibrosis was present in both (A) OP-bleo and SC-bleo models, as well as (B) human IPF. Immunohistochemical staining revealed elevated P-gp protein levels in (C) OP-bleo and in SC-bleo but not in (D) human IPF. Similarly, BCRP protein was increased in (E) OP-bleo and in SC-bleo but not in (F) human IPF. Small-cell carcinoma (SCC) sections had substantial P-gp and BCRP expression, as expected. n = 4 mice per control group and 5 mice per OP-bleo and SC-bleo groups. n = 4 for nonfibrotic human control group, 6 for patients with IPF, 2 per SCC group. Scale bars: 100 μm.

Figure 3.

Localization of Abcb1b with cell types in the lung. Fluorescent in situ hybridization (FISH) was performed with the RNAscope Multiplex Fluorescent Kit V2 on formalin fixed paraffin embedded (FFPE) lung tissue sections from 14-day OP-bleo SC-bleo and control mice. (A) FISH detected localization of Abcb1b on cells positive for Cd68 (macrophages), Sftpc (alveolar type II epithelial cells), Pecam1 (endothelial cells), Col1a1 (collagen-producing cells, such as fibroblasts and myofibroblasts), and Cspg4 (pericytes). Arrows point out colocalizations. (B) FFPE lung tissue RNA quality was assessed using housekeeping genes Polr2a (low-expressed gene) and Ubc (high-expressed gene) as positive controls. (C) FISH nonspecific background staining was assessed using the bacterial gene dapB as a negative control. n = 3 per group.

Figure 4.

Localization of Abcg2 with cell types in the lung. FISH was performed with the RNAscope Multiplex Fluorescent Kit V2 on FFPE lung tissue sections from 14-day OP-bleo, SC-bleo, and control mice. FISH detected localization of Abcg2 on cells positive for Cd68, Sftpc, Pecam1, Col1a1, and Cspg4. n = 3 per group. Arrows point out colocalizations.

Figure 5.

Bleo reduces lung exposure of a P-gp substrate drug, nintedanib. Nintedanib (30 mg/kg) was orally administered to OP-bleo and SC-bleo mice 1 hour before animals were killed, then quantified in (A) plasma and (B) lung tissue via LC-MS/MS. n = 4 mice per group. *P < 0.05 indicates significant difference from control group.

Figure 6.

Lack of antifibrotic efficacy of nintedanib in OP-bleo–induced pulmonary fibrosis. (A) Hydroxyproline content as fibrosis measurement. (B) Pressure–volume curve, (C) tissue elasticity, (D) tissue damping, and (E) FEV as a measure of lung function. n = 5 mice per group for control and 7 mice per vehicle and nintedanib-treated groups. *P < 0.05 indicates significant difference from vehicle-treated group. Ctrl = control; FEV = forced expiratory volume.

Figure 7.

LPS-induced acute lung injury increased P-gp expression, but not BCRP expression, in mice. Gene expression of (A) inflammatory cytokines and chemokines and (B) fibrogenic markers. (C) HYP content. (D) Gene expression of drug efflux–associated ABC transporters. (E) Protein expression of P-gp and BCRP proteins with immunohistochemistry. Scale bar: 100 μm. n = 3 mice per group. *P < 0.05 indicates significant difference from the control group.