Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jun;6(12):e13753.
doi: 10.14814/phy2.13753.

Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury

Thomas H Sisson  1 , Paul J Christensen  2 , Yo Muraki  3 , Anthony J Dils  1 , Lauren Chibucos  1 , Natalya Subbotina  1 , Kimio Tohyama  3 , Jeffrey C Horowitz  1 , Takanori Matsuo  3 , Marc Bailie  4 , Sham Nikam  3 , Masatoshi Hazama  3
Affiliations

Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury

Thomas H Sisson et al. Physiol Rep. 2018 Jun.

Abstract

Fibrosis of the lung constitutes a major clinical challenge and novel therapies are required to alleviate the associated morbidity and mortality. Investigating the antifibrotic efficacy of drugs that are already in clinical practice offers an efficient strategy to identify new therapies. The phosphodiesterase 4 (PDE4) inhibitors, approved for the treatment of chronic obstructive pulmonary disease, harbor therapeutic potential for pulmonary fibrosis by augmenting the activity of endogenous antifibrotic mediators that signal through cyclic AMP. In this study, we tested the efficacy of several PDE4 inhibitors including a novel compound (Compound 1) in a murine model of lung fibrosis that results from a targeted type II alveolar epithelial cell injury. We also compared the antifibrotic activity of PDE4 inhibition to the two therapies that are FDA-approved for idiopathic pulmonary fibrosis (pirfenidone and nintedanib). We found that both preventative (day 0-21) and therapeutic (day 11-21) dosing regimens of the PDE4 inhibitors significantly ameliorated the weight loss and lung collagen accumulation that are the sequelae of targeted epithelial cell damage. In a therapeutic protocol, the reduction in lung fibrosis with PDE4 inhibitor administration was equivalent to pirfenidone and nintedanib. Treatment with this class of drugs also resulted in a decrease in plasma surfactant protein D concentration, a reduction in the plasma levels of several chemokines implicated in lung fibrosis, and an in vitro inhibition of fibroblast profibrotic gene expression. These results motivate further investigation of PDE4 inhibition as a treatment for patients with fibrotic lung disease.

Keywords: cAMP; collagen; epithelium; fibroblast; pulmonary.

PubMed Disclaimer

Figures

Figure 1
Figure 1
The pharmacokinetics of Compound 1 administration. 10.0 mg/kg of Compound 1 was administered by oral gavage at time 0. Plasma and BALF were collected simultaneously from separate cohorts of mice at 0.5, 1, 2, 4, 8, and 24 h after the dosing. The plasma concentration of drug over time (1A) and the BALF cAMP levels over time (1B) are reported. A linear relationship between plasma drug level and BALF cAMP concentration was observed (1C). Data are reported as mean ± SD (n = 4/group).
Figure 2
Figure 2
Prophylactic administration of roflumilast and piclamilast to targeted type II AEC‐injured mice reduces weight loss and ameliorates pulmonary fibrosis. DTR‐expressing mice (DTR+) were administered daily I.P. PBS or DT from day 0 through Day 14. Subsets of the DTR+:DT‐injured animals were treated by oral gavage once daily beginning on day 0 with vehicle, roflumilast (1.0 mg/kg or 5.0 mg/kg), or Piclamilast at 30 mg/kg. Mice were weighed daily (A) and on day 21, lungs were harvested and analyzed for total collagen content using an assay for hydroxyproline (B). Data are reported as mean ± SEM (n = 10 per group). Weight curves are analyzed with a two‐way ANOVA + Tukey multiple comparison tests (# P ≤ 0.05 for DTR+:DT Piclamilast‐treatment versus DTR+:DT vehicle treatment. ## P ≤ 0.05 DTR+:DT piclamilast and roflumilast (1.0 mg/kg or 5.0 mg/kg) treatment versus DTR+:DT vehicle treatment), and hydroxyproline is analyzed with a one‐way ANOVA + Tukey multiple comparison tests.
Figure 3
Figure 3
Prophylactic administration of Compound 1 to targeted type II AEC‐injured mice reduces weight loss and ameliorates pulmonary fibrosis. DTR‐expressing mice (DTR+) were administered daily I.P. PBS or DT from day 0 through Day 14. Subsets of the DTR+:DT‐treated animals were treated by oral gavage once daily beginning on day 0 with vehicle, Compound 1 (1.0 mg/kg, 5.0 mg/kg, or 10.0 mg/kg) or Piclamilast at 30 mg/kg. Mice were weighed daily (A) and on day 21, lungs were harvested and analyzed for total collagen content using an assay for hydroxyproline (B). Data are reported as mean ± SEM (n = 8 per group). Weight curves are analyzed with a two‐way ANOVA + Tukey multiple comparison tests (# P ≤ 0.05 for DTR+:DT piclamilast and Compound 1‐treatments (all doses) versus DTR+:DT vehicle treatment), and hydroxyproline is analyzed with a one‐way ANOVA + Tukey multiple comparison tests.
Figure 4
Figure 4
Therapeutic administration of Compound 1 and roflumilast to targeted type II AEC‐injured mice reduces weight loss and ameliorates pulmonary fibrosis. DTR‐expressing mice (DTR+) were administered daily I.P. PBS or DT from day 0 through Day 14. Subsets of the DTR+:DT‐treated animals were treated by oral gavage once daily beginning on day 11 with vehicle, roflumilast (5.0 mg/kg), or Compound 1 (1.0 mg/kg or 5.0 mg/kg). Mice were weighed daily (A) and on day 21, lungs were harvested and analyzed for total collagen content using an assay for hydroxyproline (B) and by histopathology (C) with representative sections stained with picrosirius red (400× magnification). Data are reported as mean ± SEM (n = 10–12 per group). Weight curves are analyzed with a two‐way ANOVA + Tukey multiple comparison tests (# P ≤ 0.05 for DTR+:DT roflumilast and Compound 1 (5.0 mg/kg) treatments versus DTR+:DT vehicle treatment), and hydroxyproline is analyzed with a one‐way ANOVA + Tukey multiple comparison tests.
Figure 5
Figure 5
Compound 1 treatment of A549 human lung epithelial cell cultures increases intracellular and extracellular cAMP concentrations. A549 human lung epithelial cells were seeded in a 24 well plate for 24 h. The cultures were treated with increasing concentrations of Compound 1 (1 × 10−10 mol/L to 1 × 10−5 mol/L) for 30 min and stimulated with 10 μmol/L forskolin and 10 μmol/L prostaglandin E2 for 30 min. cAMP concentrations were measured in cell lysates (A) and culture supernatants (B). The correlation between intracellular cAMP and cAMP in the culture supernatants is shown in (C). Data are reported as mean + SD (n = 3 per group).
Figure 6
Figure 6
Prophylactic treatment of targeted type II AEC‐injured mice with Compound 1 reduces plasma SP‐D levels. DTR‐expressing mice (DTR+) were administered daily I.P. PBS or DT from day 0 through Day 14. Subsets of the DTR+:DT‐treated animals were treated by oral gavage once daily beginning on day 0 with vehicle, Compound 1 (1.0 mg/kg, 5.0 mg/kg, or 10.0 mg/kg) or Piclamilast at 30 mg/kg. Plasma was collected on day 21 and analyzed for SP‐D concentration (A). The plasma SP‐D concentration was then correlated with lung collagen content (B). Data are reported as mean + SEM with individual data points shown (n = 8 per group). # P ≤ 0.05 versus Vehicle group by two‐tailed Williams’ test. $ P ≤ 0.05 versus Vehicle group by Student's t‐test.
Figure 7
Figure 7
Therapeutic treatment of targeted type II AEC‐injured mice with Compound 1 reduces plasma cytokine levels. DTR‐expressing mice (DTR+) were administered daily I.P. PBS or DT from day 0 through Day 14. Subsets of the DTR+:DT‐treated animals were treated by oral gavage once daily beginning on day 0 with vehicle or Compound 1 at 5.0 mg/kg. Plasma was collected on day 21 and analyzed for CCL11/eotaxin, CXCL10/IP‐10, CXCL5/LIX, and CCL5/Rantes. Data are reported as mean ± SD with individual data points shown (n = 13 for control, 22 for Compound 1 per group). # P ≤ 0.05 versus Control group by Student's t‐test.
Figure 8
Figure 8
Compound 1 treatment of human lung fibroblasts cell cultures increases intracellular cAMP and decreases TGF‐β‐mediated induction of fibrotic gene expression. WI‐38 human lung fibroblasts were seeded on 24‐well plates and treated with increasing concentrations of Compound 1 (1 × 10−10 mol/L to 1 × 10−5 mol/L). After 30 min, cells were stimulated by 1 μmol/L forskolin for 30 min and cell lysates were analyzed for cAMP concentration (A). To analyze fibroblast gene expression, WI‐38 human lung fibroblasts were cultured in media containing 0.5% FBS for 24 h and treated with increasing concentrations of Compound 1 (1 × 10−10 mol/L to 1 × 10−5 mol/L) for 1 h followed by TGF‐β (3 ng/mL) and forskolin (1 μmol/L) for 24 h. Total RNA was extracted from cell lysates, cDNA was amplified, and target gene mRNA was measured. The target gene expression levels of Col1a1 (Type‐1 collagen), Fn (Fibronectin), CTGF (connective tissue growth factor), and PAI‐1 (plasminogen activator inhibitor‐1) was normalized to Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). Data are reported as mean ± SD with individual data points shown (n = 3 per group). # P ≤ 0.05 versus forskolin group by two‐tailed Williams’ test.
Figure 9
Figure 9
Therapeutic administration of Compound 1 to targeted type II AEC‐injured mice reduces the expression of TNF α within the lung. DTR‐expressing mice (DTR+) were administered daily I.P. PBS or DT from day 0 through Day 14. Subsets of the DTR+:DT‐treated animals were treated by oral gavage once daily beginning on day 11 with vehicle or Compound 1 at 5.0 mg/kg. On day 21, the left lung was harvested and homogenized, and total RNA was extracted. First‐strand cDNA was synthesized and mRNA levels for Col1a1, Fibronectin, CTGF TNF α and PAI‐1 (plasminogen activator inhibitor‐1) were assessed using SYBR Green‐based detection. The expression levels were normalized to GAPDH using the following formula: %GAPDH expression = 100/2‐ΔΔCT. Data are presented as an average ± SEM (n = 9 per DTR+:DT vehicle‐ and drug‐treated groups).
Figure 10
Figure 10
Roflumilast is equivalent to pirfenidone and nintedanib in ameliorating fibrosis following targeted type II AEC injury. DTR‐expressing mice (DTR+) were administered daily I.P. PBS or DT from day 0 through Day 14. Subsets of the DTR+:DT‐treated animals were treated by oral gavage beginning on day 11 with vehicle, roflumilast at 5.0 mg/kg once daily, pirfenidone at 100.0 mg/kg three times daily, or nintedanib 100.0 mg/kg two times daily. Mice were weighed daily (A) and on day 21, lungs were harvested and analyzed for total collagen content using an assay for hydroxyproline (B). Data are reported as mean ± SEM (n = 6–10 per group). Weight curves are analyzed with a two‐way ANOVA + Tukey multiple comparison tests (# P ≤ 0.05 for DTR+:DT roflumilast and pirfenidone‐treatment versus DTR+:DT nintedanib treatment, and hydroxyproline is analyzed with a one‐way ANOVA + Tukey multiple comparison tests.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Borensztajn, K. , Crestani B., and Kolb M.. 2013. Idiopathic pulmonary fibrosis: from epithelial injury to biomarkers–insights from the bench side. Respiration 86:441–452. - PubMed
    1. Chaudhary, N. I. , Roth G. J., Hilberg F., Muller‐Quernheim J., Prasse A., Zissel G., et al. 2007. Inhibition of PDGF, VEGF and FGF signalling attenuates fibrosis. Eur. Respir. J. 29:976–985. - PubMed
    1. Chu, Y. T. , Chang T. T., Jong Y. J., Kuo P. L., Lee H. M., Lee M. S., et al. 2010. Suppressive effects of formoterol and salmeterol on eotaxin‐1 in bronchial epithelial cells. Pediatr. Allergy Immunol. 21:345–352. - PubMed
    1. Corboz, M. R. , Zhang J., LaSala D., DiPetrillo K., Li Z., Malinin V., et al. 2018. Therapeutic administration of inhaled INS1009, a treprostinil prodrug formulation, inhibits bleomycin‐induced pulmonary fibrosis in rats. Pulm. Pharmacol. Ther. 49:95–103. - PubMed
    1. Cortijo, J. , Iranzo A., Milara X., Mata M., Cerda‐Nicolas M., Ruiz‐Sauri A., et al. 2009. Roflumilast, a phosphodiesterase 4 inhibitor, alleviates bleomycin‐induced lung injury. Br. J. Pharmacol. 156:534–544. - PMC - PubMed

Publication types

MeSH terms