Antifibrotic action of pirfenidone and prednisolone: Different effects on pulmonary cytokines and growth factors in bleomycin-induced murine pulmonary fibrosis
Introduction
Idiopathic pulmonary fibrosis is a progressive and lethal lung disease characterized by the proliferation of fibroblasts, deposition of extracellular matrix and loss of pulmonary function (Gross and Hunninghake, 2001, Selman et al., 2001, White et al., 2003). Although corticosteroids or other immunosuppressants have been used for the treatment of patients with idiopathic pulmonary fibrosis, the response to these agents is limited, and patients have a poor prognosis (American Thoracic Society, 2000, Collard and King, 2003, Warwick et al., 1980). Since there is a possibility that direct inhibition of fibrosis attenuates the development of chronic pulmonary failure, antifibrotic agents are expected to be useful for the treatment of patients with idiopathic pulmonary fibrosis.
Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone, S-7701; Shionogi & Co., Ltd., Osaka, Japan; MARNAC Inc., Dallas, TX, USA; InterMune Inc., Brisbane, CA USA) is a novel small compound with combined antiinflammatory, antioxidant and antifibrotic effects in experimental models of pulmonary fibrosis (Margolin et al., 1982, Margolin et al., 1994, Iyer et al., 1995, Iyer et al., 1999a, Iyer et al., 1999b, Iyer et al., 2000, Kehrer et al., 1997, Gurujeyalakshmi et al., 1999, Misra et al., 2000, Oku et al., 2002). Clinical studies with pirfenidone in idiopathic pulmonary fibrosis have demonstrated its effectiveness in extending survival time and improving pulmonary function (Raghu et al., 1999, Nagai et al., 2002, Azuma et al., 2005). In bleomycin-induced pulmonary fibrosis in hamsters, antiinflammatory effects of pirfenidone have also been reported (Iyer et al., 2000), in which pirfenidone suppresses bleomycin-induced infiltration of inflammatory cells into the bronchoalveolar lavage fluid. Furthermore, suppression of transforming growth factor (TGF)-β and platelet derived growth factor (PDGF) has been reported (Iyer et al., 1999b, Gurujeyalakshmi et al., 1999). In our previous study, pirfenidone was found to protect mice from endotoxin-induced lethal shock, through suppression of tumor necrosis factor (TNF)-α and enhancement of interleukin (IL)-10 (Oku et al., 2002); however, the regulatory effects of pirfenidone on other cytokines and the relationship to its antifibrotic effects have not been investigated in detail.
Many proinflammatory, antiinflammatory and Th1/Th2 type cytokines, chemokines and profibrotic growth factors are known to be associated with progression of pulmonary fibrosis. To determine the antifibrotic action of pirfenidone, we investigated its regulatory effect on several pulmonary cytokines induced by bleomycin in mice. We used ICR mice, in which bleomycin-induced pulmonary fibrosis has been highly observed (Ekimoto et al., 1984, Ekimoto et al., 1987) and the antifibrotic effect of pirfenidone has been determined (Kakugawa et al., 2004). Corticosteroids, used for the treatment of patients with idiopathic pulmonary fibrosis, are reported to be ineffective in suppressing fibrosis in bleomycin-induced animal models (Hosoya, 1997, Tamagawa et al., 2000). Therefore, it was expected that the antifibrotic action of pirfenidone and its regulatory effect on pulmonary cytokines could be clarified by comparing pulmonary cytokine levels in mice treated with pirfenidone and prednisolone.
In this study, we demonstrate that pirfenidone exhibits antifibrotic activity in a murine model of bleomycin-induced pulmonary fibrosis, while prednisolone fails to suppress fibrosis. From the analysis of cytokines in the lung tissue at the protein level, the antifibrotic effect of pirfenidone was considered to be due to a regulatory effect on lung interferon (IFN)-γ, TGF-β1, basic-fibroblast growth factor (bFGF), stroma cell derived factor (SDF)-1α and IL-18 levels in bleomycin-induced pulmonary fibrosis.
Section snippets
Animal preparation
Male ICR mice (Crlj:CD1 (ICR)) were purchased from Charles River Laboratories Japan (Yokohama, Japan). Pirfenidone (S-7701) was obtained from Shionogi & Co., Ltd., Osaka, Japan. Prednisolone (Sigma-Aldrich, St. Louis, MO) and bleomycin (Nippon Kayaku, Tokyo, Japan) were purchased from a local pharmacy. All procedures for experiments using animals were carried out according to the Guidelines of Animal Care and Use Committee of Shionogi.
Bleomycin injection and drug administration
All mice (13 weeks old) were injected intravenously (i.v.)
Effect of pirfenidone on bleomycin-induced pulmonary fibrosis (Experiment 1)
Repeated bleomycin injections significantly increased the hydroxyproline content of the lung by 40%, as compared with that of the saline-injected group. The administration of pirfenidone dose-dependently suppressed the increase (Fig. 1-A). The suppression was significant at doses of 30 and 100 mg/kg/day, t.i.d. The minimum effective dose was 30 mg/kg/day, t.i.d.
Histological examination showed focal fibroplasia with destruction of the alveolar architecture and interstitial fibrosis of the
Discussion
Idiopathic pulmonary fibrosis is an end-stage, progressive lethal pulmonary disease for which effective therapies are not readily available (Gross and Hunninghake, 2001, Selman et al., 2001). The disease is characterized by failure of alveolar re-epithelialization, the presence of fibroblasts/myofibroblasts, deposition of extracellular matrix, and distortion of the lung architecture, which ultimately results in respiratory failure (White et al., 2003). Corticosteroids and antiinflammatory
Acknowledgements
We are grateful to Mr. Takashi Fukunaga, Mrs. Masayo Igarashi, Mrs. Toshimi Kitai, Dr. Takeshi Yoshioka, Mrs. Masako Takeuchi, Dr. Masashi Furukawa, Mrs. Yoriko Iwasaki and Dr. Masatoshi Nakajima for their expert advice and for reviewing the manuscript. Also we are grateful to Mrs. Mariko Hirano and Mrs. Takako Miyoshi for their technical assistance.
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