2013 Volume 36 Issue 10 Pages 1525-1527
Pirfenidone (PFD) is the first and only clinically used antifibrotic drug for the treatment of idiopathic pulmonary fibrosis (IPF). This study evaluated the antifibrotic effects of two metabolites of PFD, 5-hydroxypirfenidone (PFD-OH) and 5-carboxypirfenidone (PFD-COOH), on WI-38 cells in an in vitro lung fibroblast model. The inhibitory effects of PFD-OH and PFD-COOH on transforming growth factor-β1 (TGF-β1)-induced collagen synthesis in WI-38 cells were evaluated by measuring intracellular hydroxyproline, a major component of the protein collagen. PFD-OH and PFD-COOH at 300 and 1000 µM concentrations significantly decreased the TGF-β1-induced hydroxyproline content in WI-38 cells. These results indicate that PFD-OH and PFD-COOH have antifibrotic activities, which inhibit collagen synthesis in fibroblasts. This study suggests that the concentrations of PFD and its metabolites should be considered in clinical therapy for IPF.
Fibrotic diseases occur in various tissue regions and can resemble scar tissue when they form in inappropriate locations such as lung, liver, heart, eye, and kidney. In particular, idiopathic pulmonary fibrosis (IPF) is devastating with an extremely low five-year survival rate (<50%).1) Currently, researchers are working to develop an antifibrotic drug that will improve the survival rate of patient with IPF.
Pirfenidone (PFD, Fig. 1), 5-methyl-1-phenyl-2-(1H)-pyridone, is the first and only clinically used antifibrotic drug for the treatment of IPF in Japan (Pirespa®), Europe (Esbriet®), and India (Pirfenex®).2) PFD has antifibrotic, anti-inflammatory, and antioxidative actions.2,3) In experimental animal models, PFD has demonstrated an antifibrotic effect in several tissues, such as lung, liver, and kidney.4–6) To date, clinical studies that have evaluated the PFD pharmacokinetics have been conducted in patients with IPF. After oral administration of PFD in humans, it is rapidly eliminated from plasma.7,8) Other researchers have shown that PFD is rapidly metabolized to 5-hydroxypirfenidone (PFD-OH) and 5-carboxypirfenidone (PFD-COOH) (Fig. 1), and the major metabolite PFD-COOH is eliminated in the urine (>87%).9) However, the antifibrotic effects of PFD-OH and PFD-COOH have not been reported. In the present study, we discussed the possible involvement of pirfenidone metabolites in the antifibrotic action of a therapy for IPF.
PFD was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). PFD-OH and PFD-COOH were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). TGF-β1 was purchased from Peprotech Inc. (Rocky Hill, NJ, U.S.A.). All other reagents were commercially available and of analytical grade. Male SD rats, 8 weeks of age and 230–270 g body weight, were purchased from CLEA Japan, Inc. (Tokyo, Japan). The care and use of animals followed “The Guidelines for the Care and Use of Animals” approved by Ohu University in accordance with the principles of the NIH guidelines (Approval number: 2012–50).
Antifibrotic Experiments in VitroWI-38 cells (Riken Gene Bank, Tsukuba, Japan), a human lung fibroblast cell line, were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) and 40 µg/mL gentamicin in a humidified atmosphere of 5% CO2 at 37°C. Cells from passage numbers 12–13 were seeded (2.0×104 cells/well) on 24-well culture plates. At confluence, the medium was replaced with DMEM containing 0.4% FBS and 50 µg/mL of ascorbate. After a 24-h incubation, transforming growth factor (TGF)-β1 (10 ng/mL) and serial concentrations of PFD, PFD-OH, and PFD-COOH were added to the WI-38 cells, and subsequently the cells were incubated at 5% CO2 at 37°C for 24 h. After incubation, the medium was removed by aspiration and washed twice with ice-cold phosphate buffered saline (PBS). The cells were then extracted with 300 µL of 2 M NaCl, and the concentration of hydroxyproline in the cell extracts was measured by HPLC as described below. The DNA concentration in the cell extracts was determined using Fluorescent DNA Quantitation Kit (Bio-Rad, Hercules, CA, U.S.A.).
Pharmacokinetics Experiment in VivoPFD dissolved in PBS was intravenously administered to rats at a dose of 30 mg/kg and the dosage volume was 1 mL/kg. At each designated time point, rats were anesthetized using intraperitoneal injections of pentobarbital sodium at a dose of 40 mg/kg and blood was collected from the jugular vein. The concentration of PFD and its metabolites was measured in each sample by HPLC as described below. The pharmacokinetic analysis was performed using the non-compartment analytical method.10)
Determination of Hydroxyproline by HPLCThe concentrations of hydroxyproline in samples were measured by HPLC following fluorescent derivatization, using the method of Hutson et al.11) The derivative in samples was subjected to HPLC using a system (Jasco Corporation, Tokyo, Japan) involving an InertSustain C18 (3.0×250 mm internal diameter; GL Sciences, Inc., Torrance, CA, U.S.A.). The mobile phase was 85 mM acetic buffer (pH 4.3)–acetonitrile (68 : 32). Separation was performed at a flow rate of 0.4 mL/min at 45°C, and the eluate from the column was monitored by fluorescence detection (excitation wavelength of 250 nm and emission wavelength of 310 nm).
Determination of PFD and Its Metabolites by HPLCThe concentrations of PFD, PFD-OH, and PFD-COOH in plasma were measured by HPLC, using the method by Wang et al.12) The prepared samples were subjected to HPLC using a system involving a Mightysil RP-18GPII column (3.0×250 mm, Kanto Chemical Co., Tokyo, Japan). The mobile phase was 0.2% acetic acid/methanol (74 : 26). The separation was performed at a flow rate of 0.4 mL/min at 45°C and the column was monitored by UV absorbance detection (absorbance wavelength of 310 nm).
StatisticsStatistical analysis was performed using the Dunnett’s t-test and SPSS software version 21 (IBM Inc., Armonk, NY, U.S.A.).
The present study evaluated the antifibrotic effects of the PFD metabolites PFD-OH and PFD-COOH. The effects of different concentrations of PFD, PFD-OH, and PFD-COOH on hydroxyproline content, a major component of the protein collagen, in WI-38 cells, a human lung fibroblast cell line, are shown in Fig. 2. PFD at 100 µM (18.5 µg/mL), 300 µM (55.6 µg/mL), and 1000 µM (185 µg/mL), and PFD-OH at 300 µM (60.4 µg/mL) and 1000 µM (201 µg/mL), and PFD-COOH at 300 µM (64.6 µg/mL) and 1000 µM (215 µg/mL) significantly decreased the TGF-β1-induced hydroxyproline content in WI-38 cells without cellular toxicity. PFD and its metabolites were stable at 37°C in medium for 24 h (data not shown). These results indicate that PFD-OH and PFD-COOH have antifibrotic activities, which inhibit collagen synthesis in lung fibroblasts. In addition, after intravenous administration of PFD to rats, the PFD-COOH concentration in plasma was comparable to that of PFD (Fig. 3). The calculated terminal elimination half-life (T1/2) of PFD, PFD-OH, and PFD-COOH were 0.74±0.12, 0.79±0.26, and 0.84±0.26 h, respectively. The areas under the concentration–time curves (AUC) for PFD, PFD-OH, and PFD-COOH were 30.5, 3.9, and 24.2 µg*h/mL, respectively. These results indicate that the antifibrotic effect because of the presence of PFD-COOH in plasma cannot be neglected. The plasma concentrations of both PFD and PFD-COOH were lower than effective concentrations of those in vitro. We reasoned that PFD can possibly exert antifibrotic effect in synergy with PFD-COOH in vivo. Recently, Huang et al. have reported that PFD-COOH concentration in plasma was approximately 60% of PFD concentration in plasma after oral administration to human.9) Thus, it is thought that PFD-COOH may participate in the antifibrotic effects of PFD in the treatment of IPF. On the other hands, PFD-OH pharmacokinetics in human are not well understood. The involvement of PFD-OH in the antifibrotic action of a therapy for IPF in human is thought to need examination in both pharmacodynamics and pharmacokinetics in human.
PFD and its metabolites with TGF-β1 (10 ng/mL) were applied to WI-38 cells, followed by incubation for 24 h at 37°C with 5% CO2. After incubation, the intracellular hydroxyproline amount was determined; the data are shown as the ratio of intracellular hydroxyproline to amount of DNA. Each point represents the mean±S.D. (n=4). * p<0.05 and ** p<0.01: significantly different from TGF-β1 alone.
PFD (30 mg/kg wt) was intravenously administered to rats. At each time point (2.5, 5, 10, 15, and 30 min, 1, 2, 4, and 6 h) after administration, plasma was collected, and the concentrations of PFD and its metabolites were determined for each sample. Each point represents the mean±S.D. (n=4).
PFD has many mechanisms of pharmacological action. PFD has been shown to reduce collagen I expression,13) block the proliferative effects of platelet-derived growth factor (PDGF),14) and inhibit the expression of heat shock protein (HSP) 47 on lung fibroblasts.13) In this study, the inhibition of collagen synthesis by PFD-OH and PFD-COOH were weaker than that of the parent compound (Fig. 3). Since many mechanisms contribute to fibrosis,13–15) the pharmacological effects may differ in the mechanisms of PFD and its metabolites. Final stage in idiopathic pulmonary fibrosis (IPF), deposition of the excessive extracellular matrix (collagen) occurs via multiple pathways such as tissue injury, various mediators activation, and chemokine imbalance.16) Therefore, we evaluated first the inhibitory effects on the collagen synthesis of PFD and its metabolites. Further studies in lung fibroblasts are warranted to determine the contribution of PDGF and HSP47 expression in the antifibrotic effects of PFD-OH and PFD-COOH.
PFD-COOH, a main metabolite of PFD, reduced the hydroxyproline content in lung fibroblasts, which resulted in reduced collagen synthesis in lung fibrosis. This study suggests that not only the concentration of PFD but also that of its metabolite should be considered in clinical therapy of IPF.
This work was supported by a Grant-in-Aid (No. 24790167) for Young Scientists (B) provided by Japan Society for the Promotion of Science.
