October 20131525

Communication to the Editor Biol. Pharm. Bull. 36(10) 15251527 (2013)

Possible Involvement of Pirfenidone for IPF.

Metabolites in the Antibrotic Action
MATERIALS AND METHODS
of a Therapy for Idiopathic Pulmonary
Fibrosis Materials and Animals PFD was purchased from Tokyo
Chemical Industry Co., Ltd. (Tokyo, Japan). PFD-OH and
Kohei Togami,a,b Yukimune Kanehira,b and PFD-COOH were purchased from Toronto Research Chemi-
Hitoshi Tada*,a,b cals Inc. (North York, ON, Canada). TGF-1 was purchased
a from Peprotech Inc. (Rocky Hill, NJ, U.S.A.). All other re-
Division of Pharmaceutics, Hokkaido Pharmaceutical University
agents were commercially available and of analytical grade.
School of Pharmacy; 71 Katsuraoka-cho, Otaru, Hokkaido
0470264, Japan: and bDepartment of Biopharmaceutics, School Male SD rats, 8 weeks of age and 230270 g body weight,
of Pharmaceutical Science, Ohu University; 311 Misumido, were purchased from CLEA Japan, Inc. (Tokyo, Japan). The
Tomita-Machi, Koriyama, Fukushima 9638611, Japan. care and use of animals followed The Guidelines for the
Received June 7, 2013; accepted July 25, 2013 Care and Use of Animals approved by Ohu University in ac-
cordance with the principles of the NIH guidelines (Approval
Pirfenidone (PFD) is the rst and only clinically used number: 201250).
antibrotic drug for the treatment of idiopathic pulmonary Antibrotic Experiments in Vitro WI-38 cells (Riken
brosis (IPF). This study evaluated the antibrotic effects of Gene Bank, Tsukuba, Japan), a human lung broblast cell
two metabolites of PFD, 5-hydroxypirfenidone (PFD-OH) and line, were maintained in Dulbeccos modied Eagles medium
5-carboxypirfenidone (PFD-COOH), on WI-38 cells in an in
(DMEM) containing 10% heat-inactivated fetal bovine serum
vitro lung broblast model. The inhibitory effects of PFD-OH
and PFD-COOH on transforming growth factor-1 (TGF-1)- (FBS) and 40 g/mL gentamicin in a humidied atmosphere
induced collagen synthesis in WI-38 cells were evaluated by of 5% CO2 at 37C. Cells from passage numbers 1213 were
measuring intracellular hydroxyproline, a major component seeded (2.0104 cells/well) on 24-well culture plates. At con-
of the protein collagen. PFD-OH and PFD-COOH at 300 and fluence, the medium was replaced with DMEM containing
1000 M concentrations signicantly decreased the TGF-1- 0.4% FBS and 50 g/mL of ascorbate. After a 24-h incubation,
induced hydroxyproline content in WI-38 cells. These results transforming growth factor (TGF)-1 (10 ng/mL) and serial
indicate that PFD-OH and PFD-COOH have antibrotic concentrations of PFD, PFD-OH, and PFD-COOH were added
activities, which inhibit collagen synthesis in broblasts. This to the WI-38 cells, and subsequently the cells were incubated
study suggests that the concentrations of PFD and its metabo- at 5% CO2 at 37C for 24 h. After incubation, the medium was
lites should be considered in clinical therapy for IPF.
removed by aspiration and washed twice with ice-cold phos-
Key words pirfenidone; 5-hydroxypirfenidone; 5-carboxypir- phate buffered saline (PBS). The cells were then extracted
fenidone; idiopathic pulmonary brosis; lung broblast with 300 L of 2 M NaCl, and the concentration of hydroxypro-
line in the cell extracts was measured by HPLC as described
Fibrotic diseases occur in various tissue regions and can below. The DNA concentration in the cell extracts was de-
resemble scar tissue when they form in inappropriate loca- termined using Fluorescent DNA Quantitation Kit (Bio-Rad,
tions such as lung, liver, heart, eye, and kidney. In particular, Hercules, CA, U.S.A.).
idiopathic pulmonary brosis (IPF) is devastating with an Pharmacokinetics Experiment in Vivo PFD dissolved
extremely low ve-year survival rate (<50%).1) Currently, re- in PBS was intravenously administered to rats at a dose of
searchers are working to develop an antibrotic drug that will 30 mg/kg and the dosage volume was 1 mL/kg. At each desig-
improve the survival rate of patient with IPF.
Pirfenidone (PFD, Fig. 1), 5-methyl-1-phenyl-2-(1H)-pyri-
done, is the rst and only clinically used antibrotic drug for
the treatment of IPF in Japan (Pirespa), Europe (Esbriet),
and India (Pirfenex).2) PFD has antibrotic, anti-inflamma-
tory, and antioxidative actions.2,3) In experimental animal
models, PFD has demonstrated an antibrotic effect in several
tissues, such as lung, liver, and kidney.46) 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 antibrotic
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 antibrotic action of a therapy
Fig. 1. Chemical Structure of Pirfenidone (PFD) and Its Metabolites
The authors declare no conflict of interest. (PFD-OH and PFD-COOH)

 To whom correspondence should be addressed. e-mail: h-tada@hokuyakudai.ac.jp

* 2013 The Pharmaceutical Society of Japan
1526
Fig. 2. Effects of PFD and Its Metabolites on TGF-1 Stimulated Increases in Hydroxyproline in WI-38 Cells
PFD and its metabolites with TGF-1 (10 ng/mL) were applied to WI-38 cells, followed by incubation for 24 h at 37C 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 meanS.D. (n=4).
* p<0.05 and ** p<0.01: signicantly different from TGF-1 alone.
nated 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 HPLC The con-
centrations 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 (3.0250 mm internal diameter;
GL Sciences, Inc., Torrance, CA, U.S.A.). The mobile phase
was 85 m M acetic buffer (pH 4.3)acetonitrile (68 : 32). Separa-
tion was performed at a flow rate of 0.4 mL/min at 45C, 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 HPLC
The 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
(3.0250 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 45C and the
column was monitored by UV absorbance detection (absor-
bance wavelength of 310 nm).
Statistics Statistical analysis was performed using the
Dunnetts t-test and SPSS software version 21 (IBM Inc., Ar-
monk, NY, U.S.A.).
RESULTS AND DISCUSSION
The present study evaluated the antibrotic 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 broblast cell line, are
Vol. 36, No. 10
shown in Fig. 2. PFD at 100 M (18.5 g/mL), 300 M (55.6 g/
mL), and 1000 M (185 g/mL), and PFD-OH at 300 M
(60.4 g/mL) and 1000 M (201 g/mL), and PFD-COOH at
300 M (64.6 g/mL) and 1000 M (215 g/mL) signicantly
decreased the TGF-1-induced hydroxyproline content in
WI-38 cells without cellular toxicity. PFD and its metabolites
were stable at 37C in medium for 24 h (data not shown).
These results indicate that PFD-OH and PFD-COOH have
antibrotic activities, which inhibit collagen synthesis in lung
broblasts. 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 0.740.12, 0.790.26, and 0.840.26 h, respectively. The
areas under the concentrationtime curves (AUC) for PFD,
PFD-OH, and PFD-COOH were 30.5, 3.9, and 24.2 g*h/mL,
respectively. These results indicate that the antibrotic 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 antibrotic
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 antibrotic effects of PFD in
the treatment of IPF. On the other hands, PFD-OH pharmaco-
kinetics in human are not well understood. The involvement
of PFD-OH in the antibrotic action of a therapy for IPF in
human is thought to need examination in both pharmacody-
namics and pharmacokinetics in human.
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 broblasts.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 mecha-
nisms contribute to brosis,1315) the pharmacological effects
October 20131527
Fig. 3. Time Courses of the Concentrations of PFD and Its Metabolites
in Plasma after Intravenous Administration to Rats
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 col-
lected, and the concentrations of PFD and its metabolites were determined for each
sample. Each point represents the meanS.D. (n=4).
may differ in the mechanisms of PFD and its metabolites.
Final stage in idiopathic pulmonary brosis (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 evalu-
ated rst the inhibitory effects on the collagen synthesis of
PFD and its metabolites. Further studies in lung broblasts are
warranted to determine the contribution of PDGF and HSP47
expression in the antibrotic effects of PFD-OH and PFD-
COOH.
CONCLUSION
PFD-COOH, a main metabolite of PFD, reduced the hy-
droxyproline content in lung broblasts, which resulted in
reduced collagen synthesis in lung brosis. This study sug-
gests that not only the concentration of PFD but also that of
its metabolite should be considered in clinical therapy of IPF.
Acknowledgment This work was supported by a Grant-
in-Aid (No. 24790167) for Young Scientists (B) provided by
Japan Society for the Promotion of Science.
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