Shinshu Med J, 67⑶：183～195, 2019

Effects of Subtype-selective E Prostanoid Receptor Agonists on
Bleomycin-induced Pulmonary Fibrosis in Rats
Takashi Ichiyama1）＊, Hiroshi Yamamoto1） and Kazuhiro Hongo2）
1） First Department of Internal Medicine, Shinshu University School of Medicine
2） Department of Drug Discovery Science, Shinshu University School of Medicine

Background : Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with limited treatment options and a poor
prognosis. In vitro research has shown that prostaglandin (PG) E2 can suppress pulmonary fibrosis via cAMP
production. EP2 and EP4, which are subtypes of the receptors for PGE2, are involved in cAMP production. The
present study was designed to examine the effects of EP2 and EP4 agonists on bleomycin (BLM)-induced pul­monary fibrosis in rats.
Materials and Methods : The EP2 and EP4 agonists were subcutaneously administered to BLM-induced pulmo­nary fibrosis rats for 21 days. The lung weight, mRNA expressions of transforming growth factor (TGF)-β1 andprocollagen genes, and degree of pulmonary fibrosis were compared between EP2 agonist, EP4 agonist, vehicle,
and pirfenidone and nintedanib administered groups. We also examined the EP2 and EP4 expressions in humanlung tissues with IPF and in rat lung tissues with BLM-induced pulmonary fibrosis by immunohistochemical
staining. The human lung tissues with IPF were obtained from autopsy cases.
Results : The EP2 agonist significantly suppressed the lung weight gain and inhibited the mRNA expressions of
TGF-β1 and procollagen genes. In addition, the fibrosis scores and hydroxyproline content tended to be lower
in the EP2 agonist administered group. However, the EP4 agonist did not show such evidence in suppression offibrosis. The enhanced expressions of EP2 and EP4 were demonstrated in both human and rat lung tissues withfibrosis relative to those in normal lung.
Conclusions : The EP2 agonist may become a novel therapeutic agent for IPF. Shinshu Med J 67 : 183―195,
2019(Received for publication October 3, 2018 ; accepted in revised form January 24, 2019)
Key words : pulmonary fibrosis, E prostanoid receptor agonists, cyclic adenosine monophosphate,transforming growth factor-β1, bleomycin-induced pulmonary fibrosis

Ⅰ Introduction

these drugs significantly improved the standardized
mean difference of change in forced vital capacity1）2）,

Idiopathic pulmonary fibrosis (IPF) is a specificrecent meta-analysis did not show significant im­

form of chronic, progressive, fibrosing, interstitial

provement in mortality3）. Development of new anti-

pneumonia of unknown causes and the prognosis isfibrotic drugs is expected to improve the prognosis

very poor. Currently, pirfenidone and nintedanib areof patients with IPF. Prostaglandin (PG) E2, the majorclinically used in Japan for its treatment. Althoughprostanoid in the lung, is an important antifibrotic
lipid mediator. PGE2 levels were reduced in the lungs＊

Corresponding author : Takashi Ichiyama
First Department of Internal Medicine, Shinshu University
School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano
390-8621, Japan
E-mail : ichiyama@shinshu-u.ac.jp

No. 3, 2019of patients with IPF4）. Preventive effects of PGE2 on
pulmonary fibrosis have been studied in an animalmodel, bleomycin (BLM)-induced pulmonary fibro­sis5）6）. Administration of PGE2 to BLM-induced pul­
183

Ichiyama・Yamamoto・Hongo

monary fibrosis in mice suppressed collagen deposi­
5）
6）

agonist and EP4 agonist were synthesized by Kissei. It was demonstrated

Pharmaceutical Co. Ltd (Hotaka, Japan). The struc­that administration of PGE2 increased cAMP leveltural formula of the EP2 agonist is shown in Fig. 1awith preventive effects of myofibroblast differentia­

and that of the EP4 agonist in Fig. 1b. The EC50 oftion and inhibition of extracellular membrane pro­each compound was calculated as follows. HEK293T

tion and reduced mortality

7）

duction of pulmonary fibroblasts . Four E prostanoid

cells forcibly expressing rat EP2 and EP4 were stim­

receptor (EP) subtypes (EP1, EP2, EP3 and EP4) haveulated with EP2 agonist, EP4 agonist and PGE2 andbeen identified as receptors for PGE2. EP2 and EP4 areanalyzed according to the protocol of cAMP screen­

coupled to Gs protein and can increase the intracellular

ing system (Thermo Fisher Scientific K. K.), using

8）9）cAMP level . We have developed an EP2 agonist and
10）

GraphPad Prism (GraphPad Software, California, USA)as candidates for novel antifibrotic

analyzed concentration effect data. Pharmacokinetics,

agents. In the present study, we examined the phar­plasma protein binding of the EP2 agonist and EP4

macological effects of the EP2 agonist and the EP4

agonist in SD rats were obtained in studies conductedagonist on BLM-induced pulmonary fibrosis in rats.

by Kissei Pharmaceutical Co. Ltd and are shown in

We speculate that the expressions of EP2 and EP4 in

Fig. 1c, d.

lung tissues of rats with pulmonary fibrosis are fun­２

an EP4 agonist

Study protocol

damentaly required in the therapeutic target of the

The experimental design is shown in Fig. 2. The

EP2 agonist and EP4 agonist on IPF. The immuno­

10-week rats were divided into 6 groups of 8 rats

histological staining was performed in lung tissues of

each. After intraperitoneal administration of mixed

human IPF patients as well as of BLM-induced pulmo­

anesthesia (1 mg/kg midazolam, 0.2 mg/kg medeto­nary fibrosis in rats in order to confirm the expressions

midine, and 2.5 mg/kg butorphanol), the rats wereof EP2 and EP4 in lung tissues. The present study was

intratracheally administered with 2.2 mg/kg BLM

designed to evaluate the pharmacological potential

dissolved in 100 μL PBS. The sham control rats re­of the EP2 and EP4 agonists on human IPF.

ceived intratracheally PBS only. On the next day,Ⅱ

Materials and Methods

Ａ Efficacy evaluation of EP2 and EP4 agonists in
BLM-induced pulmonary fibrosis rats
１

Animals and reagents
All experimental procedures were conducted ac­

cording to Shinshu University Animal Experimental

the administration of drugs, 0.3 mg/kg EP2 agonist(BLM＋EP2), 0.3 mg/kg EP4 agonist (BLM＋EP4), 30
mg/kg pirfenidone (BLM＋pirfenidone), and 1 mL/kg vehicle (50 ％ PEG400, 50 ％ DMSO) (BLM＋vehi­cle), were performed by subcutaneous injection
three times a day, and 50 mg/kg nintedanib (BLM＋nintedanib) was administered orally once a day.

Rules, reviewed according to the guidelines of the

The dosage of EP2 agonist and EP4 agonist were

National Regulation and Animal Care Committee,

set at 0.3 mg/kg subcutaneous injection 3 times per

and approved by the Shinshu University Animal Re­

day based on the pharmacokinetics and EC50 data.

search Committee (Authorization number : 270038,

The AUC for pirfenidone 600 mg oral single dose

approval date : July 1st, 2016). The 48 Wister-Hann(single dose in clinical practice) to adult males, de­

9-week rats were purchased from Japan CRLs. We

scribed in the interview form11）, was 37.03 μg・hr/mL

purchased BLM from Nippon Kayaku (Tokyo, Japan),and Cmax was 10.57 μg/mL. The AUC determined

midazolam from Fuji Pharma Co. (Tokyo, Japan), me­by a single dose administration of pirfenidone 30 mg/

detomidine from Kyoritsuseiyaku Co. (Tokyo, Japan)

kg for S/D rats conducted by Kissei Pharmaceuticaland butorphanol from Meiji Seika Pharma Co. (Tokyo,

Co. Ltd. was 33.79 μg・hr/mL and Cmax was 23.39.

Japan). We purchased pirfenidone from Tokyo

Because blood concentrations in clinically applied doses

Chemical Industry Co. (Tokyo, Japan) and nintedanibin human were maintained, the dosage and dose offrom eNOvation Chemicals LLC (NJ, USA). The EP2

pirfenidone in the present study was set to 30 mg/kg

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Effect of selective EP agonists on lung fibrosis in rats

c）

a）

d）

b）
Fig. 1ａｂｃｄStructural formula of EP2 agonist
Structural formula of EP4 agonist
Pharmacokinetic study of EP2 and EP4 agonists in rats
Functional assay of transient expression in HEK293T cellFig. 2
Study design for examining the antifibrotic effects of the EP2 and EP4 agonistson bleomycin (BLM)-induced pulmonary fibrosis rats.
No. 3, 2019

185

Ichiyama・Yamamoto・Hongo

subcutaneous injection three times a day.
The dosage of nintedanib was set according to themethod in the study for BLM-induced pulmonary
12）

fibrosis in rats performed by Wollin et al.

droxyproline content was measured by the assay kit(Chondrex Inc, WA, USA) following measurement
protocol.
６ mRNA expressions of the TGF-β1, procollage­n1a1 (Col1a1), and Col1a2 genes

Body weight measurements were taken daily be­fore the first dose. We euthanized the rats by ab­

The total RNA was extracted from right lung tis­

dominal aortic dissection under deep anesthesia onsues using a RNeasy Kit (Qiagen, Valencia, CA, USA)day 21 of the administration of intervention agents.

according to the manufacturer’s designated regimen.

We removed both lungs to evaluate the efficacy of

We synthesized cDNA from a 100ng total RNA tem­intervention by the agents.

plate via reverse transcription by using a Prime Script

３

RT Enzyme Mix I (TakaraBio Inc., Japan).

Measurement of lung weight
The right lung was removed and the weight was

The mRNA levels were quantified using the qRT-

measured. The lung specimens were quickly frozen

PCR system (Applied Biosystems StepOnePlus ;with liquid nitrogen and stored at －30 ℃ until the

Thermo Fisher Scientific K.K.). The cDNAs of themeasurements of hydroxyproline content and quan­rat Col1a1, Col1a2, and TGF-β1 were amplified fromtitative real-time polymerase chain reaction (qRT-

single-stranded cDNA by PCR using Taq DNA

PCR).

Polymerase with 0.4 μM of each primer. The PCR

４primers of the rat TGF-β1, Col1a1, Col1a2 genes, andHistological analysis
The left lung was removed, fixed by 10 ％ neu­

18S rRNA are described in Table 1. The qRT-PCR

tral-buffered formaldehyde solution and embedded

was performed using 10 μmol samples consisting ofin paraffin. The sections were stained with Masson’s

200 nM of each primer, 12.5 μL of SYBR premix Ex

trichrome and examined by light microscopy. The

Taq (TakaraBio), and 10 ng of template cDNA.

fibrosis score was evaluated using the modified

The experimental PCR protocol required 30 s of

Ashcroftʼs score13）. The lung section was examined

an initial denaturation at 96 ℃ followed by 40 cycles

at a magnification of 100×, and each field was visu­

of 5 s of denaturation at 96 ℃, 30 s of annealing at 60 ℃,

ally scored from 0 points (normal lung) to 8 points

and 60 s of extension at 60 ℃.

(complete obstruction with fibroma mass) under lightＢ Confirmation of EP2 and EP4 expressions in thelung tissues

microscopy. The average score was calculated withall the observed fields for an entire lung.
５

Measurement of hydroxyproline content

１

Rat lung tissues
The lung tissues of rats with administration of PBS

A sample of right lung tissue was homogenizedintratracheally were used as negative control. The

with 1,000 mg of lung tissue in 1 mL of PBS. Hy­lung tissues of rats in BLM＋vehicle group were used

Table 1

186

The primer sequence of cDNA used in the present study

cDNA, (ID)primers

sequence

TGF-β1

forward5’-CATTGCTGTCCCGTGCAGA-3’(RA059245)

reverse5’-AGGTAACGCCAGGAATTGTTGCTA-3’

Col1a1

forward5’-CTGCATCAGGGTTTCAGAGCAC-3’(RA069752)

reverse5’-TCCACATGCTTTATTCCAGCAATC-3’

Col1a2

forward5’-CTGGATTGACCCTAACCAAGGATG-3’(RA069679)

reverse5’-TTGACAGGTTGGGCCGGA-3’

18S rRNA

forward5’-AAGTTTCAGCACATCCTGCGAGTA-3’(RA015374)

reverse5’-TTGGTGAGGTCAATGTCTGCTTTC-3’
Shinshu Med J

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Effect of selective EP agonists on lung fibrosis in rats

for the indication of pulmonary fibrosis.

only normal lung findings were observed (Fig. 3a).

２

The pathological findings were improved in BLM＋EP2

Human lung tissues
Approval regarding using human lung materials(Fig. 3c) and BLM＋nintedanib (Fig. 3f) relative toin this study was obtained from the Shinshu Univer­those in BLM＋vehicle (Fig. 3b). On the other hand,sity Ethics committee (Authorization number : 2730,

there was no pathological improvement in BLM＋EP4

approval date : May 12th, 2014).

(Fig. 3d) and BLM＋pirfenidone (Fig. 3e).

The lung tissues with pulmonary fibrosis were

The mean fibrosis scores in groups of the shamobtained from autopsy cases of IPF in 2008 and 2009.

control, BLM＋vehicle, BLM＋EP2, BLM＋EP4, BLM

The normal lung tissues for the control were ob­＋pirfenidone, and BLM＋nintedanib were 0.07±0.06,

tained from resected lung lobes after lung lobectomy

3.10±0.75, 2.88±0.74, 3.41±0.60, 3.62±0.81, and 2.64

performed for the treatment of lung cancer in the±0.59, respectively. The mean fibrosis scores of the

Department of Thoracic Surgery of Shinshu Univer­

BLM＋EP2 and BLM＋nintedanib were lower thansity. Tissues were fixed in formalin and embeddedthat of the BLM＋vehicle ; however, no statistical signifi­

in paraffin.

cance was observed. (Fig. 4a).

３

２

Immunohistochemical staining

Effects on lung weights

We used Histofine SAB-PO kit (Nichirei Bioscienc­

The average lung weight of each experimental

es Inc., Tokyo, Japan) for immunostaining and per­group is shown in Fig. 4b. The lung weight of BLM

formed the procedure following the protocol. The＋vehicle was significantly higher than that of the

SAB-PO kit contains biotin-labeled anti-mouse IgGsham control. The average lung weights of BLM＋

＋IgA＋IgM rabbit antibody as a second antibody

EP2 and BLM＋nintedanib were significantly lower

and 3, 3-diaminobenzidine (DAB) as a chromogenic

than that of BLM＋vehicle. However, the EP4 andsubstrate. The anti-EP2 rabbit monoclonal antibody

pirfenidone did not show effects on the lung weights(ab 167171, Abcam, Cambridge, UK) was diluted atof rats with BLM-induced pulmonary fibrosis.

1 : 1,250 with PBS and the anti-EP4 rabbit polyclonal

３

antibody (ab 133170, Abcam) was diluted at 1 : 50

Hydroxyproline content in lung tissues
The hydroxyproline content in lung tissue is shownwith PBS and used as the primary antibody.

in Fig. 4c. The content was 22.7±13.2 μg/mL in theＣsham control, whereas it was increased to 85.3±52.8

Statistical analysis
Data were expressed as the mean±standard devi­

μg/mL in the BLM＋vehicle. The hydroxyproline

ation (SD) and processed by using GraphPad Prism 7

content was slightly suppressed to 71.0±43.6 μg/mL(GraphPad Software, Inc. USA). The significance ofin the BLM＋EP2 ; however, there was no significant

differences between each group was evaluated bydifference between the BLM＋EP2 and BLM＋vehi­

one-way ANOVA with post-hoc tests. A value of P

cle. No suppressive effect was observed in the other＜0.05 was considered to indicate statistical signifi­

groups, in which the hydroxyproline contents werecance.

93.2±61.4 μg/mL in the BLM＋EP4, 96.0±52.1 μg/
Ⅲ

Results

Ａ Efficacy evaluation of the EP2 and EP4 agonists
in the BLM-induced pulmonary fibrosis rats
１ Histopathological effects of the intervention

mL in the BLM＋pirfenidone, and 85.0±52.0 μg/mL
in the BLM＋nintedanib.
４ The mRNA expressions of TGF-β1, Col1a1, andCol1a2 genes
The TGF-β1 mRNA was significantly increased incompounds

BLM＋vehicle compared to that in the sham control.

The alveolitis and patchy fibrosis with destruction

On the other hand, it was significantly suppressed inaround the bronchus (representing the part surround­

BLM＋EP2, BLM＋EP4, BLM＋pirfenidone and BLM

ed by the red ellipse) were observed in the rat BLM-

＋nintedanib compared with that in BLM＋vehicle

induced lung fibrosis model. In the sham control,(Fig. 5a).

No. 3, 2019187

Ichiyama・Yamamoto・Hongo

Fig. 3
Histological observation of the left lung (Masson trichrome ; original magnification ×20).
ａ PBS only, as a sham control ｂ BLM＋vehicleｃ BLM＋EP2 agonistｅ BLM＋pirfenidone ｆ BLM＋nintedanib.
ｄ BLM＋EP4 agonist

The expressions of Col1a1 mRNA were significantly

suppressed in BLM＋EP2, BLM＋pirfenidone, andenhanced in BLM＋vehicle compared with those in

BLM＋nintedanib compared with that in BLM＋ve­

the sham controls. The mRNA expression of Col1a1

hicle (Fig. 5c).

gene was significantly suppressed in BLM＋EP2 andＢ Confirmation of EP2 and EP4 expressions in the

BLM＋pirfenidone compared with that in BLM＋ve­
hicle (Fig. 5b). The expression of Col1a2 mRNA was
increased in BLM＋vehicle compared to those in thelung tissues
１ Immunostaining of EP2 and EP4 in lung tissuesof experimental rats

sham controls without significant difference. The

The immunostaining showed very strong expres­mRNA expression of Col1a2 gene was significantly

sions of the EP2 (Fig. 6a) and EP4 (Fig. 6c) in the

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No. 3, 2019ｃ Hydroxyproline contents in 1 gram of right lung tissue

vehicle and ＊＊＊indicates p＜0.001 vs. BLM＋vehicle.

ｂ The right lung weight of experimental rats : ＊indicates p＜0.05 vs. BLM＋vehicle, ＊＊indicates p＜0.005 vs. BLM＋

Fig. 4ａ Histological score by modified Aschoff`s score method : ＊＊＊＊indicates p＜0.0001 vs. BLM＋vehicle group

Effect of selective EP agonists on lung fibrosis in rats

189

190

vehicle and ＊＊＊indicates p＜0.001 vs. BLM＋vehicle

Fig. 5ａ The mRNA expression of TGF-β1
ｂ The mRNA expression of procollagen 1a1ｃ The mRNA expression of procollagen 1a2 : ＊indicates p＜0.05 vs. BLM＋vehicle, ＊＊indicates p＜0.005 vs. BLM＋Ichiyama・Yamamoto・Hongo

Shinshu Med J

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Effect of selective EP agonists on lung fibrosis in rats

Fig. 6
Immunostaining of rat lung ; original magnification ×100.
ａ The EP2 of BLM-induced pulmonary fibrosis rat
ｂ The EP2 of rat administered with PBS i.t.
ｃ The EP4 of BLM-induced pulmonary fibrosis ratｄ The EP4 of rat administered with PBS i.t. The parts with EP2 expression reacted with DAB andshowed a brown color. The parts without expression of EP2 and EP4 exhibited a blue color.

lung tissues of rats with BLM-induced pulmonarycAMP. Increased intracellular cAMP levels were re­fibrosis compared to those in the negative control

ported to suppress the inductive activity of TGF-β(Fig. 6b, d).

to attenuate fibroblast migration14）. TGF-β1 is a key

２ Immunostaining of EP2 and EP4 in human lungcytokine whose sustained production causes the de­of IPFvelopment of tissue fibrosis. Repeated injury with

The immunostaining showed that the EP2 wascontinued autoinduction of TGF-β1 overrides the

positive in human IPF tissues (Fig. 7a) but negative

normal termination signals, thus creating a chronic

in normal lung tissues (Fig. 7b). On the other hand,vicious circle of TGF-β1 overproduction15）. In the pre­the EP4 showed weak expression in the alveolar epi­

sent study, the mRNA production of TGF-β1 genethelium of IPF tissues (Fig. 7c), but negative expres­was suppressed by the administration of the EP2sion in the normal lung tissues (Fig. 7d).

and EP4 agonists, suggesting a hindrance of the

Ⅳ Discussion

vicious cycle of TGF-β1 overproduction. The sup­pressions of EP2 on the expressions of mRNA ofAmong the four subtypes of the PGE2 receptor,

TGF-β1 gene and procollagen gene in the BLM-

EP2 and EP4 are coupled to G protein to increaseinduced pulmonary fibrosis rats were equivalent to

No. 3, 2019191

Ichiyama・Yamamoto・HongoａｂｃFig. 7
Immunostaining of human lung ; original magnification ×100.
The EP2 was expressed in human lung of IPF with a brown color.
The EP2 was not expressed in human normal lung tissue with a blue color.
The EP4 was expressed in human lung of IPF, d) The EP4 was not expressed in normal lung tissue.

the suppression of nintedanib in the rats with pul­

was calculated by the average score of about 100

monary fibrosis. Our study reproduced the same

fields under light microscopic observation for eachresults for BLM＋nintedanib as those reported byrat. The fact is that the pathological parts of the fi­

12）

Wollin . Despite the significant reductions in mRNA

brosis were not evenly distributed on the whole lungof TGF-β1 and procollagen genes, there was no sig­

fields, with focus on some of the lung fields and scat­nificant difference in the mean fibrosis scores andtering on the other fields. Thus, the significant dif­hydroxyproline contents between the BLM＋EP2

ferences in the fibrosis score vanished with the av­

and BLM＋vehicle groups.

erage calculation due to the large SD in the statistics.

A plausible explanation is the problem of the

The reason for no significant difference of hydroxy­evaluation method. In the present study, the pulmo­

proline contents between the BLM＋EP2 and BLM

nary fibrosis in rats was induced by intratracheal＋vehicle groups was similar. In addition, the weightadministration of BLM. Thus, the fibrosis was re­of the entire right lung was significantly lower inmarkably developed around the bronchus. The BLM

BLM＋EP2 and BLM＋nintedanib than in BLM＋vehi­＋vehicle formed strong fibrosis around the bronchi­cle. The weight of lung with fibrosis is heavier thanole, which was obviously improved with the adminis­that of normal lung. We believe that antifibrosis was

tration of EP2 and nintedanib. The fibrosis scoreachieved in the BLM＋EP2 and BLM＋nintedanib

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Effect of selective EP agonists on lung fibrosis in rats

administration of pirfenidone in BLM-induced pul­groups.
In addition, significant differences in the Col1a1

monary fibrosis.

mRNA expression and different tendencies of fibrosis

The present study demonstrated that EP2 andwere observed between the rats treated with EP2

EP4 were expressed in human and rat lung tissues

and EP4 agonists. EP4 was found to have inducedwith fibrosis but not normal lung tissues, which aredesensitization and internalization by stimulation ofnoteworthy new findings in pulmonary fibrosis. The

16）

PGE2 . Therefore, by stimulation with the same

expression of the receptors suggested that EP2 andamount of PGE2, the expression of cAMP in EP2-

EP4 are related to the development of pulmonary

expressing cells was observed to be 7-fold as com­

fibrosis. However, we found that EP4 over-expression

17）

pared with EP4-expressing cells . The production

was not apparent compared with that of EP2. The

of cAMP inhibits collagen synthesis and preventslack of preventive effects of EP4 agonist on pulmo­

differentiation of fibroblasts into probiogenic myofi­

nary fibrosis could be related the low expression in18）

broblasts . Therefore, the inhibitory effects of EP2/

the lung. However, we could show that the EP2 ago­

4 agonists on the procollagen mRNA can be explain­nist contributes to the prevention of fibrosis in an

ed. We speculate that a possible role of the cAMP level

experimental model. We need further studies using

may be one of the causes of the difference in theexperimental models and clinical samples ; however,efficacy between the EP2 and EP4 agonists in thethe present study indicates that the EP2 agonist

present study. Moreover, it has been reported that

may be an effective therapeutic agent for IPF.

another signaling pathway, which is independent ofConflicts of Interest

cAMP, could be present in EP416）19）20）.
Pirfenidone is clinically applied to treat patientswith IPF. In the present study, pirfenidone failed to

The authors have no conflicts of interest in thepresent study.

reduce the BLM-induced pulmonary fibrosis. Schaefer

Acknowledgments

et al. reviewed experiments on the effects of pirfeni­done on the animal model of BLM-induced pulmo­nary fibrosis

21）

The authors would like to thank our researchand stated in summary that the anti-

colleagues : Dr. Hiroyuki Owaki, Dr. Makoto Moro

22）
-25）

.

and Dr. Hideki Ohnota in the Department of Drug

In addition, pirfenidone was administered by oral

Discovery Science of Shinshu University School ofadministration in these studies. Recently, it was re­Medicine, Dr. Tomoya Onozato and Dr. Morimichi

ported that oral administration of 50 mg/kg pirfenidone

Hayashi in the Research Division, Safety Research

could reduce BLM-induced pulmonary fibrosis inLaboratory of Kissei Pharmaceutical Co., Ltd., Dr.

fibrotic effect was mainly found in mice and hamsters

26）

rats . In the present study, pirfenidone was admin­

Yasushi Takigawa and Mr. Toshihiro Nishimura inistered to rats by 30 mg/kg subcutaneous injection

the Research Division in the Central Research Labo­

three times a day. We believed that the dose would

ratories of Kissei Pharmaceutical Co., Ltd., Mrs. Hito­

be approximately equal to that when orally adminis­mi Imamura and Mrs. Yunden Droma in the First

tered to humans in clinical practice. However, our

Department of Internal Medicine of Shinshu Univer­dose and animal setting or administration route may

sity School of Medicine, and Mr. Naoyuki Tatsumi

be related to the lack of preventive effects on BLM-

and Dr. Toshihito Hiroi in the Pharmacology andinduced pulmonary fibrosis in the present study. We

Pharmacokinetics Research Lab of Kissei Pharma­need further experiments at various doses and oral

ceutical Co., Ltd.

No. 3, 2019193

Ichiyama・Yamamoto・Hongo

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