Production of Bleomycin-induced pulmonary fibrosis model miceby oropharyngeal aspiration and evaluation of the efficacy ofNintedanib○ Nahoko Matsuki，Naomi Hikawa，Kousuke Morizumi，Seiichi Katayama，
Katsuhide Nishi，Naoyuki Hironaka

Objective

Results

Idiopathic Pulmonary Fibrosis (IPF) is a progressive disease thatcauses loss of respiratory function by fibrosis. Two anti-fibroticdrugs are available in Japan, but these are not much effective.

µCT analysis
Lung aeration level*100%

Bleomycin, an anticancer drug, causes pulmonary fibrosis as aside-effect. In the present study, IPF model mice are induced byadministration of bleomycin. Nintedanib is one of the IPF drugs.
It inhibits growth and migration of fibroblasts by binding fibroblast
growth factor receptor, platelet derived growth factor receptor, andvascular endothelial growth factor receptor. In the present study,
we evaluated the efficacy of Nintedanib in the IPF model.

80%
60%
40%
20%
0%

Model 30 mg/kg 60 mg/kg 120 mg/kg

NormalNintedanib

Normally-aerated tissue
Poorly-aerated tissue

Summary in Japanese● Hounsfield Units (HU) ranges for analysis
Normally-aerated tissue: [-900, -250] HU[-250, 0] HU
Poorly-aerated tissue:

特発 性 肺 線 維症（ IPF ）は，肺の間質に線 維化が生じて呼吸不全となる疾 患であり，

Mean±SE*: p<0.05, Significant difference from Normal group (Student's t-test)

現在有用な治療薬はない．ニンテダニブは，線維芽細胞の活性化を抑制することで作用する抗線維化薬として知られている．そこで，我々は，肺線維症を引き起こすブレオマイ投与することでIPFモデルを作製し，ニンテダニブの薬効を評価した．ニンテダニブは30，60，120 mg/kgの用量を1日1回の頻度で22日間経口投与した．評価項目はµCT
解析，肺中ヒドロキシプロリン量，病理組織学的検査，SpO 2，呼吸機能検査および気管支肺胞洗浄液（BALF）中サイトカインとした．ニンテダニブ群では，モデル群と比較して，µCT解析による低通気部位の減少および病理組織学的検査による線維化の減少が認められ，ニンテダニブの抗線維化作用を捉えることができた．

Histopathological examination

Hydroxyproline
350

250150210050

Materials and Methods

**

32000

Fibrosis score

4***

300
(µg/Right lung)シン（BLM）を使用し，口腔咽頭吸引法（OPA）にてマウスの肺にBLM（1.5 mg/kg）を【Fibrosis score】

1NormalModel

30 mg/kg 60 mg/kg 120 mg/kg

Nintedanib

0Mean±SE***: p<0.001, Significant difference from Normal group(Student's t-test)

NormalModel

30 mg/kg 60 mg/kg 120 mg/kg

0: Not remarkable
1: Minimal
2: Mild
3: Moderate
4: MarkedNintedanibMean, **: p<0.01, Significant difference from Normal group (Wilcoxon test)● Animal

C57BL/6N mice, Male, 10 weeks oldNormal● Reagents

Masson Triclome staining
Blue: Collagen

Model

Bleomycin hydrochloride (BLM), Nippon Kayaku Co., Ltd.
Nintedanib ethanesulfonate, Biosynth Ltd.

● Preparation of IPF model: Oropharyngeal aspiration (OPA)

Bleomycin hydrochloride was administered tooropharynx at a dose of 1.5 mg/kg● Instruments

SpO2 (Oxygen saturation):
Pulse oximeter for laboratory animals,MouseOX® Plus (STARR Life Sciences Corp.)µCT (micro-Computed Tomography):micro-X-ray CT for laboratory animals,CosmoScan GX II (Rigaku Corp.)
CT image analysis:
Analyze 14.0 software (AnalyzeDirect, Inc.)
Respiratory function test:
FinePointe™ Resistance & Compliance(Data Sciences International Inc.)

NormalModel
Nintedanib 30 mg/kg
Nintedanib 60 mg/kg
Nintedanib 120 mg/kg

1010101010555555555530 mg/kg

SpO20714SpO2

HYP

Red:
Normally-aerated tissue
Yellow: Poorly-aerated tissue
Green: Trachea & Bronchus

1010101010

Respiratory functionµCTSpO2†††

Terminal#29p=0.058

95%

28
*90%

・ Respiratory function test
・ Bronchoalveolar lavage fluid (BALF)
・ Hydroxyproline (HYP) measurement
・ Histopathological examination

80%Day 7Day 1423Day 2030 mg/kg 60 mg/kg 120 mg/kg

ModelNintedanib

Mean±SE*: p<0.05, Significant difference from Normal group (Student's t-test)

Static Compliance [Day 22]

0.03

**

(mL/cm H2O)

(mL/cm H2O)

0.02

0.01

0.00

Normal*0.01

Normal

Model

0.00

30 mg/kg 60 mg/kg 120 mg/kg

Nintedanib

Mean±SE**: p<0.01, Significant difference from Normal group (Student's t-test)

Normal30 mg/kg 60 mg/kg 120 mg/kg

ModelNintedanib

Mean±SE*: p<0.05, Significant difference from Normal group (Student's t-test)

Quantification of cytokines
120IL-12p40 in BALF*

80

TGF-β1 in BALF

8*66080(pg/mL)

5040

60

3040Nintedanib

054
3110
Normal Model 30 mg/kg 60 mg/kg 120 mg/kg

*

22020

Eotaxin in BALF

770

100

090(pg/mL)

140(pg/mL)

Histopathological examination showed that Nintedanib
decreased fibrosis. CT image analysis revealed that thepercentage of poorly-aerated tissue was decreased by
10 percent in the Nintedanib groups. In addition, SpO2level and the minute-volume in the Nintedanib 60,
120 mg/kg groups tended to be higher than those in themodel group. In Nintedanib 60 mg/kg group, the levels of
TGF-β1, IL-12p40, and Eotaxin in BALF were decreased byapproximately 30 percent. Nintedanib was not mucheffective on the HYP level but it showed suppressive
effects on fibrosis as revealed by histopathologicalexamination and CT image analysis. In conclusion,
Nintedanib could have anti-fibrotic effects on the IPF
model.

Dynamic Compliance [Day 22]

0.02

2624

Mean±SE*: p<0.05，***: p<0.001, Significant difference from Normal group (Student's t-test)#: p<0.05, †††: p<0.001, Significant difference from Model group (Dunnett’s test)

Conclusion*2725

NormalModel
Nintedanib 30 mg/kg
Nintedanib 60 mg/kg
Nintedanib 120 mg/kg

85%

0.03

Minute Volume [Day 22]

30

***

20 21 22 Days

Nintedanib p.o., QD

120 mg/kg

Analyzed
image

100%

SpO2

60 mg/kg

Right lung

● Study schedule
OPA
BLM 1.5 mg/kg

Nintedanib(mL/min)Number ofanimals

Test group

Left lung
Histopathological
BALFexamination

CT image

Normal Model 30 mg/kg 60 mg/kg 120 mg/kg

Nintedanib

0Normal Model 30 mg/kg 60 mg/kg 120 mg/kg

Nintedanib

Mean±SE, *: p<0.05, Significant difference from Normal group (Student's t-test)