Ver. 2021.1 1

IPF model-SMC’s CRO services-

smccro-lab.com

SMC Laboratories, Inc.

29Source: IPF guideline from ATS/ERS/JRS/ALAT, 2010

Treatment / Drug Mechanism of action Route Animal modelGuideline recommendation

/evidence quality

Recommended in selected patients

Lung transplantationReplacement of fibrotic lung with

donor lungSurgery - Strongly/Low

Supplemental oxygen Reduction of hypoxemia Inhalation - Strongly/Very low

Acetylcysteine

monotherapyAnti-oxidant Oral, Inhalation

BLM (i.t.)

/prophylactic regimenWeakly/Low

Corticosteroid + AZA +

acetylcysteineImmunosuppressant + antioxidant Oral - Weakly/Low

PirfenidoneAnti-inflammatory + antifibrptic +

antioxidantOral

BLM( i.v.)

/prophylactic and

therapeutic regimen

Weakly/Low-to-moderate

Anticoagulation Anticoagulant OralBLM (i.t.)

/prophylactic regimenWeakly/Very low

Not recommended

Bosentan Endothelin receptor antagonist OralBLM (i.t.)

/prophylactic regimenStrongly not/Moderate

Corticosteroid

monotherapyImmunosuppressant Oral

BLM (i.t.)

/prophylactic regimenStrongly not/Very low

Corticosteroid +

immunomodulatorImmunosuppressant Oral - Strongly not/Low

EnterceptRecombinant soluble human TNF

receptorSC

BLM (i.t.), Silica (i.t.)

/prophylactic regimenStrongly not/Moderate

Interferon-gamma Immunomodulator IV BLM (unknown) Strongly not/High

Current therapies for IPF

39Source: clinicaltrial.gov

Company Drug Target/mechanism Route Period Endpoint Stage

Intermune PirfenidoneTGF-β signaling

Oral 72 weeks1)FVC

P3 finishedAnti-inflammatory 2)PFS, 6MWT etc.

Boehringer Ingelheim BIBF 1120FGFR, PDGFR,

VEGFROral 52 weeks

1)FVCP3 finished

2)SGRQ, SOBQ, etc.

Actelion ACT-064992 dual ERA Oral12

months

1)FVCP2 finished

Fibrogen FG-3019 CTGF IV 48 weeks1)FVC

P22)Fibrosis area

Centocor CNTO 888 CCL2 IV 48 weeks1)Pulmonary function and safety

P22)Clinical outcomes

MedImmune Tralokinumab IL-13 SC 72 weeks1)FVC

P22)Safety and clinical outcomes

Sanofi-Aventis SAR156597 IL-13/IL-4 SC 22 weeks1)Safety and tolerability

P22)FVC, DICO and dyspnea score

Gilead SciencesGS-6624

LOXL2 IV182

weeks

1)PFSP2

(Simtuzumab) 2)All-cause mortality

Bristol-Myers Squibb BMS-986020 LPA1 Oral 26 weeks1)FVC

P22)Safety

Hoffmann-La Roche Lebrikizumab IL-13 SC 2.5 years1)PFS

P22)FVC, 6MWT, etc.

GlaxoSmithKline GSK2126458 PI3K/mTOR Oral7 to 10

days

1)PD/PKP2

2)Safety and tolerability

Biogen(Stromedix) STX-100 αvβ6 integrin SC 24 weeks1)Safety

P22)FVC, HRCT assessment, etc.

Hoffmann-La Roche vismodegib SMO antagonist Oral Week 521)FVC

P22)DLCO

Promedior PRM-151 PTX-2 IV --

P1

GenzymeGC1008

TGF-β 1, 2 and 3 IV 3 years1)Safety and tolerability

P1(Fresolimumab) 2)Clinical outcomes

ImmuneWorks IW001 Col (V) Oral 24 week1)Safety and tolerability

P12)biologic effects

Drug candidates in clinical trials

4

Inflammation phase Fibrosis phase

BLM i.t.Day 0 Day 7 Day 14 Day 21 Day 28

Direct damageinitially to alveolar epithelial cells

Fibroblast proliferation Extracellular matrix synthesize Maximal responses

Development of fibrosis(biochemically and histologically)

Increase of ・neutrophils・lymphocytes

Marios A, et al., Current Opinion in Pulmonary Medicine. 17:355-361, 2011

■ Variation of the bleomycin animal model

Route of

administrationDose Advantage Disadvantage

Intratracheal (i.t.) 1.25 - 4 U/kg

Direct damage to alveolar epithelial.

The most common route of administration.

Only single instillation needed.

Short time frame for disease development.

High reproducibility.

Self-limiting response after 28 days.

High mortality.

Intravenous (i.v.) 30 U/kg, twice per week1ry damage to endothelium.

Subpleural fibrosis.

Not applicable in all animals.

Long time frame to disease development.

Intraperitoneal (i.p.) 15 - 35 U/kg1ry damage to endothelium.

Subpleural fibrosis.Multiple doses required.

Subcutaneous (s.c.) 150 U/kgResembles the progressive nature of fibrosis.

for a short time frame.

Daily injections/micro-osmotic pump

installation required

Also induced skin fibrosis.

Animal model for IPF: BLM-induced pulmonary fibrosis

5

-Sampling scheme allowing all analyses in a single mouse (histology, gene expression,

biochemistry and BALF analysis)

-Bleomycin-induced model optimized for evaluation of fibrosis severity, body weight loss

and mortality

-Increased reproducibility and uniform phenotype enabled by the use of Microsprayer

-Expert histology based on the knowledge of fibrotic/inflammatory diseases

-In-life evaluation of fibrotic lesions by CT scan as in clinical studies

- Availability of positive control – Dexamethasone and Nintedanib

■ SMC’s IPF model offers

■ Performance

■ Over 80 test substances have been evaluated

■ Both small-molecule compounds and antibodies were experienced

Advantages of SMC’s IPF model

69

Mean ± SEM

■ Uniform distribution by MicroSprayer®■ Dose optimization (Body weight)

■ Survival rate (3.0 mg/kg BLM)

Optimization of BLM model

79

■ After BALF collection, left and post-caval lobe bronchus are ligated to avoid leakage of the instilled fixative. Then, three

fixed lobes (for histological analyses) and two unfixed lobes (for gene expression analysis and collagen assay) are

harvested from each mouse.

SMC’s advantage: Sampling scheme and technique

8

Score: 1

x100 x400

x100 x400

Score: 3

x100

x400

Score: 5

Ashcroft score is

■ simple method of estimating severity of pulmonary fibrosis using MT-stained section

■ widely used in pharmacological study for anti-fibrotic efficacy in BLM model

x100

■ Representative microphotographs of MT stained lung sections

Key endpoint: Ashcroft score

9

Day 14 Day 21

PB

S i.t

BLM

i.t

:Fibrotic lesion

right left

anterior

posterior

Region of Interest (ROI)

■ High resolution CT (HRCT) scan shows increasing interstitial fibrotic lesions in the same mice

■ Hounsfield unit (fibrotic density) of the ROI is increased in BLM model time-dependent manner

Day 21 BLM i.t

Lung field Lung field

Lung field Lung field

■ Micro-CT imaging

Key endpoint: CT scan

10

■ BALF total cells were increased at inflammatory phase (Day7) and early fibrosis phase (Day 14), then decreased at late fibrosis

phase (Day21)

■ Lung hydroxyproline content significantly increased at late fibrosis phase (Day 21)

■ Ashcroft score were increased as well as hydroxyproline content

■ Collagen Type 1 gene were increased as well as Ashcroft score and hydroxyproline content and TIMP-1 gene were increased at

fibrosis phase (Day 14 and Day 21)

■ Disease progression of BALF total cells, hydroxyproline content, Ashcroft score and fibrosis-related

genes

Parameters: Time course of disease progression

Co

ntr

ol

Day 7

Day 1

4

Day 2

1

0

100

200

300

400

To

tal c

ell

(x1

04 c

ells

)

**

**

BLM

Co

ntr

ol

Day 7

Day 1

4

Day 2

1

0

1

2

3

4

5

Rela

tive q

uan

tity

**

BLM

****

Co

ntr

ol

Day 7

Day 1

4

Day 2

1

0

10

20

30

40

50

Rela

tive q

uan

tity

**

BLM

**

TIMP-1 geneCollagen Type 1 gene

Co

ntr

ol

Day 7

Day 1

4

Day 2

10

20

40

60

80

100L

un

g h

yd

rox

yp

rolin

e(µ

g/le

ft lu

ng

)**

BLM

Co

ntr

ol

Day 7

Day 1

4

Day 2

1

0

1

2

3

4

As

hc

roft

sc

ore

**

**

**

BLM

BALF total cells Lung hydroxyproline

content

Ashcroft score Gene expression

11

■ TGF- and TIMP-1 proteins, fibrosis makers, are increased in BLM model (Day 21)

BDL: below detection limit

Parameters: BALF cytokine levels

■ BALF TGF-β and TIMP-1 at Day 21

12

Day 21Day 0 Day 7

(n= 10) Group

1BLM + Vehicle

acclimation

(n= 10) Group

2BLM + dexamethasone 0.25 mg/kg

acclimation

Oral, QD

Oral, QD

Sacrifice

BLM

BLM

BLM: Bleomycin, Oral: Oral administration, QD: Once daily

2. Histopathological analyses

• MT staining (Ashcroft score)

1. Biochemistry

• Lung hydroxyproline content

Day 14

■ Study design

■ Analyses

Prophylactic positive control: Dexamethasone

13

x16x100

Ve

hic

leD

ex

am

eth

as

on

e

■ Ashcroft score in the dexamethasone group significantly decreased compared to the BLM-Vehicle group.

■ The lung hydroxyproline content significantly decreased in the dexamethasone group compared to the BLM-

Vehicle group.

Mean ± SEM

x16x100

Dexamethasone: Ashcroft score and lung hydroxyproline

14

Day 21Day 0 Day 7

(n= 10) Group

1BLM + Vehicle

acclimation

(n= 10) Group

2BLM + Nintedanib 100 mg/kg

acclimation

Oral, QD

Oral, QD

Sacrifice

BLM

BLM

BLM: Bleomycin, Oral: Oral administration, QD: Once daily

Day 14

■ Study design

■ Analyses

Therapeutic positive control: Nintedanib

2. Histopathological analyses

• MT staining (Ashcroft score)

1. Biochemistry

• Lung hydroxyproline content

n.s

Vehicle Nintedanib0

20

40

60

80

Hy

dro

xy

pro

line

(µg

/Le

ft lu

ng

)

P<0.001

Vehicle Nintedanib0

1

2

3

4

As

hc

roft

sc

ore

15

x16x100

Ve

hic

leN

inte

da

nib

■ Ashcroft score in the Nintedanib group was significantly decreased compared to the BLM-Vehicle group.

■ The lung hydroxyproline content did not change in Nintedanib group compared to the BLM-Vehicle group.

Mean ± SEM

x16x100

Nintedanib: Ashcroft score and lung hydroxyproline

16

Computed tomography

(day 21)

Homogeneity of it Sampling technique

Day 21Day 14Day 7 Saline i.t.

Day 0

BLM i.t.Control

(n= 12) Group

1

acclimation

BLM + Positive control(n= 12)

Group

4

Treatment (oral)

Alveolar epithelial damage

BLM + Test substance(n= 12)

Group

3

Treatment (oral/ i.v./ i.p./ s.c)

BLM + Vehicle(n= 12) Group

2

Treatment (oral/ i.v./ i.p./ s.c)

Alveolar inflammation

Fibrosis

Animal model: Bleomycin (BLM)-induced pulmonary fibrosis model■Animal: C57BL/6J mice, female, week 7-8

■Induction of pulmonary fibrosis: BLM i.t. (microspray) at day 0

Study design

■Aim of the study: To investigate the therapeutic efficacy of test substance on pulmonary fibrosis

■Route of administration: oral/ i.p./ i.v./ s.c.

■Arm: 4

control, vehicle, test substance, positive control (Dexamethasone or Nintedanib)

■The number of mice/group before dosing: n=12

■Baseline: day 0 (just prior to BLM inhalation) and day 6

■Randomization: Body weight at day 0 or day 6

■Treatment period: 2 or 3 weeks

■Endpoints (day 21):

The grade of fibrosis: Ashcroft score (MT staining), Lung hydroxyproline content

<Analytical items>

BAL: cell number, ELISA

Gene expression in the lung

CT (option)

General condition

MT staining (day 21)

Moore BB et al., Am J Physiol Lung Cell Mol Physiol. 2008 (294); L152-60

Kai Y et al., Med Mol Morphol. 2007 (40); 128-40

Study design example: BLM I.T. study

Publications and Presentations

■ Presentations

4. ERS International Congress 2018, “CHIT1 is a novel therapeutic target in idiopathic pulmonary fibrosis

(IPF): anti-fibrotic efficacy of OATD-01, a potent and selective chitinase inhibitor in the mouse model of

pulmonary fibrosis” OncoArendi Therapeutics SA

3. American Thoracic Society 2017 International Conference, “Resokine Modulates Immune Cell

Infiltration into the Lung and Provides Therapeutic Activity in a Bleomycin-Induced Lung Fibrosis Model”

aTyr Pharma, Inc.

2. The 18th International Colloquium on Lung and Airway Fibrosis, “MN-001 (tipelukast), a

nonselective phosphodiesterase, 5-lipoxygenase, leukotriene, phospholipase C and thromboxane A2

inhibitor, demonstrates anti-fibrotic effects in the Bleomycin-induced idiopathic pulmonary fibrosis mouse

model” MediciNova, Inc.

1. American Thoracic Society 2014 International Conference, “Solithromycin Reduces Inflammation In

Mice Caused By Bleomycin-Induced Lung Injury” Cempra Inc.

17