IPF model -SMC’s CRO services-
SMC Laboratories, Inc. smccro-lab.com
Ver. 2021.1 1 Current therapies for IPF
Guideline recommendation Treatment / Drug Mechanism of action Route Animal model /evidence quality Recommended in selected patients Replacement of fibrotic lung with Lung transplantation Surgery - Strongly/Low donor lung
Supplemental oxygen Reduction of hypoxemia Inhalation - Strongly/Very low
Acetylcysteine BLM (i.t.) Anti-oxidant Oral, Inhalation Weakly/Low monotherapy /prophylactic regimen
Corticosteroid + AZA + Immunosuppressant + antioxidant Oral - Weakly/Low acetylcysteine
BLM( i.v.) Anti-inflammatory + antifibrptic + Pirfenidone Oral /prophylactic and Weakly/Low-to-moderate antioxidant therapeutic regimen
BLM (i.t.) Anticoagulation Anticoagulant Oral Weakly/Very low /prophylactic regimen
Not recommended
BLM (i.t.) Bosentan Endothelin receptor antagonist Oral Strongly not/Moderate /prophylactic regimen Corticosteroid BLM (i.t.) Immunosuppressant Oral Strongly not/Very low monotherapy /prophylactic regimen Corticosteroid + Immunosuppressant Oral - Strongly not/Low immunomodulator Recombinant soluble human TNF BLM (i.t.), Silica (i.t.) Entercept SC Strongly not/Moderate receptor /prophylactic regimen Interferon-gamma Immunomodulator IV BLM (unknown) Strongly not/High 9 Source: IPF guideline from ATS/ERS/JRS/ALAT, 2010 2 Drug candidates in clinical trials
Company Drug Target/mechanism Route Period Endpoint Stage
TGF-β signaling 1)FVC Intermune Pirfenidone Oral 72 weeks P3 finished Anti-inflammatory 2)PFS, 6MWT etc. FGFR, PDGFR, 1)FVC Boehringer Ingelheim BIBF 1120 Oral 52 weeks P3 finished VEGFR 2)SGRQ, SOBQ, etc. 12 1)FVC Actelion ACT-064992 dual ERA Oral P2 finished months 1)FVC Fibrogen FG-3019 CTGF IV 48 weeks P2 2)Fibrosis area 1)Pulmonary function and safety Centocor CNTO 888 CCL2 IV 48 weeks P2 2)Clinical outcomes 1)FVC MedImmune Tralokinumab IL-13 SC 72 weeks P2 2)Safety and clinical outcomes 1)Safety and tolerability Sanofi-Aventis SAR156597 IL-13/IL-4 SC 22 weeks P2 2)FVC, DICO and dyspnea score GS-6624 182 1)PFS Gilead Sciences LOXL2 IV P2 (Simtuzumab) weeks 2)All-cause mortality 1)FVC Bristol-Myers Squibb BMS-986020 LPA1 Oral 26 weeks P2 2)Safety 1)PFS Hoffmann-La Roche Lebrikizumab IL-13 SC 2.5 years P2 2)FVC, 6MWT, etc. 7 to 10 1)PD/PK GlaxoSmithKline GSK2126458 PI3K/mTOR Oral P2 days 2)Safety and tolerability 1)Safety Biogen(Stromedix) STX-100 αvβ6 integrin SC 24 weeks P2 2)FVC, HRCT assessment, etc. 1)FVC Hoffmann-La Roche vismodegib SMO antagonist Oral Week 52 P2 2)DLCO - Promedior PRM-151 PTX-2 IV - P1
GC1008 1)Safety and tolerability Genzyme TGF-β 1, 2 and 3 IV 3 years P1 (Fresolimumab) 2)Clinical outcomes 1)Safety and tolerability ImmuneWorks IW001 Col (V) Oral 24 week P1 2)biologic effects Source: clinicaltrial.gov 9 3 Animal model for IPF: BLM-induced pulmonary fibrosis
■ Variation of the bleomycin animal model Route of Dose Advantage Disadvantage administration Direct damage to alveolar epithelial. The most common route of administration. Self-limiting response after 28 days. Intratracheal (i.t.) 1.25 - 4 U/kg Only single instillation needed. High mortality. Short time frame for disease development. High reproducibility.
1ry damage to endothelium. Not applicable in all animals. Intravenous (i.v.) 30 U/kg, twice per week Subpleural fibrosis. Long time frame to disease development.
1ry damage to endothelium. Intraperitoneal (i.p.) 15 - 35 U/kg Multiple doses required. Subpleural fibrosis.
Daily injections/micro-osmotic pump Resembles the progressive nature of fibrosis. Subcutaneous (s.c.) 150 U/kg installation required for a short time frame. Also induced skin fibrosis.
Marios A, et al., Current Opinion in Pulmonary Medicine. 17:355-361, 2011
Day 0 Day 7 Day 14 Day 21 Day 28 BLM i.t.
Direct damage Increase of Fibroblast proliferation Development of fibrosis initially to alveolar ・neutrophils Extracellular matrix synthesize (biochemically and Maximal responses epithelial cells ・lymphocytes histologically)
Inflammation phase Fibrosis phase
4 Advantages of SMC’s IPF model
■ SMC’s IPF model offers
-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
■ Performance
■ Over 80 test substances have been evaluated ■ Both small-molecule compounds and antibodies were experienced
5 Optimization of BLM model
■ Dose optimization (Body weight) ■ Uniform distribution by MicroSprayer®
Mean ± SEM
■ Survival rate (3.0 mg/kg BLM)
9 6 SMC’s advantage: Sampling scheme and technique
■ 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.
9 7 Key endpoint: Ashcroft score
■ Representative microphotographs of MT stained lung sections
Score: 1 Score: 5
x100 x400 x100 x400
Score: 3
x100 x400
Ashcroft score is x100 ■ simple method of estimating severity of pulmonary fibrosis using MT-stained section ■ widely used in pharmacological study for anti-fibrotic efficacy in BLM model
8 Key endpoint: CT scan
■ Micro-CT imaging Day 14 Day 21 Day 21 BLM i.t anterior
right left PBS PBS i.t
Lung field Lung field posterior
Region of Interest (ROI) BLM BLM i.t
Lung field Lung field :Fibrotic lesion
■ 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
9 Parameters: Time course of disease progression
■ Disease progression of BALF total cells, hydroxyproline content, Ashcroft score and fibrosis-related genes
BALF total cells Lung hydroxyproline Ashcroft score Gene expression content Collagen Type 1 gene TIMP-1 gene
400 100 4 ** 5 50 ** **
) ** **
e
s
l
y y n **
l **
i
t
t
l 80 4 40
i i
e e ** **
t
t
300 o
r 3
)
c
r
n
n
o
g
4
p
a
a
c ** n
0
y
s
u u 60 3 u 30
l 1 **
x
t
q
q
t
x
f
o
f ( 200 2
r
o
e
e
l
e
r
l
d
l
v
v
/
c
i
e
i y 40 2 20
g
t
t
h
c
h
µ a
a
l
s
( l
l
g
a
e
e 100 A 1
t n 1
o 20 R 10
R
u
T
L 0 0 0 0 0
l
l
l
l
l
4 1
4 1
4 1
4 1
4 1
7
7
7
7
o
7
o
o
o
o
1 2
1 2
1 2
1 2
1 2
r
r
r
r
r
y
y
t
t
y
t
y
t
y
t
y y
y y
y y
y y
y y
a
a
a
a
n
a n
n
n
n
a a
a a
a a
a a
a a
o
D
o
D
o
D
o
D
o
D
D D
D D
D D
D D
D D
C
C
C
C
C BLM BLM BLM BLM BLM
■ 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) 10 Parameters: BALF cytokine levels
■ BALF TGF-β and TIMP-1 at Day 21
BDL: below detection limit
■ TGF- and TIMP-1 proteins, fibrosis makers, are increased in BLM model (Day 21)
11 Prophylactic positive control: Dexamethasone
■ Study design
Day 0 Day 7 Day 14 Day 21
acclimation Oral, QD BLM + Vehicle (n= 10) Group 1 BLM
acclimation Oral, QD BLM + dexamethasone 0.25 mg/kg (n= 10) Group BLM 2
BLM: Bleomycin, Oral: Oral administration, QD: Once daily Sacrifice
■ Analyses
1. Biochemistry 2. Histopathological analyses • Lung hydroxyproline content • MT staining (Ashcroft score)
12
Dexamethasone: Ashcroft score and lung hydroxyproline Vehicle
x100 x16
Mean ± SEM Dexamethasone x100 x16
■ 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.
13 Therapeutic positive control: Nintedanib
■ Study design
Day 0 Day 7 Day 14 Day 21
acclimation Oral, QD BLM + Vehicle (n= 10) Group 1 BLM
acclimation Oral, QD BLM + Nintedanib 100 mg/kg (n= 10) Group BLM 2
BLM: Bleomycin, Oral: Oral administration, QD: Once daily Sacrifice
■ Analyses
1. Biochemistry 2. Histopathological analyses • Lung hydroxyproline content • MT staining (Ashcroft score)
14 Nintedanib: Ashcroft score and lung hydroxyproline
P<0.001 n.s
4 80 Vehicle
e
e
)
r 3 60
n
i x100 x16 g
l
o
n
o
c
r
u
s
l
p
t
t
f 2 y f 40
o
x e
r
L
o
/
c
r
g
h
d
µ
s
y
(
A 1 20
H
0 0
Nintedanib Vehicle Nintedanib Vehicle Nintedanib
Mean ± SEM x100 x16
■ 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.
15 Study design example: BLM I.T. study
Alveolar inflammation
Alveolar epithelial damage Fibrosis
Day 0 Day 7 Day 14 Day 21 Saline i.t.
acclimation (n= 12) Group Control 1 BLM i.t. Treatment (oral/ i.v./ i.p./ s.c) (n= 12) Group BLM + Vehicle 2 Treatment (oral/ i.v./ i.p./ s.c) (n= 12) Group BLM + Test substance 3 (n= 12) Treatment (oral) Group BLM + Positive control 4
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
Animal model: Bleomycin (BLM)-induced pulmonary fibrosis model Computed tomography ■Animal: C57BL/6J mice, female, week 7-8 (day 21) MT staining (day 21) ■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 Homogeneity of it Sampling technique ■Treatment period: 2 or 3 weeks ■Endpoints (day 21): The grade of fibrosis: Ashcroft score (MT staining), Lung hydroxyproline content
■ 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