Aragen Bioscience is a contract research organization that offers a range of in vitro and in vivo research services including protein production, antibody discovery, stable cell line development, and preclinical testing. They have 25,000 square feet of laboratory space in the San Francisco Bay Area and a team of highly skilled scientists, most holding PhDs. The document then describes a bleomycin-induced lung fibrosis mouse model where bleomycin is administered to induce pulmonary fibrosis over 2-3 weeks. Outcome measures include body weight, lung weight, BAL cell counts, and histopathology. Pirfenidone is presented as a positive control, showing it can partially rescue body weight loss and reduce lung weight and BAL cell infiltration compared to bleomycin alone

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Lung Fibrosis Model
Representative Studies

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In Vitro
Testing
Protein
Production
In Vivo
Testing
Stable
Expression
Bench Scale
PD
2
Antibody
Discovery

3. 3
• Bleomycin has been mostly used in the chemical induction of
pulmonary fibrosis in several species and is most prevalent in rodent
models.
• As a tissue injury and repair model of fibrosis, bleomycin has
contributed significantly to studies of the pathobiology of pulmonary
fibrosis
• Induction of disease is highly reproducible
and produces a fibrotic histologic symptoms
similar to human disease.
• The induction and progression of the
disease in rodents is of a short duration
making it a practical model for evaluating
test compounds in preclinical research
Introduction
Lung Fibrosis Model

4. Study Summary
• Study animals: Male C57B/L6
• Fibrosis induction: Clinical grade Bleomycin administered
via oralpharyngeal route
• Duration: Two-three weeks
• Treatment: Test compounds and/or Pirfenidone as positive
control
• Routes of administration: Oral
• Other options for route of administration: IV, SC, IM, IP
4
Lung Fibrosis Model

5. Lung Fibrosis Symptoms:
• Daily body weights
• Daily monitoring for general
activity level
• Survival
• Lung harvest
• Leukocytes count in BAL fluid
• Lung histology
5
Other Optional Readouts:
• Flexivent-lung function
measurements
• Hypoxia related parameters
• Whole-body plethysmography
• Sircol collagen assay
• Serum analysis
• Others: Organ harvest for
histopathology
Fibrotic Symptoms
Lung Fibrosis Model

6. 6
Pirfenidone as Positive Control in
Bleomycin Induced Lung Fibrosis

7. Pirfenidone Data
7
Body Weight Analysis
• Pre-treat animals on day 0
with Bleomycin.
• Vehicle or Pirfenidone
treatment on days 7-21
(oral BID).
• Bleomycin treatment
causes body weight loss.
• Prifenidone treatment for
14 days partially rescues
body weight loss.0 2 4 6 8 10 12 14 16 18 20
-4
-2
0
2
4
Study Day
PercentChange+/-SEM
Percent Change in Body Weight
No BLM
BLM + Vehicle
BLM + Pirfen

8. 8
• Average lung weights of animals in Bleomycin only exposed groups are
increased compared to untreated animals.
• Average lung weight is reduced in Pirfenidone treated animals.
Pirfenidone Data
Lung Weight Analysis
0
1
2
3
NormalizedWeight(+/-SEM)
Normalized Lung Weight
Day 21
No BLM BLM +
Vehicle
BLM +
Pirfen
P=0.0068

9. 9
• Bleomycin only treatment
increases leukocyte counts in
BAL fluid.
• Pirfenidone treatment of
Bleomycin exposed animals
results in BAL cell counts
similar to naïve animals.
Pirfenidone Data
BAL Leukocyte Analysis
0
2×105
4×105
6×105
CellCounts+/-SEM
Leukocyte Count in BAL
No BLM BLM +
Vehicle
BLM +
Pirfen

10. 10
• Significant macrophages infiltration is seen in Bleomycin only exposed
animals which is alleviated with Pirfenidone treatment.
• Pirfenidone treatment of Bleomycin exposed animals results in BAL cell
counts similar to naïve animals.
Pirfenidone Data
Differential BAL Leuocyte Analysis
M
acrophages
M
onocytesN
eutrophillsEosinophillsLym
phocytes
0
50000
100000
150000
200000TotalCellCount(+/-SEM)
BAL Leukocyte Counts
No BLM
BLM + Vehicle
BLM + Pirfen

11. 11
• Bleomycin only treatment
increases Ashcroft score as
compared to naïve animals.
• Pirfenidone treatment of
Bleomycin exposed animals
results in a reduction of Ashcroft
scores.
Pirfenidone Data
Histological Analysis
-2
0
2
4
6
Ashcroftscore(0-8)
Ashcroft Score
No Bleo Bleo +
Pirfen
Bleo +
Vehicle

12. 12
• Bleomycin administration induces lung fibrosis in C57B/L6
mice.
• Induction of lung fibrosis by Bleomycin administration
results in increased lung weight and leukocyte infiltration
into lungs compared to normal animals.
• Histopathology of tissues confirms development of lung
fibrosis in Bleomycin induced animals.
• Histopathology of tissues confirms that Pirfenidone
treatment of Bleomycin induced animals reverses lung
fibrosis.
Pirfenidone Data
Conclusions

13. 13
Case Study: Small Molecule
(Compound X)

14. 14
• Therapeutic Study: Bleomycin day 0 with compound treatment on days 7-
21 (oral QD).
• Test compound reduces Bleomycin induced increase in lung weights and
leukocyte counts.
Therapeutic Candidate Data
Lung Weight-BAL Leukocyte Analysis
0.0
0.5
1.0
1.5
2.0
2.5
NormalizedWeight(+/-SEM)
Normalized Lung Weight
+BLM
CmpdX
(mpk)
+ + + +--
-- 60 30 10 3--
0
2×105
4×105
6×105
CellCounts+/-SEM
Average Leukocyte Counts in BAL
+BLM
CmpdX
(mpk)
+ + + +--
-- 60 30 10 3--

15. 15
• Test compound reduces Ashcroft Scores when given
therapeutically or prophylactically.
Therapeutic Candidate Data
Histological Analysis
Average Ashcroft Score
(n=10/group)
Ashcroftscore
Study
1
Study
2
Study
3
(pro)
Study
3
(ther)
Study
4
Study
5
Study
6
0
1
2
3
4
5
No BLM
BLM + Vehicle
BLM/60 mpk Cmpd X
BLM/30 mpk Cmpd X
BLM/10 mpk Cmpd X
BLM/3 mpk Cmpd X

16. PBS
(Ashcroft score = 0)
Bleo/veh
(Ashcroft score 7)
Bleo/60 mpk Cmpd X
(Ashcroft score 1)
Bleo/60 mpk Cmpd X
(Ashcroft score 2)
Bleo/60 mpk Cmpd X
(Ashcroft score 3)
Bleo/60 mpk Cmpd X
(Ashcroft score 4)
Bleo/veh
(Ashcroft score 6)
Therapeutic Candidate Data
Histological Analysis

17. 17
Test compound reduces lung fibrosis in Bleomycin
induced C57/Bl6 mouse model.
– Reduced lung weights
– Reduced leukocyte counts in BAL
– Reduced Ashcroft score
Therapeutic Candidate Data
Histological Analysis

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