in vivo models - inflammation
TRANSCRIPT
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In vivo Disease Models
Inflammation
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In vivo models of Inflammation
SB Drug Discovery
Asthma
OVA-challenged Mouse Asthma Model
The OVA-challenged asthma mouse is the industry gold standardmodel used to assess pulmonary inflammation. Inhalation of OVAhas been demonstrated to induce an immune response that isassociated with increased airway hyper-responsiveness (AHR). Inaddition this model exhibits a number of other featurescharacteristic of human asthma including, cellular infiltration intothe lungs, increase in eosinophil numbers, and elevated levels of
inflammatory cytokines in BAL fluid. We include dexamethasone (acorticosteroid known to decrease inflammation) as a positive controlin this procedure.
SB can evaluate small molecule inhibitors in this model of airwayinflammation. Details of our protocol, the endpoints to be measured
and control data for theophilline, roflumilast and dexamethasoneare described below.
OVA Model Protocol
We use 6 weeks old male Balb/c mice, which are sensitised to OVAby two intra-peritoneal injections, 12 days apart (n=5 per group).Ten to eighteen days later, mice are challenged three times with anaerosol of 1% OVA in sterile saline for 30 minutes. Non-challenged
mice are exposed to aerosolised saline only. Test compounds areadministered orally for the final 10 days of the sensitisationprocedure. Each drug is administered at a dose of 3mg/Kg (or asdirected by the client). We include Dexamethasone as a positive
control in this procedure. To measure airway hyper-responsiveness
we calculate the change in enhanced pause (Penh) in response tochallenge with methacholine.
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In vivo models of Inflammation
SB Drug Discovery
The schematic below summarizes the approach taken to test
compounds in our in vivo asthma model:
21 22 23 24 25 26 27 28 29 30 31
Drug administration - oral
Day 0 12
OVA - IP
1% OVA - Nebulised
Drug Administration
Drug administration is normally by oral delivery and is carried out
over a 10-day period beginning on day 21. Study groups typicallycomprise of 6 animals per treatment group.
Negative Control Group
Non OVA-challenged mice receiving saline solution tomeasure base level of inflammatory markers in current animalbatch.
Two Positive Control GroupsOVA-challenged mice receiving saline solution to measureallergy induced pulmonary inflammation.
OVA-challenged mice receiving dexamethasone to measurethe effect of a known anti-inflammatory agent on allergyinduced pulmonary inflammation.
3 Compound Test Groups (or 3 doses of 1 compound)OVA-challenged mice receiving high dose of compound.
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In vivo models of Inflammation
SB Drug Discovery
Parameters Measured
Asthma Endpoints
This asthma in vivo model is used to undertake a comprehensiveevaluation of the effect of lead compounds. Animals will besacrificed 24 hours after final OVA challenge. The following
endpoints are measured:
1) AHR: Via Buxco whole body plethysmograph. This is a keyfeature of asthma and this method allows us to assess AHR non-
invasively in unrestrained and conscious mice.
2) Cellular Infiltration: We can determine the total cell numberrecovered from BAL fluid (by either haemocytometer or coultercounter). The total cell counts in the BAL is taken as an indicator of
inflammatory cell influx into the lung from the surrounding bloodnetwork. An increase in total cell number may be indicative ofinflammation and the data is used in conjunction with thedifferential cell counts. By differentially counting the cells retrievedfrom the lung (in BAL fluid) we can determine the specificinflammatory cell populations present and thus distinguish theinflammatory response. We measure macrophages, eosinophils andleukocyte numbers. An increase in eosinophil number would be
indicative of sensitisation and by relating this to total cell numbers,a true measurement can be gained of the inflammatory response.
3) Total Protein Levels in BAL Fluid: We measure the total proteinlevels obtained in the BAL fluid. This is used as a marker of whetherthe test compound evokes epithelial damage or inhibits micro-vascular leakage.
4) Option to measure Cytokine Levels in BAL Fluid: Themeasurement of primary BAL fluid for biochemical markers is one ofthe most important tools for understanding the mechanism of lung
inflammation, as any markers measured are those directly releasedinto the lumen of the airways to elicit an inflammatory response.
BAL fluid from each mouse will be tested in duplicate for IL-5 andIgE.
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In vivo models of Inflammation
SB Drug Discovery
OVA Model Control Compound Data
Total Cell Number
Differential Cell Counts
-veC
ontro
l
+veC
ontro
l
Theo
phyll
ine1m
g/Kg
Theo
phyll
ine3m
g/Kg
Theo
phy.
10mg/Kg
Theo
phyl.
30mg
/Kg
DEX
3mg/kg
0
20
40
60
Totalcellcounts(x104)
-veC
ontro
l
+veC
ontro
l
Roflu
m.0
.3mg/kg
Roflu
m.1
.0mg/kg
Roflu
m.3m
g/kg
Roflu
m.10m
g/kg
Dex3
mg/kg
0
25
50
75
Totalcellcounts(x104)
-vec
ontrol
+vec
ontrol
Theo
1mg/kg
Theo
3mg/kg
Theo
10mg
/kg
Theo
30mg
/kg
Dexa
metha
sone
0
10
20
30
40
Eosinophils(x104)
-veC
ontrol
+veC
ontrol
Roflu
m.0.3m
g/kg
Roflu
m.1.0m
g/kg
Roflu
m.3m
g/kg
Roflu
m.10mg
/kg
Dex3
mg/kg
0
10
20
30
40
50
Eosinophils(x104)
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In vivo models of Inflammation
SB Drug Discovery
IL-5 Measurement
-veC
ontro
l
+veC
ontro
l
Theo
1mg/k
g
Theo
3mg/k
g
Theo
10mg
/kg
Theo
30mg
/kg
Dex3
mg/kg
0
100
200
300
IL-5(pg/ml)
-veC
ontro
l
+veC
ontro
l
Roflu
m.0
.3mg/k
g
Roflu
m.1
.0mg/k
g
Roflu
m.3m
g/kg
Roflu
m.10m
g/kg
Dex3
mg/k
g0
250
500
750
1000
IL
-5(pg/ml)
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In vivo models of Inflammation
SB Drug Discovery
Acute Lung Injury
Acute LPS model
The acute LPS model is the first stage model for Type 1inflammation (neutrophil driven). This model enables the study ofanti-inflammatories and their potential to inhibit neutrophil
trafficking. The acute nature of this model means that specific time-points after LPS challenge can be studied (e.g 2, 4, 24, 48, 72
hours) to assess at what stage of the inflammatory pathway thecompound works best.
Model Overview
We have established a mouse model of acute lung injury showingpulmonary neutrophilia. Intra-tracheal instillation oflipopolysaccharide (LPS) results in an acute, Th1-type inflammatoryresponse in these mice. Cytokines such as TNF- and CXC
chemokines such as MIP-2 and KC are involved in the trafficking ofneutrophils from the circulation into the alveolar spaces. Pulmonaryoedema, as measured by an increase in lung water content, occurs
following this inflammatory response. The client would receive alung profile of cellular, biomchemical and histological markers, usingdexamethasone as a reference compound. We can also measure IP
neutrophil accumulation.
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In vivo models of Inflammation
SB Drug Discovery
Parameters Measured
1) Cellular Infiltration: We will determine the total cell numberrecovered from BAL fluid (by either haemocytometer or coultercounter). The total cell counts in the BAL is taken as an indicator ofinflammatory cell influx into the lung from the surrounding blood
network. An increase in total cell number may be indicative ofinflammation and the data is used in conjunction with thedifferential cell counts. By differentially counting the cells retrievedfrom the lung (in BAL fluid) we can determine the specific
inflammatory cell populations present and thus distinguish theinflammatory response. We measure macrophages, eosinophils,neutrophils and leukocyte numbers.
2) Total Protein Levels in BAL Fluid: We measure the total protein
levels obtained in the BAL fluid. This is used as a marker of whetherthe test compounds evoke epithelial damage or inhibits micro-vascular leakage.
3) Option to measure Cytokine Levels in BAL Fluid: The
measurement of primary BAL fluid for biochemical markers is one ofthe most important tools for understanding the mechanism of lung
inflammation, as any markers measured are those directly releasedinto the lumen of the airways to elicit an inflammatory response.BAL fluid from each mouse will be tested in duplicate for TNF-alpha,MIP-2 and IL-6.
4) Option to measure pulmonary edema by water content (only
relevant in longer time-points such as 48-72 hours)
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In vivo models of Inflammation
SB Drug Discovery
Control Compound Data
Total cell number:
MIP2 (IL-8 homologue)
Time Course Of Inflammation
salin
e
2H
ours
5H
ours
24Hou
rs
48Hou
rs
72Hou
rs
0
25
50
75
100
125
150
TC
NinBAL(x104)
Time Course Of Inflammation
salin
e
2H
ours
5H
ours
24Hou
rs
48Hou
rs
72Hou
rs
0
250
500
750
1000
MIP-2inBAL(pg/ml)
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In vivo models of Inflammation
SB Drug Discovery
Neutrophil Cell number:
Protein:
Time Course Of I nflammation
salin
e
2H
ours
5H
ours
24Hou
rs
48Hou
rs
72Hou
rs
0
25
50
75
100
125
150
Neu
trophilnumbersinBAL(x104)
Time Course Of Inflammation
salin
e
2H
ours
5H
ours
24Hou
rs
48Hou
rs
72Hou
rs
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
TotalproteininBAL(mg/ml)
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In vivo models of Inflammation
SB Drug Discovery
COPD
Chronic LPS Model
SBs COPD murine model is based on the model described byVernooy et al, 2002 (Am J Res Mol Biol (2002) 26: 152-159). This
model, in which mice were exposed to multiple intratrachealinstillations of E. coli LPS, was originally developed to study the
pathological effects of long-term LPS exposure to the lung.Repeated intratracheal LPS instillation in these mice results in
persistent chronic pulmonary inflammation with altered cytokineexpression, accompanied by airway and alveolar alterations. Theobserved inflammatory and pathological changes mimic alterations
observed in humans with chronic pulmonary inflammatory
disorders, especially COPD.
This model has the following key characteristics relevant to COPD:
Accumulation of macrophages
Increase in TNF-alpha, IFN-gamma and IL-18 mRNA levelsbut not IL-6
Increase in mucus producing cells
Metaplasia of airway mucus producing cells in larger airways
Thickened airway walls
Alveolar enlargement
Active repair process interrupted
The dose of LPS delivered (5g) is equivalent to smoking
approximately 25 cigarettes. Therefore, in contrast with smokingmodels, the amount of insult added is known.
The model shows irreversible lung damage, in contrast to someshorter exposure LPS models. There are pathological changes in theairways and parenchyma characterised by mucus producing cellmetaplasia and airway wall thickening, as well as irreversiblealveolar enlargement. Significant inflammatory cell influx is alsoobserved.
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In vivo models of Inflammation
SB Drug Discovery
Method
Chronic inflammation is induced by giving mice a non surgicalintratracheal instillation of LPS (2 doses per week) for a total of 12weeks. Test compounds can be added anytime during the study butwe recommend dosing take place during the last 2 weeks (5 dosesper week). After 12 weeks, there is a 1-week rest period beforemice are sacrificed.
Parameters Measured
Using this COPD model, we have the ability to measure thefollowing endpoints:
1) BAL Fluid: Measured for a variety of inflammatory mediators,
typical of COPD pathology, including IL-8 (as MIP-2); IL-6; TNF-alpha and other cytokines/biochemical parameters as required.
2) Cellular Infiltrates: We will perform differential cell counts on theBAL.
3) Protein levels: Determine total protein levels in BAL fluid.
4) AHR: We can investigate airway mechanics using Buxco WBPtechnology, measure regularly to determine changes in lungvolumes.
5) Histology: We can undertake histopathological analysis of thelung following treatment with the compound(s) from 2 mice pergroup (all mice of all groups can be performed on request). Leftlung embedded and sections taken for histological analysis. We willprovide all slides once the study is complete (1 H&E stained slide +2 unstained slides).
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In vivo models of Inflammation
SB Drug Discovery
Control Compound Data
Here we provide data on theophylline and Dexamethasone treated
groups in the LPS COPD murine model.
We use male CD-1 mice (12 weeks old) and repeatedly challengewith 5g/50l (per mouse) LPS (055:B5) by intratracheal instillationtwice a week for a total of 12 weeks. The animals are allowed a restperiod on the 13th week. Drugs/compounds for evaluation are
administered on week 11 and 12. Animals are then sacrificed andinvestigated for signs of COPD-like symptoms and markers. Micemay also be monitored throughout this process by using the Buxco
lung function screen.
Whole Cell Number
-veco
ntrol
+veco
ntrol
Theo
phyllin
e
Dexa
metha
sone
The
o.+Dex
.0
20
40
60
80
100
120
Macrophages
Neutrophils
Lymphocytes
CellCountsin
BAL(x104)
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In vivo models of Inflammation
SB Drug Discovery
MIP-2 Measurements (IL-8 Homologue)
Total Protein Measurement
-veC
ontro
l
+veC
ontro
l
Theo
phyll
ine
Dexa
meth
ason
e
Theo
.+Dex
.0
10
20
30
40
MIP-2(pg/ml)
(-)Con
trol
(+)C
ontrol
Theo
phylline
Dexa
meth
asone
Theo
.+Dex.
0.0
0.5
1.0
1.5
2.0
2.5
Protein(mg/ml)