In Vivo Models - Inflammation

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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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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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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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OVA Model Control Compound Data

Total Cell Number

Differential Cell Counts

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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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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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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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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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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)


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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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)


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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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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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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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

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Macrophages

Neutrophils

Lymphocytes

CellCountsin

BAL(x104)


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MIP-2 Measurements (IL-8 Homologue)

Total Protein Measurement

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Theo

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Theo

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.0

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MIP-2(pg/ml)

(-)Con

trol

(+)C

ontrol

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phylline

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meth

asone

Theo

.+Dex.

0.0

0.5

1.0

1.5

2.0

2.5

Protein(mg/ml)

In Vivo Models - Inflammation

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