In vivo Disease Models Inflammation

Asthma
OVA-challenged Mouse Asthma Model
The OVA-challenged asthma mouse is the industry gold standard model used to assess pulmonary inflammation. Inhalation of OVA has been demonstrated to induce an immune response that is associated with increased airway hyper-responsiveness (AHR). In addition this model exhibits a number of other features characteristic of human asthma including, cellular infiltration into the lungs, increase in eosinophil numbers, and elevated levels of inflammatory cytokines in BAL fluid. We include dexamethasone (a corticosteroid known to decrease inflammation) as a positive control in this procedure. SB can evaluate small molecule inhibitors in this model of airway inflammation. Details of our protocol, the endpoints to be measured and control data for theophilline, roflumilast and dexamethasone are described below.

OVA Model Protocol

We use 6 weeks old male Balb/c mice, which are sensitised to OVA by two intra-peritoneal injections, 12 days apart (n=5 per group). Ten to eighteen days later, mice are challenged three times with an aerosol of 1% OVA in sterile saline for 30 minutes. Non-challenged mice are exposed to aerosolised saline only. Test compounds are administered orally for the final 10 days of the sensitisation procedure. Each drug is administered at a dose of 3mg/Kg (or as directed 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 to challenge with methacholine.

In vivo models of Inflammation SB Drug Discovery

The schematic below summarizes the approach taken to test compounds in our in vivo asthma model:

OVA - IP

Drug administration - oral

Day 0

12

21 22

23 24 25 26 27 28 29 30

31

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 typically comprise of 6 animals per treatment group. Negative Control Group Non OVA-challenged mice receiving saline solution to measure base level of inflammatory markers in current animal batch. Two Positive Control Groups OVA-challenged mice receiving saline solution to measure allergy induced pulmonary inflammation. OVA-challenged mice receiving dexamethasone to measure the effect of a known anti-inflammatory agent on allergy induced pulmonary inflammation. 3 Compound Test Groups (or 3 doses of 1 compound) OVA-challenged mice receiving high dose of compound.

In vivo models of Inflammation SB Drug Discovery

Parameters Measured
Asthma Endpoints This asthma in vivo model is used to undertake a comprehensive evaluation of the effect of lead compounds. Animals will be sacrificed 24 hours after final OVA challenge. The following endpoints are measured: 1) AHR: Via Buxco whole body plethysmograph. This is a key feature of asthma and this method allows us to assess AHR noninvasively in unrestrained and conscious mice. 2) Cellular Infiltration: We can determine the total cell number recovered from BAL fluid (by either haemocytometer or coulter counter). The total cell counts in the BAL is taken as an indicator of inflammatory cell influx into the lung from the surrounding blood network. An increase in total cell number may be indicative of inflammation and the data is used in conjunction with the differential cell counts. By differentially counting the cells retrieved from the lung (in BAL fluid) we can determine the specific inflammatory cell populations present and thus distinguish the inflammatory response. We measure macrophages, eosinophils and leukocyte 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 protein levels obtained in the BAL fluid. This is used as a marker of whether the test compound evokes epithelial damage or inhibits microvascular leakage. 4) Option to measure Cytokine Levels in BAL Fluid: The measurement of primary BAL fluid for biochemical markers is one of the most important tools for understanding the mechanism of lung inflammation, as any markers measured are those directly released into the lumen of the airways to elicit an inflammatory response. BAL fluid from each mouse will be tested in duplicate for IL-5 and IgE.

In vivo models of Inflammation SB Drug Discovery

-v
20 40 60 0

Eosinophils (x104) 10 20 30 40 0
Co n tro l tro l g g Co n m g/ K m g/ K m g/ K -v e Th eo p hy lli n e1 e3 0 lli n .1 hy eo p eo p hy op hy g 0m g/ K D g EX 3m g/ kg l. 3 Th +v e

Total cell counts (x104)

ec on

+v

Th eo
Th Th e

Total Cell Number

Differential Cell Counts

ec on

tro l

tro l

1m g/ kg Th eo 3m g/ Th kg eo 10 m g/ Th kg eo 30 m D g/ ex kg am eth as on e

Eosinophils (x104)
0
-v e +v e m .0 .1 Ro flu Ro m flu m .3 m m .0 m .3 .1 D ex Co nt ro l g/ g/ m 0m 3m kg kg g/ k Co nt ro l
10 20 30 40 50 0

Total cell counts (x104)

25 50 75

OVA Model Control Compound Data

Ro flu Ro flu

g g/ kg g/ kg

-v eC on tro l +v e Co Ro nt flu ro l m .0 .3 m Ro g flu /k g m .1 .0 m Ro g/ kg flu m .3 m Ro g/ flu kg m .1 0m g/ kg D ex 3m g/ kg

In vivo models of Inflammation

SB Drug Discovery

IL-5 (pg/ml)
100 200 300

-v
0

eC on tro l l tro Co n 1m g/ kg g/ kg g/ kg g/ kg g/ kg 3m m m

+v e Th eo Th eo Th eo 10 30 3m Th eo D ex

IL-5 Measurement

IL-5 (pg/ml)
1000 250 500 750 0
-v eC on tro l +v eC Ro on tro flu l m .0 .3 m Ro g/ flu kg m .1 .0 m Ro g/ kg flu m .3 m Ro g/ flu kg m .1 0m g/ kg De x 3m g/ kg

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 1 inflammation (neutrophil driven). This model enables the study of anti-inflammatories and their potential to inhibit neutrophil trafficking. The acute nature of this model means that specific timepoints after LPS challenge can be studied (e.g 2, 4, 24, 48, 72 hours) to assess at what stage of the inflammatory pathway the compound works best.

Model Overview
We have established a mouse model of acute lung injury showing pulmonary neutrophilia. Intra-tracheal instillation of lipopolysaccharide (LPS) results in an acute, Th1-type inflammatory response in these mice. Cytokines such as TNF- and CXC chemokines such as MIP-2 and KC are involved in the trafficking of neutrophils from the circulation into the alveolar spaces. Pulmonary oedema, as measured by an increase in lung water content, occurs following this inflammatory response. The client would receive a lung profile of cellular, biomchemical and histological markers, using dexamethasone as a reference compound. We can also measure IP neutrophil accumulation.

In vivo models of Inflammation SB Drug Discovery

Parameters Measured
1) Cellular Infiltration: We will determine the total cell number recovered from BAL fluid (by either haemocytometer or coulter counter). The total cell counts in the BAL is taken as an indicator of inflammatory cell influx into the lung from the surrounding blood network. An increase in total cell number may be indicative of inflammation and the data is used in conjunction with the differential cell counts. By differentially counting the cells retrieved from the lung (in BAL fluid) we can determine the specific inflammatory cell populations present and thus distinguish the inflammatory 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 whether the test compounds evoke epithelial damage or inhibits microvascular leakage. 3) Option to measure Cytokine Levels in BAL Fluid: The measurement of primary BAL fluid for biochemical markers is one of the most important tools for understanding the mechanism of lung inflammation, as any markers measured are those directly released into 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)

In vivo models of Inflammation SB Drug Discovery

Control Compound Data

Total cell number:
Time Course Of Inflammation 150 125 TCN in BAL (x10 4 ) 100 75 50 25 0
rs rs l in rs rs ou ou ou ou H H sa H H H 72
rs ou H 72 48 Ho

24

MIP2 (IL-8 homologue)

Time Course Of Inflammation 1000 MIP-2 in BAL (pg/ml)

750

500

250

0
rs rs rs e lin ou H sa H H ou ou ur s

24

48

ou

rs

In vivo models of Inflammation SB Drug Discovery

Neutrophil Cell number:

Time Course Of Inflammation Neutrophil numbers in BAL (x10 4 ) 150 125 100 75 50 25 0
rs rs rs lin rs ou ou ou ou H H sa H H H
rs

24

48

Protein:
Time Course Of Inflammation 2.00 Total protein in BAL (mg/ml) 1.75 1.50 1.25 1.00 0.75 0.50 0.25
rs rs lin ou ou ou H H ou H H sa H ou rs 72 rs e

24

48

72

In vivo models of Inflammation SB Drug Discovery

ou

rs

COPD
Chronic LPS Model
SBs COPD murine model is based on the model described by Vernooy et al, 2002 (Am J Res Mol Biol (2002) 26: 152-159). This model, in which mice were exposed to multiple intratracheal instillations 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 cytokine expression, accompanied by airway and alveolar alterations. The observed 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 levels but 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 smoking models, the amount of insult added is known. The model shows irreversible lung damage, in contrast to some shorter exposure LPS models. There are pathological changes in the airways and parenchyma characterised by mucus producing cell metaplasia and airway wall thickening, as well as irreversible alveolar enlargement. Significant inflammatory cell influx is also observed.

In vivo models of Inflammation SB Drug Discovery

Method
Chronic inflammation is induced by giving mice a non surgical intratracheal instillation of LPS (2 doses per week) for a total of 12 weeks. Test compounds can be added anytime during the study but we recommend dosing take place during the last 2 weeks (5 doses per week). After 12 weeks, there is a 1-week rest period before mice are sacrificed.

Parameters Measured
Using this COPD model, we have the ability to measure the following endpoints: 1) BAL Fluid: Measured for a variety of inflammatory mediators, typical of COPD pathology, including IL-8 (as MIP-2); IL-6; TNFalpha and other cytokines/biochemical parameters as required. 2) Cellular Infiltrates: We will perform differential cell counts on the BAL. 3) Protein levels: Determine total protein levels in BAL fluid. 4) AHR: We can investigate airway mechanics using Buxco WBP technology, measure regularly to determine changes in lung volumes. 5) Histology: We can undertake histopathological analysis of the lung following treatment with the compound(s) from 2 mice per group (all mice of all groups can be performed on request). Left lung embedded and sections taken for histological analysis. We will provide all slides once the study is complete (1 H&E stained slide + 2 unstained slides).

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 challenge with 5g/50l (per mouse) LPS (055:B5) by intratracheal instillation twice a week for a total of 12 weeks. The animals are allowed a rest period on the 13th week. Drugs/compounds for evaluation are administered on week 11 and 12. Animals are then sacrificed and investigated for signs of COPD-like symptoms and markers. Mice may also be monitored throughout this process by using the Buxco lung function screen.

Whole Cell Number

120 Cell Counts in BAL (x104) 100 80 60 40 20 0
Th eo ph yll D in ex e am et ha so ne Th eo .+ D ex . co nt ro l co nt ro l

Macrophages Neutrophils Lymphocytes

-v e

+v e

In vivo models of Inflammation SB Drug Discovery

Protein (mg/ml)

MIP-2 (pg/ml) 10 20 30 40 0
-v eC +v eC Th eo ph yl lin D e ex am et ha so ne Th eo .+ D ex . on tro l on tro l

0.0

0.5

1.0

1.5

2.0

2.5

Total Protein Measurement

(-) Co nt

ro l

MIP-2 Measurements (IL-8 Homologue)

ro l Th eo ph yl lin D e ex am et ha so ne Th eo .+ D ex .

(+ )C on t

In vivo models of Inflammation

SB Drug Discovery