Biology and basic science of COPD

Professor Maria G Belvisi

Respiratory Pharmacology Group
Imperial College NHLI, Imperial College London Royal Brompton Hospital

m.belvisi@imperial.ac.uk

http://www.irpharma.co.uk/
 ASTHMA AND COPD PATHOLOGY

+++ Inflammation +++

+++ ASM +

+++ BM -

+ +++
Fibrosis
- +++
Alveolar
Asthma death disruption Severe COPD
Courtesy of Jim Hogg
 ASTHMA AND COPD
ASTHMA COPD

Airway Inflammation
Mast cells Macrophages
Inflammatory gene
Eosinophils expression Neutrophils

Th2 cells Tc1 cells

AIRFLOW LIMITATION

Steroid sensitive Steroid resistant

 What we do….. COPD MODEL-Endpoints Lung function
Biomarker-protein 100
Biomarker- gene 75

50
Symptoms
25 Disability
Death
0
25 50 75
Mechanistic Age (years)
biomarkers

Functional
Phenotype Exercise Testing
Steroid-resistant inflammation
600
Cells x103/ml

400
*

200
0
3 0.1 0.3 1 3 Imaging (MRI)
Saline Elastase

Mucus Air spaces Histopathology-

Isolated tissues-bioassay Histopathology Cellular inflammation
Profiling of Drugs in Human and Animal Models

Models involving assessment of compounds on,

• Inflammation
• Lung damage and emphysema
• Airflow limitation-bronchodilators
• Sensory nerve activity and the cough reflex
 NF-B IN ASTHMA AND COPD
URTI Viruses ASTHMA COPD
rhinovirus Allergen TNF-, IL- Cigarette smoke Oxidative
influenza virus 1ß Irritants stress

IKK2 IKK2
IB IB
NF-B NF-B
Inflammatory Inflammatory
genes genes
B B

mRNA mRNA

Chemokines Enzymes Adhesion Cytokines

RANTES iNOS, COX-2 molecules Chemokines Adhesion Enzymes
TNF-, IL-1ß
MIP-1, MCP-3 cPLA2 IL-8, GRO-, molecules MMP-9
ICAM-1, GM-CSF
eotaxin MCP-1 ICAM-1, E-selectin
VCAM-1
EFFECT OF TPCA-1 ON LPS-INDUCED INFLAMMATION

NF-kB DNA binding

LPS - + + + +
TPCA-1 - - 3 10 30
(mg/kg)

Vehicle/saline Vehicle/LPS

TPCA-1 (60mg/kg) Budesonide (3mg/kg)

Birrell et al., 2006, Mol Pharmacol. 69(6):1791-800
 NF-kB in LPS and CS-induced lung inflammation
NF-
kB(p65):DNA association following acute LPS NF-
kB(p65):DNA association following acute CS exposure

Absorbance (450nm)
3 0.8
*
Absorbance (450nm)

Air * Air
Smoke (500ml/min) 0.6 Smoke (500ml/min)
2
0.4
* *
1
0.2
*
0 0.0

72
24
24
48
48
72

96
96
2
2
6
6

168
168

LPS 2hr
Saline 2hr

24
24
48
48
72
72
96
96
2
2
6
6

168
168
Time after challenge (hours)
Time after challenge (hours)

NF-kB subunit:DNA association following NF-

kB(p65):DNA association following Sub-chronic CS exposure
LPS and acute CS exposure
1.0 1.5
Absorbance (450nm)

* Saline 2hrs

Absorbance (450nm)
0.8 LPS 2hrs *
* Air 2hrs 1.0
0.6 Smoke 2hrs Air
Smoke 500m//min)
0.4
0.5
0.2

0.0 0.0 Saline 2hr

LPS 2hr

72
72
96
96
24
24
48
48

168
168
2
2
6
6
Time after challenge (hours)
NF-kB subunit
Human p65:DNA association
p65:DNA association in human lung
nuclear extract
2.5
*
Absorbance (450nm)
2.0

1.5

1.0

0.5

0.0

Disease Status
 Epithelial IKK-2/NF-kB signalling following LPS exposure
• Aerosolized LPS (1mg/ml), 30 mins
kB(p65):DNA association in epithelial cell IKK-2 KO
NF-
Wildtype
1.5
f/f
IKK-2
Absorbance (450nm)

1.0 #
IKK-2 f/f Cre +
* IKK-2 f/f Tet +
0.5
IKK-2 f/f Cre +Tet +
0.0
Saline LPS
C. BALF neutrophils BALF KC
800 # 800 #
600
Cell (10 3/ml)

600
*
KC (pg/ml)
400 * 400

200 200

0 0
Saline LPS Saline LPS
 Myeloid-derived cell IKK-2/NF-kB signalling following LPS exposure

NF- kB(p65):DNA association

in myeloid cell IKK-2 KO

0.25
# Wildtype
Absorbance (450nm)

0.20
f/f
IKK-2
0.15
*
0.10
IKK-2 f/f Cre +
0.05
0.00

Saline LPS

BALF neutrophil number BALF KC

800
* 300
*
Cell (10 3 /ml)

600 # KC (pg/ml) 200

400
100 #
200

0 0

Saline LPS Saline LPS

 The Smoke System
Side-stream Cigarette

Exhaust

Pinch Valve Animal Cages

Smoke Chamber

Pump
 CS dose-dependently increase CINCs, neutrophils and
mucin in BAL after an acute exposure
7L
•Rats exposed to 50 mLs smoke every 30 s
•2-5 cigarettes (2R4F Research cigarettes)
•Once a day for 3 days
•Controls exposed to air

BAL mucin(units/mL)Goblet Cell Density

CINC-1/GRO (pg/ml)

8.0 *
400
* * 6.0
300
* * *
1.2E+05 4.0
Neutrophils (cells/ml)
200
* * *
1.0E+05 2.0
100
8.0E+04 0.0 0 2 3 4 5
0
0 2 3 4 5 * Cigarette Dose
Cigarette Dose 6.0E+04
*
CINC-2 (pg/ml)

8.0
1600 * 4.0E+04
1200 2.0E+04
* 6.0
* * * 4.0
*
800 * 0.0E+00
400 0 2 3 4 5 2.0
0 0.0
0 2 3 4 5 Cigarette Dose 0 2 3 4 5
Cigarette Dose
Cigarette Dose
Stevenson et al., AJP-LCMP 2005, 288:L514
 Notes
Short term exposure to cigarette smoke (once
a day for 3 days) induces a dose-dependent
increase in neutrophil chemokines, BALF
neutrophil numbers, mucous cell metaplasia
and mucous in the lavage fluid. These are the
primary changes associated with the acute
smoking model and when a steroid is tested in
this system….
BAL Neutrophils (x104 cells/mL) BAL Neutrophils (x104 cells/mL) CS induces dose-dependent increase in PMNs

BAL Neutrophils (x104 cells/mL) BAL Neutrophils (x104 cells/mL)

balb/c c57/bl6
18 18
16
** 16
14 14
12
** 12
10 10
8 8
6 6
***
4 4 **
2 2
0 0
sham 2 3 4 5 sham 2 3 4 5

A/J sv129
18 18
16 *** 16
14 14
12 *** 12
10 10
8 8
6 6
*** ***
4 4
2 2
0 0
sham 2 3 4 5 sham 2 3 4 5
Number of cigarettes Number of cigarettes
Morris et al., JPET 2008 273: 851-862.
 Notes

Another important feature to recognise in these smoking models, in

particular in mouse smoking models, is that there is variation
amongst strains in how they respond to smoke exposure. Smoke
exposure induces similar acute and chronic inflammatory changes
in different mouse strains – This is represented here by a dose-
dependent increases in airway neutrophilia after 3 days of smoke
exposure in 4 strains of mice that are commonly used in models of
airway inflammation or strains commonly used to generate GM
mice. It is clear, however, that the degree of this response vary
amongst the strains which has been well characterised now by us
as well as others. The consequences of these changes have yet to
be fully elucidated, but it suggests that there may be different
mechanisms involved in mediating these responses which can
impact on the efficacy of certain mechanisms being investigated
using these models; thus potentially impacting of drug discovery
efforts if care is not taking in the design of the experiments.
 Neutrophil Chemokines are Elevated in BAL and
Tissue in chronic models

CINC-1/GRO (pg/mg protein)

1400
** 800
***
CINC-1 BAL
CINC-1/GRO (pg/mL)

1200 700 CINC-1 Tissue

1000 Sham 600
Sham
Smoked 500 Smoked
800
*** *** 400
n=1 ***
600 ***
** *** 300
**
400 *** * ** 200
* *** ** *** ***
***
200 ** ** 100

0 0
3500
CINC-2 BAL CINC-2 Tissue
600
** ***

CINC-2 (pg/mg protein)

3000
Sham
CINC-2 (pg/mL)

Sham
500
Smoked *** ***
2500 Smoked ***
400
*** ***
2000 *** ** **
*** 300 *** ***
1500 *** ***
*** *** 200 *** *
1000 *** ** **

500
* 100

0 0
1d 3d 5d 2w 3w 4w 6w 8w 12w 16w 26w 34w 26+8w 1d 3d 5d 2w 3w 4w 6w 8w 12w 16w 26w 34w 26+8w

Stevenson et al., AJP-LCMP 2007, 293:L1183 2 sample t-test * = p<0.05, ** = p<0.01, *** = p<0.001
 Notes
Smoke exposure drives a bi-phasic response
characterised by an initial acute phase that lasts for
approximately one week, followed by a intermediate
“sub-chronic” phase where there is a partial resolution
of some of the inflammatory response that lasts up to
month of exposures. After 1 month, a progressive,
chronic inflammation becomes established. The
kinetics of these changes are represented here by
changes in the GROa homologues CINC-1 and -2 in the
lavage fluid and lung tissue over a period of 8 months
of exposures.
 CS-induces neutrophil infiltration in the lung
BAL Neutrophils Tissue Neutrophils

Neutrophil sham
Neutrophil smoke
1.8 100
*** *** *** ***
1.6 90

Neutrophils / mm2
1.4 80 **
Cells x 10^6

1.2 ** 70 **
** *
1 *** 60 **
0.8 *** 50
40
0.6 30
0.4 20
***
0.2 10
0 0
1d 3d 5d 6w 12w 16w 26w 34w 26w + 1d 3d 5d 3w 4w 6w 8w 16w 26w 34w 26w
8w rec + 8w

2 sample t-test * = p<0.05, ** = p<0.01, *** = p<0.001

Stevenson et al., AJP-LCMP 2007, 293:L1183

 Notes
• The acute phase again is characterised by increases in airway/ling neutrophilia,
neutrophil chemokine production and mucous cell metaplasia. After 1 month of
exposure neutrophils, macrophages and lymphocytes begin to accumulate in the
airways mostly in the alveolar spaces and terminal bronchi. Eventually
patholoogical changes (namely emphysema and airway fibrosis) become
evident; although these changes are very mild by comparison to what is
observed clinically and only small changes inlung function have been observed
as a result.

• Another feature of the model is that like the clinic, the inflammation is slowly
resolving as you can see in this figure, after 6 months (or 26 weeks) of cigarette
smoke exposure there is a considerable number of neutrophils recovered in the
lavage fluid. If smoke exposures are terminated at this point and then assessed
2 months later you can see there is still an elevated level of neutrophilia in the
airways albeit much lower than when the animals were being actively exposed.
 Budesonide has no inhibitory effect on CS-induced neutrophilia

BAL cells Tissue cells

Sham
Smoke
Smoke Bud (0.3)
1.0E+05 Smoke Bud (1.0)
1.2E+06

Total cells/mg tissue

9.0E+04 Smoke Bud (3.0)
8.0E+04 1.0E+06
7.0E+04
Cells / mL

6.0E+04 8.0E+05
5.0E+04 #
6.0E+05
4.0E+04
#
3.0E+04 4.0E+05
2.0E+04 *
2.0E+05
1.0E+04
0.0E+00
*
0.0E+00
Mac Neut Eos Mac Neut Eos

# = p < 0.05 compared to sham control * = p < 0.05 compared to smoke control

Stevenson et al., Proc Am Thor Soc, 2004; 169: A205

 Notes
• …you can see it has no effect on the smoke-induced
increases in inflammatory cell infiltration recovered in the
lavage fluid and lung tissue. In this study budesonide was
dosed intratracheally once a day 1 hour before each
exposure to cigarette smoke at doses the completely
attenuate airway inflammation in the LPS; however as you
can see budesonide has no effect in this system.

• Sidenote - unlike the elastase model, NF-kB is activated in

this system; thus mechanisms of steroid resistance could
potentially be different in the two systems
Hypothesis
 Caspase 1 activity in human lung tissue

0.5 *
Absorbance (405nm)
Caspase activity

0.4

*
0.3

0.2

0.1

0.0

Non-Smoking Smoking Donor Emphysema

Donor Eltom et al., 2011, PLoS One 6(9): e24097
 Effect of acute CS exposure in P2X7 KO mice

0.4
# 4 #
Caspase-1 Activity

IL-1b ( pg/ml)
(Absorbance ~ 405nm)

0.3 3
* *
0.2 2

0.1 1

0.0 0
WT P2X7 -/-WT P2X7 -/- WT P2X7 -/-WT P2X7 -/-

Air Smoke Air Smoke

Eltom et al., 2011, PLoS One 6(9): e24097

Effect of P2X7 inhibitor on acute CS induced inflammation
100
Vehicle
Neutrophils (10 3/ml)

80 A438079 mg/kg
#
60

40 *
20 *
*
0
1000 30 100 300 1000 P2X7-/-

Air Smoke
Eltom et al., 2011, PLoS One 6(9): e24097
 Protease (PPE) Instillation:
Inflammation/Emphysema/compromised lung function
• Instillation of proteases
– Pulmonary Inflammation (acute 48h)
• Lung neutrophils, lymphocytes, mucus production, and
inflammatory cytokines.
– Emphysema development (8 weeks)
• Increased residual volume and decreased flow
Inflammatory cells Mucus containing cells Air spaces
Saline

Elastase

Birrell et al., Am J Respir Crit Care Med. 2005 Jul 1;172(1):74-84.

 Notes
• The peak of inflammatory cell burden in the
• airway appears between 24 and 48 hours after
PPE insult, with
• levels returning to that of the controls by Day 7
• Lung function changes after 2 weeks continue
for 10 wks
• LM at 8 weeks – BUD either dosed from b4
PPE or 4wks after dosing
Profiling of Drugs in Human and Animal Models

Models involving assessment of compounds on,

• Inflammation
• Lung damage and emphysema
• Airflow limitation-bronchodilators
• Sensory nerve activity and the cough reflex
Functional assays: isolated tissue bioassays

0
% 100 mM Papaverine

-10 DMSO
100nM ONO-AE3-208
response

-20

-30

-40
EP4 receptor
-50
-novel bronchodilator target
-9 -8 -7 -6 -5
log [PGE2] (M) Buckley et al., 2011, Thorax
 DURATION OF ANTICHOLINERGIC EFFECTS
10min
Control
9h

Atropine
Ipratropium
Tiotropium
5h

7h
9h

Guinea-pig trachea
Electrical field simulation
 DURATION OF ANTICHOLINERGIC ACTION

Guinea-pig bronchi in vitro: inhibition of cholinergic nerves

Tiotropium

Ipratropium
Atropine

Takahashi et al: AJRCCM 1995

 DURATION OF ANTICHOLINERGIC ACTION
Human bronchi in vitro: inhibition of cholinergic nerves

Onset Wash-out

Tiotropium
(1nM)

Ipratropium
(10nM)

Takahashi et al: AJRCCM 1995

Profiling of Drugs in Human and Animal Models

Models involving assessment of compounds on,

• Inflammation
• Lung damage and emphysema
• Airflow limitation-bronchodilators
• Sensory nerve activity and the cough reflex
 Airway Sensory Nerves
C-fibres ‘Cough receptors’
RARs Capsaicin, BK, Mechanosensitivity
low pH, PGs
Hypertonic saline Low pH
Ad nociceptors
Mechanosensitivity Capsaicin, BK, low pH

Airway epithelium
RAR C-fibre
SP, NKA,
CGRP
Bronchial vessel
CNS
Airway smooth muscle

Nasra, Belvisi 2009,

Pharmacol & Ther, 180: 923-8.
SAR
 Coexpression of TRP channels in lung-labelled
airway neurons
A 1 2 3 4 5 6 7 8 9 10 11 12 + –
DiI i.t. 238 bp ß-Actin

TRPV1
229 bp
393 bp TRPA1

313 bp
TRPM8

B 1 2 3 4 5 6 7 8 9 10 + –

238 bp ß-Actin

TRPV1
229 bp
393 bp
TRPA1

313 bp TRPM8
Single cell
RT-PCR
Nassenstein et al., J Physiol
2008
Response to capsaicin

Intensity A.U.
MUSTARD OIL ACTIVATES VAGAL C-FIBERS
IN THE GUINEA PIG LUNGS

MUSTARD OIL
AITC 30 µM
Functional assays: isolated tissue bioassays
Isolated vagal nerve preparation; sensory nerve activation
Chart Recorder

Krebs / Drugs Vaseline

Nerve Reference
Electrode

1 mV 5 min

Human
4 % saline 3 mM capsaicin low pH

2 % saline 10 mM bradykinin

Belvisi et al. 2008, Br J Pharmacol., 155(4):547-57

 Effect of a TRPA-1 ligands or receptor KO on
0.60
Depolarisation to acrolein and capsaicin
0.60
0.45 0.45
mV

mV
0.30 0.30
0.15 0.15
0.00 0.00
-4.5 -4 -3.5 -3 -2.5 -2 -4.5 -4 -3.5 -3 -2.5 -2
Log [Acrolein (M)] Log [Cinnamaldehyde(M)]

Vehicle Log [HC-030031 (M)]

100 TRPA KO mice
Inhibition (%)

75 0.5
0.4
50

mV
0.3
0.2
25
0.1
0 0
-6.5 -6 -5.5 -5 -4.5 -4.5 Wild type KO Wild type KO
Capsaicin Acrolein
Acrolein Capsaicin
Birrell et al., 2009, Am J Respir Crit Care Med. 180(11):1042-7.
 TRPA1 Ligands induce cough in conscious guinea-pig
model and in normal volunteers
17.5
Cough/10 minutes

15.0
12.5 7 Human
10.0
7.5 6

Number of coughs
5.0
2.5
5
0.0
10 30 100 300 4
Acrolein (mM) 3
6 2 Baseline
Cough/10 minutes

5 HC-030031
1 One hour later
4
(300 mg/kg)
3
0
2
* 0 200 400 600 800 1000
1 Cinnamaldehyde (mMoles)
0
Acrolein (100mM)
Birrell et al., 2009, Am J Respir Crit Care Med. 180(11):1042-7.
Andre et al., 2009, Br. J. Pharmacol, 158: 1621-1628.
 Cough Model
A)

Box Flow
(16 ml/s)
BoxFlow

(16(16ml/s)
Flow

ml/s)

Time (2 sec)
Box

Time (2 sec)
Time (2 sec)
COUGH AS A MAJOR UNMET MEDICAL NEED
• Commonest symptom for medical consultation
• Chronic cough: 10-38% of pulmonary out-patients
• No effective therapy apart from opiates
• High over the counter sales (>$4bn pa)
• Troublesome symptom
• Physical basis understood
• Relevant animal models
• Logical mechanistic approach possible
REST OF
WORLD
23%

EUROPE
26%
AMERICA
51%

WORLD MARKET: 4 billion USD

 TIOTROPIUM BLOCKS LATE RESPONSE TO ALLERGEN
Conscious Brown Norway rats (n=5-8)
Allergen Challenge Effect of tiotropium
12 Late response
2000
10
Average Penh

Abolished by anaesthesia
8 1500
Early

Penh (AUC)
response
6
Sensitized 1000
4

2 *
Non-sensitized 500
0

0 50 100 150 200 250 300 350 0

Time after challenge (min) Control OVA OVA+ Tio
Ovalbumen
(0.1mg/kg)
Raemdonck K et al: Thorax 2011
 TRPA1 INHIBITOR BLOCKS LATE RESPONSE TO ALLERGEN

TRPA1 inhibitor (HC-030031)

TRPV1 inhibitor (JNJ-17203212) CNS
2500

2000
Penh (AUC)

TRPA1
1500

Mediator X
1000 * Tiotropium
Mast cell

500
ACh
M3R
0 Allergen
Control OVA TRPA1 TRPV1 Bronchoconstriction
Raemdonck K et al: Thorax 2011
 Effect of budesonide on LAR in mice
8 following exposure to air or cigarette smoke

6 air/ova/veh
air/ova/bud
smoke/ova/veh
Penh

smoke/ova/bud
4

0
0 60 120 180 240 300 360 420 480 540 600
 CHOLINERGIC CONTROL OF AIRWAYS
CNS
Nodose
ganglion

Laryngeal Vagus nerve

oesophageal Parasympathetic nerve
afferents

A-fibre ACh
Airway wall C-fibre Parasympathetic ganglion
Muscarinic
C-fibre ACh receptors
receptors ACh
Submucosal
Irritant gland
receptors Mast cell
Airway
epithelium
Allergens
Irritants (e.g. cigarette smoke)