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Animal models and the search for antirheumatic drugs
Objectives
To understand the problems with existing therapies
for RA
The reasoning behind screening cascades and the
position of in vivo pharmacology
To appreciate the specific problems related to animal
experimentation
To understand how to manipulate in vivo models
(immune/non immune etc)
To give an example of a tertiary model of arthritis its
similarities and differences to RA
Rheumatoid arthritis
Diagnosis of rheumatoid arthritis
Any 4 of the following must be present to allow diagnosis of RA:Morning stiffness  1h
Three or more joints involved
Present for  6wk
Arthritis of hand joints
Symmetric arthritis
Rheumatoid nodules
Rheumatoid factor (positive < 5% normal subjects)
Radiographic changes (must show erosion/decalcification)
Pathology of rheumatoid arthritis
Traditional pyramid for treatment of RA
Assumptions:1) RA relatively benign disease
2) NSAIDs less toxic than DMARDs
Exper
imental
therapy
High dose
corticosteroids
Cyclophosphamide
Methotrexate, azathiaprine
Hydroxychloroquine
Sulphasalazine
NSAIDs
Patient
education
Physical
therapy
Gold
salts
Low dose prednisone
Occupational
therapy
High dose
salicylates
Sawtooth strategy for treatment of RA
Typical course of disease
Sawtooth course of disease
1
2
3
4
5
6
DMARD
0
5
10
15
Years
20
25
7
Current therapies: Treatment cascade &
limitations
NSAIDs
Volterol, Vioxx, Celebrex
(GI ulceration & Bleeding. Not DMARD)
DMARDs
Methotrexate, Leflunamide, Hydroxychloroquine
Efficacy (refractory), Safety – Myelosuppression, Hepatic toxicity
BIOLOGICALS
Enbrel, infliximab, Adalimumab, Anakinra (IL-1r)
Expensive, inconvenient Admin, Partial/Non-responders?,
Safety – TB, Op infection, CHF, Demyelinating Disease, Lymphoma
NCEs and NBEs
• NCEs generally work well across species
– tend to target well-conserved sites eg enzyme
active site
• NBEs are often species specific
– Large molecular interactions are less likely to be
well-conserved
– May need parallel reagent for animal models with
final testing of human reagent in primates
– Use human transgenic animal
– Use human explant tissue
Screening cascade for NCE
Isolated protein
Functional cell assay
Counter screens for selectivity
Ex vivo assay
In vitro toxicology
CYP induction/inhibition
Mini Ames
Micronucleus test
In vivo PK
In vitro Metabolism
Microsomal stability
CaCO-2 studies
In vivo efficacy
Primary model
Secondary model
Tertiary model
Screening cascade for NBEs: concept of
parallel reagents
Murine parallel reagent
Human therapeutic
In vitro studies isolated target
and murine cell systems
In vitro studies isolated target
and human cell systems
Furry test tube
Furry test tube?
Primary model
Hu-Mouse transgenic?
Single dose PK
Human explant studies?
Medium term secondary models
Primate studies
Multidose PK
Clinical trial
Tertiary disease model
Animal models: considerations for
use and interpretation of data
• Ethical concerns: When to use. Distress scoring.
• Legal: Licensing issues.
• Practical concerns: Is this a good experiment?
• Statistical concerns: Group sizes. Most
appropriate analysis. What to do with data from
culled animals.
Effects of compound on plasma TNF 90
mins after challenge with LPS
Plasma TNF ng/ml
30
20
*
10
**
0
0
3
10
mg/kg p.o.
30
Paw oedema
•Sub plantar injection of irritant eg carrageenan
•Reaction usually maximal by 3 hours
•Readily quantified by plethysmometry
Disadvantages
•Not an arthritis
•Not amenable to histological or biochemical assay
•End point is crude
•Very popular in industry!
Measurement of paw oedema
using plethysmometry
Results expressed as
paw volume (ml)
or
change in paw volume
(Δml)
Cavity models
•Peritonitis
•Pleurisy
•Air pouches
Advantages
•Exudate volume
•Total and differential cell counts
•Amenable to biochemistry
Exudate volume(ml)
Effects of experimental compound on exudate
volume recovered from a 4 hour carrageenan
pleurisy in the rat
0.75
Vehicle= 10% DMSO and 90% Labrafil
Drugs were administered 30 mins before
carrageenan injection
0.50
**
***
0.25
***
0.00
-
Vehicle
0.1
1
10
+1%carrageenan
(mg/kg)
Means  s.e.m., n=8/9 per treated group, n=4 in untreated group
Statistical analysis: One way ANOVA followed by a Bonferroni's multiple comparisons test
** p<0.01, ***p<0.001comparison with vehicle control
Effects of experimental compound on total cells
recovered from a 4 hour carrageenan
pleurisy in the rat
Vehicle= 10% DMSO and 90% Labrafil
Drugs were administered 30 mins before
carrageenan injection
6
Total cells (x10 )
20
*
***
10
***
***
0
-
Vehicle
0.1
1
10
+1%carrageenan
(mg/kg)
Means  s.e.m., n=8/9 for treated groups, n=4 in untreated group
Statistical analysis: One way ANOVA followed by a Bonferroni's multiple comparisons test
p<0.05, ***p<0.001comparison with vehicle control
Air pouches
•No interfering visceral organs
•Pouch lining cells have similarities to synovial cells
•Prolonged inflammation can be induced
Disadvantages
•Preformed cavities (6-day-old-pouches) give best results
Accumulation of cells in a murine 6-day-old
air pouch in response to LPS
Total cells x106
10
5
0
0.000
0.025
0.050
0.075
0.100
LPS mg/air pouch
0.125
Manipulation of cavity models
•Non-immune models
-complement dependent
-complement independent
•Immune models
-active (sensitisation and challenge)
-passive (adoptive transfer of serum or cells)
Models of arthritis
•Matrix degradation
•Polyarthritis
-Adjuvant arthritis
-Collagen II arthritis
•Monoarticular arthritis
-non-immune
-Immune
Collagen induced arthritis
Similarities to RA
Differences to RA
• Susceptibility linked to
MHC
• immunologically
mediated
• Erosions of cartilage &
bone
• Autoimmunity to collagen
seen in some RA
patients
• Arthritic rather than
multisystem
• Disease is induced
• The model “resolves”
• New bone formation
more marked
• No synovial vasculitis
• No cycles of relapse &
remission
• No RF
• No sex predilection
Collagen induced arthritis
•Animals (rats or mice) sensitised to collagen ll in adjuvant
•Booster injection (mice)
•Distress scoring
•Examine for clinical signs
•Measurement of hind paw volume (rats)
•Histology
Assessment of CIA
Clinical assessment
Disease score
Body weight
Analgesia
Drug specific
Plasma levels
Bioactivity markers
Joint pathology
Histology
Radiographs
Serum markers of disease
progression
Acute phase protein
COMP
Bone sialoprotein
Immunology
CII Ab
Ex vivo lymphocyte proliferation
Hypersensitivity to CII
Clinical presentation of
murine CIA
Score
0
1
2
3
Pictures Remi Okoye
NORMAL
WRIST SWOLLEN
WRIST + PAD
SWOLLEN
WRIST + PAD + DIGITS
SWOLLEN
Effects of an experimental compound on
rat CIA
Paw volumes
Vehicle
0.03mg/kg
0.3mg/kg
3mg/kg
10mg/kg
1.3
1.2
AUC Dpaw volume (Days 14-24)
Paw volume (ml)
1.4
Change in paw volumes
AUC Days 14-24
1.1
1.0
3
Means  s.e.m., n=10/group
Statistical analysis ANOVA
With Bonferroni post test
**p<0.01 vs Vehicle
2
1
**
**
0
0.9
0
5
10
15
20
Days post sensitisation
25
Vehicle
0.03
0.3
3
mg/kg u.i.d.
10
Effects of leflunomide on murine CIA
Clinical score
8
Vehicle
Leflunomide
6
**
4
2
0
12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Animals dosed day -1
Leflunomide 3mg/kg
PO once daily
Vehicle = 1% Methyl
Cellulose
DBA/1 mice N = 15
Day
P= <0.05 Day 31/32 *
P= <0.01 Day 30/33-36**
Dosing strategies in CIA
Dosing through
sensitisation
period
Boost
Clinical score
Therapeutic dosing
Sensitisation
Time
Prophylactic dosing
CIA Histopathology
Score 3
0
1
2
3
normal
inflammatory cell influx,
cell influx & focal erosion cartilage & bone
loss of joint architecture
Score 0
X-ray changes in CIA
CLINICAL SCORE = 0
CLINICAL SCORE = 3
Serological markers
• Anticollagen II antibodies and their isotypes
• Acute phase proteins
• Cartilage oligomeric matrix protein (COMP)
• Bone sialoprotein
Summary
• There remains an unmet need in the treatment of
RA
• In vivo models of inflammation are an essential
part of drug discovery
• The strategy for NCEs and NBEs is different
• Efficacy in models helps define species for
toxicology
• Models can help with calculation of therapeutic
dose in man and with identifying surrogate
markers of drug activity for use in the clinic