Animal models and the search for anti-rheumatic drugs

ObjectivesTo understand the problems with existing therapies

for RAThe reasoning behind screening cascades and the

position of in vivo pharmacologyTo appreciate the specific problems related to animal

experimentationTo 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

Morning stiffness 1h

Three or more joints involved

Arthritis of hand joints

Symmetric arthritis

Rheumatoid nodules

Rheumatoid factor (positive < 5% normal subjects)

Radiographic changes (must show erosion/decalcification)

Present for 6wk

Any 4 of the following must be present to allow diagnosis of RA:-

Pathology of rheumatoid arthritis

Exper

imental

therapy

High dose

corticosteroids

Cyclophosphamide

Methotrexate, azathiaprine

Hydroxychloroquine

Sulphasalazine

Gold

salts

NSAIDs Low dose prednisone

Patient

education

Physical

therapy

Occupational

therapy

High dose

salicylates

Sur

gery

Intra-articular corticosteroids

Traditional pyramid for treatment of RA

Assumptions:-

1) RA relatively benign disease

2) NSAIDs less toxic than DMARDs

Sawtooth strategy for treatment of RA

0 5 10 15 20 25

Dis

ab

il ity

i nd

ex

Years

Typical course of disease

Sawtooth course of disease

DMARD

1 4 532 6 7

Current therapies: Treatment cascade & limitations

NSAIDs

Volterol, Vioxx, Celebrex(GI ulceration & Bleeding. Not DMARD)

DMARDs

Methotrexate, Leflunamide, HydroxychloroquineEfficacy (refractory), Safety – Myelosuppression, Hepatic toxicity

BIOLOGICALSEnbrel, 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 NCEIsolated protein

Functional cell assay

Ex vivo assay

In vitro toxicology

CYP induction/inhibition

Mini Ames

Micronucleus test

In vitro Metabolism

Microsomal stability

CaCO-2 studies

In vivo PK

In vivo efficacy

Primary model

Secondary model

Tertiary model

Counter screens for selectivity

In vitro studies isolated target and murine cell systems

Single dose PK

Medium term secondary models

Tertiary disease model

Furry test tube

Primary model

Multidose PK

Screening cascade for NBEs: concept ofparallel reagents

In vitro studies isolated target and human cell systems

Human explant studies?

Furry test tube?

Hu-Mouse transgenic?

Primate studies

Murine parallel reagent Human therapeutic

Clinical trial

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

0 3 10 300

10

20

30

mg/kg p.o.

***

Pla

sma

TN

F n

g/m

l

Paw oedema

•Sub plantar injection of irritant eg carrageenan

•Reaction usually maximal by 3 hours

•Readily quantified by plethysmometry

•Not an arthritis

•Not amenable to histological or biochemical assay

•End point is crude

•Very popular in industry!

Disadvantages

Measurement of paw oedema using plethysmometry

Results expressed as paw volume (ml)

or

change in paw volume (Δml)

Cavity models

•Peritonitis

•Pleurisy

•Air pouches

•Exudate volume

•Total and differential cell counts

•Amenable to biochemistry

Advantages

Effects of experimental compound on exudate volume recovered from a 4 hour carrageenan

pleurisy in the rat

- Vehicle 0.1 1 100.00

0.25

0.50

0.75

+1%carrageenan

(mg/kg)

**

***

***Ex

ud

ate

vo

lum

e(m

l)

Means s.e.m., n=8/9 per treated group, n=4 in untreated groupStatistical analysis: One way ANOVA followed by a Bonferroni's multiple comparisons test** p<0.01, ***p<0.001comparison with vehicle control

Vehicle= 10% DMSO and 90% LabrafilDrugs were administered 30 mins beforecarrageenan injection

Effects of experimental compound on total cells recovered from a 4 hour carrageenan

pleurisy in the rat

- Vehicle 0.1 1 100

10

20

+1%carrageenan

(mg/kg)

****

***

***

To

tal c

ells

(x1

06)

Means s.e.m., n=8/9 for treated groups, n=4 in untreated groupStatistical analysis: One way ANOVA followed by a Bonferroni's multiple comparisons testp<0.05, ***p<0.001comparison with vehicle control

Vehicle= 10% DMSO and 90% LabrafilDrugs were administered 30 mins beforecarrageenan injection

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

0.000 0.025 0.050 0.075 0.100 0.125

0

5

10

LPS mg/air pouch

To

tal

cell

s x1

06

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

•Polyarthritis

•Monoarticular arthritis

-Adjuvant arthritis-Collagen II arthritis

•Matrix degradation

-non-immune-Immune

Collagen induced arthritis

• 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

Similarities to RA Differences to RA

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

Clinical assessmentDisease scoreBody weightAnalgesia

ImmunologyCII AbEx vivo lymphocyte proliferationHypersensitivity to CII

Serum markers of disease progressionAcute phase proteinCOMPBone sialoprotein

Assessment of CIA

Joint pathologyHistologyRadiographs

Drug specificPlasma levelsBioactivity markers

Clinical presentation of murine CIA

Score 1 2 3

Pictu

res Rem

i Ok

oye

NORMAL WRIST SWOLLEN WRIST + PAD SWOLLEN

WRIST + PAD + DIGITS SWOLLEN

0

Effects of an experimental compound onrat CIA

0 5 10 15 20 250.9

1.0

1.1

1.2

1.3

1.4 Vehicle0.03mg/kg0.3mg/kg3mg/kg10mg/kg

Days post sensitisation

Paw

vo

lum

e (m

l)

Vehicle 0.03 0.3 3 100

1

2

3

**

**

mg/kg u.i.d.

AU

C p

aw v

olu

me

(Day

s 14

-24)

Paw volumes Change in paw volumesAUC Days 14-24

Means s.e.m., n=10/groupStatistical analysis ANOVAWith Bonferroni post test**p<0.01 vs Vehicle

Effects of leflunomide on murine CIA

VehicleLeflunomide

Animals dosed day -1Leflunomide 3mg/kgPO once dailyVehicle = 1% MethylCelluloseDBA/1 mice N = 15

12 14 16 18 20 22 24 26 28 30 32 34 36 38 400

2

4

6

8

**

Day

Clin

ica

l sc

ore

P= <0.05 Day 31/32 *P= <0.01 Day 30/33-36**

Dosing strategies in CIA

Clin

ica

l sc

ore

SensitisationB

oost

Prophylactic dosing

Therapeutic dosing

Dosing through sensitisation period

Time

CIA Histopathology

Score 3 Score 0

0 normal1 inflammatory cell influx, 2 cell influx & focal erosion cartilage & bone 3 loss of joint architecture

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