Baker 3 rs york pdf

Historical and Personal Perspective on Scientific and 3Rs Challenges of Modelling

Multiple SclerosisDavid Baker

[email protected]

www.ms-res.org

BAKER NC3Rs 2016

3Rs RESEARCHER:- RESEARCHING 3Rs ISSUES

RESERCHER: RESEARCHING SCIENCE USING 3Rs TO ADDRESS ISSUES

TRANSLATIONAL NEUROSCIENCE –BENCH TO BEDSIDE- GOOD SCIENCE WITH 3Rs BENEFIT

BIOLOGY OF MULTIPLE SCLEROSIS-MS IS A BAD DISEASE - KNOW YOUR DISEASE

FAILURE TO TRANSLATE: HUMAN PROBLEMS IN THE TRANSLATIONAL PROCESS

SCIENCE-BASED APPROACH TO 3Rs

BAKER NC3Rs 2016 PLAN-OBJECTIVES

BIOLOGY OF ANIMAL MODEL - EAE IS A SEVERE DISEASE - KNOW YOUR DISEASE

MODELS MOVING FORWARD-UNMET CHALLENGES

Multiple sclerosis (MS) is a chronic (auto)immune-mediated CNS-

confined demyelinating disease affecting 2,500,000 people worldwide

Disease onset is usually between 20 and 40 years of age

(F:M ratio, 2:1)

MS is clinically characterized by a relapsing-remitting course usually

followed by a progressive disability

MS is pathologically heterogeneous – inflammation, demyelination and

neurodegeneration (axonal loss and neuronal damage)

Spontaneous repair occurs but MS invariably progresses (ambulatory

problems in 70-80% people with MS at 25 years from onset)

As a consequence of nerve damage eople can develop many

troublesome symptoms and reduce quality of Life

KNOW YOUR DISEASE-BIOLOGY OF MS

NERVE LOSS

PROGRESSIVE MSRELAPSING-REMITTING MS

DISABILITY

Frequent inflammation, demyelination,axonal transections, plasticity and

remyelination

Inflammation, Persistent Demyelination & Gliosis

Infrequent inflammation, Gliosis, Chronic Neurodegeneration

CLINICAL THRESHOLD

INFLAMMATION

Symptoms

Clinical Effects are Due to Altered Nerve Conduction

CLINICAL COURSE

Immune-Mediated

Beta-interferons, Alemtuzumab (CD52),

Cladribine, Bone Marrow Transplantation,

Fingolimod (S1P1-modulator) , Rituximab

(CD20) are ACTIVE

Neurodegeneration

Beta-interferon, Alemtuzumab,

Cladribine, Rituximab

Bone Marrow Transplantation,

Fingolimod are INACTIVE


White Blood Cells

No Repair

• INFLAMMATORY, DEMYELINATING, NEURODEGENERATIVE DISEASE OF CNS

•MULTIFOCAL OLIGODENDROCYTE LOSS

MS

plaques

Myelin (blue)

stain


NORMAL AGING

ATROPHY RATE 0.1% p.a.

MULTIPLE SCLEROSIS

ATROPHY RATE 0.4-1.0 %

MS is a Neurodegenerative

Disease


MS is a Bad Disease

AssistanceRequired to

Work

DisabilityAffects Daily

Routine

Restricted toWheelchair

RelativelySevere

Disability

ModerateDisability

MinimalDisability

NoDisability

Restricted To Bed or

Wheelchair

Confined To Bed

0.0 1.0 3.02.0 4.0 5.0 6.0 7.0 8.0 9.0

Time (Years)

QUALITY OF LIFE WORSE THAN DEATH

0.4

Mean

Uti

lity

±9

5%

CI

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

0.5

0.6

0.7

0.8

0.9

1.0


PERIPHERAL IMMUNITY Slow RELAPSE RATE

NEURODEGENERATION Slow PROGRESSION

SYMPTOM CONTROL Improve QUALITY OF LIFE

REPAIR Reverse Deficits

OUTCOME OF CONTROLDISEASE PROCESS

KNOW YOUR MODELLING DESIRES

RRR

eplacementefinementeduction

of Animals in Research

THE 3Rs CENTRAL TO USE OF ANIMALS IN EU

RRR

eplacementefinementeduction

National Centre of3Rs

of Animals in Research

THE 3Rs CENTRAL TO USE OF ANIMALS IN EU

www.ms-res.org

Drug-The Game

DRUG DEVELOPMENT

WORKS & SAFE IN PEOPLE WITH DISEASEPHASE III

APPROVALPOST-MARKETING SURVELLIENCE PHASE IV

10-15 Years$1-2 billion

Warning Video

ANIMALS CENTRAL TO DRUG DEVELOPMENT

http://www.ms-res.org/

No Clinical Disease-No Spinal Cord Infiltration- No Cytokine X, Y or ZLimp tail

Impaired

righting reflex

hindlimb paralysis

Moribund

partial paralysis

Normal

Remission

0

1

2

3

4

5

(1)

Clinical Score

Day 7

Spinal cord homogenate in Freund’s complete adjuvant

Day 0

Spasticity & Tremors

Develop

KNOW YOUR MODEL-BIOLOGY OF EAE

Video is Coming Next

43

No Clinical Disease-No Spinal Cord Infiltration- No Cytokine X, Y or ZLimp tail

Impaired

righting reflex

hindlimb paralysis

Moribund

partial paralysis

Normal

Remission

0

1

2

3

4

5

(1)

Clinical Score

Day 7

Spinal cord homogenate in Freund’s complete adjuvant

Day 0

Spasticity & Tremors

Develop


Video is Coming Next

43

MULTIPLE SCLEROSIS MODELS


Normal Remission 1

Remission 2 Remission 4

NfH

(µ

g/m

g t

ota

l p

rote

in)

Number of AttacksN RM1 RM2 RM3

50

100

150

200

250

300

350

400Axonal Content Assessed By Neurofilament ELISA

www.msbrainhealth.org

No-evidence of Disease Activity (NEDA)


Stop Relapses Save Brain

1868 Jean Martin Charcot Describes Multiple Sclerosis1885 Louis Pasteur Rabies Vaccine1888 Encephalitis due to Rabies Vaccine

1925 Encephalitis in rabbits due to repeated inoculation of spinal cord1933 Encephalitis in macaques with repeated inoculation

1942 Freunds Adjuvant developed1947 Induction of EAE

1960 Adoptive Transfer of EAE

1962 Induction of EAE with myelin 1970 Induction of EAE with myelin peptides

1981 Adoptive Transfer of Relapsing EAE in Mice

1995 Induction of EAE in Transgenic Mice1996 Induction of EAE in Knockout Mice

2001 EAE in Fluorescent Mice

Hartley Guinea pig hyper-acute EAEJuvenile Strain 13 guinea pig EAELewis Rat (non-demyelinating EAE)

SJL, PL, ABH Mouse EAE

1994 Induction of EAE with MOG C57BL/6 EAE, DA Rat EAE, Marmoset EAE


HISTORICAL PERSPECTIVE

1993 Spontaneous EAE in TCR Transgenic Mice

• PRE-CLINICAL FAILURE

• Model does not reflect human disease biology

• Drug does not target biology relevant to human application

• Lack of appreciation of human disease

• Dogma & overstating effect

• Model used in a way that does not reflect human indication

• Drug doses are not used in at physiological doses

• Drugs are not delivered in a way appropriate to how used in humans

• Studies are not transparent & not reproducible (Ineffective Study Design)

• CLINICAL FAILURE

• Lack of clear understanding of human pathology

• Drug is seldom investigated by scientists developing the Idea.

• Over-interpretation of significance of pre-clinical studies

• Drug is not used at a dose relevant to the pre-clinical studies

• Population does not respond as predicted. (Ineffective Trial Design)

• Dose-limiting side-effects

• Study Underpowered, too short or unrealistic expectations

• Measurement Instruments Inadequate Clinical Outcomes and Surrogate Markers

• Wrong Group of pwMS studied (IneffectiveTrial Design)

• Commercial Interests

Mechanism is all Important.

Relevance of Slight Delay of a Few Days, Slight Diminution

Prophylactic/Therapy

“Toxicity leading to Stress”

“Route & Timing”

Reporting Issues

KNOW THE LIMITATIONS-FAILURE TO TRANSLATE

Two thousand drugs tested in EAE, only ten classes of drugs approved

Time Post Induction (Days)

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Mean C

linic

al S

core

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Vehicle n= 10/10

Test Drug n = 10/10

Test Drug 2 n=0/10

or n = 7/10

ARRIVE Guidelines (UK)

Kilkenny C, Browne WJ, Cuthill IC, Emerson M

& Altman DG. Improving bioscience research

reporting: the ARRIVE guidelines for reporting

animal research. PLoS Biol. 2010;

8(6):e1000412 (2010)

National Research Council (USA). Institute for

Laboratory Animal Research. Guidance for

the Description of Animal

Research in Scientific Publications. National

Academies Press, Washington (DC) (2011).

Guidelines on EAE Studies

Baker D, Amor S. Publication guidelines for

refereeing and reporting on animal use in

experimental autoimmune encephalomyelitis. J

Neuroimmunol. 2012 242:78-83

LIMIT YOUR LIMITATIONS-EXPERIMENTAL DESIGN

RANDOMISATION

BLINDING

SAMPLE-SIZE

DATA HANDLING

REPLICATION

NINDS GUIDELINE Nature 2012; 490:187

>10%

>20%

>1%

Parametric

(47%)t- test

(46%)

Non

Parametric

39%Not

Reported

(14%)

Parametric

(65%)

t test (67%)

Non-Parametric (4%)

Not

Reported

(31%)

All EAE Publications 6 months

1:12:2011-31:5:2012 n=175

All EAE Publications 2 Years

1:1:2010-11:9:2012 n=26

All PUBMED PUBLICATIONS

BIG 6 PUBLICATIONS

Nature, Nat Med, Nat Immunol,

Nat. Neurosci, Science, Cell

T-test Criteria

Continuous

Normally Distributed

Equal Variances


Figure legend. Clinical scores of two independent

EAE experiments at d23 post disease induction.

Individual scores as well as the mean score of two

independent experiments are shown. Control: n=10,

vehicle: n=13, xxxxx-345: n=11. Control versus

vehicle: P=0.620, control versus xxxxx-345: P=0.017,

vehicle versus xxxxx-345 P=0.029. * indicate P

values <0.05 and ** indicate P values <0.005 based

on a non-paired Student’s t test. Error bars are s.e.m

Nature Paper

2015Drug-treated animals the scores appear to be: 0, 0

,0, 0.5, 0.5, 2, 2.5, 3, 3.5, 3.5, 3.5 n=11

Vehicle scores appear to be: 0.5, 2.5, 2.5, 2.5, 2,75,

2.75, 3, 3.5, 3.5, 3.5, 3.5, 3.5, 3.5 n=13.

Do a t test drug verses vehicle p=0.029

The assumptions of a t test (a) Data is normally

distributed. You test this and it passes the test

p=0.152,

(b) data groups have equal variances

Test for that and it fails P<0.05.

(c) Data is Parametric. Fail. It is non-parametric.

t test is not a valid test

Mann Whitney test. P=0.082.........Ooooops.


Limp tail

Paralysed

No reported Detail of

Randomisation,

Blinding or Sample Size

Scores of Animals

C57BL/6 malesMOG peptide with PTX

SJL mice

malesPLP peptide without

PTX

C57BL/6 malesMOG peptide without

PTX

Krementsov DN et al. 2015 FASEB J 29:3446

LIMIT YOUR LIMITATIONS-QUALITY CONTROL

LIMIT YOUR LIMITATIONS-QUALITY CONTROL

• PRE-CLINICAL FAILURE

• Model does not reflect human disease biology

• Drug does not target biology relevant to human application

• Lack of appreciation of human disease

• Dogma & overstating effect

• Model used in a way that does not reflect human indication

• Drug doses are not used in at physiological doses

• Drugs are not delivered in a way appropriate to how used in humans

• Studies are not transparent & not reproducible (Ineffective Study Design)

• CLINICAL FAILURE

• Lack of clear understanding of human pathology

• Drug is seldom investigated by scientists developing the Idea.

• Over-interpretation of significance of pre-clinical studies

• Drug is not used at a dose relevant to the pre-clinical studies

• Population does not respond as predicted. (Ineffective Trial Design)

• Dose-limiting side-effects

• Study Underpowered, too short or unrealistic expectations

• Measurement Instruments Inadequate Clinical Outcomes and Surrogate Markers

• Wrong Group of pwMS studied (IneffectiveTrial Design)

• Commercial Interests

Mechanism is all Important.

Relevance of Slight Delay of a Few Days, Slight Diminution

Prophylactic/Therapy

“Building Site Effect”

“Route & Timing”

“Placebo Effect”

Less Circuitry so Less Compensation Capacity

Non-Responders

Immune (T/B cell) or Neurodegeneration

Professional Trialists

Reporting Issues

Two thousand drugs tested only ten classes of drugs approved


• CD4 T cell depletion inhibits EAE

• CD4 T cell deletion inhibits virtually every T and B Cell mediated autoimmunity

• CD4 T cell deletion does not inhibit multiple sclerosis(Lindsey et al. 1994 Neurol 44:413 : 810; van Oosten et al. 1998 Mut Scler 1:339)

.

• CD4 T cell deletion does not inhibit other human autoimmune diseases

Problem with the Study

Planned <60% Depletion

Naïve CD45RA preferentially affected

Primed CD45RO T cells relatively unaffected (Llewellyn-Smith et al. 1997 Neurol 48:810; 48)

MS IS NOT CONTROLLED BY T CELL DEPLETION

No effect on Gadolinium enhancing lesions

Time post-disease onset (days)

-1 0 1 2 3 4 5 6 7 8

Me

an

Ne

uro

logic

al S

co

re

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

250µg YTS191

25µg YTS191

5µg YTS191

Vehicle

250µg YTA3

85% deletion

60%

deletion

20%

deletion

If 60% T cell Depletion does not Stop EAE,

Why would it be expected to stop MS?

The CD4 trial in MS was Probably doomed before it even started

Day 0 Day 1 Day 3 Day 7 Day 14 Day 21

% o

f C

on

tro

l

0

10

20

30

40

50

60

70

80

90

100

110

120

130

Untreated

250µg CD4 mAb

50µg CD4 mAb

25µg CD4 mAb

5µg CD4 mAb


Degree of CD4 depletion was important with regard to treatment efficacy.

There was a significant 41% reduction in relapses.






KNOW YOUR DISEASE/MODEL-CHALLENGES

Adaptive Immune-Dependent Neurodegeneration

Innate Immune-Dependent Neurodegeneration

Post-Demyelination Repair Chronic Demeylination in Gliosis Repair

Symptomatic Motor SensorySymptomatic Cognitive

Relapsing T B cell Immunity

Red = abundant CB1 receptors Black = moderately abundant CB1 receptors

PHARMACEUTICAL CANNABIS

BENCH TO BESIDE – SYMPTOM CONTROL

Time Post-Injection (Min)

0 10 20 30 40 50 60 70 80 90 100 110 120

Ch

an

ge

in

Hin

dlim

b S

tiff

ne

ss

(%

) ± S

EM

-50

-40

-30

-20

-10

0

10

20

Sativex

Vehicle







This extreme close-up with a scanning

electron microscope shows

oligodendrocytes cultured in a well among

the tapered glass micropillars.

Mei et al. Nat. Med., 2014 20:954

A cluster of antimuscarinic compounds

scored high in cell differentiation and

membrane wrapping in the high-

throughput screen.

EAE

CUPRIZONE FEEDING

(OLIGODENDROCYTE TOXIN)

MONONUCLEAR

CELL INFILTRATE

In vivoModels

Focal Model

BENCH TO BESIDE – REPAIR







Optic Neuritis is

Often First Sign of MS

Most accessible part of

Human CNS

Courtesy of Roy Weller

BENCH TO BESIDE – NEUROPROTECTION

EYE

OPTIC NERVE

VISUALCORTEX

The eye is the window to the brain…

OCCIPITALLOBE

The Visual SystemX

Damage

in MS

Optic Neuritis

Common First

Sign of MS

Occurs in Over

50% of MS

X

OPTIC NEURITIS IN MULTIPLE SCLEROSIS


Develops Spontaneous/Induced Optic Neuritis.Subclinical Spinal Cord DiseaseLess Severe than Classical EAE

Fluorescent Retinal Cells Detection of Nerve Loss in Living Eye

Repeated Monitoring Not Requiring Histology

Disease is Concentrated in the Visual SystemHuman Relevant Outcome Measures

Visual Acuity, Electrophysiology (Demyelination Nerve Loss), Imaging the Living Eye


Day 0 (before disease)

Day 21 (after disease)

After Optic Nerve Head

OCT imageHistology image

Eye from Living AnimalEye from Dead Animal

Outer

Nuclear

Layer

Photo-

Receptors

Ganglion

Cell Layer

Inner

Nuclear

Layer

Before

Carbamazepine

Me

an

Nu

mb

er

of R

etin

al G

an

glio

n C

ells

/mm

2

800

900

1000

1100

1200

1300

1400

1500

1600

1700

1800

1900

Non-Induced

Optic Neuritis + Control

Optic Neuritis + Sodium Channel Blocker

OxcarbazepineBaseline

* *

A

D

CB

Carbamazepine

Me

an

Nu

mb

er

of R

etin

al G

an

glio

n C

ells

/mm

2

800

900

1000

1100

1200

1300

1400

1500

1600

1700

1800

1900

Non-Induced




* *

A

D

CB

VehicleNa Channel

Blocker

Carbamazepine

Mean N

um

ber

of R

etinal G

anglio

n C

ells

/mm

2

800

900

1000

1100

1200

1300

1400

1500

1600

1700

1800

1900

Non-Induced




* *

A

D

CB

Low Power

Normal

Inhibition of Retinal Ganglion Cell Loss

Reduced Nerve Damage

Normal mouse

Mea

n r

etin

a ce

ll d

ensi

ty (

cells

/mm

2)

1000

1100

1200

1300

1400

1500

1600

1700

1800

1900

OPTIC NEURITIS+ Vehicle

OPTIC NEURITIS +drug

Na Channel

BlockerVehicle

Al-Izki S et al. 2014


Neuroprotection in Optic Neuritis in Humans

Gabilondo I et al. 2015

Raftopoulos R et al. 2016

30-35% Less Nerve Loss








SEVERITY EXPERIENCED BY ANIMALS INCREASES

IMPACT-PAPERS INFLUENCING EUROPEAN POLICY ON ANIMAL RESEARCH

MAKE SURE YOU BASE YOUR 3Rs ON SCIENCE

Wolfensohn S, Hawkins P, Lilley E, Anthony D, Chambers C, Lane S, Lawton M,

Voipio HM, Woodhall G. Reducing suffering in experimental autoimmune

encephalomyelitis (EAE). J Pharmacol Toxicol Methods. 2013; 67(3):169-76.

WEIGHT LOSS CORRELATES WITH SEVERITY BUT IS NOT AN INDICATOR OF MORTALITY

C57BL/6 Strain (MOG peptide-Induced)Animals with Weak Signs (Limp tail, Impaired righting reflex) 2.1 ± 3.7% loss n=3/88Animals with Strong Signs (Hindlimb paralysis) 17.1 ± 4.7% loss n=36/88Animals with EAE-related Mortality or Euthanasia Not Applicable n=0/88

HA Strain (Spinal cord Homogenate Induced)Animals with Weak Signs (Limp tail, Impaired righting reflex) 22.1 ± 8.1% loss n=11/54Animals with Strong Signs (Hindlimb paralysis) 28.8 ± 4.2% loss n=20/54Animals with EAE-related Mortality or Euthanasia 28.8 ± 7.0% loss n=23/54

Biozzi ABH Strain Spinal Cord Homogeneate Induced)Animals with Weak Signs (Limp tail, Impaired righting reflex) 26.4 ± 4.6% loss n=9/65Animals with Strong Signs (Hindlimb paralysis) 30.8 ± 3.5% loss n=60/65Animals with EAE-related Mortality or Euthanasia 31.8 ± 3.0% loss n=12/1978


WEIGHT LOSS CORRELATES WITH SEVERITY BUT IS NOT AN INDICATOR OF MORTALITY

WEIGHT LOSS CORRELATES WITH SEVERITY BUT IS NOT AN INDICATOR OF MORTALITY_________________________________________________________________________

Weight Distribution of Surviving ABH Mice_________________________________________________________________________

Weight loss Frequency

> 5% 300/300 (100%)>10% 295/300 (98%)>15% 290/300 (97%)>20% 272/300 (91%)>25% 213/300 (82%)>30% 105/300 (35%)>35% 10/300 ( 3%)>40% 0/300 (0%)

_________________________________________________________________________Weight collected pre-1993 Weight loss is maximum related EAE verses day 11/Maximum weight


• Use lower doses of antigen

• Reduce all stressors

• Reduce noise levels

• Raise ambient temperature – use heating blankets, extra litter and nesting material,

Do not isolate sick animals – house with well animals, so they can huddle with the other mice to keep warm

• Reduce study duration if possible

• Apply a humane end point appropriate to the study


TEMPERATURE AS AN ENDPOINT• QUANTITATIVE• NON-SUBJECTIVE• CAN BE DONE NON-INVASIVELY• DETECTABLE (HOURS) PRIOR TO DEATH

• CAN BE DONE REMOTELY WITHOUT ANIMAL HANDLING-TELEMETRY CHIP• CAN BE DONE IN HOME CAGE –TELEMETRY CHIP• CAN REMOTELY ALARMED TO TRIGGER ENDPOINT-SOFTWARE EXSISTS


TELEMETRY CHIP 1BATTERY POWERED-TRANSMITTING CHIPSURGICALLY IMPLANTED DISPOSABLE (£130-£1,000/chip)REMOTE DETECTOR-DETECTS MULTIPLE ANIMALS

TELEMETRY CHIP 2BASE POWERED-PASSIVE CHIPNON-SURGICALLY IMPLANTED (£10-£15-chip)BASE STATION UNDER HOME CAGE-SINGLE ANIMALS AT MULTIPLE TIME POINTS

1.6g - 6months


UCL-INSTITUTE OF NEUROLOGY

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Baker 3 rs york pdf