1

Steven G. Gilbert, PhD, DABTDirector, INND

Affiliate Associate Professor, U of W

Animal Models in Non-clinical Drug and Device Development

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ThalidomideIntroduced in 1956 as sedative (sleeping pill) and to reduce nausea and vomiting during pregnancyWithdrawn in 1961

Discovered to be a human teratogen causing absence of limbs or limb malformations in newborns5000 to 7000 infants effectedResulted in new drug testing rules

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“The Commons”

The Tragedy of the CommonsBy Garrett Hardin, Science, 1968

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Technical Solutions

“It is our considered professional judgment that this

dilemma has no technical solution.”

The Tragedy of the CommonsBy Garrett Hardin, Science, 1968

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Problems – Solutions?

Lead and kidsFetal alcohol syndromeNuclear disarmamentBioterrorismOcean FisheriesPersistent chemicalsThe Commons

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“When an activity raises threats of harm to human health or the environment, precautionary measures

should be take even if some cause and effect relationships are not fully established scientifically.”

Wingspread Conference, 1998.

Precautionary Principle

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FDA regulations of Drugs (1938)FDA regulations of Dietary Supplements (Dietary Supplement Health and Education Act of 1994 (DSHEA))Ephedra present an unreasonable risk of illness or injury (Dec, 2003)

Safety & Efficacy vs Harm

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INTERLOCKING PLAYERS

• Investigates• Cautious• Incremental• Truth Seeking

• Regulatory• Settle Disputes• Seeks Compromise• Forced Decisions• Mandated to Protect

• Profit• Directed Research• Seeks to Raise Funds• Seeks Access

Science Government Business

PUBLIC / CONSUMERS

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SCIENTIFIC DRIVERS

Mechanism of ActionGenomics – Chromosome 21Gene therapyTransgenic animals• Morphometric analysis• ICC, PCR, Molecular biology• Image analysis• Flow Cytometry

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TOXICOLOGY VS MECHANISM

0123456789

1 3 5 7 9 11 13 15 17 19 21

Time

Impo

rtanc

e

toxmechanism

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GOVERNMENT AGENCIES INVOLVED

• FDA - Food and Drug Administration• EPA - Environmental Protection Agency• OSHA - Occupational Safety and Health

Administration• CPSC - Consumer Product Safety

Commission

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REGULATORY DEMANS

• GLPs - Good Laboratory Practices• GMPs - Good Manufacturing Practices• GCPs - Good Clinical Practices

• ICH – EC, MHW, FDA

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• Animal Care –AAALAC, USDA, NIH

• QAU / QC• Validation• CDC (animal imports)

REGULATORY DEMANDS

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2 Drugs Return a Profit

100 Tested in Humans

100,000 NewCompounds

Preclinical Evaluation

10 Marketed Drugs

CRO- Accelerates selection- Refines efficacy- Saves time & money

Clinical Evaluation

NEW DRUG ATTRITION RATE

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Years 6.1 1.5 2.4 3 2.3 Total=15.3

Test Population

Lab and Animal Studies

20-80 Healthy

Volunteers

100-300 Patient

Volunteers

1000-3000 Patient

Volunteers

Post-Marketing

Safety

Purpose

Safety and

Biological Activity

Determine Safety and

Dosage

Efficacy and Safety

Efficacy and Safety

of Long Term Use

Review Process

Large-Scale Manu-

facturing

Distribution

Education

File IND File NDA

Goal - reduce preclinical development to 3-4 years by employing state of the art biological

sciences & technology

DRUG APPROVAL PROCESS

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Year Pre-Clinical P Clinical PhaseApproval Phase 960's 3.2 2.5 2.4970's 5.1 4.4 2.1980's 5.9 5.5 2.8990's 6.1 6.9 2.3000's 4 5.5 2.3

3.25.1 5.9 6.1

4

2.5

4.45.5

6.9

5.52.4

2.1

2.82.3

2.3

0

2

4

6

8

10

12

14

16

18

1960's 1970's 1980's 1990's 2000's

Year

s 8.1

11.6

14.215.3

11.8

DRUG DEVELOPMENT TIME

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REGULATED PRODUCTS

• Drugs/Chemicals• Biologics• Medical Devices

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BIOLOGICAL PROPERTIES

• Acute Toxicity• Cumulative Toxicity• Absorption• Elimination• Penetration of Barriers• Milk Excretion

• Teratogenicity• Mutagenicity• Carcinogenicity• Sensitization• Local Irritation• Biocompatibility

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TOXICOLOGY STUDY ASSESSMENTS

In-Life Phase• Hematology• Clinical Chemistry• Urinalysis• Organ Weights• Neurotoxicity• Other• Histopathology

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TYPICAL TOXICOLOGY STUDIES TO SUPPORT REGISTRATION

• Genetic Toxicology• Acute Toxicology• Subchronic Toxicology• Chronic Toxicology• Reproductive/Developmental• Carcinogenicity Studies• Neurotoxicology• Immunotoxicology

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Preliminary Evaluations

Genetic Toxicology

PK & TK Kinetics(rodent and non-rodent)

Acute and Multidose Pilot(rodent and non-rodent)

In Vitro Toxicology

PharmacologicalEvaluations

Genetic Toxicology Battery

DEVELOPING A TOXICITY PROFILE I

EFFICACY

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Acute Toxicology(Several species, various routes)

Pharmaco- and Toxicokinetics

Subchronic Toxicology(2 weeks to 90 days, rodent and non-rodent)

Teratology

Chronic Toxicology(6 months to 1 year, rodent and non-rodent)

Reproductive ToxicologyEnvironmental Assessment

Oncogenic Studies(2 year, 2 rodent species)

DEVELOPING A TOXICITY PROFILE II

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WHAT IS THIS?

CH3

N

N N

N

CH3

CH3

O

O

1

3

7

24

0

2

4

6

8

10

12

14

Con

cent

ratio

n (µ

g/g)

0 1 2 3 4 5 6 7 8 9 10

Time (hrs)

How Long It Takes To Go

PK - HALF-LIFE

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MECHANISTIC APPROACH

• Specific Endpoints• Animal Models of Disease• Transgenic Mouse Models• Functional Genomics• Focused Study Design

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COMPLIANCE & ACCREDITATION

• GLP Compliant• AAALAC Accredited (1991)• USDA Registered• OLAW Approved (OPRR)• Radioisotope License

Quality Assurance Manager

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PERSONNEL• Ph.D. Level Scientists• Attending Veterinarian• AALAS Certified Animal Technicians• Accredited Laboratory Staff• Technical Staff on Duty 7 Days a Week• Certified QA Staff

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(CIA) Model forRheumatoid Arthritis (RA)

Collagen-Induced Arthritis

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MECHANISTIC APPROACH

Immunomodulating compoundsor

Anti-inflammatory compounds

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METHODS

• Heterologous type II collagen is used as an immunizing agent

• Results in the generation of anti-type II collagen autoantibody

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TWO PHASES

Immune response - cross-reacting antibodies(ELISA to test for anti-collagen antibodies)

Inflammatory phase - the antibodies bind to the animal’s type-II collagen in cartilage and induce inflammation in the joint

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SUBJECTS AND EXPOSURE

• 6 to 7 week-old male BB/WOR diabetic-resistant rats

• Subcutaneous injection of either bovine type I or type II collagen

• Arthritis was evaluated daily on days 8-21

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G roup

T reatm ent

Paw Score

P aw M easurem ent

A nti-T ype II C ollagen

A ntibodies 1 B I im m unized norm al norm al -

2 B II im m unized elevated elevated +

3 B II im m unized + p iroxicam

norm al norm al +

RESULTS

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CONCLUSION

Discriminate between a compound’s immunomodulating AND anti-inflammatory effect in the CIA model.

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MODELS OF CNS DISEASE

• Parkinson’s Disease • Alzheimer’s Disease• ALS• Neurodegenerative Disorders

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DEVELOPMENT OF CNS DRUGS

• MPTP (1-Methyl-4-phenyl-1,2,3,4-tetrahydropyridine)

• Behavioral Effects of MPTP• Morphometric Analysis of Effected

Areas of the Brain - Cell Counts• Subjects Mice C57-Bl

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BEHAVIORAL DATA

Grid Crossings and Exposure

0.00

20.00

40.00

60.00

80.00

100.00

120.00

-6 0 2 4

10 17 24 29 31 39 57

Study Day

0.00

5.00

10.00

15.00

20.00

25.00

Num

ber o

f Cro

ssin

gs

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MORPHOMETRICANALYSIS OF THE BRAIN

Brain Cell Counts

0

2000

4000

6000

8000

10000

12000

14000

16000

SNC SNR

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0

500

1,000

1,500

Normal MPTP

Loco

mot

or a

ctiv

ity

(cou

nts

/5h

r)

EFFECTS OF MPTP ON THE LOCOMOTOR ACTIVITY IN COMMON MARMOSETS

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CARDIOVASCULAR BIOLOGY

• Animal Modeling Capabilities- Large and small animals; Acute, chronic, long term

studies; Cardiac, coronary, peripheral vascular models; Physician training; Radioactive materials license

• Imaging and Monitoring- Digital fluoroscopy, intravascular ultrasound

(IVUS), consulting echo; ECG, IBP, CO, SaO2, EtCO2, blood gases

• Laboratory Support- Hematology, clinical chemistry, histology,

pathology, morphometry, digital imaging

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SWINE HOUSING

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SHEEP HOUSING

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SATELLITE FACILITY

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LARGE ANIMAL SURGERY SUITE

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SURGERY IN ACTION

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SWINE CORONARY ARTERIES

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SWINE ILIAC REGION

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RABBIT ILIAC REGION

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IVUS IMAGE

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Normal Coronary Artery Balloon Injured Artery

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RESTENOSIS DEFINED

• A complex injury response leading to the narrowing or occlusion of a treated vascular segment

• A result of performing a PTCA, atherectomy or stent procedure in an atherosclerotic vessel

• The major limitation of coronary interventional procedures

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THE SCOPE OF THE PROBLEM

• PTCA is a very common in hospital procedure - 800,000+ per year worldwide- 500,000+ per year in the U.S.

• Restenosis occurs in - 30-50% of lesions treated with PTCA- 20-40% of lesions treated with stents = “in-

stent” restenosis

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RESTENOSISA Result of at Least Four General

Processes

Stents &Antithrombotics

Neointima formation

Thrombus formationElastic recoil of the vessel wall

Geometric “negative” remodeling

?

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NEOINTIMA FORMATION

• A Combination of Three Processes- Cell migration- Cell proliferation (hyperplasia)- Extracellular matrix deposition

• Current and potential therapy options address one or more of these processes

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THERAPY AVENUES

• Mechanical (Treatment and Prevention)- Repeat PTCA or Stenting- Rotational atherectomy / Directional atherectomy- Cutting balloon- Energy (ultrasound, cryotherapy, ELCA, PDT)

• Radiation (Prevention)- Beta and Gamma emitters- Radioactive stents and catheter based delivery

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THERAPY AVENUES

• Pharmacological (Prevention)- Systemic delivery- Drug eluting stents- Local vascular delivery- Biodegradable stents

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ANIMAL MODELS OF RESTENOSIS

• There is a similarity in the basic elements of arterial injury response across species

• Neointimal response differs for a given injury- Rat < Dog < Rabbit < Human < Swine

• Coronary versus peripheral arteries

• Adult atherosclerotic versus juvenile normal arteries

• Limitations with human size devices

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SWINE MODEL• Species readily available• Vessel access site• Vessel size• Coronary vascular anatomy• Develop lesions quickly• Hemostasis properties• Similar lipoprotein metabolism including

spontaneous development of atherosclerosis

• US FDA recommendation

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NORMAL CORONARY ARTERY

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NORMAL CORONARY ARTERY

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BALLOON INJURED ARTERY

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BALLOON INJURED ARTERY

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BALLOON INJURED ARTERY

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MODEL CHARACTERISTICS

• 20-30% PTCA balloon overstretch• Break(s) in the IEL, medial damage• 2-4 weeks to complete the injury

response process• 25-75% luminal occlusion• 10% mortality rate• Excellent for screening potential

anti-restenosis (anti-proliferative) therapies

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RADIATION FORPREVENTION OF RESTENOSIS

• Also called vascular brachytherapy• Beta and Gamma emitters are being tested• Radioactive stents and removable sources• Positive and encouraging results in pre-clinical

and clinical trials• Limitations are emerging

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RADIATION IS EFFECTIVE

Control Artery Radiation Treated

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POTENTIAL LIMITATIONS• Delayed healing of endothelium• Thrombogenic surface• “Edge effect”• Possible risk for accelerated atherosclerosis

(seen in animal models)• Dosage and centering difficulties• Patient and operator exposure• Need for radiation oncologist and radiation

physicist• Radioactive waste storage and disposal 68

“EDGE EFFECT” ???

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PHARMACOLOGICAL ALTERNATIVES

• Agents under pre-clinical investigation- Heparin, LMWH- Oligonucleotides- Anti-inflammatories- Nitric Oxide- Taxol- Rapamycin- Ethanol- Anti-growth factor agents- Many others… 70

ELEMENTS FOR OPTIMAL PHARMACOLOGICAL THERAPY

• Achieve a therapeutic concentration in the vessel wall (i.e. efficient delivery)

• Achieve sufficient vessel retention time• Avoid systemic effects/toxicity• Limit duration of effect to time course of

restenosis• Avoid delays of reendothelialization and normal

healing

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ADVANTAGES OFPRECLINICAL ANIMAL TESTING

• Saves Time• Saves Money• Prototype Testing • Information Gathered to

Plan Clinical Trials• Mechanism of Action

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STUDY DESIGN

• Simple

• Specific Endpoints

• Appropriate Animal Model

• Appropriate Number of Animals

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SUMMARY• Its Science not Toxicology • Advances in Science• Education• Flexibility• More Investment• More Consolidation

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Cluster Analysis Example

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Normal - Abnormal Tissues

GenePlus™ Analysis

Dis

cove

red

Gen

es

Patients76

GenePlus™ Example

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REFERENCES• FDA Red book – (regulatory guideline for study

design) • FDA – www.fda.gov• Society of Toxicology – www.toxicology.com• American Association for Laboratory Animal

Science (AALAS) – www.aalas.org• Washington Biotechnology & Biomedical

Association (WBBA) – www.wabio.com

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Steven G. Gilbert, PhD, DABTtel: 206.527.0926 / fax: 206.525.4301

sgilbert@innd.org

Animal Models in Preclinical Drug and Device Development