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1
Steven G. Gilbert, PhD, DABTDirector, INND
Affiliate Associate Professor, U of W
Animal Models in Non-clinical Drug and Device Development
2
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
3
“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
15
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
17
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
25
MECHANISTIC APPROACH
• Specific Endpoints• Animal Models of Disease• Transgenic Mouse Models• Functional Genomics• Focused Study Design
26
COMPLIANCE & ACCREDITATION
• GLP Compliant• AAALAC Accredited (1991)• USDA Registered• OLAW Approved (OPRR)• Radioisotope License
Quality Assurance Manager
27
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
28
(CIA) Model forRheumatoid Arthritis (RA)
Collagen-Induced Arthritis
29
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
31
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
32
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
33
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
34
CONCLUSION
Discriminate between a compound’s immunomodulating AND anti-inflammatory effect in the CIA model.
35
MODELS OF CNS DISEASE
• Parkinson’s Disease • Alzheimer’s Disease• ALS• Neurodegenerative Disorders
36
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
37
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
38
MORPHOMETRICANALYSIS OF THE BRAIN
Brain Cell Counts
0
2000
4000
6000
8000
10000
12000
14000
16000
SNC SNR
39
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
40
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
42
SHEEP HOUSING
43
SATELLITE FACILITY
44
LARGE ANIMAL SURGERY SUITE
45
SURGERY IN ACTION
46
SWINE CORONARY ARTERIES
47
SWINE ILIAC REGION
48
RABBIT ILIAC REGION
49
IVUS IMAGE
50
Normal Coronary Artery Balloon Injured Artery
51
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
52
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
53
RESTENOSISA Result of at Least Four General
Processes
Stents &Antithrombotics
Neointima formation
Thrombus formationElastic recoil of the vessel wall
Geometric “negative” remodeling
?
54
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
55
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
56
THERAPY AVENUES
• Pharmacological (Prevention)- Systemic delivery- Drug eluting stents- Local vascular delivery- Biodegradable stents
57
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
58
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
59
NORMAL CORONARY ARTERY
60
NORMAL CORONARY ARTERY
61
BALLOON INJURED ARTERY
62
BALLOON INJURED ARTERY
63
BALLOON INJURED ARTERY
64
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
65
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
66
RADIATION IS EFFECTIVE
Control Artery Radiation Treated
67
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” ???
69
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
71
ADVANTAGES OFPRECLINICAL ANIMAL TESTING
• Saves Time• Saves Money• Prototype Testing • Information Gathered to
Plan Clinical Trials• Mechanism of Action
72
STUDY DESIGN
• Simple
• Specific Endpoints
• Appropriate Animal Model
• Appropriate Number of Animals
73
SUMMARY• Its Science not Toxicology • Advances in Science• Education• Flexibility• More Investment• More Consolidation
74
Cluster Analysis Example
75
Normal - Abnormal Tissues
GenePlus™ Analysis
Dis
cove
red
Gen
es
Patients76
GenePlus™ Example
77
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
78
Steven G. Gilbert, PhD, DABTtel: 206.527.0926 / fax: 206.525.4301
Animal Models in Preclinical Drug and Device Development