Type 1 Diabetes Pre-Clinical Research

Biomedical Research Models,
Inc.
Founded 1996
www.brmcro.com
67 Millbrook St., Suite 422
Worcester, MA 01606

Diabetes Mellitus is Not a
Single Disease
ABSOLUTE
INSULIN
DEFICIENCY
TYPE 2 (NIDDM)
MODY
(Maturity Onset
Diabetes of the Young) TYPE 1b (IDDM)
TYPE 1a (IDDM)
LADA
(Latent Autoimmune
Diabetes in Adults)
RELATIVE
INSULIN
DEFICIENCY
DIABETES
SPECTRUM
HYPERGLYCEMIA

Genetics, Environment and T1A Diabetes
• The environment seems to be important in
human T1Diabetes
• Monozygotic twin with the disorder: ~50%
concordance
• The incidence of type 1 diabetes is increasing
• Finland, Sardinia, the Baltic States, Poland
• Is this genetics, environment, or both?

Candidate Environmental Agents
• Toxins
• e.g. nitrosoureas
• Diet
• e.g. cow milk protein
• Toll-like receptor ligands (e.g. LPS)
• Infection
• e.g. mumps, rubella, measles
• “Hygiene”

Epidemiology of Type 1A Diabetes
• Seasonal Variation
• Lower incidence in Finnish boys in June with a
spike in November-December
• Correlations of Type 1A diabetes onset with:
• Coxsackie B
• Mumps
• Rubella
• CMV,EBV, Retrovirus are less clearly associated

Viruses and Human T1D
• Epidemiological studies suggest association
• “Causality” is unproven
• No evidence of direct viral infection of islets
• Other putative mechanisms include:
• Bystander T-cell activation
• Molecular mimicry
• T cell activation by viral superantigen

Changing Incidence of AutoimmunityIncidenceofInfectionsDiseases(%)
1950 1990198019701960 2000
Rheumatic
fever
Mumps
Measles
Tuberculosis
Hepatitis A
0
100
50
IncidenceofImmuneDiseases(%)
1950 1990198019701960 2000
Multiple
sclerosis
Crohn’s
Disease
Asthma
Type 1
diabetes
100
200
300
400

“Spontaneous” Type 1 Diabetes
Human NOD BBDP Rat
MHC Multiple I-Ag7 RT1B/Du
Other Loci ~18 ≥27 >5
Gender M=F F>M M=F
Insulitis + + +
DKA + +/- +
AutoAbs + + +

Rat Models of Autoimmune Diabetes:
• Spontaneous
• BBDP/Wor
• BBZDP/Wor
• Long Evans
Tokushima Lean
(LETL)
• Komeda Diabetes
Prone (KDP)
• LEW.1AR1/
Ztm-iddm
• Induced
• YOS
• PVG
• PVG.RT1u
• PVG.R8
• WAG
• BBDR/Wor
• WF.iddm4
congenic
• Both
• MAD
(LEW.1WR1)
• Transgenic
• None
Strains in blue are maintained by BRM

Insulitis in Human, Rat, and Mouse
BBDP/Wor
Human Pancreas
NOD Mouse

The Diabetes Prone BB Rat
• Oldest Model of Spontaneous Type 1
Diabetes
• Evidence for Autoimmunity
• Pancreatic insulitis
• Class II MHC associated (RT1u)
• T cell dependent
• Preventable by immunosuppression
• Transferable to adoptive recipients
• Autoantibodies
• Lymphocytic thyroiditis

Tolerance in the Presence of
Genetic Susceptibility
A
AA
R
β
Autoimmunity
CD4+ART2+ Treg Transfusion
A
A R
R
RA
β
Tolerance
BBDP/Wor Rat

Environmental Considerations
• Viruses have been shown to alter the tempo of diabetes among
BBDP/Wor rats
• Some viruses delay the onset of diabetes
• (SDAV,LCMV and Sendai)
• Others accelerate (RCMV)
• Incidence of diabetes in virus free colony ~85%
• In BBDR rats virus can induce resistant rats to develop
autoimmune disease
• Hashimoto’s Thyroiditis
• Type 1 Diabetes
• No spontaneous diabetes among virus free rats

• Parvovirus in the BBDR Rat
• Kilham’s rat virus (KRV)
• Single stranded DNA virus
• 3 overlapping structural proteins, VP1, VP2 and VP3
• 2 overlapping nonstructural proteins, NS1 and NS2
• Parvovirus B19 associated with autoimmune
rheumatoid arthritis in humans (Simpson et al 1984
Science 223:1425)
• BBDR rats are an animal model for Rheumatoid
Arthritis (Watson et al. 1990 J.Exp.Med 172:1331)

Susceptibility and Mechanism
of Parvovirus Induced Diabetes
• Protocol
• 21-25 day old male and female animals
• 1 x 107 PFU KRV i.p. on day 0
• Monitor plasma glucose and body weight
• Experimental data demonstrates that the virus
• working through TLR-9
• creates and immune imbalance

Diabetes in Parvovirus (KRV)
infected BBDR Rats
Days After Infection
0 10 20 30 40
CumulativeDiabetes-Free
Survival(%)
0
20
40
60
80
100
KRV Alone (N=24)

Immunological Balance
Tolerance and Autoimmunity in BB rats
A
AA
R
β
Autoimmunity
CD4+ART2+ Transfusion
“Treg” Cells
A
A R
R
RA
β
Tolerance
ART2+ Treg Depletion
Virus Infection
BBDP BBDR
TLR Ligation

Diabetes in Parvovirus vs. H-1
Infected BBDR Rats
0 10 20 30 40
Survival(%)
0
20
40
60
80
100
KRV Alone (N=24)
H-1 Alone (N=6)

Parvovirus Reduces
Splenic Treg Cells
BBDR WF
CD4+CD25+
TregSpleenCells(%)
0%
1%
2%
No Virus
KRV
H-1
N.D.

BBDR Summary 1
• BBDR rats show virus-specific susceptibility
to the triggering of T1D
• Virus-induced Treg modulation may be one
mechanism of viral induction of diabetes

Innate Immune Responses
Trigger T1D in Rats
• Adaptive Immunity
• Pathogen-specific defense
• Involves the MHC and T Cell Receptor
• Long lasting immunity
• Innate Immunity
• First line of defense
• Activation of immune responses through toll-like
receptors (TLRs) that recognize pathogen
associated molecular patterns (PAMPs)

Hypothesis and Method
• Innate immune responses produced by TLR
ligation will induce T1D in resistant rats
• Polyinosinic:polycytidylic acid (poly I:C)
– A synthetic double-stranded polyribonucleotide
– Ligand of toll-like receptor 3 (TLR3)
– Strong inducer of cytokines
– A simple tool for testing multiple strains

TLR3 Ligation Can Induce Diabetes
Strain RT1 B/D Diabetes
MAD (LEW.1WR1) u 22/22
BBDR u 20%-100%
PVG.RT1u u 26/30
LEW.1AR1 u 4/20
PVG.R8 u 6/9
LEW.1AR1-iddm u 0/10
WF u 1/22
WAG u 1/9
Treatment with poly I:C alone for 2-3 weeks; insulitis data concordant
Data from Ellerman and Like, Tirabassi et al., and Hedrich et al.

Synergy: Diabetes After Virus Infection
and Activation of Innate Immunity
0 10 20 30 40
Survival(%)
0
20
40
60
80
100
KRV Alone (N=24)
KRV after Poly I:C (N=6)
H-1 Alone (N=6)
H-1 after Poly I:C (N=10)

Parvovirus (KRV) Itself May Act via TLR9
Stimulus
CpG KRV Poly I:C
IL-12p40(%ofControl)
0
20
40
60
80
100
0.1 µg/ml
1 µg/ml
10 µg/ml
The ability of spleen cells to respond to KRV by producing IL12-p40
is inhibited by iCpG, an inhibitor of TLR9 (Dr. D Zipris)

BBDR Summary 2
• Innate immune activation can trigger T1D in
many but not all rat strains with a high risk
MHC haplotype
• Innate immune activation can synergize with
viral infection to increase the penetrance of
T1D
• One diabetogenic virus, KRV, may act in part
via activation of TLR9

New Model of Type1 Diabetes
The MAD Rat (LEW.1WR1)
• Recombinant inbred congenic strain
• Same MHC class II RT1B/Du haplotype required for autoimmune
diabetes in BB, Komeda, and LEW.1AR1/Ztm-iddm rats
• In the colony of rats maintained at BRM, Inc.:
• Spontaneous diabetes absent from acquisition in 1989 until 1999
• Now occurs at low frequency (0.5-3%)
• Develop autoimmune insulitis and diabetes when treated with
poly I:C, a ligand of toll-like receptor 3 (TLR3)

Extending the Scope of Viral Triggers:
The MAD Rat
• The BBDR + KRV combination is not unique
• LEW.1WR1 Rats
– MHC-Congenic LEW rats
– High risk MHC class II RT1B/Du haplotype
– Normal immunological phenotype
– Diabetes occurs at consistently but at low
frequency (~2.5%)

Hypothesis
• Given its genetic predisposition to
spontaneous T1D:
• Viral infection will trigger T1D in
MAD rats

Rat Cytomegalovirus
(RCMV)
• Beta-herpesvirus homologous to human
cytomegalovirus
• Persistent and latent infections
• No reported associated diseases
• Stocks are prepared from salivary glands

Relevance to Human Disease
• Human Cytomegalovirus (HCMV)
• Associated with autoimmune diseases
• Ubiquitous pathogen causing unapparent infections in
immunocompetent individuals
• Peptide from HCMV protein stimulate CD4+ T cells
that recognize GAD
• Organ transplantation
• Currently no effective vaccine

MAD Rats are Sensitive to
Virus-Induced Diabetes
Treatment N % Diabetic Grade
None 6 0 n.a.
Kilham Rat Virus 8 38 3.3
H-1 Virus 20 0 n.a.
Rat Cytomegalovirus 16 44 4.0
Vaccinia virus 10 0 n.a.
Coxsackie B4 10 0 n.a.
Tirabassi, et. al. 2004. Diabetes. 53 Supplement 2:A301

Diabetes in BBDR and MAD Rats
Summary of Induction Studies
Method BBDR/Wor MAD
Spontaneous - +
Anti-Art2a - +
TLR3 ligands - +
Parvovirus 21days + +
RCMV -/+ + +
TLR 3 + Art2a + +
Parvovirus 45days - +

Diabetogenicity of five TLR
agonists in MAD rats
Group Test
Article
TLR Induction
of
Diabetes
Induction
Of
Arthritis
Gender
Bias
1 Poly I:C (primarily HMW) TLR3 ++++
No ♀ = ♂
2 LMW poly I:C (purified) TLR3 ++++
3 HMW poly I:C (purified) TLR3 ++++
4 Zymosan TLR2 +++
5 R848 TLR7 ++
6 CpG oligodeoxynucleotide TLR9 -
The dosing regimen was 3 times weekly by intraperitoneal injection beginning
at 21-24 days of age. Compounds were administered to 6 female and 6 male
rats at each dose of compound. Animals were treated over a 30 day period and
were monitored for diabetes from 7 to 40 days after the initiation of treatments

Serial Viral Infection Alters
Diabetes Incidence
Virus
% Diabetic
BBDR MAD
Parvovirus 41% 38%
RCMV 6.3% 44%
Parvovirus→
RCMV
0% 40%
RCMV→KRV 75% 90%

RCMV accelerates diabetes onset
in Diabetes Prone BB rats
0 5
0
20
40
60
80
100
30 40 50 60 70 80 90 100 110 120
RCMV/mock
infection
+ RCMV
- RCMV
RCMV INFECTED (n = 47)
CONTROLS (n = 29)
Age (days)
%DiabeticBB-DPrats
p = .0043
(Kaplan-Meier Log Rank)
van der Werf et al 2003: Clin Dev Immunol. 10:153
Hillebrands et al 2003: Clin Dev Immunol 10:133

Virus N Diabetes Virus N Diabetes
None 6 0 (0%) H-1 20 0 (0%)
Parvo 32 11 (34%) Coxsackie B4 18 0 (0%)
RCMV 38 14 (37%) Vaccinia 10 0 (0%)
Triggering of T1D in MAD Rats
is Virus-Specific
Latency to onset 16-30 Days
Dose of virus varied from 104 to 107 PFU

RCMV Infected Cells in Salivary Glands
But Not in Pancreatic Islets
Immunohistochemistry for RCMV early antigen (mAb 8)
Salivary Gland (mAb8) Islet (H&E) Islet mAb8

Conclusions
Virus Triggering of T1D in Resistant Rats
• T1D can be triggered in the rat by:
– Specific viral infections
– Two infections that are synergistic
– TLR ligation
– Certain viruses only in an immune system “pre-
activated” by TLR ligation

Topic 2
Prevention by Immunization
• If viral infections do promote diabetes onset
in susceptible rats with normal immune
systems…
• Then immunization may prevent the disease

“Immunized Pups”
X
Immunized
Female
Naïve
Male
Virus
Non-Diabetic
Virus
Diabetes?
A “Maternal Immunization” Protocol
MAD rat
Females

Maternal Immunization Protects Weanling
MAD Rats from Virus-Induced Diabetes
0
Survival(%)
0
20
40
60
80
100
10 20 30
Pups born to RCMV+KRV
Immunized Dam (N=10)
KRV+RCMV co-infection
Ordinary pups (N=10)
Pups born to RCMV
Immunized Dam (N=26)
Ordinary pups (N=13)
RCMV

Conclusion
• Maternal immunization can provide effective
and specific protection from virus-induced
diabetes in weanling rats
• Diabetes may be preventable by vaccines that
target candidate pathogens
• Rats like the MAD can be used to test diabetes
vaccination strategies

T-1D RAID Programs
• Rapid Access to Interventional Development for Type 1
diabetes
• BRM responded to two USPHS RFP’s in June of 2005.
• Our requested budgets totaled > $11.3 MM
• BRM was notified in December 2005 that it was
technically competent and within the Competitive
Range for Both RFP’s
– Only company competing for both contracts

T-1D RAID Programs
• Rapid Access to Interventional Development for Type 1
diabetes
• Preclinical Studies of Efficacy in Animal Models of
Type 1 Diabetes (Pathogenesis) N01-DK-6-2909
– BRM Awarded $4.9Million Contract 2006-2012
– Selected over a Top 25 Research Institute

T1D RAID Process
Potential therapeutics
submitted by US scientists
5 selected yearly by T1D RAID board
Successful compounds screened
and identified by BRM
Entry into TrialNet
New successful drug identified

Trials to Prevent or Treat Type 1 Diabetes:
Clinical Considerations
 Tight glycemic control is correlated with C-peptide
preservation in type 1 diabetes clinical trials.
 Interventions are more likely to succeed if done early, while
some beta cell function is preserved.

NOD Model Problem 1:
Treatment of diabetes
in new onset NOD mice
 Diabetic NODs are treated with poorly-
characterized insulin pellets or by insulin
injection resulting in variable or inadequate
glycemic control.
 Poor control may contribute to rapid beta cell
demise or loss of functionality, and therefore a
poorer response to immune interventions
designed to reverse disease may be observed.

NOD Model Problem 1:
Solutions
 Contract is developing better ways to
control glycemia in NOD diabetic mice
 Contract is developing ways to predict
imminent diabetes in order to intervene
prior to beta cell loss.

Interventions to Maximize Success
• Tight glycemic control of diabetic animals for periods
of 3-6 weeks
– Provides immune modulatory therapies maximum
opportunity for success
• Contract is developing ways to predict imminent
diabetes to intervene prior to complete beta cell loss

Model Standardization
• Use optimized rodent models for standardized
preclinical testing of agents to prevent or reverse
diabetes
– Testing in multiple models (mice and rats)
– Development of standardized testing protocols
– Development of insulin treatment protocols for
reversal studies

Example Testing Schemes
Treat 21-110 days
Treat 21-160 days
Follow 50 days
Preventative Protocol
Read-outs
Preventative/Reversal Protocol
Treat when turns diabetic
Treat 2 nondiabetic littermates Read-outs
Pulse Protocol
Read-outs

Year BRM Jackson
2006 68% 65%
2007 (at 27 weeks) 86% 95%
2008 (at 23 weeks) 75% 75%
Incidence of Diabetes Among NOD Mice

Therapeutic Evaluations in the
NOD Mouse Study Designs
Reversal
Initiate treatment after
frank diabetes onset.
Insulin therapy to
maintain euglycemia.
Late Prevention
Initiate treatment to
animals with impaired
GTT prior to frank
diabetes. Insulin therapy
not used.
Early Prevention
Initiate treatment prior to
disease onset. Insulin therapy
not used.

Control and Prediction
• Contract is developing better ways to control
glycemia in NOD diabetic mice (and BB rat)
• Contract is developing ways to predict imminent
diabetes to intervene prior to complete beta cell loss

Using the GTT test to predict
diabetes onset (Protocol 2)
• Impaired glucose tolerance (IGT) expected to
be early manifestation of impending diabetes
– Perform GTT (ip injected glucose) on fasted
nondiabetic female mice at 12, 14, or 16 weeks of
age, then follow for 30 days for diabetes onset
– Analyze AUC of animals that became diabetic or
remained non-diabetic
– Determine the best parameters for prediction

*** P<0.001
* P<0.05
*** *
NOD Mice with Impaired Glucose
Tolerance Develop Diabetes

NOD Mice with Impaired Glucose
Tolerance Develop Diabetes
GTT performed on animals at 14 weeks of age.
IGTT= Impaired Glucose Tolerance, NGTT= Normal Glucose Tolerance.
9/19 animals with IGTT turned diabetic within 30 days after test.
4/31 animals with NGTT turned diabetic within 30 days after test.

GTT Conclusions
• GTT has high predictive value for diabetes
when performed at 14 weeks of age
Sensitivity Specificity Pos. Pred. Value
14 Weeks 0.69 0.73 0.47

Study Design Late
Intervention
Perform GTT
at 14 weeks
Select mice with
impaired IGTT
Randomize into
groups: Rx, vehicle
or no rRX

Development of Protocols
to Optimize Treatment of
Spontaneous Type 1
Diabetes in NOD Mice

Metabolic Control in NOD Mice
• Factors to consider
– Total daily dose (2- 4 Units) 25 g mouse
– Type of Insulin
– Frequency of dosing QD, BID, TID
– % of daily dose administered each injection
– Injection time relative to “fed state”
• Many pilots done to test insulin formulation
(Humulin 50/50, Humulin 70/30, PZI), dose,
use of diluted insulin and BID dosing

Continuous Insulin Release to Control
Blood Glucose in Diabetic NOD Mice
• Continuous release of insulin using Alzet osmotic
pumps
– Implant subcutaneously
– Use Humulin R at 0.2, 0.3, 0.4U/day
– Monitor BG daily, with an intensive (every 3 hours)
24 h monitoring on Days 3, 7 and 14
• Continuous release insulin controls blood
glucose rapidly with few glycemic excursions

Daily Blood Glucose Averages
0
100
200
300
400
500
600
0 5 10 15 20
BloodGlucose(mg/dL)
Day Post Pump Insertion
0.2U
0.3U
0.4U
Mice implanted subcutaneously with pumps releasing 0.2-0.4U Humulin R/day.
Daily blood glucose measured approx. 8-9 hours after lights on.
N=6 for 0.2 and 0.3U groups; N=12-15 for 0.4U group.

Intensive BG Monitoring – 0.4U
Mice implanted subcutaneously with pumps releasing 0.4U Humulin R/day.
BG measured every 3 hours for 24 hours on Days 3, 7 and 14 post pump insertion.
N=15 for Days 3 and 7; N=12 for Day 14.
0
100
200
300
400
500
600
0 2 4 6 8 10 12 14 16 18 20 22 24
BloodGlucose(mg/dL)
Hour in Study
3 Day
7 Day
14 Day

Intensive BG Monitoring – 0.3U
0
100
200
300
400
500
600
0 2 4 6 8 10 12 14 16 18 20 22 24
BloodGlucose(mg/dL)
Hour in Study
3 Day
7 Day
14 Day
Mice implanted subcutaneously with pumps releasing 0.3U Humulin R/day.
BG measured every 3 hours for 24 hours on Days 3, 7 and 14 post pump insertion.
N=15 for Days 3 and 7; N=12 for Day 14

Daily Blood Glucose Mean +/- SEM
0
100
200
300
400
500
600
-1 1 2 3 4 5 6 7 8 9 10 11 12 13 14
BloodGlucose(mg/dL)
Days Post Pump Insertion
.2U .25U .3U Diluent
Mice implanted subcutaneously with 1002 Alset pumps releasing 0.2-0.3U Humulin R/day.
Daily blood glucose measured approx. 5-7 hours after lights on.
N=12 for 0.2U and 0.25U groups; N=14 for 0.3U group; N=7 for Diluent group.

Day 3 Intensive BG (Mean +/- SEM)
0.0
100.0
200.0
300.0
400.0
500.0
600.0
3 3.5 4
BloodGlucose(mg/dl)
Daily blood glucose measured 3 days post insertion over 24 hours.

0.0
100.0
200.0
300.0
400.0
500.0
600.0
7 7.5 8
BloodGlucose(mg/dl)

0.0
100.0
200.0
300.0
400.0
500.0
600.0
14 14.5 15
BloodGlucose(mg/dl)

Digitized images of islets following 3
weeks of insulin Rx by Alzet pumps

Impact of insulin pumps on β-cell mass
Diluent 0.2U 0.3U 0.4U
0.00
0.03
0.06
0.09
Treatment Group
FractionalInsulinArea(%)

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