The ZDSD Rat as a Translational Model for the Development of Drugs for Obesity, Metabolic Syndrome and Diabetes that Demonstrates Many of the Serious Complications of Diabetes.

PreClinOmics, Inc.

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Metabolic Syndrome affects a large proportion of the population and is becoming increasingly important in adolescents. The syndrome has many components including central obesity, insulin resistance, dyslipidemia and hypertension. In addition, the syndrome features a chronic low grade inflammatory state, vascular endothelial dysfunction, and a prothrombotic environment. Long standing metabolic syndrome can thus pre-dispose to atherosclerosis, microvasculature disease (retina), stroke, renal injury and diabetes. Due to the complicated mechanisms involved in the syndrome and its sequelae, current standard of care reflects poly-pharmacy and is aimed at controlling atherogenic dyslipidemia, hyperglycemia and hypertension as well as intervening in secondary diseases such as renal dysfunction, stroke, and micro-vascular disease related to retinopathy. Development of new chemical entities with the potential to control more than one risk factor is hampered by currently available animal models. To that end, the ZDSD rat was designed to spontaneously develop a phenotype that mimics many aspects of the human metabolic syndrome, including hypertension and the progression to frank diabetes with long-standing disease.

ZDSD as a Preclinical Model of Metabolic Syndrome

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Development Scheme: Zucker Diabetic-Sprague Dawley Rat (ZDSD)• Produced by crossing diet induced obese (DIO) rats

derived from the Crl:CD (SD) strain (exhibiting polygenetic obesity and insulin resistance) with homozygous lean ZDF/Crl rats (which expresses beta cell failure with the Leprfa/Leprfa genotype).

• Selectively bred for obesity and diabetes.• Selected for genetically matched breeders to

develop phenotypic homogeneity.• Studied male rats at different ages.

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Characteristics of Metabolic Syndrome seen in the ZDSD Rat

• Increased body weight with increased abdominal fat• Increased fed and fasting glucose and HbA1c levels• Insulin resistance / Glucose intolerance• Hyperlipidemia• Increased blood pressure --> Hypertension• Increased Serum Biomarkers of Coagulation,

Inflammation and Vascular Disease

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Comparative Growth Curvesin SD and ZDSD Rat Fed 5008 chow

ZDSD rats were significantly (15%) heavier than their SD counterparts at 8 weeks of age. In addition, the rate of body weight gain was increased in ZDSD rats as evidenced by an 82% vs 62% weight gain when compared to SD rats over 24 weeks of observation.

Study # 09-550-170

All time points statistically different

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Spontaneous Development of Obesity in ZDSD Rats Fed 5008 Chow

Study # 09-550-170

All time points statistically different

Body composition was assessed using QNMR . The percentage of body weight identified as fat was 50 % higher in ZDSD rats compared to SD controls as early as 8 weeks of age. Body fat percentage continued to increase throughout the study and remained significantly higher than control rats at each time-point.

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Spontaneous Development of Hyperglycemia in ZDSD Rats Fed 5008 Chow

Study # 09-550-170

All time points statistically different

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Spontaneous Development of Glucose Intolerance Shown by OGTT in ZDSD Rats Fed 5008 Chow

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ZDSD rats become increasingly more insulin resistant with age as evidenced by the calculated HOMA-IR. The insulin resistance is evident compared to SD rats as early as 8 weeks of age (fed Purina 5008 chow).

Progressive Development of Insulin Resistance (HOMA-IR) in ZDSD Rats

All time points statistically different

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Representative Islets from 28 wk old ZDSD Rats

Pre-diabetic Diabetic

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Blood pressure data produced in collaboration with Dr. Subah Packer’s Laboratory, IU School of Medicine

Blood Pressure in ZDSD vs CD Rats8-16 weeks of age

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60 70 80 90 10080

100

120

140

160ZDSDCD

Age in Days

Syst

olic

BP

(mm

Hg)

Urinary albumin

10 20 22 24 26 300

25

50

75

100

125

150SDZDSD

Age (weeks)

Urin

ary

albu

min

(mg/

day)

beta-2 microglobulin

10 20 22 24 26 300

500

1000

1500

2000 SDZDSD

Age (weeks)

Urin

ary

b-2 m

icro

glob

ulin

( mg/da

y)

Cystatin C

10 20 22 24 26 300

10

20

30SDZDSD

Age (weeks)

Urin

ary

cyst

atin

C ( mg/

day)

KIM-1

10 20 22 24 26 300.0

2.5

5.0

7.5

10.0

12.5

15.0SDZDSD

Age (weeks)

Urin

ary

KIM

-1 (n

g/da

y)

Time Course of Urinary Biomarkers

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Glomerular Basement Membrane Thickness

Time of Diabetes in the ZDSD Rat

Thic

knes

s (m

m)

CD Control 12 Weeks 16.5 Weeks0

100

200

300

400

500

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Kidney Histopathology of the ZDSD Rat

Glomerulopathy Tubular dilation Protein casts Inflammation

His

topa

thol

ogy

Sco

re (0

-5)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0Non-diabetic Diabetic

* * **

* compared to Non-diabetic animals(t-test p<0.05)

/degeneration

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Wound Healing in the ZDSD Rat

This figure demonstrates the wound healing in the three groups of animals. There were no differences between diabetic and non-diabetic ZDSD animals. There were several statistically significant differences between the SD group and the ZDSD groups (* p<0.05). Since the data were not different in the ZDSD groups there were also analyzed as a combined group.

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Separated Diabetic and Non-Diabetic ZDSD

4 7 9 11 14-100

-80

-60

-40

-20

0 SDDiabetic ZDSD

Non-Diabetic ZDSD

**

**

*

*

*

*compared to SD (Dunnett's)

Time(days) post-wounding

% C

hang

e Fr

om In

iital

Wou

nd

Combined ZDSD Data

4 7 9 11 14-100

-80

-60

-40

-20

0SD

ZDSD

*

*

* *

*

*compared to SD (t-test)

Time(days) post-wounding

% C

hang

e Fr

om In

iital

Wou

nd

Structural Properties L4 vertebraeP-values for differences in diabetic rats vs. respective controls

Mean ±SEMn=12-17/group

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0102030405060708090

ZDF (fa/fa) ZDF (+/fa)

Ene

rgy

to U

ltim

ate

Load

(mJ)

*

Biomechanical Test – axial compression ZDF ZDSD Yield Force (N) p<0.050 p<0.001Stiffness (N/mm) p<0.001 p<0.005

Ultimate Load (N) p<0.001 p<0.001Energy to Ultimate Load (mJ) p<0.050 p<0.001

0

10

20

30

40

50

60

70

8090

ZDSD Controls

Ene

rgy

to U

ltim

ate

Load

(mJ)

*

ZDSDObesity

Metabolic SyndromeDiabetes

Obesity Modelbefore

diabetes develops,5-16 weeks of age

Metabolic Syndrome

Insulin Resistance

Hyperlipidemia

Obesity

Hypertension

Delayed Wound Healing

Diabetes Models

SpontaneousDevelopment

(LabDiet 5008; Slower & more random)

Diabetic Nephropathy

Osteoporosis

Cardiovascular/ Inflammatory Biomarkers

Delayed Wound Healing

Diet Synchronized

(RD D12468 or TestDiet 5SCA)

Diabetic Nephropathy

Osteoporosis

Cardiovascular/ Inflammatory Biomarkers

Delayed Wound Healing

The ZDSD Rat: One Rat – Many Models

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ZDSD Summary- One Rat, Many Models

• Polygenic phenotype that can be modulated by diet• Functional Leptin Pathway• Early onset of hyperglycemia with slower

progression to frank diabetes when compared to the ZDF rat

• Mirrors human development of type II diabetes• Manifests diabetic complications:

• Diabetic nephropathy• Hypertension• Inflammation• Osteoporosis• Delayed wound healing

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