WuXi Biology

Diabetes Service Platforms

Full scale of diabetes assays and models

To accelerate your drug discovery process

www.wuxiapptec.com

Research Service Division (RSD)

Enabling Unbounded Possibilities

Prevalence

2000 2010 20300

100

200

300

400

500

151

285

438

No

. o

f D

iab

ete

s (

Millio

n)

• 6.4% of adult population in 2010

• 54% increase 2010 to 2030

• 36% in India and China alone

China: 2007-2008

(NEJM 362;12, 2010)

China: 2010

(JAMA. 2013, 310:948)

9.7% Diabetes

15.5% Prediabetes

11.6% Diabetes

50.1% Prediabetes

AMPK, PPARragonist

Blood glucose

5.6 mM/100 mg/dl

3.9 mM/70 mg/dl

Normal range

Insulin

Insulin analog

GLP-1 , GIP

&Aanalog

DPPV inhibitor

SGLT2 inhibitor

Hepatic Glucose

output Glucose uptake and

utilization

Glucose

reabsorption

GLP-1 agonist

KATP channel

inhibitor

AMPK agonist

GCGR

antagonist

Glucose absorption

-glucosidase

inhibitorHyperglycemia

Reference: DRCP, 2010 , 87:4

Diabetes Service Platforms

Mode of Action in Treatment of Diabetes

In Vitro Assay Services Applications

New drug R&D

In vitro pharmacological

profiling of compounds,

insulin and/or biosimilars

Biosimilar equivalence

assessment, quality testing

IND filling

• Complete recording and

achieving of samples, assay

procedures, raw data and

results

• Data authenticity

• Assay traceability

• CFDA on site inspection1

Category

Name/Target

(human if not

specified)

Cell lineLigand binding

/Enzyme assayFunction assay

INS & Analog

Insulin-R A

(short)CHO I-125 phosphorylation

Insulin-R B

(long, exon 11)CHO I-125 phosphorylation

IGF-1R H19-7 I-125 phosphorylation

IR/GLUT4

3T3-L1

adipocytes

[3H]Deoxy-D-

glucoseglucose uptake

IR [14C]-glucose lypogenesis

IR [14C]-glucoseGlycogen

formation

GLP1R GLP-1R HEK293 FRET/I-125 cAMP

GCGR Glucagon R HEK293 I-125 cAMP, Ca

SGLT

SGLT1 CHO [14C]Methyl a-D-

glucopyranoside

(AMG)SGLT2 CHO

SGLT2 (rat) CHO

DPP DPP4，8， 9 Luminescent

NHR PPAR-gamma Fluorescent

2

Diabetes Service Platforms

Insulin Assay

Affinity: Binding Assay

Method: Radioligand filtration binding assay

Format: 96-well

Insulin receptor-A binding assay

-2 -1 0 1 2 3 4

0

20

40

60

80

100

120

Insulin

IC50 = 1.4 nM

Log Con. [nM]

% In

hib

ito

n

Insulin receptor-B binding assay

-3 -2 -1 0 1 2 3

0

20

40

60

80

100

120

Insulin

IC50 = 1.2 nM

Log Con. [nM]

% In

hib

ito

n

Stable cell line: CHO-INSRA, WuXi Stable cell line: CHO-INSRB, WuXi

Function: Phosphorylation Assay

Method: Alpha screen assay (phosphorylation)

Format: 384-well

Insulin Receptor-A function assay

-2 -1 0 1 2 3 4

0

20

40

60

80

100

120

Insulin

EC50 = 10.6 nM

Log Con. [nM]

% A

cti

vit

y

Insulin Receptor-B function assay

-2 -1 0 1 2 3 4

0

20

40

60

80

100

120

Insulin

EC50 = 15.1nM

Log Con. [nM]

% A

cti

vit

y

Stable cell line: CHO-INSRA, WuXi Stable cell line: CHO-INSRB, WuXi

Glucose Uptake Assay

Cell line: 3T3-L1, ATCC

Method: Radioactive assay

Format: 96-well

Glucose Uptake Assay

Glucose uptake in 3T3-L1 adipocytes

-3 -2 -1 0 1 2 3-20

0

20

40

60

80

100

120Insulin

EC50 = 2.0 nM

Log Con. [nM]

% A

cti

vit

y

Adipocytes

Induction

3T3-L1 (ATCC, CL-173™)

Cell line: 3t3-L1, ATCC

Method: Radioactive assay

Format: 96-well

Lipogenesis Assay Glycogen Synthesis Assay

Lipogenesis in 3T3-L1 adipocytes

-4 -2 0 2 4-50

0

50

100

150Insulin

EC50 = 0.71 nM

Log[Con.nM]

Acti

vit

y%

Glycogen synthesis in 3T3-L1 adipocytes

-4 -2 0 2 4-50

0

50

100

150Insulin

EC50 = 1.42 nM

Log[Con.nM]

Acti

vit

y%

Cell line: 3t3-L1, ATCC

Method: Radioactive assay

Format: 96-well

Diabetes Service Platforms

Function: cAMP Assay

Method: TR-FRET cAMP assay

Format: 384-well

-16 -14 -12 -10 -8 -6 -40

2

4

6Exenatide

GLP-1 receptor binding assay (Tag-lite)

IC50 = 6.2 nM

Log Con. [nM]

HT

RF

Rati

o (

665/6

20)

GLP-1 receptor cAMP assay

-6 -4 -2 0 2 4-20

0

20

40

60

80

100

120

140

Exenatide

EC50 = 0.06 nM

Log Con. [nM]

% A

cti

vit

y

Affinity: Binding Assay

Method: Tag-lite assay

Format: 384-well

Stable cell line: HEK-GLP-1R, WuXi Stable cell line: HEK-GLP-1R, WuXi

Affinity: Binding Assay

Method: Radioligand filtration binding assay

Format: 96-well

Function: cAMP Assay

Method: TR-FRET cAMP assay

Format: 384-well

GCGR receptor binding assay

-2 -1 0 1 2 3 4

20

40

60

80

100

120

Glucagon

IC50 = 7.7 nM

Log Con. [nM]

% In

hib

ito

n

GCGR receptor cAMP assay-antagonist mode

-2 0 2 4 6-20

0

20

40

60

80

100

120

MK-0893

IC50 = 37.7 nM

Log Con. [nM]

% In

hib

ito

n

Stable cell line: HEK-GCGR, WuXi Stable cell line: HEK-GCGR, WuXi

GLP-1R Assay

Glucagon Receptor Assay

Primary Screening

Enzyme: DPP4

Method: Luminescent assay

Format: 384-well

DPP4 inhibition assay

-14 -12 -10 -8 -6

0

20

40

60

80

100

120

Linagliptin

IC50 = 0.29 nM

Log Con. [M]

% C

on

tro

l

DPP Assay

PPAR-gamma Binding Assay

0 1 2 3 4 50

25

50

75

100

125

150

175Troglitazone

IC50 = 408 nM

Log Con. [nM]

Po

lari

zati

on

(m

P)

PPAR-γ Assay

3

Primary ScreeningEnzyme: PPARγ-LBDMethod: Fluorescence polarization

Format: 384-well

Transporter assay

Method: Radioactive assay

Format: 96-well

hSGLT1 transporter assay

-2 0 2 4 6-20

0

20

40

60

80

100

120

Dapagliflozin

IC50 = 417.5 nM

Log Con. [nM]

% In

hib

itio

n

hSGLT2 transporter assay

-4 -2 0 2 4-20

0

20

40

60

80

100

120

Dapagliflozin

IC50 = 0.74 nM

Log Con. [nM]

% In

hib

itio

n

Stable cell line: CHO-hSGLT2, WuXi Stable cell line: CHO-hSGLT1, WuXi

SGLT2/1 Assay

Diabetes Service Platforms

NHP Diabetes Resource

Fat NHP:

High fat diet induced, spontaneous

Diabetes study:

• Oral Glucose Tolerance Test (OGTT)

• Mixed Meal Tolerance Test (MMTT)

• Intravenous Glucose Test (IVGTT)

• Insulin Tolerance Test (ITT)

• Graded Glucose Infusion (GGI)

• Glucose Clamping

Glucose Clamping

20% Glucose

solution

Continuous i.v.

infusion Serial blood sampling

Rapid plasma

glucose measurement

Glucose infusion rate adjustment to achieve plasma steady state

Insulin

solution

4

Imaging:

• MRI liver fat content quantitation

• MRI liver fibrosis detection

• MRI blood vessel imaging

• Body composition measurement

by DEXA

Tissue biopsy:

• Liver

• Kidney

• Muscle

• Fat

• Brain

Graded Glucose Infusion

Diabetes Service Platforms

Glucose Tolerance Test (Acute)

Introduction:

An assay to assess glucose tolerance including oral glucose

(OGTT), intraperitoneal glucose(IPGTT) or intravenous glucose

(IVGTT) to fasted animals

Species: Mouse, rat and NHP

Endpoint: Blood glucose and Insulin

Throughput: 8 animals/group; 5~8 groups

Groups in a standard assay: Vehicle, Positive control, Testing

compounds with 3 doses

Turnaround time : ~5 days/each study

Effect of Cpd A (s.c)on Blood Glucose lowering

time course in IPGTT

0

10

20

30

40

0 30 120-60

Glucose

20 40

Test article

60

Cpd A High

VehicleCpd A LowCpd A Medium

Exenatide

Time(min)

Blo

od

glu

co

se(m

mo

L/L

)

Effect of Cpd A (s.c) on reducing Glucose AUC 0-120min in IPGTT

Data were presented as Mean ± SEM, n=5 per group, ***p<0.001

vs. vehicle, one way ANOVA followed by Dunnett’s test

Urine Glucose Assay

Introduction:

An assay to measure urine glucose

Species: Rat and NHP

Endpoint: Urine volume; Urine glucose; Blood glucose

Groups in a standard assay: Naïve(Vehicle), Positive

control, Testing compounds with 3 doses; n=4~6

animals/group

Turnaround time : 2 days/each study

0

1 0

2 0

3 0

4 0

Urin

e v

olu

me

(m

L/2

00

g B

W) ***

E ffe c t o f C a n a g lif lo z in (p .o .) o n u r in e v o lu m e

in 2 4 h rs p o s t tre a tm e n t

C a n a g liflo z in 1 m p k

V e h ic le

C a n a g liflo z in 3 m p k

C a n a g liflo z in 1 0 m p k

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

Urin

e g

luc

os

e (

mg

/20

0 g

BW

)

***

***

***

E ffe c t o f C a n a g lif lo z in (p .o .) o n th e u r in e g lu c o s e e x c lu s io n

in 2 4 h rs p o s t t re a tm e n t

C a n a g lif lo z in 3 m p k

C a n a g lif lo z in 1 0 m p k

C a n a g lif lo z in 1 m p k

V e h ic le

V eh ic le ≈0

Data were presented as Mean±SEM, n=5 per group, *** p＜0.001 vs vehicle

group, one way ANOVA followed by Dunnett multiple comparison test.

5

Diabetes Service Platforms

db/db mouse model

Introduction:

A commonly used model to assess the anti-diabetic

effect in db/db mice

Species: Mouse

Model: C 57 BL/KsJ.db male mice

Endpoint:

Body weight, food intake, water intake, blood

glucose, GTT, insulin, GLP-1, GTT,

HBA1C/fructosamine, pancreases histology stain

Throughput: 10~12 animals/group; 5~8 groups

Groups in a standard assay: Vehicle, Positive control,

Testing compounds with 3 doses

Turnaround time : ~6 weeks/each study

Effect of Rosiglitazone and DMBG (p.o) on Blood Glucose (mmol/L)in db/db mice

0

5

10

15

20

25

30

35

db/m wild type vehicle

db/db vehicle

db/db Rosiglitazone

db/db DMBG

****** ******

*** *** *** ***

Blo

od

Glu

co

se (m

mm

ol/L

)

Baseline Week1 Week2 Week3

Data were presented as Mean ± SEM, n=12 per group, *p<0.05,**P<0.01,

***p<0.001 vs. vehicle, one way ANOVA followed by Dunnett’s test

7-8 weeks old db/db mice were treated with Cpds for three weeks.

ZDF rat model

Introduction:

A commonly used model to assess the anti-diabetic

effect in ZDF rat

Species: Rat

Model: Zuker fa/fa male rats

Endpoint:

Body weight, food intake, water intake, blood glucose,

insulin, GLP-1, GTT, HBA1C/fructosamine, pancreases

histology stain

Throughput: 8 animals/group; 5~8 groups

Groups in a standard assay: Vehicle, Positive control,

Testing compounds with 3 doses

Turnaround time : ~6 weeks/each study

Data were presented as Mean ± SEM, n=8 per group, *p<0.05

vs. vehicle, one way ANOVA followed by Dunnett’s test

7-10 weeks old ZDF rats were treated with Rosiglitazone for three weeks.

Effect of Rosiglitazone on Blood Glucose in ZDF rats

High Fat Diet Induced Obesity (DIO) model

Introduction:

• Mice fed with HFD (Research Diet D12492 ) for 12~14

weeks to assess anti-obesity agents

• 200 DIO mice /month ready to use

Species: Mouse and NHP

Endpoint:

Body weight, food intake, GTT, total Cholesterol (TC),

Triglyceride (TG), ALT, AST, TG in, liver/muscle Insulin,

GLP-1, HBA1C ,Leptin, HE/Oil red O stain, Fat pad, etc.

Throughput: 8 mice/group; 5~8 groups

Groups in a standard assay: Vehicle, Positive control,

Testing compounds with 3 doses

Turnaround time : ~6 weeks/each studyAnti-obesity effect of Rimonabant chronic treatment

Data were presented as Mean ± SEM, n=8 per group, *p<0.05, **p<0.01 vs.vehicle, one way ANOVA followed by Dunnett’s test

6

High Fructose Diet model

Introduction:

A model induced by feeding with High Fructose Diet (HFD)

to assess dyslipidemia

Species: Rat and NHP

Endpoint: Body weight, food intake, Total cholesterol (TC),

Triglyceride (TG), HDL-C, LDL-C, etc.

Throughput: 8 rats/group; 5~8 groups

Groups in a standard assay: Vehicle, Positive control, Testing

compounds with 3 doses

Turnaround time : ~4 weeks/each study

Data were presented as Mean ± SEM, n=8 per group, *p<0.05,**P<0.01,

***p<0.001 vs. vehicle, one way ANOVA followed by Dunnett’s test

Effect of EPA on Plasma TG levels

Diabetes Service Platforms

Research Service Division (RSD)

Enabling Unbounded Possibilities

Contact

Henry Lu

Vice President, Biology

Henry_lu@wuxiapptec.com

Changqing Cao

Executive Director

Cao_changqing@wuxiapptec.com

Wei Liang

Director

Liang_wei_sb066@wuxiapptec.com

Longji Xu

Director, Business Development

Xu_longji@wuxiapptec.com