Selected Examples of In-vivo Models Enabled at Jubilant

1

A publicly listed integrated global pharmaceutical & life sciences company

Jubilant Life Sciences revenue for FY’19 $ 1,310 Mn

Jubilant Life Sciences – An Integrated Global Life Sciences Company

Drug Discovery and Development Solutions• Drug Discovery Services

• Development: GMP and Non-GMP NCE supply

• Integrated Discovery Collaborations

• Proprietary Innovation Assets for out-licensing

• Machine Learning

Life Science Ingredients

Specialty Intermediates• Advance Intermediates• Fine Ingredients• Crop Science IngredientsNutritional Products• Vitamins• Animal Nutrition

Life Science Chemicals • Life Science Chemicals• Ethanol & Specialty Gases

Pharmaceuticals

Generics• API’s • Dosage Formulations • Indian Branded PharmaceuticalsSpecialty Pharmaceuticals• CMO-Sterile Injectable • Radiopharmaceuticals• Allergy Therapy Products

1

2

Company

Employees

Customers

Founded in 2000; part of Jubilant Life Sciences

Global top 10, mid-size and virtual pharmaceutical and biotech companies

750+ employees. Most PhDs have >10 years of US/EU experience

Jubilant Biosys: an Innovation Driven CRO…

2

Integrated discovery capabilities from target to clinical candidate and Digital

Drug Discovery ServicesPre-clinical Chemistry capabilities

including GMP and Scale-up

Track Record Delivered over 75 integrated projects

Bangalore, India Noida, India

3

Target to Lead Generation

Lead Optimization Candidate Selection (IND)

Structural Biology (Protein Science, Crystallography) Medicinal and Computational

Chemistry Screening & Profiling Early ADMET & PK In vitro / In vivo Pharmacology

Medicinal Chemistry DMPK SBDD (Co-crystallization,

Computational Modelling) Target Engagement & Disease

Models Safety Profile Pre-formulation

Integration of Discovery Process Designed for Speed

Process Development Scale-up & GMP API Supply Genotox Non-GLP & GLP Tox D2M Predictions (WinNonlin)

3

4

Gained experience through a broad portfolio of programs which drive successful outcomes

Target to Hit Ph I / IIHit to Lead Lead Op Candidate IDTV

4

Non Enzymatic

Enzyme

GPCR

Kinase

Disease Biology Expertise

Oncology Metabolic Disorder CNS Pain & Inflammation (P&I)

20 + Programs

15 + Programs

15 + Programs

15 + Programs

5

Why Jubilant Biosys?

A highly experienced scientific team; delivered >75 integrated discovery programs

New technologies applied to accelerate drug discovery and improve decision-making quality

A boutique CRO with a talented team of >550 scientists giving your program priority

Investing in the future of drug discovery and development through expansion

Leadership with global pedigree in US, EU and Japan

5

“We share your passion of Drug Discovery & Development”

6

JDDS Flexible Business Models made for SpeedSustainable Innovation & Outcomes to Our Clients

Build to alignSolutions

Integrated DiscoverySolutions

Proprietary Innovation for

Licensing

Discovery, Preclinical & Clinical Solutions (FTE / FFS)

Medicinal & Synthetic Chemistry, Structural Biology, Molecular Modeling, Discovery Biology, Pharmacology, Bioinformatics, Bio analysis, Toxicology, Scale-up, cGMP

Jubilant Shared risk for Integrated Drug Discovery Programs

Focused across therapeutic areas– Oncology, CNS , MD and P&ITarget to Preclinical Candidate, Candidate to POC

Jubilant Innovation for Licensing / Partnerships

Proprietary drug discovery programs funded by Jubilant to create small molecule assets (up to IND) to be partnered or licensed

6

7

CNS/Pain Models

7

8

CNS/ Pain Models

• Neuropathic Pain Models• CCI model• SNL model (acute and morphine tolerance)• Formalin induced phase II response• Capsaicin induced secondary sensitization and flinching

• Disease Specific Pain Models• Chemotherapy induced mechanical allodynia• Bone cancer pain model• Streptozocin induced diabetic neuropathy• Post-operative pain model

• Alzheimer’s Disease/ neurodegeneration Models• Amyloid beta measurement from plasma and CSF• Total and phospho tau measurement (ex vivo)• Unfolded protein response (UPR) studies in brain: tunicamycin, thapsigargin induced UPR measure using

stress markers or de novo protein synthesis (ongoing)• Stroke model (stress marker and de novo protein synthesis)• Target engagement models

• PK-PD Correlation using free brain concentration and pharmacological response and / or target engagement

8

9

Glucoselevels

Glucoselevels

GlucoseLevels + allodynic

Inclusion, randomization dosingSTZ inj

45mpk i.v.

9

Neuropathic Pain Models

Day 6

Paclitaxel 1 mg/Kg ip onDay 0,2,4 and 6 Basal Allodynia

Inclusion of animalsDosing and compound testing

STZ Compound DRCPaclitaxel model Tramadol DRC

PK-PD Correlation

cmpd-1 (mpk, p.o.)

% M

PE (m

echa

nica

l, Von

Fre

y)

Conc ( µM)

Vehicle 3 10 30 1000

20

40

60

0.01

0.1

1

10

100Total Pl Total Brain

0.65µMrIC50

**Free plasma Free Brain

Paclitaxel induced neuropathic pain in male SD ratsTramadol DRC

1hr 2hr0

22

44

66

88

110

% M

PE (m

echn

ical

allo

dyni

a)

*

****

*p<0.05,****p<0.0001 Vs Vehicle postBonferroni test

*

**** ******** **** **** Vehicle(n=6)Tramadol-0.1mpk (n=5)Tramadol-0.3mpk (n=6)Tramadol-10mpk (n=6)Tramadol-20mpk (n=6)Tramadol-40mpk (n=6)

Tramadol (p.o. pre-treatment time)

Objective:To optimize and validate neuropathic pain models modeled based on diabetic neuropathy or cancer chemotherapy

• Developed STZ induced neuropathic pain model and validated with standard compound• Established paclitaxel induced neuropathic pain model and validated with Tramadol

10

Spinal Nerve Ligation (SNL) Model: Intrathecal Catheterization for Drug Delivery

10

Baseline testing andselection of animals

Compd testingI.T catheterization + SNL surgery

Double surgery

50%

PWT

(g) m

ean±

SEM

CL Basal 0.5 hr 1 hr 3 hr0

5

10

15

20

y ( )

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

96% 96%86%

GBP(300µg/10µl)

Saline(10µl)

Reversal

Objective:To optimize double surgery of spinal nerve ligation for induction of neuropathic pain and intrathecal catherization to locally deliver drugs in spinal cord

• Optimized combined SNL surgery and intrathecal surgery in SD rats• The model was validated with 300 ug gabapentin directly delivered in spinal cord• Time course of the study indicated efficacy gabapentin over three hours of the study period

11

Bone Cancer Pain (BCP) Model in Mice: Gabapentin DRC

Mechanical Allodynia (%MPE) Day 7 Cold Allodynia (%MPE) Day 7

Baselinerecording

Compd testingB16-F10 cells inj

Baselinerecording

Compd testing

Objective:To develop bone cancer pain model in mice by injecting a melanoma cell line and validation of the model using gabapentin

• Mouse melanoma cell line was injected in tibia of mice• Tumor was observed on day 6 and robust mechanical allodynia was induced in the animals• The model was validated with gabapentin in mechanical and cold read out on day 7 and day16

11

12

PK-PD (Rat)

30 mpkCmpd A (p.o.)

% p

S262 T

auR

educ

tion

vsve

hicl

e Conc

(µM)

Vehicle 100 mpk

0

10

20

0.01

0.1

1

10Hippocam pus (µM)Plasm a (µM)

**

***

Cell IC50 0.11µM

Ctrl

+ Dox

Dox+ Cmpd A

Tau Kinase: PK-PD Correlations

12

Objective:To test the reduction in phospho-tau level upon treatment with a tau kinase inhibitor and establishment of PK-PD correlations

• SD rats were treated with vehicle or compounds and brain was collected to isolate hippocampus• Phospho-tau and total tau level was measured by western blot• PK-PD correlation was established by plotting reduction in phospho-tau vs total drug level in hippocampus or

plasma

13

Target Engagement (TE) Model for a CNS indication

Objective:To develop TE model with no precedence and utilize it to discover inhibitors to address unmet needs in a CNS indication

• Established robust target engagement model• Model was successfully employed for integrated drug discovery project

13

Liver to Brain

Expression of down stream mRNA transcripts were profiled in exhaustive time-course and dose response experiments

Biomarker-A showed good dose proportional increase and was chosen to be tracked

Develop acute TE model

No literature precedence

Identify reliable biomarker

ChallengesTE model

Concentration of trigger (mpk, i.p.)

mR

NA c

hang

es in

liver

Fold

ove

r veh

icle

con

trol

0.1 0.3 1

0

40

500

1000

1500

Bio-B

Bio-A

Bio-C

Bio-G

Bio-FBio-E

Bio-D

Up-regulation of Kinase of interest activity in liverAnalysis of down stream biomarkers Biomarker -A in liver

mRN

AFo

ld o

ver n

orm

al c

ontro

l

0

50

100

VehNaive 3mpk 10mpk 30mpk 100mpkCompound-X

Biomarker-A in brain

mR

NA

Fold

ove

r ve

hicl

e co

ntro

l

20

40

60

80

Naive Veh 50mpk 100mpk

Compound X

Biomarker-B in brain

mR

NA

Fold

ove

r ve

hicl

e co

ntro

l

0

5

10

15

VehNaive 50mpk 100mpk

Compound X

14

Nociceptive/ Inflammatory Pain Models

• Neuro-inflammation Model

• MOG induced multiple sclerosis in mice

• Acute Pain Models

• Acetic acid induced writhing

• Tail flick

• Hot plate model

• Inflammatory Pain Models

• Carrageenan induced inflammation (paw thickness, paw volume, thermal hyperalgesia, mechanical allodynia and hyperalgesia)

• CFA Induced pain (mechanical allodynia, thermal and mechanical hyperalgesia

• Collagen induced arthritis

• IL12/ IL18 Induced IFNγ response in c57BL/6 mice

• PK-PD correlation using plasma free drug concentration and pharmacodynamic response

14

1515

CFA Induced Pain Model

Inclusion, randomization dosing

CFA inj75ug/150ul

CFA induced Mechanical HyperalgesiaGabapentin DRC

% M

PE (

CFA

mec

hani

cal)

0

20

40

60

80

100VehicleGBP- 10 mpkGBP- 30 mpkGBP- 100 mpk*

***

***P<0.001 and *P<0.05 VsVehicle, One Way ANOVA,Dunnets Test

Veh 10 mpk 30 mpk 100 mpk

Gabapentin (i.p.)

CFA induced themal hyperalgesiaTramadol DRC

0

5

10

15

20Veh

3mpk

10mpk

30mpk

*****

**P<0.01,***P<0.001VsVehicle; One-way ANNOVAfollowed by Dunnett's posttestPa

w w

ithdr

awal

Late

ncy(

Sec.

)

Veh 3 mpk 10 mpk 30 mpkTamadol (i.p.)

Objective:To establish inflammatory pain model by intraplantar injection of CFA and validation of the model with standard of care

• Intraplantar injection of CFA in rats induced pain as measured by mechanical allodynia or thermal hyperalgesia• Mechanical allodynia was reversed by gabapentin in a dose dependent manner• Thermal hyperalgesia was reversed by Tramadol in a dose dependent manner

16

MOG Induced EAE in C57Bl6 Mice

Therapeutic dosing of Figolimod (3mpk, p.o): Day 14- Day 35

Prophylactic dosing of Figolimod (3mpk, p.o): Day0- Day35

D3

MOG Immunization

Booster Pertussis inj

D0 D14 D35

16

Days

AVG

Clin

ical S

core

0

1

2Normal ControlVehicleTherapeutic groupProphylactic group

0 7 14 21 28 35

Objective:To develop a model of multiple sclerosis in mice by immunization with MOG peptide and its validation using Fingolimod

• Robust disease was induced in mice by MOG as indicated by clinical score after day 14• Therapeutic treatment with 3 mpk Fingolimod from day 14 reduced disease symptoms• Treatment with Fingolimod from day 0 delayed onset of the disease and reduced clinical score

1717

PKPD Relationship (Determination from Terminal Exposure)

Plasma Ligand (ng/ ml)Ligand Dose (mg/ Kg)Veh Std 3 10 30

Objective:To measure brain target engagement and establish a PK-PD correlation with total and free brain and plasma drug level

• Compound showed dose dependent target engagement in brain as indicated by reduced radioligand binding• Excellent correlation between in vivo target engagement and behavioral read out observed• Free brain conc/IC50 value was established to determine the efficacy parameter

18

Metabolic Disorders Models

18

19

Metabolic Disorders Models

• nSTZ - NASH Mice Model (STAM Model)

• CDA/HFD induced NASH Model in mice

• Non-Alcoholic Fatty Liver Disease (NAFLD) mice model

• Acute and Chronic OGTT, IPGTT (Glucose & Insulin) in mice and rat

• Pyruvate challenge test in mice

• Acute model for malonyl CoA lowering in rats

• DIO mouse: weight, glucose, insulin, lipids, and pair-fed studies

• Streptozotocin-Type 1 diabetic model in mice and rats

• Lean diabetic model(STZ + Nicotinamide)

• ZDF model

• ob/ob model

• db/db model19

2020

nSTZ - NASH Mice Model (STAM Model)

Objective:To develop STZ induced NASH (STAM) model in mice and its validation using Telmisartan

• STZ 200µg was administered s/c in 2 day old male mice pup and fed with high fat diet 60%kcal% fat diet post weaning

• Telmisartan show reversal in few key parameters in this model

*p<0.05 , **p<0.01 ,***p<0.001and ****p<0.0001 as compared to Control and $p<0.05 as compare to STZ+HFD control (One way ANOVA followed by, Bonferroni’s post-hoc test)

Data Shown as Mean ± SEM

2121

CDA/HFD Induced NASH Model in Mice

Objective:To develop CDA/HFD NASH model in mice and its validation using Telmisartan

• CDA/HFD Diet was fed to 8-10weeks old male C57BL/6 mice• Telmisartan show reduction in few key parameters in this model

*p<0.05,**p<0.01.***p<0.001 Vs CDA diet , One way ANOVA followed by Dunnett’s test and ##p<0.01,###p<0.001, Vs Control diet ,One Tailed Unpaired T testData Shown as Mean ± SEM

22

Non-Alcoholic Fatty Liver Disease (NAFLD) Mice Model

22

Objective:To develop NAFLD model in mice and its validation using Amlexanox

• 5-6 week old C57BL/6 mice fed on HFD for 14 weeks were treated with amlexonox for 4 week• Amlexanox show efficacy in this model

Data Shown as Mean ± SEM#p<0.05,####p<0.0001 Vs Control diet and *p<0.01,****p<0.0001 Vs DIO . Two tailed unpaired t test Data was analyzed by Graph pad Prism 5

2323

Oral Glucose Tolerance Test (OGTT) in Mice

Objective:To optimize and validate Oral Glucose Tolerance Test (OGTT) in mice

• Developed Oral Glucose Tolerance Test (OGTT) in mice and validated with Sitagliptin

Data shown as Mean ± SEM, *p<0.05 and ***p<0.001 Vs vehicle , Two-tailed Unpaired student’s t test

Blood Glucose in C57BL/6mice Plasma insulin in C57BL/6 mice

2424

Pyruvate Tolerance Test in Mice

Objective:To optimize and validate Pyruvate Tolerance Test in Mice

• Developed Pyruvate Tolerance Test in Mice and validated with Metformin

Data shown as Mean ± SEM*p<0.05,***p<0.001 and****p<0.0001 vs Vehicle, Two way ANOVA followed by Bonferroni's Post test

2525

Diet Induced Obesity (DIO) Mouse Model

Objective:To optimize and validate Diet Induced Obesity (DIO) mouse model

• Developed Diet Induced Obesity (DIO) mouse model and validated with Amelexanox

Data shown as Mean ± SEM*p<0.05, **p<0.01, ***p<0.001 vs Vehicle (HFD), Two way ANOVA

followed by Bonferroni's Post test

Rodent diet vehicleVehicle (HFD)Amelexanox 3mpk

Amelexanox 10mpkAmelexanox 30mpk

2626

ob/ob Mouse Model

Objective:To optimize and validate ob/ob mouse model

• ~ 12 weeks old male ob/ob mice were treated with metformin for 4 weeks • Model was validated using metformin @ 150 mpk

Data Shown as Mean ± SEM**p<0.01, ***p<0.001 and ****p<0.0001 when compared with ob/- vehicle and $$$ p<0.001 and $$$$ p<0.0001 when compared with ob/ob Vehicle (two-way and One way ANOVA followed by, Bonferroni’s post-hoc test)

2727

db/db Mouse Model

Objective:To optimize and validate db/db mouse model

• ~ 8 weeks old male db/db mice were treated with metformin for 4 weeks OGTT done on day 28 • Model was validated using metformin @ 200 mpk

Data Shown as Mean ± SEM****p<0.0001 when compared with db/- vehicle and $$$ p<0.001 and $$$$ p<0.0001 when compared with db/db Vehicle (two-way and One way ANOVA followed by, Bonferroni’s post-hoc test)

2828

ZDF Rat Model

Objective:To optimize and validate ZDF Rat model

• Developed ZDF Rat modeland validated with Vildagliptin

*p<0.05, ***p<0.001 Vs ZDF Control (Vehicle) Two way ANOVA followed by Bonferroni post tests

2929

STZ induced Type 1 Diabetes Model in Mice

Objective:To optimize and validate STZ induced type 1 Diabetes model in Mice

• Male mice were made diabetic by injecting STZ from day 1 to day 5 • Developed STZ induced type 1 Diabetes model in Mice and validated using Insulin

C57BL/6 mice

Data shown as Mean ± SEM, *p<0.05 & , ****p<0.0001 vs Vehicle control, Two Way ANOVA Followed by Bonferroni post test.

BALB/c mice

30

STZ induced Type 1 Diabetes Model in Rats

30

Objective:To optimize and validate STZ induced type 1 Diabetes model in Rats

• Male Sprague Dawley rats mice were made diabetic by injecting STZ on day 1 • Developed STZ induced type 1 Diabetes model in rat and validated using Insulin

Data shown as Mean ± SEM, ***p<0.001 vs STZ control, One WayANOVA Followed by Bonferroni post test.

3131

Streptozotozin + Nicotinamide Induced Type 2 Non-Obese Diabetic Mouse Model

Objective:To optimize and validate Streptozotozin + Nicotinamide Induced Type 2 Non-Obese diabetic mouse model

• Developed Streptozotozin + Nicotinamide Induced Type 2 Non-Obese diabetic mouse model and validated with veldagliptin and Metformin

*p<0.05, **p<0.01, ***p<0.001 vs Vehicle diabetic. One way ANOVA followed by Dunnett’s Post test.

32

Acute Malonyl CoA Lowering Model in Rats

32

Objective:To optimize and validate Acute Malonyl CoA Lowering Model in Rats

• Sprague Dwaley rats were used in this study. Malonyl CoA Lowering 5 hrs post dosing of Compound A,B,C,D

*p<0.05, ***p<0.001 Vs Control (Vehicle) Two way ANOVA followed by Bonferroni post tests

0

6

12

18

24 Vehicle

Compound A_30mpk

Compound C_100mpk

Compound D_100mpk

*

** **

5 h post dose

***

***P<0.001.**P<0.01,*P<0.05 Vs Vehicle Bonferroni posttest (One way ANOVA)

Compound B_100mpk

MCo

A(p

mol

es)

Plasma exposure data of Compound A, B, C, D

0 0.5 1.0 5.00

2500

5000

7500

10000

12500 Compound A_100mpk Compound B_100mpk Compound C_30mpk Compound D_100mpk

Time(h)

Plas

ma

Conc

.Mea

n±

SD

(ng/

mL)

Target Inhibition and PK/PD Study

33

Inflammation Models

33

34

Inflammation Models

• Experimental Autoimmune Uveitis model• Rat, Rabbit models

• Fibrosis Models• Lung, liver, skin, kidney, NAFLD and NASH

• Inflammatory Bowel Disease• DSS, TNBS and oxazalone-induced colitis in mice (acute and chronic)

• Gastric emptying and intestinal Transit • Mouse model of Gastric emptying and intestinal Transit using Phenol red• Mouse model of Gastric emptying

• Respiratory and Atopic Allergy Models• Steroid sensitive and resistant asthma in mice• LPS-induced neutrophilia in rats

• Inflammatory Pain • Formalin, CFA, Carageenan, Capsaicin-induced nociception

• Arthritis• Collagen induced arthritis

• Skin models• Psoriasis, Contact dermatitis

34

3535

Experimental Autoimmune Uveitis (EAU) in rats

Clinical Scores (R+L Eyes) ofIRBP (R16) + Ptx Induced EAU

in female Lewis Rats

Days post IRBP (R16, 30 ug)

Clin

ical

Sco

re_B

oth

Eyes

8 10 12 14 16 180

1

2

3

4

###

***

###

***

###

***

Clinical Scores (R+L Eyes) ofIRBP (R16) +Ptx Induced EAU in

female Lewis Rats

Days post IRBP (R16, 30 ug)

Clin

ical

Sco

re_B

oth

Eyes

8 10 12 14 16 180

1

2

3

4 ###

***

###

* #####

Induction: Day 0, Immunize with IRBP R16+PTx,Female Lewis rat (6-7 wks)

Objective:To develop Experimental Autoimmune Uveitis (EAU) model in rats using retinal antigens (IRBP)

3636

Experimental Autoimmune Uveitis (EAU) in Rats_HP scores

Based on infiltrates Based on 6 point scoring

Combined_D-16

HP

Scor

e

0

2

4

6###

**

Combined_D-17H

P Sc

ore

0

1

2

3

4

###

• Experimental autoimmune uveitis (EAU) was examined byhistopathology after Day 16 (tofa arm) &17 (dexamethasonearm) post sensitization.

• Fixatives : Davidson’s (48h) & IPA till HP analysis. H & Estaining tq, 10X magnification.

• HP evaluation has shown significant disease induction inAC, PC and ICB region on comparison to vehicle control.Tofa arm (with osmotic pump) has shown significant diseasereduction both in 6 point and infiltrate based scoring.

• HP evaluation has shown significant disease induction inAC, PC and ICB on comparison to vehicle control indexamethasone arm. Marginal reduction of disease was seenin ICB and AC & significant reduction in PC.

3737

Cytokine Induced Uveitis in Rabbits (CIU)_Acute (4 days)

IP-1

0 le

vel (

pg/m

l)

0

10000

20000

30000

40000

50000

LLQ=780 pg/ml after dilution factor is incorporated

Level of IP-10 in AH

IP-1

0 le

vel (

pg/m

l)

0

100000

200000

300000

400000

##

*

Level of IP-10 in VH

70%

LLQ=780 pg/ml after dilution factor isincorporated

IP-1

0 le

vel (

pg/m

l)

0

20000

40000

60000

80000

100000

LLQ=390 pg/ml after dilution factor isincorporated

##

*

Level of IP-10 in R+C

86%

Data shown as Mean ± SEM. One Way ANOVA, Dunnett’s Post Test. ### p<0.001 vs Vehicle Control and **p<0.01 vs Disease Control.

Male NZW rabbits quarantine & Health check

Randomization; body weight &

Basal Eye Observation / Red Reflex

Day (0)

1st IVT injection (Vehicle/Treatments)

Day 1

Scoring & Terminal sacrifice:

BW, Plasma. AH,VH, R/C, ICB from right/left eye.

Animal monitoring: Cage side

observations

Dosing regimen as indicated below

Day 3 (48 hrs)Day 2 (24 hrs)

2nd IVT injection (Vehicle/IFNg)

Objective:To develop an acute model for Cytokine induced uveitis (CIU) in rabbits

3838

Cytokine Induced Uveitis in Rabbits (CIU)_ HP scores (Acute)

Combined (AC, ICB, PC)

0

1

2

3

4

5

#

HP S

core

s

• Eyes with optic nerve were collected in Davidson’s fixative & kept for 48 hrs & then transferred to 50% isopropyl alcohol, andprocessed for histopathological analysis.

• Cytokine induced uveitis (CIU) was examined for histopathology post 24hrs of Tofa treatment.

• H & E staining tq, 10X magnification.

Data shown as Mean ± SEM.# p<0.05 vs Vehicle Control.

One Way ANOVA, Dunnett’s Post Test

Based on infiltrates

3939

Level of IP-10 in AH

IP-1

0 le

vel (

pg/m

l)

0

20000

40000

60000

LLQ=718 pg/ml after dilution factor is in corporated

###

Level of IP-10 in VH

IP-1

0 le

vel (

pg/m

l)

0

200000

400000

600000

800000

###

***

LLQ=718 pg/ml after dilution factor is in corporated

81%

Level of IP-10 in R+C

IP-1

0 le

vel (

pg/m

l)

0

50000

100000

150000

200000

250000

LLQ=390 pg/ml after dilution factor is in corporated

###

***80%

Data shown as Mean ± SEM. ### p<0.001 vs Vehicle Control and **p<0.01 vs Disease Control, One Way ANOVA, Dunnett’s Post Test

Cytokine Induced Uveitis in Rabbits (CIU)_ Chronic (10 days)

Male NZW rabbits quarantine & Health check

Randomization; body weight &

Basal Eye Observation / Red Reflex

Day (-8)

1st IVT injection (Vehicle/Treatments)

Day -7

Scoring & Terminal sacrifice:

BW, Plasma, AH,VH, R/C, ICB from right/left eye.

Animal monitoring: Cage side

observations

Dosing regimen as indicated below

Day 1 (post 24 hrs of IFNg)Day 0 (7 days pretrx)

2nd IVT injection (Vehicle/IFNg)

Objective:To develop a chronic model for Cytokine induced uveitis (CIU) in rabbits

4040

Ocular Models - Summary

Occular models in rats :• Experimental Autoimmune Uveitis (EAU) in rats (Bovine IRBP R16 peptide)Occular models in rabbits :• Endotoxin (E.coli LPS_O111:B4) induced uveitis• Poly I:C induced uveitis• Cytokine + Poly I:C induced uveitis• Cytokines (IL-6, IFN-g) induced uveitis

Compound X deposits in VH in posterior chamber

Rabbit ocular observation using Indirect ophthalmoscope at JBL

Note:• We carry out regular clinical scoring (blinded) of rat/rabbit uveitic eyes using an indirect ophthalmoscope with 28D Lens (Keeler

Vantage or Heine Omega500DV1) on a scale of 0 to 4.• We have expertise for intravitreal, subconjunctival, intracameral route of injection in rabbit eyes.• We regularly collect the following from rabbit eyes - Aqueous Humor, Iris-ciliary body, Vitreous Humor, Retina + Choroid, Sclera,

Conjunctiva, Cornea & TM. In rats, we collect AH, VH & RC. • We do both wet dissection & frozen method of samples collection. • Drug concentrations can be analyzed in the above tissues using LC-MS/MS.• Jubilant Biosys has histopathology support for rat/rabbit ocular uveitis studies.

Clinical appearance of EAU in the Lewis rat by anterior chamber examinationRef: Agarwal et al, Methods Mol Biol. 2012

41

CCl4 Induced Liver Fibrosis in Male Balb/c Mice

41

Study Design: Animals treated with vehicle (olive oil) or CCl4 (emulsion in olive oil) twice/ week. Dexamethasone is administered i.p.

(2.5 mpk, twice weekly) and test compounds are dosed orally (bid everyday). During the study, body weights are recorded twice weekly in the initial stages, with more frequent eights as the study

progresses. After 5th week blood samples are collected for plasma ALT and AST estimation. At end of 6th or 7th week, blood samples are collected for plasma ALT and AST estimation. Animals are sacrificed and

livers collected for histopathological and biochemical analyses.

Twice weekly i.p. injections of CCl4 emulsion (0.4 ml/kg);Compound dosing

Day 36 initial AST/ALT from plasma Day 1 Day 40-48

Necropsy (liver harvested)

Objective:To develop Liver Fibrosis model in mice using CCl4

42

Effect of CCl4 Administration on Liver Collagen Levels

42

Control CCl4 for 5 or 6 w

Significant changes in liver texture on gross observation

0.0

0.5

1.0

1.5

2.0

n=6

n=9

n=9

Fold

cha

nge

in c

olla

gen

over

con

trol

Control CCl4(5 wks)

Increase in collagen levels observed with CCL4 treatment

CCl4(6 wks)

Control mice with normal Gomori trichrome staining, 20x

Representative images from mice treated with CCl4 for 5 or 6 (lower panel) weeks. Note the radiating, stellate branching of collagen deposits in the tissue. These experiments have been performed with a time course to determine progression.

4343

Total Collagen

Data shown as Mean ± SEM ***p<0.001 vs Vehicle, One way ANOVA

followed by Dunnett's Post test

CCl4 Induced Liver Fibrosis, Evaluation disease induction

Liver weight Plasma ALT

Plasma AST

Note: RNA and protein-based studies of key cytokines such as TGFβ, and other proteins such as procollagens, MMPs, FGF and PDGF are part of secondary/mechanistic studies for liver fibrosis.

AU

C_B

od

y W

eig

ht

500

1000

1500

2000

##

**p=0.0531

Hyd

roxy

prol

ine

(µg/

mg)

0.0

0.5

1.0

1.5

• Administration of CCl4 twice weekly for 5th and 6th week showed induction of Liver fibrosis. • Significant increase in liver enzymes and liver weight• Improvement in body weight after AM095 and Dexamethasone treatment as compare to Disease control

44

Day (-1)

Randomization

according to body weight

Day (0)

Terminal :sacrifice

Day 8 / 14 LPS (40µg per mouse) / Bleo (50µg per mouse): single

dose : Oropharyngeal . Dexamethasone thrice weekly.

Body Weight thrice Weekly

Dosing regimen as indicated below

End Point Assays• Collagen

• Histopathology

• RT-PCR

Bleomycin/LPS Induced Lung Fibrosis

LPS (E.coli serotype 055:B5) : SigmaBleomycin sulphate : Alfa Aesar

Collagen

RNA

RNA

Reserve

Histopathology

44

Objective:To develop Lung Fibrosis model in mice using Bleomycin/LPS

4545

Data shown as Mean ± SEM# p<0.05 vs Vehicle control ; ** p<0.01 * p<0.05 vs Disease control; One Way ANOVA, Dunnett’s Post Test.

Collagen Data - LPS/ Bleomycin Induced Lung Fibrosis

0

50

100

150

200

Col

lage

n co

nc. /

Lef

t lun

g lo

be(µ

g/m

L)

Bleomycin induced lung fibrosis

#

0

1

2

3

Col

lage

n µg

/ m

g tis

sue

Bleomycin induced lung fibrosis

#

0

50

100

150

200

Col

lage

n co

nc. /

Lef

t lun

g lo

be(µ

g/m

L)

LPS in Lung Fibrosis

*

0

1

2

3

Col

lage

n µg

/ m

g tis

sue

LPS induced lung Fibrosis

#

**

4646

Histopathology: Trichrome Gomori Staining – Bleomycin/LPS Induced Lung Fibrosis

Histopathological assessment using modified Ashcroft scale

100X magnification

0

1

2

3

4

5

6

7

8

His

topa

th s

core

s

LPS induced Lung Fibrosis

*

0

1

2

3

4

5

6

7

8

His

topa

th s

core

s

Bleomycin induced Lung Fibrosis

#

*

4747

RT-PCR : LPS-Induced Group - Bleomycin/LPS Induced Lung Fibrosis

RT-PCR_Col-1

Fold

ove

r co

ntro

l

Untreate

dLPS

Dexa

0

1

2

3RT-PCR_αSMA

Fold

ove

r co

ntro

l

Untreate

dLPS

Dexa

0

20

40

60

80

RT-PCR_TGFβ

Fold

ove

r co

ntro

l

Untreate

dLPS

Dexa

0

2

4

6

8

• Bleomycin/LPS Induced Lung Fibrosis Model was established using single dose of Bleomycin/LPS • The model was validated with reference compound (Dexamethasone)

48

Acute DSS Induced Colitis Model in Mice

Data shown as Mean ± SEM* p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 vs Disease control, Two way ANNOVA followed by Bonferroni test and One way ANNOVA followed by Dennett's test

48

Objective:To develop Acute DSS Induced Colitis model in mice and its validation using Cyclosporine-A

Disease activity indexFecal consistency score

• Colitis was induced in male C57bl/6 mice using 2.5% DSS in drinking water from days 1-5, Days 6-10 animals were on drinking water.

• Cyclosporine A show dose dependent efficacy in this model.

4949

Chronic DSS Induced Colitis Model in Mice

Objective:To develop Chronic DSS Induced Colitis model in mice and its validation using Fingolimod

Disease activity indexFecal consistency scoreBody weight

Data shown as Mean ± SEM* p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 vs Disease control, Two way ANNOVA followed by Bonferroni test and One way ANNOVA followed by Dennett's test

• Colitis was induced in C57bl/6 mice using 3 cycles of 2% DSS in drinking water (day 1-5, 11-15, 21-25). • The model was validated using sphingosine-1-phosphate receptor modulator Fingolimod

50

MDR1 Alpha KO Mice Model

50

Objective:To develop MDR1 Alpha KO Mice Colitis model and its validation using reference compound

Disease activity indexFecal consistency scoreBody weight

Data shown as Mean ± SEM* p<0.05 and ** p<0.01 vs Disease control, Two way ANNOVA followed by Bonferroni test and One way ANNOVA followed by Dennett's test

• 13-14 Week old Multidrug resistance 1 alpha (MDR1 alpha) gene knockout animals were treated orally with Reference compound

• Reference compound show modest efficacy in aggravated MDR1 alpha KO mice model

51

Oxazolone Colitis Model in Mice

0

1

2

3

4

5

6

AUC

_DAI

###

**

***

3-parameter DAI Calculated based on % change in Body Weight score with Day 0 as starting reference weight

0

10

20

30

40

50

60

70

Col

on W

eigh

t/Len

gth

(mg/

cm)

***

Data shown as Mean ± SEM### p<0.001 vs Sham Control, * p<0.05, ** p<0.01, *** p<0.001 vs Disease control, One Way ANOVA, Dunnett’s Post Test

Effect of Oxazolone on Colon weight/ length Ratio

51

• Significant disease induction,• Significant disease reduction with

Tofacitinib at both the doses,• Significant reduction in colon length-

weight ratio with tofacitinib.

Objective:To develop Oxazolone Mice Colitis model and its validation using Reference compound

• Oxazolone colitis model is validated with reference compound ; more data can be shared on request

52

TNBS Induced Colitis Model in Rats: Prophylactic Mode Protocol

52

Data shown as Mean ± SEM* p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 vs Disease control, Two way ANNOVA followed by Bonferroni test and One way ANNOVA followed by Dennett's test

Colon weight/length ratioFecal consistency scoreBody weight

Objective:To develop TNBS Induced Colitis model in rats and its validation using reference compound

• Colitis was induced by single dose of Intra-rectal TNBS in Male Sprague Dawley rats, animals were treated in prophylactic mode with reference compound.

• The model was validated using Reference compound

5353

TNBS Induced Colitis Model in Rats: Therapeutic Mode Protocol

Objective:To develop TNBS Induced Colitis model in rats and its validation using Dexamethasone

• Colitis was induced by single dose of Intra-rectal TNBS in Male Sprague Dawley rats, animals were treated in therapeutic mode with Dexamethasone.

• The model was validated using Dexamethasone

Data shown as Mean ± SEM* p<0.05, ** p<0.01 and **** p<0.0001 vs Disease control, Two way ANNOVA followed by Bonferroni test and One way ANNOVA followed by Dennett's test

Colon weight/length ratioFecal consistency scoreBody weight

54

Mouse Model of Gastric Emptying and Intestinal Transit using Phenol Red

Small Intestinal transitGastric Emptying

Data shown as Mean ± SEM*p<0.05 and ***p<0.001 vs Vehicle control, One Way ANNOVA Followed by Dunnett’s post test

54

Objective:To develop Mouse model of Gastric emptying and intestinal Transit and its validation using loperamide and metoclopramide

• Phenol Red 0.05% W/V was used to monitor GE and SIT in Male C57bl/6 mice .• The model was validated using Loperamide and Metoclopramide

55

Mouse Model of Gastric Emptying

Data shown as Mean ± SEM***p<0.001 vs Vehicle control, One Way ANNOVA Followed by Dunnett’s post test

55

Objective:To develop Mouse model of Gastric emptying and its validation using GLP-1 agonist, Exendin-4

• Phenol Red 0.05% W/V was used to monitor GE and SIT in Male C57bl/6 mice .• The model was validated using GLP-1 agonist, Exendin-4 and Metoclopramide

56

Ovalbumin Mediated Steroid-resistant and -sensitive Atopic Allergy Model in Mice

56

Objective:To develop steroid-resistant and -sensitive atopic allergy model in mice using ovalbumin

Day 0 & 12

Priming,Ovalbumin with CFA/Alum

Day 20

Sacrifice,BALF & Lungs, post 48 hrs

Challenge with Ovalbumin,Dexa (i.p.) 2.5hrs prior to challenge

17 1816

BLUE BARS: - DexamethasoneGREY BARS: + Dexamethasone

Flow cytometric analysis of lavage fluid from lungs of mice treated to mimic steroid

resistant (OVA-CFA priming) and sensitive (OVA-ALUM priming) asthma.

Study design : Harlan Male Balbc mice (5-6 weeks) were used in the study

• Ovalbumin mediated Steroid-resistant and sensitive atopic allergy model was established in mice • Model was validated with reference compound (Dexamethasone)

57

Collagen-Induced Arthritis Model in Mice

57

Objective:To develop Arthritis model in mice using Collagen

• Immunization of mice with an emulsion of complete Freund’s adjuvant and type II collagen (CII) Arthritis as evident by clinical scores

• Arthritis was significantly reversed by reference compound, Dexamethasone

58

Imiquimod-Induced Psoriasis Model in Mice

Disease induction & treatment : Prophylactic topical application of Dexa(100µg, 300µg)Readouts : Regular body weight, ear thickness, clinical observation (scoring 5 scale)

5% Imiquimod cream application on back &

ear of animal

D1 D10

Experimental plan Readouts available: Clinical scores, histopathology

Days

Scor

e (0

-4)

1 2 3 4 5 6 7 8 9 100

1

2

3

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

****

******

********

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

Effect of Dexamethasone on Imiquimod induced skin thickness in male Balbc mice

1 2 3 4 5 6 7 8 9 10

0.30

0.35

0.40

0.45

Effect of Dexamethasone on Imiquimod induced ear thickness in male Balbc mice

Days

Ear T

hick

ness

(mm

)

** ****

*****

****

****

****

****

**** Normal controlImiquimodDexamethasone (100µg)Dexamethasone (300µg)

*p<0.05,**p<0.05,****p<0.0001 VsImiquimod post Bonferroni test

58

Objective:To develop Psoriasis model in mice using Imiquimod

• Imiquimod application in mice induced Psoriasis as evident by ear thickness/clinical scores

• Psoriasis was significantly reversed by reference compound, Dexamethasone

59

TPA Induced Contact Dermatitis Model in BALB/c Mice

Hr

Ear t

hick

ness

(mm

)

0 3 6 10 24

0.30

0.35

0.40

0.45

Vehicle ControlTPA (2.5 µg/ear)Tofa(250µg)/ear +TPA

** ****$$$$

$ Control Vs TPA*TPA Vs Tofa,postBonferroni test

$$$$

$$$$

$$$$

****

Effect of Tofacitinib on TPA induced contact dermatitis

Ear w

t (m

g)

0

5

10

15

20

$$$

***

$ Control Vs TPA*TPA Vs Tofa,post

Dunnett's post test

Vehicle ControlTPA (2.5 µg/ear)Tofa (250µg)/ear +TPA

Effect of Tofacitinib on TPA induced contact dermatitis

Ear Thickness

Tofacitinib (0.5% in DMSO:Acetone (1:7)/ Ear.) 30

min before TPA

Experimental plan

Basal Ear Thickness TPA 2.5 μg/ear

3 6 1024

Hr

Ear Weight, Collection of ear tissue

Ear thickness (Mitutoyo Model # PK-0510,Serial # 006749); Ear Weight: Punch Biopsy (6mm)

59

Objective:To develop Contact Dermatitis in mice using TPA

• TPA induced contact dermatitis model in BALB/c mice model was validated with multiple reference compounds (Tofacitinb; Dexamethasone)

60

In vivo Efficacy Models for Cancer

60

61

Preclinical Mouse Tumor Models: Representative Examples

61

Cancer type ModelsLung adenocarcinoma Calu61, A5491, H19751, H821, HCC8271, KLN-2055

Colorectal cancer HT291, HCT1161, Colo2051, LoVo1, CT263

Melanoma A3751, B16F104

Breast cancer MDA-MB-2312, 4T13, MCF7 (3D spheroids)Renal cancer RENCA3

Glioblastoma U87-MG1

Ovarian cancer A27801, SKOV31

Gastric cancer Hs746T1, SNU52, MKN451

Prostate cancer PC31, DU145 1

Pancreatic cancer MiaPaCa21, BxPc31

Multiple myeloma MM.1S / MM.1R2 (Isogenic and Pomalidomide resistant)Mantle cell lymphoma Jeko11, Mino1, Z1382

Burkitt’s lymphoma Ramos2

B myelocytic leukemia MV4.111

Syngeneic- In bold; 1Nude, 2SCID, 3Balbc, 4C57BL/6, 5DBA/2

62

CT26: Colon Cancer Syngeneic Allograft Model

62

CT26 Syngenic tumor model in Balb/c mice (F)

Days post treatment initiation

Tum

or V

olum

e ( m

m3 )

0 3 6 9 12 150

300

600

900

1200

1500

1800

2100

Vehicle ControlAnti mouse PD1 Ab (0.1 mg/mouse)Epacadostat (30 mpk, PO-BID)

CT26 Syngenic tumor mModel in Balb/c mice (F)

Days post treatment initiation

% C

hang

e in

Bod

y w

t

3 6 9 12 15

-15

-10

-5

0

5

10

15

20

Vehicle Control

Anti mouse PD1 Ab(0.1mg/mouse)

Epacadostat (30 mpk, PO-BID)

Objective:To establish and validate CT26 syngeneic mouse allograft model

• Established CT26 syngeneic allograft model in BalbC mice• Mouse anti PD-1 antibody (0.1 mg/mouse, IP)/ Epacadostat (30 mg/kg, PO, BID) were evaluated in the model

which showed a tumor growth inhibition (TGI) of 52.6 % and 34.2 %, respectively.• Similar studies have been performed in B16-F10 and 4T1 syngeneic tumor models

63

Renca: Renal Cancer Syngeneic Allograft Model

63

Renca s.c. Syngenic Tumor Model in Balb/c (F)

Days post treatment initiation

Tum

or v

olum

e (m

m3 )

0 3 6 9 120

500

1000

1500

2000

2500Vehicle Control

PD-L1 100 µg/mouse

Renca s.c. Syngenic Tumor Model in Balb/c (F)

Days post treatment initiation

% C

hang

e in

bod

y w

eigh

t

0 3 6 9 12-10

-5

0

5

10

Vehicle Control

PD-L1 100 µg/mouse

Objective:To establish and validate Renca syngeneic mouse allograft model

• Established Renca syngeneic allograft model in BalbC mice• Mouse anti PD-L1 antibody (0.1 mg/mouse, IP) was evaluated in the model which showed a tumor growth

inhibition (TGI) of ~47 %

64

Calu 6: Non Small Cell Lung Cancer (NSCLC) CDX model

64

Calu-6 s.c. Xenograft Model in Nude Mice (F)

Days after treatment initiation

Tum

or V

olum

e ( m

m3 )

0 3 6 9 12 15 180

200

400

600

800

1000 VNB-ControlVNB (2.5 mg/kg )VNB (5 mg/kg )VNB (10 mg/kg)VNB (15 mg/kg)

Calu-6 s.c. Xenograft Model in Nude (F) Mice

Days after treatment initiation

% C

hang

e in

Bod

y w

t.

0 3 6 9 12 15 18-6

-3

0

3

6

9

12

15

18 VNB-ControlVNB (2.5 mg/kg )VNB (5 mg/kg )

VNB (10 mg/kg)VNB (15 mg/kg)

Objective:To establish and validate Calu6 lung cancer xenograft model

VNB: Vinorelbine

• Established Calu6 xenograft model in nude mice• Validated with chemotherapeutic agent Vinorebine at different doses and a clear dose response was

established.

65

NCI-H1341: Small Cell Lung Cancer (SCLC) CDX model

65

H1341 s.c. xenograft model in nude (F) mice

Days post treatment initiation

Tum

or v

olum

e (m

m3 )

0 3 6 9 12 150

700

1400

2100

2800Vehicle control

Test agent PO Q2D

Days post treatment initiation

0 5 10 15-20

-10

0

10

20

% Change in body weight

Vehicle control

Test agent PO Q2D

Objective:To establish NCI-H1341 small cell lung cancer xenograft model

• Established NCI-H1341 xenograft model in nude mice• Validated the model with HDAC1 inhibitor

66

A2780: Ovarian Cancer CDX model

66

% Change in Body wt

Days post treatment initiation

0 5 10 15-20

-10

0

10

Vehicle Control

Gemcitabine(120mpk),IP, Q4D x2

A2780 s.c. Xenograft Model in Nude Mice (F)

Days post treatment initiation

Tum

or V

olum

e ( m

m3 )

0 5 10 150

1000

2000

3000

4000 Vehicle ControlGemcitabine(120mpk),IP, Q4D x2

Objective:To establish A2780 ovarian cancer xenograft model

• Established A2780 xenograft model in nude mice• Validated the model with Gemcitabine, one of the first line therapies

67

SNU5: Gastric Cancer CDX model

67

Days post treatment initiation0 3 6 9 12 15 18 21

-15

-10

-5

0

5

10

15 Vehicle Control PO, QIDTest agent, PO, QD

% Change in body weight SNU5 s.c. xenograft model in SCID mice (F)

Days post treatment initiation

Tum

or v

olum

e (m

m3 )

0 3 6 9 12 15 18 210

300

600

900

1200

Vehicle Control POTest agent PO QD

Objective:To establish SNU5, gastric cancer xenograft model

• Established SNU5 xenograft model in SCID mice• Validated the model with MET inhibitor

68

MV-4-11: B Myelocytic Leukemia CDX model

68

0

250

500

750

1000

1250

1500

1750

2000

1 4 6 8 11 13 15

Tum

or v

olum

e (m

m3 )

Days post tumor initiation

MV.4.11 xenograft

Vehicle control

Test agent

10

15

20

25

30

35

1 4 6 8 11 13 15

Days post tumor initiation

Vehicle control

Test agent

Body Weight (g)

Objective:To establish MV-4-11 B myelocytic leukemia xenograft model

• Established MV-4-11 xenograft model in nude mice• Validated the model with BET inhibitor

69

Thank You for your Time

Our Values

For More Information:

www.jubilantbiosys.combd@jubilantbiosys.com

Office and Research Sites:

Jubilant Biosys Limited#96, Industrial Suburb 2nd Stage, Yeshwantpur Bangalore - 560 022 Karnataka India.Tel. : +91 80 66628400

Jubilant Chemsys LimitedB-34, C Block, Sector 58, Noida, Uttar Pradesh 201301Tel: +91 120 409 3300

69