selected examples of in-vivo models enabled at jubilantdiscovery, preclinical & clinical...
TRANSCRIPT
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:
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