Drug Discovery Collaboration
Discovery Research
Suven Life SciencesSerene Chambers, Road-5, Avenue-7, Banjara Hills,
Hyderabad-500034, India.Contacts: [email protected], [email protected]
Drug Discovery Collaboration
VisionSearch for new CNS therapies
Mission
Become the leading company focused on the treatments for unmet medicalneed in Mental Health
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MissionHealth for Patients and Value for Partners
Excellent Pipeline of CNS drugs to improve Cognition, treat Depression and PainNovel Mechanism of action and first in class preclinical data
Clinical CandidatesSUVN-502, SUVN-G3031, SUVN-D4010 and SUVN-911
Index
Slides Slides
• Suven Company Overview 04 - 05 • In-vitro Biology(Assay Development, Validation, Screening, Profiling)
32 – 59
• Suven Discovery Profile 06 • In-vitro ADME & In-vivo PK 60 - 74
• Integrated Drug Discovery 08 - 09• Target engagement : Microdialysis,
Receptor Occupancy and EEG75 – 89
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33
Receptor Occupancy and EEG
• Suven Discovery Pipeline 10 - 11• In-vivo Efficacy Pharmacology &
Safety Pharmacology90 - 99
• Suven Patent Pipeline 12 - 15• Preformulation, Biopharmaceutics
and Early Formulations100 - 119
• Suven Discovery Scientists(Qualifications / Experience)
16 - 17 • Discovery Toxicology 120 - 126
• Flow Scheme 18 • Contacts 127
• Results/Data -Communication
19
• Medicinal Chemistry 20 - 31
Suven Life Sciences
27 years of Pharmaceutical Leadership
1989
2005Drug Discovery & Development
Support Services (DDDSS)Major Pharma & Biotech (USA, Europe)
2006Collaborative Research Program
Eli Lilly, USA
1989Incorporated by Mr. Venkat Jasti
1995IPO, Listed on NSE and BSE in India
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
4
1989Contract Research & Manufacturing
Services (CRAMS)FDA Approved Plant
(Handled more than 500 projects)
2009Formulation Development
Clinical Trial Supplies
2002Suven Discovery
(Cognition, Psychosis, Anxiety,Depression, Pain, Obesity)
2013First US IND for SUVN-502
2015First Clinical Proof-of-Concept Study in USA for SUVN-502
Relationship with many (~30) Global Life Science andBiotech companies
Drug Discovery
LeadOptimizationHit Lead Pre Clinical Clinical Trials
ProcessResearch
CustomSynthesis
FormulationDevelopmentAnalytical &Regulatory
Clinical SuppliesManufacturing &
Packaging
Suven Life Sciences - Business Model
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OptimizationHit Lead Research SynthesisRegulatoryServices
Packaging
Drug Discovery & DevelopmentSupport Services (DDDSS)
Contract Research AndManufacturing Services (CRAMS)
Collaborative Research Partner (CRP) …Seamless transition
5
Suven Discovery - Profile
More than 14 years of Discovery and Development experience
Worked on 8-10 Central Nervous System targets in drug discovery
First discovery project delivered SUVN-502 (5-HT6 antagonist for Alzheimer's Disease). Phase II POC for
Alzheimer's Disease in aged population is currently ongoing in USA
Two full spectrum Discovery Collaborative Projects with Eli Lilly, USA.
First project: CNS target - Hit to Candidate Identification
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First project: CNS target - Hit to Candidate Identification
Second Project: Dual CNS Target Pharmacology involving Hit Identification to Candidate Selection over a period of
3 years and then to GLP tox and clinical development
Filed first Investigational New Drug (IND) application on SUVN-502 in 2007
Phase -1 First in Human Clinical Trials for SUVN-G3031 has been completed
Phase-II POC studies for SUVN-502 are initiated in 2015
Phase -1 First in Human Clinical Trials for SUVN-D4010 has been completed
66
Drug Discovery and Developmental Research
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77
Suven Integrated Drug Discovery
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8
Suven Discovery Research
Medicinal / Organic / Scale-up ChemistryAnalytical ChemistryPhysicochemical Properties and Biopharmaceutical characterization
Assay development and validationIn-vitro Screening; Functional AssaysIn-vitro ADME Assays
In-vivo PharmacokineticsIn-vitro, In-vivo Metabolism
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In-vitro, In-vivo MetabolismIn-vivo MicrodialysisIn-vivo Receptor OccupancyEEG
CNS (Cognition, Depression, Psychosis, Anxiety),Pain and Obesity Pharmacology
CNS, CV, GI, Renal Safety Pharmacology
Preformulation, Biopharmaceutics and Early Formulations
Toxicology (In-vitro & In-vivo)
99
Suven Discovery Pipeline
Neurodegenerative Diseases
MCI / Cognition / Alzheimer's /
Schizophrenia
Dis
cove
ryP
recl
inic
al
IND
Pha
se-I
Phase
-II
Pha
se-III
Exp
ecte
dLau
nch
SUVN-501 (5-HT6 Antagonist)
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1010
SUVN-502 (5-HT6 Antagonist) 2015 2020
SUVN-507 (5-HT6 Antagonist)
SUVN-512 (5-HT6 Antagonist)
Muscrainic M1 (PAM) 2017
SUVN-D4010 (5-HT4 Partial Agonist) 2015 2017
SUVN-G3031 (Histamine-3 Antagonist) 2014 2017
Pain / Major Depressive Disorder(MDD) / Psyciatric Discorders
Dis
cove
ryP
reC
linic
al
IND
Phase
-I
Phase
-II
Phas
e-III
Exp
ecte
dLau
nch
Suven Discovery Pipeline
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1111
nAChR Alpha-4 Beta-2 Antagonist (Pain)
SUVN-911 (nAChR Alpha-4 Beta-2) (MDD) 2016 2016
CB2 Agonist (Pain)
Multi Modal (Psychiatric disorders) 2017
PCT and National
Phase
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
2015
2016
Tota
l
Discovery Research
Applications 1 8 4 3 2 3 3 4 4 1 2 1 5 _ 2 43
Published _ 3 7 3 1 5 3 2 2 5 2 1 2 2 3 41
National Phase
Suven Patent Pipeline
Company ConfidentialCopyright © 2016 Suven Life Sciences LimitedAs on Jun - 2016 1212
National Phase
entered_ _ 3 3 1 1 2 3 1 2 5 3 1 1 2 28
Process Research
Applications _ 2 2 2 1 2 1 _ _ _ _ 2 _ 3 _ 15
Published _ _ _ 3 1 3 _ 2 1 _ _ _ _ 5 _ 15
National Phase
entered_ _ _ _ 1 2 2 _ 1 _ _ _ _ 1 1 8
Patents Granted
Euro
pe
USA
Aust
ralia
Japan
Sin
gapore
New
Zeala
nd
Isra
el
AR
IPO
Discovery (Product Patents) 21 24 23 18 23 24 9 2
Process Patents 2 5 2 2 _ 1 1 _
South
Afr
ica
India
Sri
Lanka
Kore
a
Bra
zil
Mexic
o
Maca
u
Suven Patent Pipeline
Company ConfidentialCopyright © 2016 Suven Life Sciences LimitedAs on Jun - 2016 1313
South
Afr
ica
India
Sri
Lanka
Kore
a
Bra
zil
Mexic
o
Maca
u
Discovery (Product Patents) 23 18 12 19 _ 21 8
Process Patents _ 6 _ 3 _ _ _
Canada
Chin
a
Eura
sia
Norw
ay
Hongkong
Russ
ia
Discovery (Product Patents) 23 20 19 5 21 3
Process Patents 2 2 1 _ _ _
Suven Published PCT Patent ApplicationsDiscovery Research
Year Publication Numbers Number of Publications
2016 WO2016027275, WO2016027276, WO2016027277 3
2015 WO2015083179, WO2015092804 2
2014 WO 2014/030170, WO 2014/147636 2
2013 WO 2013/042135 1
2012 WO 2012/029070, WO 2012/114348 2
2011WO 2011/030349, WO 2011/061751, WO 2011/080751, WO 2011/080750,
WO 2011/0834875
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1414
WO 2011/083487
2010 WO 2010/032257, WO 2010/032258 2
2009 WO 2009/034581, WO 2009/053997 2
2008 WO 2008/084492, WO 2008/084491, WO 2008/136017 3
2007WO 2007/020653, WO 2007/020652, WO 2007/046111, WO 2007/046112,
WO 2007/1386115
2006 WO 2006/095360 1
2005 WO 2005/066184, WO 2005/066157, WO 2005/005439 3
2004WO 2004/108671, WO 2004/055026, WO 2004/048331, WO 2004/048330,
WO2004/048328, WO 2004/041781, WO 2004/0832027
2003 WO 2004/000849, WO 2004/000845, WO 2004/000205 3
Total as on Jun - 2016 41
Suven Published PCT Patent ApplicationsProcess Research
Year Publication NumbersNumber of
Publications
2015WO 2015/008294, WO2015037013, WO/2015/155664,
WO/2015/162506, WO/2016/0272775
2010 WO 2010/061398 1
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1515
2010 WO 2010/061398 1
2009 WO 2009/007995, WO 2009/007998 2
2007 WO 2007/026375, WO 2007/020654, WO 2007/094007 3
2006 WO 2006/038220 1
2005 WO 2005/063693, WO 2005/030738, WO 2005/111010 3
Total as on Jun - 2016 15
Suven Discovery Scientists Qualifications / Experience
Scientists Breakdown
Ph.D.Average Years
ExperienceMS /
M.PharmAverage Years
ExperienceTechnician /Lab Support
Chemistry 10 6 - 9 Years 60 2 - 6 years 12
Biology 12 >10 Years 95 2 - 8 years 25
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Biology 12 >10 Years 95 2 - 8 years 25
DMPK 7 >10 Years 45 2 - 5 years 12
Management 2 (more than 20 years of discovery experience)
Office / Admin 25
Senior and Core Group Leaders have pursued their education from reputed academic institutionslike IIT (Indian Institute of Technology), IISc (Indian Institute of Science), IICT (Indian Institute ofChemical Technology), Osmania University, Manipal University, MS University Baroda, LM Collegeof Pharmacy, Hyderabad Central University etc.
Suven Discovery Scientists
Most of the Scientists regularly attend International conferences like ACS, AAIC, SFN,AAPS, CRS, SOT, In-vivo and ISSX
Most of the Scientists from Assay development, Assay Validation, ADME, Microdialysis,Receptor Occupancy, PK, Toxicology had spent 2-3 weeks in training / skill upgradation,GRP etc at Collaborators / Sponsors Labs (Lilly, BMS and AstraZeneca)
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Most of the methods, assays, histopathology etc are cross validated with collaborators /sponsors Labs. Several blinded studies were conducted and data compared across.
Data generated on most of the Reference Compounds (both clinical and pre-clinicalcandidates) and compared with the published data.
17
Suven Discovery Process: Hit-to-Candidate Selection
Hit
Stage 1
Synthesis
100 mg
Primaryscreening,
Binding and/orFunctional
(rat & human)1w, 5 mg Lead
Rat TargetTissue
Distribution1w, 30 mg
500 mg
Primary In-vivoEfficacy Assay
1w, 200 mg
Confirmatory
In-vivo Assay3
1w, 200 mg
ADMESurrogateAssays1
1w, 5 mg
RodentBioavailability
2w, 25 mg
Mini SelectivityPanel2
1w, 10 mg
Rat ReceptorOccupancy1w, 35 mg
In-vivofunctional
Assay1w, 75 mg
Hit to Lead = 9 to 14 months
Lead to Candidate Identification = 6 months
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1 ADME ProfilingMetabolic Stability (Rodent and Human)(Microsomes / Hepatocytes)CYP Phenotyping (3A4, 2D6)Time Dependent InhibitionMetabolite IdentificationIn-vitro CaCO2 Permeability
2 Mini Selectivity PanelRelated Efficacy and Safety TargetshERG (Astemizole Binding)
Salt / CrystalForm Screen
1w, 150 mg/com
Preformulation forToxicity Studies
1w, 2 g
AdditionalIn-vivo Assays3
2w, 250 mg
Chronic In-vivo
Efficacy Assay
4w, 2 g
Critical Safety
Pharmacology
5w, 100 mg Non-Rodent7-day Toxicity
Study3w, 30 g
Rodent 14-dayToxicity Study
4w, 30 g
AMES1w, 2 g
Non Rodent
Dose Escalation
Toxicity Study
2w, 15 g
Candidate
Selection
Stage 3
Synthesis
150 g
Stage 2
Synthesis
10 g
Non-RodentBioavailability
2w, 750 mg
Metabolite IDProfiling
2w, 250 mg
Rodent 7-day
Toxicity Study
3w, 12 g
SelectivityFull Panel,
4 w, 100 mg
Candidate
Identification
Candidate Identification to Selection = 6 months
Lead to Candidate Identification = 6 months
Hit to Candidate Selection = 24 to 30 months
Collaborative Projects
Data upload in dedicated eRoom on real time
Secured Tunnel for safe email exchanges
Weekly WebEx for Chemistry, Biology and project progression
Regular teleconference with the collaborative partners
Access to structures is restricted to group leaders
Results/Data - Communication
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Access to structures is restricted to group leaders
Internal Projects
Secured tunnel for email exchanges between different divisions
Internal project progress meeting for Chemistry and Biology
All compound related communications with internal Suven code numbers
Access to structures is restricted to group leaders
Suven Discovery Research
Medicinal Chemistry
Core Capabilities
Hit Generation and Hit follow up
Hit to lead generation with consideration of potential IP issues
Lead Optimization and selection of clinical development candidate
Discovery Research - Medicinal Chemistry
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Scale-up Chemistry Support
Identification, synthesis and characterization of metabolites
Synthesis of reference standards
Dedicated analytical support laboratory
Collaborative Capabilities
Hit Generation and Hit follow up
Independent or Customer supported Hit to lead and Lead generation
Independent or Synergistic work for Lead Optimization with the Collaborator
Scale-up Chemistry Support for developmental program
Discovery Research - Medicinal Chemistry
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Scale-up Chemistry Support for developmental program
Identification, Synthesis and characterization of metabolites
True collaborative Research Program, with pre-determined objectives andgoals
Case study -1: Internal Discovery Project - Histamine H3 antagonistsOptimization of ADME properties
hH3 Ki < 5 to 10 nM, Initial hits Poor brain penetration Longer half life Poor oral exposure
NH
ON
R1
N
ON
R1
Confirmational constrainedto improve ADME properties
hH3 Ki < 5 to 10 nM, Initial hits Improved brain penetration Longer half life Poor oral exposure
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N
O
Second basic centre
HN
Second basic centre
O
HN
Cyclic rings with reduced pKa
O
Ring opening to increase number of sitesof modifcations & less chemistry demanding targets
Transpose the amide functionality
Focused SAR to inroduce several rings with reduced log P and pKa
hH3 Ki < 5 to 10 nM, Poor brain penetration Longer half life Poor oral exposure
hH3 Ki < 10 nM, Poor brain penetration Half life ~ 10 h Moderate oral exposure
Lead series; Excellent brain penetration;Acceptable half life; Improved oral exposure
23
The People - Core Strength
Well qualified, Trained and Committed Scientific Staff
Well versed with Medicinal & Contemporary Chemistry
Skilled in various synthetic techniques
Discovery Research - Medicinal Chemistry
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Highly interactive with biologists
Upto-date knowledge of IP issues, highest level of confidentiality
Well acquainted with GLP & safety practices
Trained for data mining and literature searches
The Facilities - Labs
Spacious and well equipped air conditioned laboratories
Spacious 8 fume-hoods per lab
Integrated write up area and IP secure areas within the lab for scientists.
State of the art purification systems like Combiflash
Discovery Research - Medicinal Chemistry
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Non-conventional synthesis equipments like Microwave synthesizer
GRP operational environment with safety consciousness
Separate hydrogenation laboratory (Capacity 100 mL to 5 L)
Highly organized chemical inventory store
Reaction capability between -78 ºC to + 250 ºC
Expertise in multi-step synthesis (mg to multi-gm) of metabolites and standards
natural product-like heterocyclic scaffolds building blocks, intermediates and
impurities
The Facilities - Chiral Chemistry Capabilities
Expertise in designing asymmetric synthesis
Expertise in traditional chiral resolutions via diastereoisomeric salt preparation and
crystallizations
Analytical chiral columns for chiral analysis
Method development capabilities (identify appropriate column, mobile phase and
technique)
Discovery Research - Medicinal Chemistry
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technique)
In-house facilities for semi-preparative chiral separations
Availability of various chiral columns like CHIRALPAK-AD-H, CHIRALPAK-OD-H
Collaboration with The Daicel chiral technologies at Hyderabad for SFC methods
High speed rotary evaporators with high vacuum pumps and chillers
Rapid turn around time
The Facilities - Literature
Online access to major Medicinal and Organic chemistry journals
Access to STN and Scifinder
In-house library
Discovery Research - Medicinal Chemistry
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Access to printed literature and databases
Discovery Research - Medicinal Chemistry
Analytical Support Facilities
Analytical Testing (API, Impurities, Intermediates, Starting Materials, DrugProduct)
Stability studies (API and Drug Product)
Analytical Method Development & Validation
(Chemical, Chromatographic and Chiral)
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(Chemical, Chromatographic and Chiral)
(Starting Materials, Intermediates, API, Impurities, and Drug Product)
Analytical Characterization and Certification
Analytical Support for Process Development
2828
Analytical Support Facilities
400 MHz NMR (Bruker) with three different probes – dual, multinuclear and fluorine
Dedicated mass spectrometer for med chem - LC MS/MS API-2000 (Applied biosystems)
Five Agilent HPLC systems with UV, PDA, Fluorescence and RI detectors
Discovery Research - Medicinal Chemistry
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Semi-preparative scale chromatographic system
Analytical and semi-preparative chiral columns
Polarimeter, DSC, TGA, UV spectrophotometer and FTIR
Chemistry, Manufacturing and Control (CMC)
• Good expertise in the synthesis and scale up of the APIs, intermediates, fine
chemicals, impurities and metabolites
• Excellent laboratory facilities for scale up, good manufacturing facilities, inclusive of
FDA approved production facilities
• Expertise in route selection and process controls at each stage backed by state of
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• Expertise in route selection and process controls at each stage backed by state of
the art analytical facilities for characterization and identification of the product and
the impurities. The analytical facilities include 400 MHz Proton NMR, 13C-NMR,
LCMS, HPLC systems for purity assessment, chiral separations and semi
preparative automated separation facilities, DSC, Thermogravimetric analysis, UV,
FTIR, Polarimeter, XRD etc
• Thorough characterization and certification of drug substance and intermediates
3030
• Control of the impurities through diligent planning and route selection. Identification
of the formation of solvates, hydrates etc
• Expertise in crystallization studies, polymorph identification and control
• Salt screen and selection based on solubility and exposure levels
• Dedicated Scientists with good experience for chemical manufacture and their keen
Chemistry, Manufacturing and Control (CMC)
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observation at every stage
• Competitive time lines and speedy deliveries ensuring the quality
• Demonstrated CMC capabilities by manufacturing Phase-2 POC candidate SUVN-
502 at 75 Kg scale in our FDA approved plant. Other Phase-1 clinical candidates
(SUVN-G3031 and SUVN-D4010) were manufactured at ~ 8 kg level
• Our expertise in chemical manufacture, impurity controls and speedy delivery of
products were highly appreciated by our multinational collaborators
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In-vitro Biology
Suven Discovery Research
In-vitro Biology
A group of dedicated scientists with overall expertise in:
Biochemistry, Molecular Biology and Cell Biology
Encompassing drug target class:
GPCRs, Nuclear Receptors, Monoamine transporters, ion channels and enzymes
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Expertise:
Assay Development and Validation, Screening and Profiling of Compounds, and
Diverse Mechanistic Studies of Lead/ Lead Like Molecules
With significant experience in:
Drug Discovery
3333
Biochemistry and Molecular Biology
Human cDNA Synthesis and Cloning
Expression of Recombinant Proteins in E. coli, Baculovirus and
Mammalian Cells
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Mammalian Cells
Purification and Characterization of Recombinant Proteins
Establishment, identification and characterization of stable cell lines
(both CHO and HEK293)
3434
Target Class
GPCRs
Radioligand binding assays
cAMP Measurement and Reporter Gene Driven Assays
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Functional Cell-based Assays (Signal Transduction)
3535
Target Class
Ion Channels
Radioligand binding assays
Patch clamp assay using automated electrophysiology instrument
Patchliner from Nanion, for ligand gated ion channel: P2X7 as well as
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3636
voltage gated ion channels: Sodium Channels - Nav 1.4, 1.5, 1.6, 1.7 and
Potassium Channels - Kv 10.1 and Kv11.1 (hERG)
Monoamine Transporters
Radioligand binding assays
Functional uptake assays
Nuclear Receptors
Reporter gene based assays
Target Class
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Reporter gene based assays
Enzyme-based assays
Historical expertise in establishment and validation of assays and
screening of compounds using kinase, peptidase and phosphatase as drug
target
3737
Protein - Protein Interactions and Other Assays
Yeast Two-Hybrid System and other yeast based assays
His Tag Pull Down
Immunohistochemistry
CYP Induction: PXR transactivation assay
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CYP Induction: PXR transactivation assay
In-vitro Cytotoxicity and Phospholipidosis assay
3838
Compound Run 1 Run 2 Ratio
Outside
Limits
3.5 2.5 1.400
83.74 101.2 0.827
26.66 30.5 0.874
89.5 103 0.869
18.6 16.7 1.114
95.35 141.3 0.675
17.57 26.81 0.655
2632 2900 0.908
2.1 2.3 0.913
2.7 2.2 1.227
145.7 143.1 1.018
83.24 66.66 1.249
3.4 3.5 0.971
766.8 933.4 0.822
66.74 81.78 0.816
689.1 903 0.763
7.3 5.2 1.404
10.1 5.4 1.870 #
36.14 22.17 1.630 Statistical Analysis of "Bland-Altman" Plot
Potency Spreadsheet
. . . . Assay
. . . . Assay Run 1 & Run 2
Potency Ratio vs Geometric Mean Potency
0.1
1
10
1 10 100 1000 10000Geometric Mean
Ratio
Ratio
MR
RLs
LsA
RefLine
Page
Lim
itBoundaries
Assay Validation TRT
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36.14 22.17 1.630 Statistical Analysis of "Bland-Altman" Plot
417.9 372.6 1.122 Equivalence Test Results Reproducibility Test Results
4.5 3 1.500 N 24
18.7 14.8 1.264 MR 1.04 MSR (Within-Run) 1.74
12.3 10.4 1.183 RLs 0.93 LsA 0.60
4.6 4.9 0.939 1.17 1.82
Sig Diff Between SD 0.0850
1.084 Runs Test, p = 0.4531
Validation Checklist
1 No trends in above plot (you must check visually)
2 MSR (for within-run studies) <= 3 Yes
3 LsA within 0.33 - 3.0 Yes
Correlation Coefficient of LOG-LOG Plot
= 0.993
12 points below the 45-degree line
13 points above the 45-degree line
No validation requirements exist for this plot
Run 1 vs. Run 2 with 45-degree Line
Meets Criterion ?
Page
Lim
itBoundaries
1
10
100
1000
10000
1 10 100 1000 10000
Run 2
Run
1
3939
Day 2, Plate 1
row 1 2 3 4 5 6 7 8 9 10 11
A 2362 1637 142 2384 1580 145 2388 1601 147 2300 1612
B 2369 1601 131 2328 1615 156 2387 1625 151 2380 1663
C 2391 1580 132 2338 1582 140 2392 1630 156 2338 1658
D 2360 1636 138 2367 1614 145 2358 1674 141 2376 1613
E 2310 1609 142 2309 1668 146 2344 1626 144 2311 1634
F 2391 1603 150 2350 1687 149 2377 1645 146 2355 1626
G 2286 1591 143 2369 1653 137 2300 1625 137 2377 1627
H 2315 1619 154 2362 1675 142 2383 1635 149 2405 1637
H M L H M L H M L H M
SW Z' %Spec
64.30 0.95 93.8
Day 2, Plate 2
row 1 2 3 4 5 6 7 8 9 10 11
A 129 2390 1585 157 2556 1620 158 2465 1656 121 2518
Max/Min
16.20688172
Assay Validation PUT
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A 129 2390 1585 157 2556 1620 158 2465 1656 121 2518
B 141 2294 1582 152 2438 1629 150 2524 1621 153 2540
C 168 2333 1603 110 2379 1677 174 2440 1623 157 2546
D 180 2336 1603 151 2413 1708 130 2493 1676 154 2452
E 121 2242 1656 153 2399 1693 167 2442 1613 156 2574
F 190 2262 1560 139 2300 1637 136 2518 1685 143 2589
G 160 2270 1552 161 2277 1623 156 2414 1680 163 2440
H 135 2259 1610 147 2252 1619 144 2334 1689 146 2265
L H M L H M L H M L H
SW Z' %Spec
17.25 0.83 93.8
Day 2, Plate 3
row 1 2 3 4 5 6 7 8 9 10 11
A 1528 135 2364 1650 128 2301 1585 156 2391 1610 152
B 1575 136 2303 1577 174 2352 1621 159 2324 1608 174
C 1484 152 2344 1627 159 2438 1567 162 2446 1675 172
D 1538 144 2319 1624 154 2330 1555 139 2554 1663 163
E 1616 119 2382 1567 168 2352 1629 142 2350 1691 177
F 1573 137 2273 1510 127 2246 1519 133 2387 1678 148
G 1530 159 2319 1461 144 2180 1574 159 2433 1575 132
H 1581 160 2288 1493 172 2238 1625 148 2407 1321 145
M L H M L H M L H M L
SW Z' %Spec
25.21 0.87 93.5
16.02540608
Max/Min
Max/Min
15.50320977
4040
Day 1 Plate 1
0
500
1000
1500
2000
2500
3000
0 12 24 36 48 60 72 84 96
Well - Row Oriented
Response
Max
Mid
Min
Day 1 Plate 1
0
500
1000
1500
2000
2500
3000
0 8 16 24 32 40 48 56 64 72 80 88 96
Max
Mid
Min
Well - Column Oriented
Day 1 Plate 2
3000
Day 1 Plate 2
3000
Assay Validation PUT
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0
500
1000
1500
2000
2500
0 12 24 36 48 60 72 84 96
Well - Row Oriented
Response
Max
Mid
Min
0
500
1000
1500
2000
2500
0 8 16 24 32 40 48 56 64 72 80 88 96
Max
Mid
Min
Well - Column Oriented
Day 1 Plate 3
0
500
1000
1500
2000
2500
3000
0 12 24 36 48 60 72 84 96
Well - Row Oriented
Response Max
Mid
Min
Day 1 Plate 3
0
500
1000
1500
2000
2500
3000
0 8 16 24 32 40 48 56 64 72 80 88 96
Max
Mid
Min
Well - Column Oriented
4141
Plate 1 - Plate 1 - Plate 2 -
Plate 2 Plate 3 Plate 3
Day 1 1.1 1.0 1.0 Meets Criterion
Day 2 1.1 1.1 1.0 Meets Criterion
Day 3 1.0 1.1 1.0 Meets Criterion
Day 1 - Day 2
1.0 Meets Criterion
Day 1 - Day 3
1.0 Meets Criterion
Day 2 - Day 3
Ratio EC50/IC50/Ki between Days (larger over smaller)
Ratio EC50/IC50/Ki within Days (larger over smaller)
Assay Validation PUT
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Day 2 - Day 3
1.0 Meets Criterion
Validation Checklist
Intra-Plate Tests
1 Check for drift / edge effects in all plates (you must check manually)
2 All max (HI) signal CV's < 20% Yes
3 All mid signal (unnormalized) CV's < 20% Yes
4 All normalized mid signal (mid %) SD's < 20 Yes
5 All min (LO) SD's < Min(max (HI) SD, mid SD) Yes
6 All SW's >2 Yes
7 All Z' Factors > 0.4 (and < 1 ; must pass one of 6 or 7) Yes
Inter-Plate Tests
1 All within-day fold shifts < 2 Yes
2 All Average (between-)Day fold shifts < 2 Yes
Meets Criterion ?
4242
A reporter gene driven cell based assay
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
4343
0
25
50
75
100Full Agonist
Antagonist
Partial Agonist
Inverse Agonist
Inactive Compound
%A
cti
vit
y
Cell Based Assay Validation
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
-1 0 1 2 3 4-25
Log[Compound]nM
Compounds representing various class and varying affinities are selected for an
assay validation
EC50, IC50, and Kb values are derived and compared with the published values
4444
Patch Clamp Assay
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
45
Patchliner with 8 Channel Ion Channel
45
Voltage gated ion channel: Potassium – Kv 11.1 (hERG)
Cell line : Stably expressing recombinant HEK293 hERG cell line
Test item: Astemizole, Haloperidol and Quinidine
Cells with membrane resistance of > 1 GOhms; Currents of > 200 pA; without
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
rundown of current were taken into consideration for the Analysis
-80 mV
+40 mV
-40 mV
500 ms
500 ms 500 ms
-40 mV
-80 mV
hERG I-V protocol
Tail Currents
46
Voltage gated ion channel: Potassium – Kv 11.1 (hERG)
Astemizole
200
100
0
-100
pA
'Control''Astemizole 10 nM''Astemizole 100 nM''Astemizole 300 nM''Astemizole 1 uM''Astemizole 10 uM'
1.0
0.8
0.6
0.4
0.2
Astemizole
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
4747
Haloperidol
-100
1.51.00.50.0
s
1.0
0.5
0.0
-0.5
nA
1.51.00.50.0
s
'Control''Haloperidol 10 nM''Haloperidol 100 nM''Haloperidol 1 uM''Haloperidol 10 uM'
0.0
100 pM 1 nM 10 nM 100 nM 1 µM 10 µM
1.0
0.8
0.6
0.4
0.2
0.0
100 pM 1 nM 10 nM 100 nM 1 µM 10 µM
Haloperidol
Voltage gated ion channel: Potassium – Kv 11.1 (hERG)
Quinidine
600
400
200
0
-200
pA
'Control''Quinidine 300 nM''Quinidine 1 uM''Quinidine 3 uM''Quinidine 10 uM'
1.0
0.8
0.6
0.4
0.2
Qunidine
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
4848
S.No CompoundObtained IC50 value
(nM)Reported IC50 value
(nM)
1 Astemizole 29.6 ± 7.9 1 - 26
2 Quinidine 404.4 ± 80.7 300 - 1000
3 Haloperidol 117.4 ± 23.8 174
-200
-400
1.51.00.50.0
s
0.2
0.0
10 nM 100 nM 1 µM 10 µM
Voltage gated ion channel: Sodium – Nav 1.5
Cell line : Stably expressing recombinant HEK293 Nav 1.5 cell line
Test item: Tetracaine, Lidocaine, Proparacaine and Tetrodotoxin
Cells with membrane resistance of > 1 GOhms; Currents of > 2 nA; without
rundown of current were taken into consideration for the Analysis
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rundown of current were taken into consideration for the Analysis
Nav 1.5 I-V Protocol5 ms
-30 mV
40 ms
5 ms
-100 mV-100 mV
49
Voltage gated ion channel: Sodium – Nav 1.5
Tetracaine
-5
-4
-3
-2
-1
0
nA
ControlTetracaine 100 nMTetracaine 300 nMTetracaine 1 uMTetracaine 3 uMTetracaine 10 uMTetracaine 30 uMTetracaine 100 uM
1.0
0.8
0.6
0.4
0.2
Tetracaine
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Lidocaine
-5
403020100
ms
-5
-4
-3
-2
-1
0
nA
403020100
ms
ControlLidocaine 10 uMLidocaine 30 uMLidocaine 100 uMLidocaine 300 uMLidocaine 1 mM
0.0
1 nM 10 nM 100 nM 1 µM 10 µM 100 µM
1.0
0.8
0.6
0.4
0.2
0.0
100 nM 1 µM 10 µM 100 µM 1 mM
Lidocaine
50
Voltage gated ion channel: Sodium – Nav 1.5
Proparacaine
-6
-5
-4
-3
-2
-1
0
nA
ControlProparacaine HCl 100 nMProparacaine HCl 300 nMProparacaine HCl 1 uMProparacaine HCl 3 uMProparacaine HCl 10 uMProparacaine HCl 30 uMProparacaine HCl 100 uM
0.8
0.6
0.4
0.2
Proparacaine_HCl
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Tetrodotoxin
51
-6
403020100
ms
0.0
1 nM 10 nM 100 nM 1 µM 10 µM 100 µM
-8
-6
-4
-2
0
nA
403020100
ms
ControlTetrodotoxin 30 nMTetrodotoxin 100 nMTetrodotoxin 300 nMTetrodotoxin 1 uMTetrodotoxin 3 uMTetrodotoxin 10 uM
1.0
0.8
0.6
0.4
0.2
0.0
1 nM 10 nM 100 nM 1 µM 10 µM
Tetrodotoxin
Voltage gated ion channel: Sodium – Nav 1.5
S.No CompoundObtained IC50 value
(µM)Reported IC50 value
(µM)
1 Tetracaine 1.3 1.7
2 Lidocaine 258 260
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3 Proparacaine HCl 1.8 1.9
4 Tetrodotoxin 5.3 4.3
52
Ligand gated ion channel: ATP – Purinergic P2X7
Cell line : Stably expressing recombinant HEK293 P2X7 cell line
Test item: Agonist – ATP and Bz ATP, Antagonist - Suramin
Cells with membrane resistance of > 1 GOhms; Currents of > 1 nA; without
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rundown of current were taken into consideration for the Analysis.
Whole cell patchclamp recordings using the stacked solution protocol by
holding cells at -80 mv
53
Ligand gated ion channel: ATP – Purinergic P2X7
ATP
-1.5
-1.0
-0.5
0.0
nA
ATP 250 uMATP 500 uMATP 1 mMATP 2 mMATP 3 mM
1.0
0.8
0.6
0.4
0.2
ATP
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5454
Bz ATP
543210
s
-5
-4
-3
-2
-1
0
nA
543210
s
BZ ATP 100 uMBZ ATP 250 uMBZ ATP 500 uMBZ ATP 750 uMBZ ATP 1 mMBZ ATP 1.5 mM
0.03 4 5 6 7 8 9
0.0012 3
M1.0
0.8
0.6
0.4
0.2
100 µM2 3 4 5 6 7 8 9
1 mM
Bz_ATP
Ligand gated ion channel: ATP – Purinergic P2X7
Suramin-1.5
-1.0
-0.5
nA
Bz ATP 1mMSuramin 10 uM + Bz ATP 1mMSuramin 100 uM + Bz ATP 1mMSuramin 1 mM + Bz ATP 1mMSuramin 10 mM + Bz ATP 1mM
1.0
0.8
0.6
0.4
Suramin
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5555
S.No Compound Obtained EC50 value (nM) Reported EC50 value (nM)
1 ATP 890 µM 780 µM
2 Bz ATP 320 µM 100 µM
S.No Compound Obtained IC50 value (nM) Reported IC50 value (nM)
1 Suramin 48 µM 40 µM
-2.0
543210
s
0.2
0.0
100 nM 1 µM 10 µM 100 µM 1 mM 10 mM
Cyototoxicity assessment
Tamoxifen Acetaminophen Compound-10
25
50
75
100100 M10 M
MTT Assay
1 M
%V
iabilit
y
100100 MLDH Assay
Test System: HepG2 (Human hepatocellular Carcinoma),HEK293 (Human embryonic kidney) andIMR32 (Human neuroblastoma)
Test item: Tamoxifen (Positive control)AcetaminophenTest compound
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
5656
Tam oxifen Acetam inophen Com pound-10
25
50
75
100 M10 M
LDH Assay
1 M
%C
ell
death
Tamoxifen Acetaminophen Compound-10
25
50
75
100100 M10 M
Trypan blue exclusion
1 M
%V
iabilit
y
Test compound
Test Conc.: 1 µM, 10 µM and 100 µM
Incubation: 24 hour
Assessment: MTT (Cellular Metabolic activity)LDH (Lactate dehydrogenase activity)Trypan blue (Cell Membrane Integrity)
Phospholipidosis
Test System: CHO-K1 (Chinese Hamster Ovary cell line)and CHL/IU (Chinese Hamster Lung)
Test item: Amiadarone (Positive control)Acetaminophen (Negative Control)Fluoxetine
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5757
Test Conc.: 1 µM - 100 µM
Incubation: 24 hour
Assessment: Fluorescent MicroscopyFluorometry (Fluorescence Plate Reader)
Phospholipidosis
Vehicle Acetamoinophen (100 µM)(Negative Control)
Fluoxetine (30 µM)(Positive Control)
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
5858
30 M 20 M 15 M 10 M 5 M 3 M0
20
40
60
80
100
120AMIODARONE
ACETAMINOPHEN
FLUOXETINE
PhospholipidosisFluorometric Assay
PLD
Inducin
gpote
nti
al(%
)
List of In-vitro Assays
Target Assay Type Target Assay Type
GPCRs Transporters
Adenosine A2A Reporter Gene Driven Cell Based SERT Radioligand binding/ Functional Uptake
Adrenoceptor alpha 1B Radioligand binding/ Reporter Gene Driven Cell Based DOPT Radioligand binding/ Functional Uptake
Adrenoceptor alpha 2C Reporter Gene Driven Cell Based NET Radioligand binding/ Functional Uptake
5-HT1A Radioligand binding/ Reporter Gene Driven Cell Based Nuclear Receptors
5-HT2A Radioligand binding LXR alpha Reporter Gene Driven Cell Based
5-HT2C Radioligand binding LXR beta Reporter Gene Driven Cell Based
5-HT4 Reporter Gene Driven Cell Based PPAR gamma Reporter Gene Driven Cell Based
5-HT6 Reporter Gene Driven Cell Based RXR alpha Reporter Gene Driven Cell Based
5-HT7 Radioligand binding/ Reporter Gene Driven Cell Based Ion Channels
In Vitro Assay Inventory at Suven
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
5959
5-HT7 Radioligand binding/ Reporter Gene Driven Cell Based Ion Channels
Dopamine D1 Reporter Gene Driven Cell Based Nicotinic Alpha4Beta2 Radioligand binding
Dopamine D2 Radioligand binding 5-HT3 Radioligand binding
Dopamine D5 Reporter Gene Driven Cell Based Kv 10.1 Electrophysiology
Histamine H1 Radioligand binding/ Reporter Gene Driven Cell Based Kv 11.1 (hERG) Radioligand binding/ Electrophysiology
Histamine H3 Radioligand binding/ Reporter Gene Driven Cell Based Nav 1.4 Electrophysiology
Histamine H4 Radioligand binding Nav 1.5 Electrophysiology
Muscuranic M1 Radioligand binding/ Reporter Gene Driven Cell Based Nav 1.6 Electrophysiology
Muscuranic M2 Radioligand binding/ Reporter Gene Driven Cell Based Nav 1.7 Electrophysiology
Muscuranic M3 Radioligand binding/ Reporter Gene Driven Cell Based P2X7 Electrophysiology
Muscuranic M4 Radioligand binding ADME
Muscuranic M5 Reporter Gene Driven Cell Based A → B and B → A Permeability
CB1 Radioligand binding/ Reporter Gene Driven Cell Based P-gp Substrate and Inhibitor assessment
CB2 Radioligand binding/ Reporter Gene Driven Cell Based CYP Induction assay Reporter Gene based (PXR Transactivation )
Prostaglandin EP2 Reporter Gene Driven Cell Based Toxicity
Cyototoxicity assay Trypan blue, LDH and MTT assay
Phospholipidosis assay Microscopy and Fluorometry (Plate reader)
Caco2 assay
In-vitro ADMEIn-vivo PKRegulatory Bioanalysis
Suven Discovery Research
• Solubility
• Lipophilicity
• Permeability assay (PAMPA & CACO-2)
• Metabolic stability assay (Hepatic, intestinal microsomes and hepatocytes)
• CYP Inhibition assay – 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 & 3A4 (Supersomes and microsomes)
• Time Dependent Inhibition assay (Human liver microsomes) – 1A2, 2B6, 2C19, 2D6 and 3A4
In-vitro ADME
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
• Time Dependent Inhibition assay (Human liver microsomes) – 1A2, 2B6, 2C19, 2D6 and 3A4
• In-vivo approach for evaluation of MBI of CYP3A in rats
• MAO Inhibition assay – MAO-A & MAO-B (Recombinant enzymes – Florescent based)
• In-vitro Protein binding studies – Plasma, Brain & Microsomes (RED / HT Dialysis method)
• CYP isoform fingerprinting / Reaction phenotyping studies (rCYP’s and Liver microsomes)
• CYP induction assay (Cryopreseved inducible hepatocytes)
• Metabolite identification using LC-MS/MS (Phase I & II)
• Reactive metabolite identification using LC-MS/MS
61
In-vivo PK
In Vivo Studies− Bioavailability− Bioequivalence− PK in target site (CNS or peripheral)− Single and multiple-dose pharmacokinetics− Tissue distribution (cold and 14C labelled)− Biliary vs. urinary excretion (cold and 14C labelled)− PK/PD studies in animal models
In Vivo Dosing− IV bolus (single and cassette)− IV infusion− Oral− Sucutaneous− Intra-nasal− Sublingual− Topical/ Intradermal− Specialized CNS drug delivery/ Sampling
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62
− PK/PD studies in animal models
Species− Mice, rat, rabbit, guinea pig, hamster− Beagle dogs
Biological Matrices− Blood, plasma, serum− Bile, urine, feces− Synovial fluid, cerebrospinal fluid− Microdialysis samples (brain, dermal, blood)− Tissues (skin, muscle etc)
− Specialized CNS drug delivery/ Sampling
Intestinal Permeability models− In situ single/ double/ triple pass method− In situ closed loop method− Everted gut sac, intestinal ring method
Bio-analytical capabilities− LC-MS/MS API 6500, 6500 Qtrap, 4000,
4000 Qtrap, API 3000− HPLC-VU/ECD/ FLD/ PDA
− Scintillation counter Tri-carb 3110TR
− Specialized tissue processing equipment.
• Jugular vein Cannulated Rats, Guinea pigs & Hamsters
• Femoral vein Cannulated Rats & Guinea pigs
• Portal vein Cannulated Rats and Guinea pigs
• Carotid artery Cannulated Rats
Surgical Models
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
• Abdominal aorta Cannulated Rats
• Bile Duct Cannulated Rats
• Duodenal, Jejunum, Ileum and Colon Cannulated Rats
• CSF collection from Rats
• Synovial fluid collection from Rats
63
Bi-directional Permeability assay
Test System: Caco-2
Test item: Digoxin (Standard)and test compounds
Test Conc.: 10 µM
Incubation: 1 hour
Permeability125
150
0.0
2.5
5.0
7.5 Efflux Ratio
Dig
oxin
TC
#2
TC
#3
TC
#4
Threshold
TC
#1
Eff
lux
Rati
o
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
6464
Incubation: 1 hour
Membrane TEER and LuciferIntegrity: Yellow
Analysis: LC-MS/MS
0.0
2.5
5.0
A-
B
B-
A
Digoxin TC #1 TC #2 TC #3 TC #4
A-
B
A-
B
A-
B
A-
B
B-
A
B-
A
B-
A
B-
A
25
50
75
100
TC: Test compound
Pap
p(1
0-6
cm
/sec)
P-glycoprotein (P-gp) Substrate assessment
Test System: Caco-2
Test item: Digoxin(P-gp Substrate)
Test Conc.: 10 µM
Assessment: - / + Verapamil16
0
10
20
30
A - B
B - A
1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10Days
Pap
p(x
10
-6cm
/sec)
Digoxin assessed on ten different days
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
6565
Assessment: - / + Verapamil(P-gp Inhibitor)
Inhibitor Conc.: 10 µM
Incubation: 1 hour
Membrane TEER and LuciferIntegrity: Yellow
Analysis: LC-MS/MS
Day
1
Day
2
Day
3
Day
4
Day
5
Day
6
Day
7
Day
8
Day
9
Day
10
0
2
4
6
8
10
12
14
16
- Verapamil
+ Verapamil
Threshold
Eff
lux
Rati
o
- Verapamil + Verapamil
P-glycoprotein (P-gp) Inhibitor assessment
Test System: Caco-2
Test item: Verapamil(Positive control)Phenelzine(Negative control)
0
5
10
15
20DigoxinEffluxRatio
ThresholdDig
ox
inE
fflu
xR
ati
o
0
5
10
15
20
25
30
35
A-
B
B-
A
A-
B
A-
B
B-
A
B-
A
D igoxin
10 M
Verapamil
50 M
Phenelz ine
50 M
Digoxin Permeability
Dig
oxin
Papp
(10
-6cm
/sec)
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
6666
P-gp Substrate: Digoxin
Incubation: 1 hour
Membrane TEER and LuciferIntegrity: Yellow
Analysis: LC-MS/MS
0
Digoxin
10M
Verapamil
50M
Phenelzine
50M
0
25
50
75
100
125 Degree of Inhibition
Verapamil50 M
Phenelzine50 M
%In
hib
itio
no
fD
igo
xin
Eff
lux
Rati
o
CYP3A Time Dependent Inhibition (Single point screen)
Matrix : Pooled Human Liver Microsomes (Mixed Gender)
Test Concentration : range spanning 10 fold to IC50 µM
Protein concentration : 0.5 mg/ mL 0.25 mg/mL
Incubation time : 10 & 30 min ± NADPH
Substrate : Midazolam
Incubation time : 2 min
Metabolite : 1-OH Midazolam
Method of Detection : LC-MS/MS
DrugNormal
CYP3A
High activity
CYP3A
Indinavir 0 0
Ketoconazole 0 0
Fluconazole 0 0
Zolpidem 5.5 2.54
Fluoxetine 14.24 0
Terfenadine 8.15 0
Nifedipine 16.58 0
Atomoxetine 0 0
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
TDI (%) - Normal CYP3A Vs High activity CYP3A
y = 1.0272x
R2
= 0.9446
0
20
40
60
80
100
0 20 40 60 80 100
Normal CYP3A - TDI (%)
Hig
ha
cti
vit
yC
YP
3A
-T
DI(%
)
Positive control : Troleandomycin and Erythromycin
Results : % TDI @ 30 min
Paroxetine 0 6.42
Risperidone 0 8.99
Clozapine 0 7.4
Duloxetine 0 14.49
Fluvoxamine 9.66 15.5
Erythromycin 44.04 47.67
Diltiazem 44.9 46.93
Domperidone 45.84 41.74
Dasatinib 33.94 49.64
Saquinavir 49.7 54.74
Troglitazone 50.95 55.29
Troleandomycin 65.21 67.89
Mibefradil 89 89
Verapamil 81.42 84.33
Nefazodone 91.79 93.07
67
CYP3A Time Dependent Inhibition (IC50 shift)
Matrix : Pooled Human Liver Microsomes (Mixed Gender)
Test Concentration : range spanning 10 fold to IC50 µM
Protein concentration : 1.0 mg/ mL 0.1 mg/mL
Incubation time : 10 & 30 min ± NADPH
Substrate : Midazolam
Incubation time : 5 min
Metabolite : 1-OH Midazolam
Method of Detection : LC-MS/MS
% Activity (+ NADPH) 10 Min
% Activity (+ NADPH) 30 Min
% Activity (-NADPH) 30 Min
% Activity (-NADPH) 10 Min
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
Positive control : Verapamil
Results : IC50 shift (fold change)
0.001 0.01 0.1 1 10
0
40
80
120
% Activity (-NADPH) 30 Min
Verapamil (µM)
Perc
en
to
fC
on
tro
l(%
)
68
CYP3A4: Time dependent Inhibition (Inactivation kinetics)
Preincubation assay-
Test compound: Troleandomycin
Protein concentration: 0.5 mg/mL
Test Compound Concentration: 0, 0.312, 0.625, 1.25, 2.5, 5, 10 µM
Preincubation time points: 0, 2.5, 5, 10, 20 and 30 min
Dilution in to activity assay: 20 fold
Activity assay:
Incubation time: 2 min
Midazolam concentration: 25 µM (approximately 10 times Km)
Measured: 1-OH midazolam
No compound
y = -0.009x + 4.61
R2 = 0.6928
0.312 µM
y = -0.0497x + 4.61
R2 = 0.9303
0.625 µM
y = -0.0895x + 4.61
R2 = 0.9388
1.25 µM
y = -0.1614x + 4.61
R2 = 0.9385
0.00
1.00
2.00
3.00
4.00
5.00
Na
tura
lL
og
of
%A
cti
vit
yR
em
ain
ing
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
0 2 4 6 8 10
0.0
0.1
0.2
0.3
KINACT
KI
0.2880
1.169
CLinact = 246 mM-1 min-1
Troleandomycin (µM)
Ko
bs
(min
-1)
Measured: 1-OH midazolam
Analysis: LC-MS-MS2.5 µM
y = -0.206x + 4.61
R2 = 0.9152
5.0 µM
y = -0.2301x + 4.61
R2 = 0.882
10.0 µM
y = -0.253x + 4.61
R2 = 0.9137
-5.00
-4.00
-3.00
-2.00
-1.00
0 5 10 15 20 25 30 35
Na
tura
lL
og
of
%A
cti
vit
yR
em
ain
ing
No compound0.3120.625 µM1.25 µM2.5 µM5.0 µM10. 0µMLinear (No compound)
69
CYP Induction: Fold Change in Gene Expression of CYP1A2, CYP2B6 &
CYP3A4 in Three Donors
44.6
33.8
50.5
48
60
72
84
Fo
ldch
ang
ere
lati
ve
to
Co
ntr
ol
Omeprazole (CYP1A2 inducer)
Phenobarbital (CYP2B6 inducer)
Rifampicin (CYP3A4 inducer)
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
33.8
10.8
6.9 6.7
10.7
5.97.5
0
12
24
36
HH
1032
HH
1007
HH
1031
Fo
ldch
ang
ere
lati
ve
to
4 fold change in gene expression relative to control is considered positive for screening purposes.
70
In-vitro Reactive Met ID evaluation (Ticlopidine)
Compound : Ticlopidine
Matrix : SD Rat liver microsomes
Instrumentation and Analytical Method
LC-MS/MS : API-4000 Qtrap (Applied Biosystems)
Ion source : Turbospray
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
HPLC : Shimadzu SIL HTc
Column : Atlantis dC18, 4.6 x 150 mm, 5 µ
Mobile phase : 0.1 % Formic acid in water (A) :: 0.1 % Formic acid in water:Acetonitrile (B) [100::15:85 %]
Flow rate : 0.3 mL/min
Gradient program : 0.1-1.5 min (5%B), 25 min (85%B), 30 min (85%B), 30.5-35min (5%B)
Sample Preparation : 100 µM of test compound incubated at 37°C with 1 mg/mL of microsomal protein, 2 mM of NADPH
and 2 mM of GSH. After 45 min of incubation, precipitated with 50 µL of 30 % (w/v) trichloroacetic acid (TCA)
in water. Centrifuged at 10,000 rpm for 10 min at 4°C and supernatant was injected into LC-MS/MS system
71
Cl
N
S
Epoxidation S-oxidation
Cl
N
S
O
Cl
N
S
O
Ticlopidine
In-vitro Reactive Met ID evaluation (Ticlopidine)
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
•Both M1 and M2 were identified at 100µM of substrate.
P: m/z 264 (Parent); RT = 19.85
M1: m/z 280 (P+16); RT = 18.50M2: m/z 587 (P+16+307); RT = 15.55
•Fragmentation consistent with the proposed structure.
P : 125, 154M1: 262, 252, 154, 125M2: 569, 539, 494, 458, 440, 416, 410, 341, 308, 296, 287, 280.
Summed ion chromatogram of Parent, M1 and M2
Cl
N
SOHGS
Proposed structure of Ticlopidine-GSH conjugate
GSHGSH
72
Dog PK: Assessment of pH and food effect on absorption of Test Compound
Test compound (as suspension in capsule) administered by oral
gavage in male Beagle dogs. All animals pretreated with pentagastrin
or famotidine with one week washout period between each
pretreatment.
Test compound, 1 mg/kg, p.o.10000
Pentagastrin
Test compound, 1 mg/kg, p.o.
10000
Test compound administer by oral gavage in male Beagle dogs at
fasted and fed conditions with one week washout period between
each pretreatment.
Effect of Food on the Absorption of Test CompoundsEffect of Food on the Absorption of Test Compounds
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
Notes: Pentagastrin (6 µg/kg, i.m/each dog) was administered 20 minutes prior to test compound (1
mg/kg, p.o) administration. Famotidine (40 mg Capsule/dog) was administered 3 hours prior to test
compound (1 mg/kg, p.o) - administration
1
10
100
1000
0 4 8 12 16 20 24 28 32 36 40 44 48
Time (hr)
Mean
pla
sm
aC
onc.
(nM
)
Pentagastrin
Famotidine
1
10
100
1000
0 4 8 12 16 20 24 28 32 36 40 44 48
Time (hr)
Mean
pla
sm
aC
onc.
(ng/m
L)
Fasted
Fed
73
Regulatory Bioanalysis and Formulation Analysis
Exclusive advanced analytical laboratory accredited with ISO:IEC 17025 for quality
systems since 2005
Developed more than 150 sensitive and challenging bioanalytical methods
Performed bioanalysis for Phase-1 first in human studies and submitted dossiers for
regulatory agencies
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
74
Performed bioanalysis and formulation analysis for IND enabling toxicology studies
Cross Validated Bioanalysis Method and Data Analysis with Collaborator Laboratory
Conducted Bioanalysis for various biopharmaceutical projects to many clients both
domestic, US and Europe clients for clinical development and regulatory submission
Successfully completed number of regulatory and client audits (Vertical and System
Audits)
Target Engagement:Receptor OccupancyMicrodialysisElectroencephalography in Rodents (EEG)
Suven Discovery Research
Electroencephalography in Rodents (EEG)
LC-MS/MS (or) LSA Based Receptor Occupancy Methodology
Formulation
Radiolabelled ([3H]) tracer (i.v.) dose at Tmax of testNon-radiolabelled tracer (i.v.) dose at Tmax of test
Animal model: rat/ mouse/guinea pig
Test compound dosingp.o./s.c./ i.p./i.v.
LC-MS/MS based Liquid scintillation analyzer (LSA) based
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
76
Receptoroccupancyandexposurecorrelation
Radiolabelled ([ H]) tracer (i.v.) dose at Tmax of testcompound
Cervical dislocation
Brain regional isolation
Sample homogenation & filtration or tissue digestion
Radioactivity determination by Liquid scintillationcounter -Tricarb 3110TR (CPM or DPM)
max
compound
Cervical dislocation & trunk blood collection
Brain regional isolation
Sample homogenation, protein precipitation ¢rifugation
LC-MS/MS quantification of tracer/ test compound(ng/g or ng/mL) - API 6500 Q Trap
Receptor Occupancy calculation (ratio /
specific binding / positive control method)
Receptor Tracer Specific region
#, * 5-HT1A WAY-100635■ Frontal cortex
5-HT1B AZ10419369 Hypothalamus
* 5-HT2A MDL-100907 Frontal cortex
5-HT2C SB242084 Choroid plexus
5-HT4 SB207145 Striatum
Receptor Tracer Specific region
A2A SCH442416 Striatum
nAChR α4β2 ZW-104 Thalamus
nAChR α7 Methyllycaconitine Hypothalamus
Histamine H3 GSK-189254 Frontal cortex
PDE10 AMG 7980 Striatum
List of Validated Receptor Occupancy Assays
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77
5-HT6 Lu AE60157■ Striatum
NERT S,S-MeNERHypothalamus/
Thalamus
DAT Nomifensine Striatum
#SERT DASB Frontal cortex
GABAA Flumazenil Cortex
CB1 AM251Cerebellum/ Brain
stem
D1 SCH39166 Striatum
* D2 Raclopride■ Striatum
NK1 GR205171Striatum and
Habenula
mGluR5 MEPyHippocampus/
Striatum
adrenergic α1A Prazosine Frontal cortex
5-HT: Serotonin; NERT: Nor epinephrine reuptake transporter; DAT: Dopamine reuptake transporter; SERT: Serotonin reuptake transporter; GABA: Gamma amino butyric acid:CB: Cannabinoid; A: Adinosine; nAChR: nicotinic acetylcholine receptor; PDE: Phosphodiesterase; D: Dopamine; NK: Neurokinin; mGluR: metobotropic glutamate receptor
All tracer are non-radiolabeled tracers; ■ Radiolabeled tracers; *# Dual or triple target receptor occupancy assay
Receptor Occupancy using radio or non-radiolabelled tracer
0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 00
2 0
4 0
6 0
8 0
1 0 0
P e r c e n t D ( s t r ia tu m ) r e c e p to r o c c u p a n c y o f h a lo p e r id o l in
N o n - r a d io la b e lle d r a c lo p r id e a s tr a c e r
H a lo p e r id o l ( m g / k g , p .o . )
%D
2re
cepto
roccupancy
0 .0 0 1 0 .0 1 0 .1 1 1 00
2 0
4 0
6 0
8 0
1 0 0
P e r c e n t D ( s t r ia tu m ) r e c e p to r o c c u p a n c y o f h a lo p e r id o l in
R a d io la b e lle d r a c lo p r id e a s t r a c e r
H a lo p e r id o l ( m g / k g , p .o . )
%D
2re
cepto
roccupancy
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
78
P e r c e n t D 2 ( s t r ia tu m ) r e c e p to r o c c u p a n c y o f h a lo p e r id o l in
fa s te d r a t s u s in g r a c lo p r id e a s a t r a c e r
P e r c e n t D 2 ( s t r ia tu m ) r e c e p to r o c c u p a n c y o f h a lo p e r id o l in
fa s te d r a ts u s in g [ 3 H ] r a c lo p r id e a s a t r a c e r
0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 00
2 0
4 0
6 0
8 0
1 0 0
P e r c e n t 5 - H T 6 ( s t r ia tu m ) r e c e p to r o c c u p a n c y o f S B - 7 4 2 4 5 7
in r a t s u s in g [ 3 H ] L u A E 6 0 1 5 7 a s a t r a c e r
R a d io la b e lle d [ 3 H ] L u A E 6 0 1 5 7 a s tr a c e r
S B - 7 4 2 4 5 7 ( m g /k g , s . c . )
%5-H
T6re
cepto
roccu
pancy
0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 00
2 0
4 0
6 0
8 0
1 0 0
P e r c e n t 5 - H T 6 ( s t r ia tu m ) r e c e p to r o c c u p a n c y o f S B - 7 4 2 4 5 7
in r a ts u s in g n o n - r a d io la b e lle d L u A E 6 0 1 5 7 a s a t r a c e r
N o n - r a d io la b e lle d L u A E 6 0 1 5 7 a s tr a c e r
S B - 7 4 2 4 5 7 ( m g /k g , s . c . )
%5-H
T6re
cepto
roccupancy
Single and multi target receptor occupancy assay
0.01 0.03 0.10 0.30 1.00 3.00 6.000
25
50
75
100 ED50 = 0.11 mg/kg
D2RO (mean ± SEM) of ziprasidone in male Wistar rat
Ziprasidone (mg/kg, i.v.)
%D
2re
ce
pto
ro
ccu
pa
ncy
0.03 0.10 1.00 3.00 6.000
25
50
75
100 ED50 = 10.92 mg/kg
5-HT1ARO (mean ± SEM) of ziprasidone in male Wistar rat
(n=4) using single non-radiolabelled WAY-100635 tracer
Ziprasidone (mg/kg, i.v.)
%5
-HT
1Are
ce
pto
ro
ccu
pa
ncy
0.01 0.03 0.10 0.30 1.00 3.00 6.000
25
50
75
100 ED50 = 0.03 mg/kg
5-HT2ARO (mean ± SEM) of ziprasidone in male Wistar rat
(n=4) using single non-radiolabelled MDL-100,907 tracer
Ziprasidone (mg/kg, i.v)
%5
-HT
2Are
ce
pto
ro
ccu
pa
ncy
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
79
0.01 0.03 0.10 0.30 1.00 3.00 6.000
2 5
5 0
7 5
1 0 0
R O ( m e a n ± S E M ) o f z ip r a s id o n e in m a le W is ta r r a t ( n = 4 ) u s in g c o c k ta il n o n - r a d io la b e lle d t r a c e r s
5 - H T 2 A : M D L - 1 0 0 ,9 0 7 c o c k ta il, E D 5 0 = 0 .0 4 m g /k g
D 2 : R a c lo p r id e c o c k ta il, E D 5 0 = 0 .0 8 m g /k g
5 - H T 1 A : W A Y 1 0 0 6 3 5 c o c k ta il, E D 5 0 = 9 .5 7 m g /k g
Z ip r a s i d o n e ( m g / k g , i . v . )
%re
ceptorocc
upancy
(n=4) using single non-radiolabelled raclopride tracer(n=4) using single non-radiolabelled WAY-100635 tracer (n=4) using single non-radiolabelled MDL-100,907 tracer
PK/PD: Prediction of Human exposure-RO Relationship
1. Predict Human PKcharacteristics
2. Assume PK in plasmais proportional to PK in
brain
Parameter Value
E0 (%) 5.6
EC50 (ng/mL) 4100
Emax 75
gamma 1.3
Simple Emax model
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80
3. Determine rat PKPDrelationship
4. Assume human PKPDrelationship is similar to
rat
5. Simulate human ROgiven ‘known’ PK
In-vivo Brain Microdialysis Capabilities
Study Designs
Parallel Treatment Groups
Cross-over Treatment Design
Mechanism of Action Studies using various Antagonists/Blockers.
Pharmacokinetic, Pharmacodynamic (PK/PD) Studies in-vivo, by simultaneous monitoring of neurotransmitters and drug concentrations.
Species
Rat (Wistar/Sprague Dawley)
Guinea pig (Dunkin Hartley) AcclimatizationStereotaxic
surgery
Preparation of animals for study
RecoveryAcclimatizationStereotaxic
surgery
Preparation of animals for study
AcclimatizationStereotaxic
surgery
Preparation of animals for study
AcclimatizationStereotaxic
surgery
Preparation of animals for study
Recovery
Typical experimental protocol:
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81
Guinea pig (Dunkin Hartley)
Mouse (Under Validation)
Neurotransmitters:
Acetylcholine
Glutamate
Histamine
Monoamines (NE, DA and 5-HT) and Metabolite
Gamma-amino butyric acid (GABA)
Routes of administration
Systemic (p.o., i.p., s.c., i.v. – bolus and infusion), Prolonged infusion using osmotic infusion pumps
Local application (retrodialysis), Intracerebroventricular (ICV) injection
4-5 days0.5 - 0.75 h
Acclimatization surgery
Guide cannula implantation
2-5 days
Stabilization Basal collection
1-2 h
Probe implantation
~ 16 h before
1-2 h
Test compound administration
Modulation of neurotransmitters in
dialysates and test compound levels in
dialysates and/ plasma
Based on the protocol
Recovery
Experimental Day
4-5 days0.5 - 0.75 h
Acclimatization surgery
Guide cannula implantation
2-5 days
Stabilization Basal collection
1-2 h
Probe implantation
~ 16 h before
1-2 h
Test compound administration
Modulation of neurotransmitters in
dialysates and test compound levels in
dialysates and/ plasma
Based on the protocol
4-5 days0.5 - 0.75 h
Acclimatization surgery
Guide cannula implantation
2-5 days
Stabilization Basal collection
1-2 h
Probe implantation
~ 16 h before
1-2 h
Test compound administration
Modulation of neurotransmitters in
dialysates and test compound levels in
dialysates and/ plasma
Based on the protocol
4-5 days0.5 - 0.75 h
Acclimatization surgery
Guide cannula implantation
2-5 days
Stabilization Basal collection
1-2 h
Probe implantation
~ 16 h before
1-2 h
Test compound administration
Modulation of neurotransmitters in
dialysates and test compound levels in
dialysates and/ plasma
Based on the protocol
Recovery
Experimental Day
In-vivo Brain Microdialysis Facility
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82
Neurotransmitter Measurement Using In-vivo Brain Microdialysis in rats
Acetylcholine: SB-277011A, 10 & 30 mg/kg,s.c. - mPFC
0
50
100
150
200
250
300
350
400
Ace
tylc
ho
line
(%C
ha
ng
e)
Vehicle
SB-277011A 10 mg/ kg s.c.
SB-277011A 30 mg/kg, s.c.
*
*
**
**
*
***
Glutamate: SB-271046 , 2.5 mg/kg,s.c. – Frontal cortex
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
83
-60 -30 0 30 60 90 120 150 180 210 240
Time (min)
0
50
100
150
200
250
300
-80 -40 0 40 80 120 160 200 240
Time (min)
His
tam
ine
(%ofm
ean
basalv
alu
e)
GSK189254 10 mg/ kg, s.c. (n=6)
Vehicle (n=6)
Histamine: GSK-189254, 10.0 mg/kg,s.c. – prefrontal Cortex
Arrow indicates the point of treatment. Data expressed as mean ± SEM.
83
GABA: Phenetzine, 30 mg/kg, i.p. prefrontal cortex
200
400
600
800Dopamine: Vehicle
Dopamine: Atomoxetine
Norepinephrine: Vehicle
Norepinephrine: Atomoxetine
%c
ha
ng
efr
om
me
an
ba
sa
lv
alu
e
Monitoring Neurotransmitters using In Vivo Brain Microdialysis
100
200
300
400
500
600Serotonin: Fenfluramine
5-H
T(%
chan
gefr
om
mea
nb
asal
valu
e)
Serotonin (5-HT)Fenfluramine 10 mg/ kg, i.p. (Guinea Pig- striatum)
Norepinephrine & DopamineAtomoxetine 3 mg/ kg, i.p. ( Rat- prefrontal cortex)
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-90 -60 -30 0 30 60 90 120 150 180 210 2400
50
100
150
200
250
300Glycine: Vehicle
Glycine: ALX-5407
Time (min)
Gly
cin
e(%
cha
ng
efr
om
me
an
bas
al
va
lue)
-90 -60 -30 0 30 60 90 120 150 180 210 2400
Time (min)
-90 -60 -30 0 30 60 90 120 150 180 210 2400
Time (min)
-120 -90 -60 -30 0 30 60 90 120 150 180 210 2400
200
400
600
800
Norepinephrine: Venlafaxine
Dopamine: Venlafaxine
Serotonin: Venlafaxine
TIme (min)
%ch
ang
efr
om
me
anb
asal
valu
e
Norepinephrine, Dopamine & SerotoninVenlafaxine 20 mg/ kg, i.p. ( Rat- prefrontal cortex)
GlycineALX-5407 10 mg/ kg, p.o. ( Rat- prefrontal cortex)
84
400
600PGE2: ELISA
PGE2: LC-MS/MS
PG
E2
(%c
ha
ng
efr
om
me
an
ba
sa
lv
alu
e)
Prostaglandin E2 (PGE2)Formalin (5%), 50 µL ( Rat- Dorsal Horn)
200
300
Substance P: Formalin (5 %, 50 µL)
Substance P: Vehicle
Su
bs
tan
ce
P(%
ch
an
ge
fro
mm
ea
nb
as
al
va
lue
)
Substance PFormalin (5%), 50 µL ( Rat- Dorsal Horn)
Monitoring Endogenous Peptides using In Vivo Microdialysis
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
Data expressed as mean ± SEM.
Arrow indicates the point of treatment.
-45 -30 -15 0 15 30 45 60 75 90 105 1200
200
Time (min)
PG
E2
(%c
ha
ng
efr
om
me
an
ba
sa
lv
alu
e)
-60 0 60 120 180 240
100
Time (min)
Su
bs
tan
ce
P(%
ch
an
ge
fro
mm
ea
nb
as
al
va
lue
)
85
PK/PD Studies in Rats Using In-vivo Brain Microdialysis / CSF Pharmacokinetics
50
100
150
200
250
300
350
400
Acety
lcholin
e(%
of
baselin
e)
-4
0
4
8
12
16
20
24
GS
K189254
conc
(nm
ol)
Modularion in cortical Aetylcholine
Dialysate conc of GSK189254
Dialysate profile and simultaneous Acetylcholine changes after GSK189254
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
86
0
50
-80 -40 0 40 80 120 160 200 240
Time (min)
-8
-4
Telemetry Systems with Implantable Telemetry- Data Sciences International (DSI)
Acquisition Platform- Ponemah
Data Analysis Software- NeuroScore v3.0 with automated sleep scoring, seizure detection, video synchronization and batch processing
modules
Grass S88 stimulator for stimulating specific regions of brain in anesthetized animals
In-Vivo Electroencephalography at Suven
Infrastructure
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87
Sleep/ Wake activity
Temperature and wake promoting activity
Total EEG power
Power Analysis in Frequency Bands
Seizure Detection
Assessment of cognitive biomarkers (Theta and Gamma Activity)– Under Validation
Parameters
In-Vivo Electroencephalography Facility
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88
Modafinil: Dose Dependent Increase in Wakefulness
Results Generated at Suven Edger and Seidel 1997
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
*p<0.05, **p<0.01, ***p<0.001 Vs Vehicle
Results Generated at Suven are Comparable to the Results Reported in Literature
89
In-vivo Efficacy Pharmacology
&
Suven Discovery Research
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
Safety Pharmacology
Therapeutic areas
• Cognition
• Depression
• Psychosis
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91
• Parkinson’s Disease
• Anxiety
• Pain
• Obesity
Rodent Models of cognition
• Novel object recognition task
(Scopolamine & Time induced episodic memory deficit)
• Morris water maze
(Scopolamine induced spatial memory deficit)
• Radial arm maze
(Scopolamine induced working memory deficit)
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92
• T-Maze
(Scopolamine induced working memory deficit)
• Fear Conditioning (Under validation)
Rodent Models of Depression
• Dominant submissive assay
• Forced swim test
(Rat & Mice)
• DRL-72s
• Tail suspension test
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93
Rodent Models of Psychosis
• MK-801 and Amphetamine induced hyperlocomotion & stereotypy
• Prepulse inhibition
• Condition avoidance response
• Dominant submissive assay (Mania)
• Resident Intruder Test (Aggression)
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94
Rodent Models of Anxiety
• Elevated Plus Maze
• Vogel conflict test
• Hole board
• Novelty induced hypophagia
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95
Rodent Models for Pain Disorders
• Formalin Induced Nociception, Hot Plate & Acetic acid
induced writhing
• Complete Freund’s Adjuvant-Induced Mechanical
Hyperalgesia
• Chemotherapy-Induced Neuropathic Pain,
Streptozotocin-Induced Diabetic Neuropathic Pain,
Chronic constriction injury, Partial sciatic nerve
• Capsaicin induced mechanical allodynia
• Osteoarthritis & Rheumatoid arthritis induced pain
models
• Reserpine induced Myalgia in rats
• Chronic post-Ishemia pain (CPIP)
• Burrowing behavior of rats for pain assessment
• Pain: In-Vivo electrophysiology
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96
ligation, Spinal Nerve (L5) Ligation models for
neuropathic pain
Rodent Models for Obesity
• Acute food intake in adapted rats
• Diet induced obesity
(Rat & Mice)
• Visceral sickness test
• Behavioral satiety sequence
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
9797
Rodent Models for Parkinson's Disease
• MPTP model in C57 mice
CNS Safety Pharmacology
(as per ICH S7 safety guidelines)
Test SpeciesCompound Used For
StandardisationIRWIN test / Functional
Observation Battery (FOB)Rat or Mouse MK-801 and Diazepam
Rota Rod Rat or Mouse Chlordiazepoxide
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98
Open Field (Locomotor Activity) Rat or Mouse MK-801 and Haloperidol
Proconvulsant effect Rat or Mouse Pentetrazole
Antagonism of Seizures induced
by electric shockRat or Mouse Carbamazapine
Antagonism of Seizures induced
by pentetrazole (PTZ)Rat or Mouse Diazepam
Catalepsy Rat or Mouse Haloperidol
CV, GI, Renal & Respiratory Safety Pharmacology
(as per ICH S7 safety guidelines)
Test SpeciesCompound Used For
Standardisation
QT, QTc, RR interval and Heart
rate, Mean arterial blood
pressure, systolic blood pressure
and diastolic blood pressure
Anesthesized
Guinea pigTerfenadine
Gastric Emptying Rat Sibutramine & Metoclopromaide
CV Safety
GI Safety
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99
Intestinal or colonic Transit Rat or Mouse Atropine and prucalopride
Gastric Secretion (SHAY’s
method)Rat Cimetidine
Ulcerogenic effect Rat Indomethacin
Saline load diuresis Rat Furosemide
Renal function Rat Furosemide
Head out plethysmography Rat Chlordiazepoxide & Theophylline
Whole body plethysmography Rat Chlordiazepoxide & Theophylline
Respiratory Safety
Renal Safety
Suven Discovery Research
Preformulation, Biopharmaceutics and Early
Formulations
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
Preformulation
Screening
• Salt and co-crystal screening
• Crystallization screening
• Polymorph screening
Selection
• Hygroscopicity
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
• Solubility
• Chemical stability
• Physico Chemical Parameters (Log D, Log P, pKa etc.,)
• Hydrates / Solvates
Solid State
• Thermal properties
• Surface morphology, Particle size
• FTIR, Raman, XRD, Cross Polarized Microscopy, DSC, TGA, DVS etc.,
• Compatibility101
Salt form screening and selection
Crystallinity
Hygroscopicity
No
Yes
Yes
No
No
Solubility enhancement
Unacceptable
Continue crystallizationattempts
Company ConfidentialCopyright © 2016 Suven Life Sciences Limited
SolubilityYes
No
Stability
No
Yes
PolymorphismNo
Yes
Salt for PK study Final salt candidate
Control
Salt for PK study Final salt candidate
No
Secondary candidate
Stability enhancement
102
Crystal Forms
Compoundfrom
Crystalline Screening
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103
fromChemistry
Amorphous Screening
Minimum Maximum MeanAgglomeration
tendencyCrystal habit
Length(μm) 23.1 54.6 36.28No Sticks
Width (μm) 8 12.18 10.18
Powder properties
• Powder properties are measured using USP bulk density apparatus andangle of repose is calculated by funnel method
• Bulk density
• Tapped density
• Hausner ratio
Flow Property Angle of repose Carr’s Index Hausner Ratio
Excellent 25-30 <10 1.0-1.11
Good 31-35 11.0-15.0 1.12-1.18
Fair 36-40 16-20 1.19-1.25
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• Hausner ratio
• Angle of repose
Parameters Doxofylline Gabapentin Nitazoxanide Nimesulide
Bulk density 0.428 0.384 0.468 0.405
Tapped density 0.6 0.583 0.652 0.625
Carr's index 28.66 34.69 28.22 35.2
Hausner Ratio 1.4 1.529 1.39 1.541
Angle of repose 45 40.7 35.52 36.31
104
Passable 41-45 21-25 1.26-1.34
Poor 46-55 26-31 1.35-1.45
Very Poor 56-65 32-37 1.46-1.59
Extremely Poor >66 >38 >1.6
Compatibility studies
Analysis of mixture was carried out
at regular intervals
Physical mixture of drug excipient ratio was taken whichRepresents to final formulation
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Physical Changes(by DSC, IR etc.,)
Chemical Changes(by HPLC)
Compatible / Incompatible
105
Solubility & Stability Studies
• Solubility
• Solubility in different pH buffers
• UV profile
• Thermal properties (DSC & TGA)
• Solution state stability
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• Hydrolysis (water, pH buffers 2, 4, 6 and 8)
• Acid stress condition (0.01N HCl, 0.1N HCl)
• Oxidation (peroxide oxidation at pH 2 and 8)
• Solid state stability
• Accelerated (40°C/75% RH)
• Thermal condition (60°C and 80°C)
• Photo stability (UV, Sun light)
106
Formulations for Preclinical studies
• Vehicle selection and exclusion for PK and PD Studies
• High dose formulations for early Tox studies
• Solutions and Suspensions formulations for Rodent and Non rodents
• Solubilization
• Complexation
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• Complexation
• Enabled formulations
• Dose formulation analysis
• Formulation stability evaluation
107
Vehicle selection & formulation preparation
Vehicle selection&
Formulation support
PhysicochemicalEvaluation
Pharmacokinetic Pharmacology studiesToxicology studies
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108108
Pharmacokineticstudies
Pharmacology studies(Activity / Efficacy)
Toxicology studies
BiopharmaceuticalEvaluation
Developability Assessment
108
Vehicle selection decision tree in preclinical studies
Stage 3
Stage 2
HEC,CMC & Methyl cellulose
Stage 1
Water , PBS, Pharmasolve (i.v.)Aqueous Vehicles
Suspending agents (p.o)
Complexing agents (p.o & i.v)
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Stage 6
Labrafil, Capmul, Caprol PGE, MCM
Stage 5
Tween 80,Cremophore EL, Alkamul EL
Stage 4
PEG 400 & Propylene glycol
HPBCD ,Captisol
109
Complexing agents (p.o & i.v)
Co-solvents (p.o & i.v)
Lipids
Surfactants (p.o)
Biopharmaceutics
• Vehicle selection and exclusion for in-vivo studies
• Formulations for PK, Efficacy and Tox studies
• Study of morphological changes in slurry formulations of Tox studies
• Study of affect of stress like milling etc., on NCE during formulations
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• Biopharmaceutics characterization of NCE’s
• Biopharmaceutics Risk Assessment
• Solubility vs Permeability Evaluations
• In situ single pass intestinal perfusion studies
• Selection of dosage form for development
110
Biopharmaceutics Risk Assessment
• Solubility in SGF & SIF
• Solubility in FaSSIF & FeSSIF fluids
• pH Solubility Profile
• Stability in GI fluids
• Chemical stability
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• Chemical stability
• Effect of food on bioavailability
• Solubility estimation
• Permeability evaluation
• Absorption issues in PK studies
Solubility and Permeability affects oral absorption and bioavailability
111
Drug Absorption Studies
• In situ single/double/triple pass method
• In situ closed loop method
• Everted gut sac method
• Intestinal rings method
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• Intestinal diffusion method
• Segmental dependent perfusion studies
• pH dependent perfusion studies
• Mesentric blood sampling method
• Pre hepatic & Post hepatic blood sampling method for liver extraction
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Biopharmaceutics Evaluation
Solid Solid Solid
X SolidX Solid X Solid
Dissolution ratelimited
Permeabilitylimited
Solubilitylimited
Solubility
Dissolution rate
Drug molecules Body
Tablet Suspension Solution I.V. injection
MRT disint MRT diss MRT abs MRT iv
Disintegration Dissolution Absorption Elimination
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X SoluX Solu
DissolvedDissolved
Absorbed
Dissolved
Absorbed
Sensor
X abs
Absorbed
X absX abs
Permeation rateX Solu
MAT
MRT po
• Dosage form selection for Development
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Early Formulations
“API – Focused”
Characterize and Monitor API Properties to Enable Strong Drug Product Development
• Physical
• Chemical
• Mechanical
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• Mechanical
• Biopharmaceutical
Minimal Drug-Product Development
• “Fit-for-use” formulation that meets the needs for early clinical evaluation
• “Fit-for-use” analytical methods that meets compliance needs appropriate for
early stage
Exploratory Formulation Approach
Use the simplest possible formulation consistent with the clinical goalof the early phase studies
• API only: “Powder in a bottle or capsule approach”
“Exploratory formulations”
Early Formulations
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• “Exploratory formulations”
The goal is to achieve adequate exposure from a “dosage form” ofadequate quality.
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Oral formulations with varying degrees of complexity
API in Bottle - solution preparation in clinic
API in Capsule
API in Bottle - suspension prep in clinic
Solution
APIonly
Increasing Complexity
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Solution
Aqueous suspension
Simple Formulated Capsule
Simple Formulated Tablet
Solubilized formulation
Solid dispersion
Modulated release
ExploratoryFormulations
Phase 1 & Phase 2A Formulations
• Formulation for Phase-I & II studies
• Dosage form Selection
• Immediate Release Formulations
• Solution, Suspensions
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• API-in-Capsules and Tablets
• Taste masking formulations
• Solid Dispersions
• Clinical supplies manufacturing
• eCTD IND documentation
Novel Formulations
• SEDDS / SMEDDS Formulations
• Nano Suspension Formulations
• Microemulsion Formulation
• Micro Capsules
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• Nano Crystal formulation
• Liposomes
• Extended Release Formulations
• Timed Release Formulations
Formulation Analysis Support
• Analytical support to drug product development, clinical supplies
manufacturing and stability studies
• State of art HPLC instrumentation lab with six Agilent HPLCs having various
detectors of UV, PDA, RI and ELSD
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• Dissolution testing systems equipped with 12+2 and 6+2 stations
• Disintegration tester, Hardness tester, Friabilator, KF titrator and pH meters
• Stability chambers for accelerated, longterm and refrigerated conditions.
• Stress studies of drug products
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Suven Discovery Research
Discovery Toxicology Services
Aggressive Timelines together with high Scientific Quality
Decision making for NCE progression
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Decision making for NCE progression
Suven Life Sciences introduces
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Wherein Toxicity studies are conducted on new chemical entities in the early stage of
discovery. Data are generated in extremely rapid timelines at the same time with highest
quality and scientific integrity for your decision making of molecule progression.
4-Day Tox in 14 Days
7-Day Tox in 17 Days
Toxicity studies Acute toxicity studies in rodents
4-Day / 7-Day repeat dose discovery tox studies in rodents
Repeat dose (14 / 28 days) toxicity studies in rodents
In vivo & In vitro Genotoxicity studies
Bacterial reverse mutation test (Ames Test)
Discovery Toxicology Capabilities
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In vitro chromosomal aberration test
In vivo and in vitro micronucleus test
In vivo chromosomal aberration test
Clinical Pathology Services
Histopathology Services
Toxicokinetic studies
Formulation analysis
Bio-analysis
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Animal Facility
Vivarium spreading over 6000 sft
Double corridor system
HEPA filtered 100% clean air supply
Individually ventilated cages
Necropsy room
State of art instruments
Discovery Toxicology - Facilities
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Clinical Pathology Lab
Histopathology Lab
Documentation Room
Well Equipped Genotox Lab
Spacious Archives
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SOPs
Planning and initiation of study, live phase of toxicity study, necropsy and organ collection, clinical and
histopathology, training of personnel, archiving of study data and specimens, equipments
Formats
Logbooks
Individual training records
Equipment records
Discovery Toxicology – Good Research Practices
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Equipment records
Equipment ID & List, installation reports, master schedule for calibration, quality controls, responsible person
for each equipment
Test item and chemical records
Record for receipt, storage, utilization & disposal
Quality assurance
Archives
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Advantages with Suven Discovery Toxicology
Aggressive timelines together with high scientific excellence and quality data are critical in
drug discovery. Having years of experience in discovery research, Suven Tox offers quality
results of preclinical toxicity studies. The tox division is well equipped to handle toxicity
studies with rigor timelines to offer results of 4-day tox in 14 days and 7-day tox in 17 days
(from day 1 of treatment to submission of the draft report).
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Expertise in discovery research
Cost effective
Rapid turnaround time with quality results
Flexibility in study conduct as per Sponsor’s requirement
Follow GLP principles
Suven Discovery Tox expertise and contribution to progression of NCEs
Identified phospholipidosis in early stage of discovery by conducting4-day tox
Identified potential platform toxicity
Identified potential back-up compounds with good margin of safety
Discovery Toxicology
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Contributed towards critical decision making in drug discovery of severalPharma and Biotech companies in India, Europe, and USA
Preparation of comprehensive toxicity/safety report for IND filing
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Contacts:
Venkat Jasti
Chairman & CEO
E-mail: [email protected]
Ramakrishna Nirogi
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Ramakrishna Nirogi
Vice President, Discovery Research
E-mail: [email protected]
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