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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

Company ConfidentialCopyright © 2016 Suven Life Sciences Limited

22

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


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


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


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


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


77

Suven Integrated Drug Discovery


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


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)


1010

SUVN-502 (5-HT6 Antagonist) 2015 2020



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


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


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


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


16

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)


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


18

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


19


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



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


21

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



22

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


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



24

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



25

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)



26

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



27

Access to printed literature and databases


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)


(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



29

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


• 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)


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

3131

In-vitro Biology


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


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


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


Functional Cell-based Assays (Signal Transduction)

3535

Target Class

Ion Channels


Patch clamp assay using automated electrophysiology instrument

Patchliner from Nanion, for ligand gated ion channel: P2X7 as well as


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


Functional uptake assays

Nuclear Receptors

Reporter gene based assays

Target Class


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


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


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


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



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


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


4141

Plate 1 - Plate 1 - Plate 2 -

Plate 2 Plate 3 Plate 3

Day 1 1.1 1.0 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)



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


4343

0

25

50

75

100Full Agonist

Antagonist

Partial Agonist

Inverse Agonist

Inactive Compound

%A

cti

vit

y

Cell Based Assay Validation


-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


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


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


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


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


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


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




Nav 1.5 I-V Protocol5 ms

-30 mV

40 ms

5 ms

-100 mV-100 mV

49


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


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


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


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


S.No CompoundObtained IC50 value

(µM)Reported IC50 value

(µM)

1 Tetracaine 1.3 1.7

2 Lidocaine 258 260


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


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


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


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


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


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


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


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)


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


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


• 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


• 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


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


• 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


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


6565

Assessment: - / + Verapamil(P-gp Inhibitor)

Inhibitor Conc.: 10 µM

Incubation: 1 hour


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)


6666

P-gp Substrate: Digoxin

Incubation: 1 hour


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


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



Positive control : Verapamil

Results : IC50 shift (fold change)

0.001 0.01 0.1 1 10

0

40

80

120


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


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)


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


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)


•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


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


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)


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


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 &centrifugation

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


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


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


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


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


AcclimatizationStereotaxic

surgery


AcclimatizationStereotaxic

surgery


Recovery

Typical experimental protocol:


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



2-5 days


1-2 h

Probe implantation

~ 16 h before

1-2 h






4-5 days0.5 - 0.75 h



2-5 days


1-2 h

Probe implantation

~ 16 h before

1-2 h






4-5 days0.5 - 0.75 h



2-5 days


1-2 h

Probe implantation

~ 16 h before

1-2 h






Recovery

Experimental Day

In-vivo Brain Microdialysis Facility


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


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)


-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


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


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


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


88

Modafinil: Dose Dependent Increase in Wakefulness

Results Generated at Suven Edger and Seidel 1997


*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

&



Safety Pharmacology

Therapeutic areas

• Cognition

• Depression

• Psychosis


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)


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


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)


94

Rodent Models of Anxiety

• Elevated Plus Maze

• Vogel conflict test

• Hole board

• Novelty induced hypophagia


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


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


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


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


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


Preformulation, Biopharmaceutics and Early

Formulations


Preformulation

Screening

• Salt and co-crystal screening

• Crystallization screening

• Polymorph screening

Selection

• Hygroscopicity


• 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


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


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


• 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


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


• 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


• Complexation

• Enabled formulations

• Dose formulation analysis

• Formulation stability evaluation

107

Vehicle selection & formulation preparation

Vehicle selection&

Formulation support

PhysicochemicalEvaluation

Pharmacokinetic Pharmacology studiesToxicology studies


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)


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


• 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




• 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


• 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

112

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


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

113

Early Formulations

“API – Focused”

Characterize and Monitor API Properties to Enable Strong Drug Product Development

• Physical

• Chemical

• Mechanical


114

• 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


115

• “Exploratory formulations”

The goal is to achieve adequate exposure from a “dosage form” ofadequate quality.

115

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


116

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


117

• 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


118

• 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


• 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

119


Discovery Toxicology Services

Aggressive Timelines together with high Scientific Quality

Decision making for NCE progression


Decision making for NCE progression

Suven Life Sciences introduces


121

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


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

122122

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


Clinical Pathology Lab

Histopathology Lab

Documentation Room

Well Equipped Genotox Lab

Spacious Archives

123

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


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

124

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).


125

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


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

126126

Contacts:

Venkat Jasti

Chairman & CEO

E-mail: [email protected]

Ramakrishna Nirogi


127

Ramakrishna Nirogi

Vice President, Discovery Research

E-mail: [email protected]

drug discovery collaboration - suven · drug discovery collaboration discovery research suven life...

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