In Vitro Safety Profiling During Lead Optimisation

Murray BrownManager, Data Interpretation and Business ProcessScreening and Compound Profiling

Drug Discovery Process

Basic Research

Lead Discovery

Preclinical Development

Clinical Development

FDA Filing

Target Selection

CandidateSelection

INDfiling

NDAfiling

3 1 6 1.5Years

Once a candidate is selected the pharmacological properties of the molecule are fixed

High Throughput Screening approaches apply within Lead Discovery to:– Reduce the cycle time from target selection to candidate selection– Increase the number of candidates per program– Increase the quality of candidates selected

Strategies to Improve the Quality of Lead and Candidate Generation

Robust screening infrastructure– Automation quality control– Statistical methods for hit selection

Quality “drug-like” compound libraries– Low molecular weight and cLogP

New Screening Paradigms– Fragment Screening– Encoded Library technology

Relevant assay biology– Native cellular systems– Biophysical Screening

Pharmacology of lead series– Ability to assess on-target and off-target activity

Causes of Attrition – Safety & Efficacy

EARLY LATEStrategic 243 18

Resources 19 2Efficacy 229 25Safety 457 8

Technical 93 0Unknow n 42 3

Total 1083 56

Termination ReasonsEARLY LATE

Strategic 29 3Resources 0 1

Eff icacy 25 2Safety 65 4

Technical 7 0Unknow n 5 0

Total 131 10

GSK

KMR Group R&D General Metrics Study Final Report July 1, 2010

KMR TerminologyEarly Dev: Preclin up to Ph III StartLate Dev: Start Ph III to Launch

Reasons For NME Termination By Stage2005-2009 Industry Portrait

Addressing the Challenge of Drug Safety in Early Discovery

Vision• To increase candidate quality and probability of clinical success

through the identification and mitigation of safety hazards in chemical series prior to candidate selection

Strategy• Using high throughput techniques, implement panels of assays for use

in early discovery that identify likely safety liability in hits, leads and candidates

Attrition Reduction Activities in GSK

Strategic Intent– Implement assays during H2C to identify and manage compound series

likely to cause toxicity in preclinical or clinical studies

Assays configured during 2009-2010 with capacity to screen 1200 hits, leads and candidates/year

eXP Cardiotoxicity Hepatotoxicity Genotoxicity

A bi-weekly panel of 50 molecular target assays with known

clinical liability

A panel of IonChannel assaysenabled by high

throughputelectrophysiology

GreenScreen assay licensed to

identifyGenotoxicants

Cell Health assaydetects 70% of

known hepatotoxicants

eXP (enhanced cross screen panel)

Cardiac/vascular:KCNQ1/minKL-type CaVR4M2Adenosine 2aβ2 adrenergicα1b adrenergicα2a adrenergic

Gastro-intestinal:5-HT3PDE4BGSK3bPI3k

Neuromuscular:a1bgd nAChR

Neuronal:a1b3g2 GABASERTDATNETM1MAO-BμopioidκopioidDopamine 1Dopamine 2Histamine 1NK1

Immunological:LCKCannabinoid 2

Nav1.5Kv1.5

5-HT1B5-HT2A5-HT2C

COX2V1a

Hepatic:OATP1B1PXR

11 point dose response curveFunctional/activity assays

Interpharma Safety Profiling

Knowledge-Sharing - Secondary Pharmacology Screening

AstraZenecaJoanne Bowes

NovartisSteven

WhitebreadJacques Hamon

PfizerGareth Waldron

GlaxoSmithKlineAndrew Brown

Arun Sridhar• What Targets?• What Technologies?• What Process?• Shared Case Studies

PharmaxisWolfgang Jarolimek

2009-present Outcomes:• Poster at SPS meeting:

• Rational design of an in vitro safety profiling panel to reduce undesired secondary pharmacology of drug candidates. Steven Whitebread, Joanne Bowes, Andrew Brown, Jacques Hamon, Wolfgang G. Jarolimek, Gareth Waldron and Arun Sridhar Journal of Pharmacological and Toxicological Methods; 64(1), July-August 2011, Page e18.

• Manuscript - in preparation

CNS(GABAA)

(Barracuda)

Cardiac Ion Channels

High Throughput Electrophysiology Assays to predict Functional Cardiotoxicity

See posters by Metul Patel et al on hERG IonWorks® population patch clamp and Joanna Taylor et al on stem cell derived Cardiomyocytes

hERGNaV1.5CaV1.2KV1.5

KCNQ1(IonWorks® and PatchXpress®)

Genetic Toxicology: The GreenScreen Assay

Genotoxic agents either react directly with DNA or disrupt the cellular apparatus which regulate the fidelity of the genome

Regulations require a minimum of 3 GLP tests:– a test for gene mutation in bacteria (for

example, the Ames test),– a test for chromosomal aberrations in vitro or the

MLA– an in vivo test for chromosomal damage in

rodent haematopoietic cells.

The GADD45a gene is upregulated in response to DNA damage in the GreenScreen assay (Gentronix)

Reporter transfected into Human p53 competent lymphoblastoid cells

15% of pre-candidates are terminated due to Genotoxicity

GSK has licensed the GreenScreen HC genotoxicity assay for profiling of hits, leads and candidates

Early stage (HitID and SoC) identification of GreenScreen HC actives enable LO chemistry to focus on molecules without this liability

BlueScreen HC has now been implemented to allow higher throughput

31 of 34 known genotoxic agents induced GADD45a

reporter

41 of 41 non-genotoxic agents did not induce the GADD45a reporter

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Genetic Toxicology: GreenScreen Assay

See poster by Kate Simpson et al on BlueScreen assay validation

Cell Health Assay to Detect Hepatotoxicants

Drug-Induced Liver Injury (DILI) is a recurrent problem in pharmaceutical developmentIdiosyncratic hepatotoxicity is one of the leading causes of drug withdrawals, non-approvals and warnings (Kaplovitz 2005)Can we identify hepatotoxicants prior to candidate selection and reduce attrition due to pre-clinical or clinical hepatotoxicity?

• 96well assay using HepG2 (Human liver carcinoma) cells

GSK Cell Health Assay Description

Measures cytotoxic effect of compounds in human liver-derived HepG2 cells in 384-well format3 parameter automated imaging assay Using fluorescent staining, the key parameters measured in this assay are : -· Nuclear Condensation

Hoechst 33342Cell permeable DNA binding dye

· Mitochondrial membrane potentialTMRM Accumulates in healthy Mitochondria but leaks out when mitochondrial membrane potential is discharged

· Membrane permeabilityTOTO-3Cell membrane impermeable nuclear stain

Impaired mitochondrial function is an early indicator of cell injury whereas loss of membrane integrity and changes in nuclear morphology are indicators of acute or late stage cytotoxicity. Quantification is carried out using the InCell

Example images

NucleiMitochondrial

PotentialMembrane

Permeability

Negative Control

Postive Control

Typical dose response curves

Correlation between Cell Health readouts

Compounds usually show very similar IC50s in all 3 readouts, but there are exceptions where toxicity is specific to a single readout

Cell Health Assay to Detect Hepatotoxicants

The Cell Health assay for profiling of hits, leads and candidates

Early stage elimination of Cell Health actives enable LO chemistry to focus on molecules without this liability

Concentration5E-7 1E-6 5E-6 1E-5 5E-5 0... 0.0... 5E-7 1E-6 5E-6 1E-5 5E-5 0... 0.0... 5E-7 1E-6 5E-6 1E-5 5E-5 0... 0....

-50

-25

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Compound A failed due to liver toxicity.

No reported hepatotoxicity forCompounds B and C.

A CB

Negative compounds 28/28Human and rat hepatotoxicants 29/30Cytotoxicants 4/4Idiosyncratic human hepatotoxicants 3/18

Frequency of toxicity in Cell Health for marketed drugs and failed clinical candidates

Drug FC0%

10%

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40%

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59Failed Candidates twice as likely to be active in Cell Health than marketed drugs

Significant proportion of marketed drugs show toxicity in Cell Health

Drugs FailedDevelopmentCandidates

Active in Cell Health

Inactive in Cell Health

Toxic compounds? Toxic dose

Alle Ding' sind Gift, und nichts ohn' Gift; allein die Dosis macht, daß ein Ding kein Gift ist. "All things are poison and nothing is without poison, only the dose permits something not to be poisonous."

Phillippus Aureolus Theophrastus Bombastus von Hohenheim (1493-1541)

Paracelsus

Cell Health cytotoxicity vs normal and toxic exposure levels

Cell Health pIC50100 uM 10 uM

Exp

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Therapeutic or ‘normal’ blood concn

Toxic blood concn

Therapeutic and toxic blood concentrations of more than 800 drugs and other xenobiotics M. Schulz, A. Schmoldt Pharmazie 58(7) 2003 447-474 http://fscimage.fishersci.com/webimages_FSC/downloads/winek.pdf

10 nM

100 uM

1 uM

What drives cytotoxicity?i) Physchem properties - clogP

What drives cytotoxicity?ii) Physchem properties – rotatable bonds

What drives cytotoxicity?iii) chemical series

Cell Health cytotoxicity by chemical cluster

clogP distribution for each cluster

clogP vs Cell Health pIC50

Cel

l Hea

lth p

IC50

Cell Health Cytotoxicity is SAR-able in lead optimisation

Physicochemical properties can be manipulated to reduce likelihood of Cell Health cytotoxicity

– ↓clogP, ↓# aromatic rings, ↓heavy atom count (or ↑ >50!), ↑ heteroatoms, ↓ rotatable bonds

Cell Health cytotoxicity is a feature of chemical series beyond their physicochemical properties (toxicophores)

– Early screening in Cell Health assay at HitID allows selection of series with lower likelihood of cytotoxicity

Cell Health Assay Related to Promiscuity in eXP

x ≤ 0.10 0.10 < x ≤ 0.20 0.20 < x ≤ 0.30 0.30 < x ≤ 0.40 0.40 < x0%

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Fraction assays with pXC50 >5Green = Inactive in Cell HealthRed = Active in Cell Health

In vitro cross screening profile of selected drugs

Assay

Non-promiscuous, highly tolerable, non-toxic in Cell health

a1B a2Cb2

Low promiscuity, mechanism based toxicity detected by eXP, non-toxic in Cell Health

Highly promiscuous, low tolerability, toxic in Cell Health

Moderately promiscuous and highly tolerable, non-toxic in Cell health

CB2 Ag

Attrition Reduction Toolkit: An annotated one-stop-shop for all attrition reduction assays at GSK

Attrition Reduction Toolkit: An annotated one-stop-shop for all attrition reduction assays at GSK

Compound Structure

Profile similarity in eXP

Conclusions

We have implemented a panel of assays in early discovery to assess toxicity hazards in hit and lead seriesAssays are annotated according to likely clinical effectEach assay is not decision making in isolation

– Data enables comparative decisions between chemical series– Activity in multiple assays needs to be considered

Assays can be used to drive SAR Cell Health assay in HepG2 cells concords well with physicochemical properties shown to be important in clinical attrition and clinical in vivo tolerabilityExposure levels are key for toxicology (as well as efficacy) expected dose is important factor to be included in interpretation of early Safety Profiling data

AcknowledgementsSteve Rees (formerly Screening and Compound Profiling)Andrew Brown (Screening & Compound Profiling)Dave Morris (Screening & Compound Profiling)Wolfgang Jarolimek (formerly Screening and Compound Profiling)Joanna Taylor (Screening & Compound Profiling)Kate Simpson (Screening & Compound Profiling)Metul Patel (Screening & Compound Profiling)Rob Jepras (Screening & Compound Profiling)Rob Eagle (Screening & Compound Profiling)Darren Green (Computational & Structural Chemistry)Cerys Lovatt (Safety Assessment)Julie Holder (Safety Assessment/Stem Cells)Nick McMahon (Safety Assessment)Paul Hastwell (Safety Assessment)Patrick Wier (Safety Assessment)Steve Clarke (DMPK)Bob Hertzberg (Screening & Compound Profiling)Many other GSK scientists responsible for generating the assays and data