1. In Vitro Safety Profiling During Lead Optimisation
Murray Brown
Manager, Data Interpretation and Business Process
Screening and Compound Profiling

2. Drug Discovery Process
Target
Selection
Candidate
Selection
IND
filing
NDA
filing
Basic
Research
Lead
Discovery
Preclinical
Development
Clinical
Development
FDA
Filing 3 1 6
1.5
Years
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

3. 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

4. Causes of Attrition – Safety & Efficacy
Reasons For NME Termination By Stage
Industry Portrait
KMR Terminology
Early Dev: Preclin up to Ph III Start
Late Dev: Start Ph III to Launch
KMR Group R&D General Metrics Study Final Report July 1, 2010

5. 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

6. 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 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 Ion
Channel assays
enabled by high
throughput
electrophysiology
GreenScreen
assay licensed to
identify
Genotoxicants
Cell Health assay
detects 70% of
known
hepatotoxicants

7. eXP (enhanced cross screen panel)
Neuronal:
a1b3g2 GABA
SERT
DAT
NET M1
MAO-B
μopioid
κopioid
Dopamine 1
Dopamine 2
Histamine 1
NK1
Cardiac/vascular:
KCNQ1/minK
L-type Ca
VR4 M2
Adenosine 2a
β2 adrenergic
α1b adrenergic
α2a adrenergic
Nav1.5
Kv1.5
5-HT1B
5-HT2A
5-HT2C
COX2
V1a
Hepatic:
OATP1B1
PXR
Gastro-intestinal:
5-HT3
PDE4B
GSK3b
PI3k
Neuromuscular:
a1bgd nAChR
Immunological:
LCK
Cannabinoid 2
11 point dose response curve
Functional/activity assays

8. Interpharma Safety Profiling
Knowledge-Sharing - Secondary Pharmacology Screening
AstraZeneca
Joanne Bowes
GlaxoSmithKline
Andrew Brown
Arun Sridhar
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
2009-present
What Targets?
What Technologies?
What Process?
Shared Case Studies
Pfizer
Gareth Waldron
Novartis
Steven Whitebread
Jacques Hamon
Pharmaxis
Wolfgang Jarolimek

9. (IonWorks® and PatchXpress®)
High Throughput Electrophysiology Assays to predict Functional Cardiotoxicity
Cardiac Ion Channels
CNS
(GABAA)
(Barracuda)
hERG
NaV1.5
CaV1.2
KV1.5
KCNQ1
(IonWorks® and PatchXpress®)
See posters by Metul Patel et al on hERG IonWorks® population patch clamp and Joanna Taylor et al on stem cell derived Cardiomyocytes

10. 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

11. Genetic Toxicology: GreenScreen Assay
15% of pre-candidates are terminated due to Genotoxicity
GSK has licensed the GreenScreen HC genotoxicity assay for profiling of hits, leads and candidates
31 of 34 known genotoxic agents induced GADD45a reporter
41 of 41 non-genotoxic agents did not induce the GADD45a reporter
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
See poster by Kate Simpson et al on BlueScreen assay validation

12. Cell Health Assay to Detect Hepatotoxicants
Drug-Induced Liver Injury (DILI) is a recurrent problem in pharmaceutical development
Idiosyncratic 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

13. GSK Cell Health Assay Description
Measures cytotoxic effect of compounds in human liver-derived HepG2 cells in 384-well format
3 parameter automated imaging assay
Using fluorescent staining, the key parameters measured in this assay are : -
Nuclear Condensation
Hoechst 33342
Cell permeable DNA binding dye
Mitochondrial membrane potential
TMRM
Accumulates in healthy Mitochondria but leaks out when mitochondrial membrane potential is discharged
Membrane permeability
TOTO-3
Cell 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

14. Mitochondrial Potential Membrane Permeability
Example images
Mitochondrial Potential
Membrane Permeability
Nuclei
Negative Control
Postive Control

15. Typical dose response curves

16. 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

17. Cell Health Assay to Detect Hepatotoxicants
The Cell Health assay for profiling of hits, leads and candidates
Negative compounds /28
Human and rat hepatotoxicants 29/30
Cytotoxicants 4/4
Idiosyncratic human hepatotoxicants 3/18
Early stage elimination of Cell Health actives enable LO chemistry to focus on molecules without this liability
Concentration
Compound A failed due to liver toxicity.
No reported hepatotoxicity for
Compounds B and C. A B C
150
125
100 75 50 25
-25
-50
5E-7
1E-6
5E-6
1E-5
5E-5
0...
0.0...
5E-7
1E-6
5E-6
1E-5
5E-5
0...
0.0...
5E-7
1E-6
5E-6
1E-5
5E-5
0...
0....

18. Frequency of toxicity in Cell Health for marketed drugs and failed clinical candidates
Inactive in Cell Health
Failed Candidates twice as likely to be active in Cell Health than marketed drugs
Significant proportion of marketed drugs show toxicity in Cell Health
Active in Cell Health
Drugs
Failed
Development
Candidates

19. Toxic compounds? Toxic dose
Phillippus Aureolus Theophrastus Bombastus von Hohenheim
( )
Paracelsus
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."

20. Cell Health cytotoxicity vs normal and toxic exposure levels
Therapeutic or ‘normal’ blood concn
Toxic blood concn
100 uM
Exposure level
1 uM
10 nM
100 uM
Cell Health pIC50
10 uM
Therapeutic and toxic blood concentrations of more than 800 drugs and other xenobiotics M. Schulz, A. Schmoldt Pharmazie 58(7)

21. What drives cytotoxicity? i) Physchem properties - clogP

22. What drives cytotoxicity? ii) Physchem properties – rotatable bonds

23. What drives cytotoxicity? iii) chemical series
Cell Health cytotoxicity by chemical cluster
clogP distribution for each cluster
clogP vs Cell Health pIC50
Cell Health pIC50

24. 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

25. Cell Health Assay Related to Promiscuity in eXP
Green = Inactive in Cell Health
Red = Active in Cell Health

26. In vitro cross screening profile of selected drugs
Low promiscuity, mechanism based toxicity detected by eXP, non-toxic in Cell Health
Non-promiscuous, highly tolerable, non-toxic in Cell health
a2C
a1B b2
Moderately promiscuous and highly tolerable, non-toxic in Cell health
Highly promiscuous, low tolerability, toxic in Cell Health
CB2 Ag
Assay

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

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

30. Profile similarity in eXP

31. Conclusions
We have implemented a panel of assays in early discovery to assess toxicity hazards in hit and lead series
Assays are annotated according to likely clinical effect
Each 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 tolerability
Exposure levels are key for toxicology (as well as efficacy) expected dose is important factor to be included in interpretation of early Safety Profiling data

32. Acknowledgements
Steve 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