Early safety assessment of drug candidates:

Utility of in vitro pharmacological profiling

Laszlo Urban, Steven Whitebread, Jacques Hamon, Gul

Erdemli , Eugen Lounkine & Patrick Mueller1

Preclinical Safety Profiling & PreClinical Safety, NIBR

Cambridge

F.D.A. Issues Strictest Warning on Diabetes Drugs

Safety Risks: Mitigation in support of risk

management

• Moving from idiosyncratic towards target based ADRs:– Links between chemical structure – target – molecular mechanism –disease (human)

– Data analysis and interpretation based on learning processes: modeling & simulation;

SAR, predictive in silico tools to cluster in vitro–in vivo–clinical data with structures

• Introduction of parallel, iterative optimization of potency/

efficacy, drug-like properties, including ADRs

Toxicities leading to drug withdrawal from the US market (1976-2005)

Wilke RA et al. Nature Revs Drug Discovery 6 (2007)

1

2

3

4

5

6

Hepatotoxicity (6)

Nephrotoxicity (2)

Cardiotoxicity (2)

TdP (6)

Rhabdomyolysis (2)

Other (10)

Risk mitigation and risk management

Chemical

series / scaffold

Single

molecule

Risk

management

Risk

mitigation

Efficacy

Safety

EARLY and SHORT iterative cyclesTo maximize safety profile of lead molecules

LEAD STAGE

ACTIVELY optimizing safety

profile is of utmost importance

LATER STAGES

Safety-related activities largely

restricted to recognition,

assessment and management

of adverse effects.

Tailored mitigation strategies:

Off-target safety profiling

EARLY and SHORT

iterative cycles

Test

Evaluate

Design • MinimizedAttrition atlater stages

• MaximizedSafetyprofile

What are the Chances of Success?Pergolide Ropinirole

What are the Chances of Success?Pergolide

Withdrawn:

Valvular heart disease

Ropinirole

GPCRs 38

Transporters 4

Ion channels 8Enzymes 5

Nuclear receptors 5

Proteases 4

Kinases 8

In vitro Safety Profiling Assay Panels: What & Why to Include?

Primary assay panel (24 targets)

Full assay panel (72 targets)GPCRs 12

Transporters 3

Ion channels 1

Enzymes 4

Nuclear receptors 2

• Targets associated with serious ADRs & high hit rates

• Biochemical & functional cellular assays

• + assays with lower hit rates• + additional functional cellular

assays• + kinases & proteases

In vitro arsenal for early safety assessment Human target based assays

Clinical

Development

Lead

identification

Lead

Selection

Lead

Optimization

CCPre-clinical

Development

Early identification of

potential liabilities &

promiscuity.

Assessment based

on chemical series

Full characterization

before administration in

humans – Mechanistic

understanding

Design liability out

using SAR on

individual targets

In vitro safety pharmacology panelsWhen and how are they used ?

ManageMitigate

Primary Full Primary Full, Selectivity

Individual assays on request

Determining association with clinical ADRs

9

Selecting drugs,

adverse effects, and

subpopulationsStrong signal

identification

Distribution in

subpopulations

Compound

structure and

names

Selected adverse

reactions

Observed number of reports

Exp

ecte

d n

um

be

r o

f re

po

rts

Adolescents

Females

Determining association with clinical ADRs

10

Selecting drugs,

adverse effects, and

subpopulationsStrong signal

identification

Distribution in

subpopulations

Compound

structure and

names

Selected adverse

reactions

Observed number of reports

Exp

ecte

d n

um

be

r o

f re

po

rts

Adolescents

Females

O O

NHS

O

O

OH

Cardiac ADRs of marketed PPAR agonists

Rosiglitazone Troglitazone Pioglitazone

Common features

Same target and same indication

All drugs are in the same chemical class

All generate cardiovascular ADRs , however to different extent

All are “clean” of off-target cardiovascular effects

Rosiglitazone & Pioglitazone: “Clean” off-target profile....

AERS reports:

Cardiac disorders (leading)Eye disorders

GI disorders

Hepatobiliary disorders

Metabolism and nutrition

Musculoskeletal and connective tissue

Nervous system (cerebrovascular)

Psychiatric

Renal and urinary

Respiratory, thoracic

Social

Vascular

....however, the clinical

picture is different:

ADR profile of competitor: same indication/different target Sitagliptin (Januvia)

Cardiac Renal/urinary (no cancer) Hepatobiliary

No reports No reports No reports

Skin disorders Pancreatitis

Target: DPP4 (inhibitor) Indication: Type II Diabetes

Targets Potential ADRs Clinical evidence

• hERG inhib. Ventricular arrhythmia Terfenadine, astemizole, cisapride

• 5HT2B ag. Cardiac valvulopathy Fenfluramine, Pergolide

• PDE4 inhib. Emesis, vasculitis- PDI 747

• H1 antag Sedation Antihistamines

• M2 antag Bronchoconstriction Rapacuronium

• NMDA Channel inhib. Hallucination PCP (Phencyclidine)

• Dopamine D1 ag. Dyskinesia Levodopa

• EGFR inhib. Acneiform RashGefitinib, Erlotinib, Lapatinib,

Panitumumab (Black box)

Compounds withdrawn from market or failed during clinical development due to safety

Examples of targets associated with clinical ADRs

In vitro profiling is informative but limited.....(The entire relevant target space cannot be profiled in vitro)

....in silico approaches can predict potential off-targets based on chemical similarity to known ligands

Profiling panels Potential (off)-targets

Hopkins & Groom, Nature Rev. Drug

Disc. 1, 727

in vitro in silico

15

Compound sets

Drugs with adverse effect

Compounds binding

target XY

Linking targets and ADRs using chemical featuresStatistical models as a “common language”

Structural

features

Score

Score

Score

SScore

Scheiber et al. 2009, J Med Chem 52, p. 3103

Shared chemical features

How to interpret off-target data?Introduction of Off-target coverage

IC50 = 1µM Cmax = 1µM

Cmax = 10µM

Cmax = 0.1µM

10x coverage(ie. exposure 10x above IC50

= 0.1x MARGIN)

1x coverage(ie. exposure just at IC50

= 1x margin)

0.1x coverage(ie. exposure 10x below IC50

= 10x MARGIN)

10x

10x

Case study: Balanced early assessment of cardiosafety

From hERG inhibition to cardiac off-target panel

!

PDE3 is expressed in cardiomyocytes & platelets

Increases inotropy in the heart

Indication: Cardiotonic in heart failure (Milrinone) - Thrombocytopenia (Anagrelide)

ADR: PDE3 inhibitors increase mortality in ~10% of chronic heart failure patients

PDE3: An off-target associated with serious ADR in CHF

FPC

[nM]

hPDE3 IC50

[nM]

Fractional Coverage

(Cther, free / IC50)

Milrinone (Primacor® – discontinued) 284 290 0.98

Enoximone (Perfan® - ↑ mortality) 846 1300 0.65

Inamrinone (Inocor® – discontinued) 8974 15900 0.56

» Conclusion: For clinically significant PDE3 inhibition free therapeutic plasma

concentrations at or even below the hPDE3 IC50 are sufficient.

» Fractional target coverage of marketed drugs can determine likelihood of off-

target specific effects of drug candidates at TFPC (AUC or Cmax, free)

c-Met inhibitors show strong PDE3 inhibition – myocyte necrosis

PF-04254644

PDE3: 0.2µM°

°AGUIRRE, SA. Et al, Toxicologic Pathology, 2010

N

N NN

N

N

N Chiral

*Fibrosis of the myocardial interventricular

septum

Control rat myocardium

7 day treatment with PF

10

(Off-)target coverageExample using marketed PDE3 inhibitors

Human PDE3 IC50

[µM]

Total drug:

Cth

[µM]

PPB

[%]

Free drug:

Cth

[µM]

Total drug:

Coverage

(Cmax ÷ IC50)

Free drug:

Coverage

(Cmax ÷ IC50)

Inamrinone(US: Inocor® – discontinued)

15.9 12.8 30 8.97 0.81 0.56

Milrinone(US: Primacor® – discontinued)

0.29 0.95 70 0.28 3.3 0.98

Enoximone(EU: Perfan® - increased mortality)

1.88.9

(Cmax)85

1.3

(Cmax)4.9 0.72

CPD X(terminated)

0.280.722

(Cmax)94

0.043

(Cmax)2.6 0.15

» Conclusion: For clinically significant PDE3 inhibition plasma concentrations just at the hPDE3 IC50 are sufficient.

» Target coverage of marketed drugs can be used to interpret PK/PD of off-target-specific effects

• Consider (pharmacological) plasma concentrations (Cmax) of drug candidates showing SPECIFIC off-target activity at 1° target of marketed drugs.

Indicated for the short-term

intravenous treatment of patients

with acute decompensated heart

failure (inotropic vasodilator).

(Off-)target coverageExample using marketed PDE3 inhibitors

CPD X dog

Dog, upon single dose:

• Inotropic effect (LVEF ↑)

• Chronotropic effect (HR ↑)

• Endocardial hemorrhage

Recommended activity flowchart for off-target

mitigation and management

THR < 30%

Metabolites > 10%

abundance of parent

Cold Met-ID (MAP) Plasma protein binding

(rodent, NON-RODENT, human)

kinase panel

(for kinase inhibitors

only)Full panel & hERG

FOR METABOLITES

hERG RLB

Primary panel

CANDIDATE DeclarationPrimary panel

ADME assays

Select panel

Lead Nomination

SELECTIVITY determination

Series-specific risk assessmt.

(refer to decision strat. flow chart

& Profiling Wiki)COVERAGE determination

(refer to decision strat. flow chart)

SAFETY INDEX

assessment

(based on in vivo tox / safety

pharmacology studies)

Green lines depict "yes", red lines depict "no"; Diamonds: decisions; Boxes: activities

#1: special request for assay in case off-target only in Full panel

Chemical series

(Risk mitigation)Single molecule

(Risk management)

SAR-based off-target

mitigation

"Candidate Selection"

Full panel

ADME assays

Example of early assessment and mitigation: The chemistry viewCorrelation of target LipE with major off-target activities

High LipE predicts for favorable hERG, solubility and DDI profile

Bottom 8 (LipE <5)

100% hERG active

75% insoluble

50% CYP3A4 active

Top 8 (LipE >8)

38% hERG active

0% insoluble

0% CYP3A4 active

Middle 13 (LipE 5-8)

77% hERG active

15% insoluble

23% CYP3A4 active

Target LipE

Chemistry considerations for successful mitigation

To improve chances of success and speed of lead optimization, focus on PROPERTIES of leads and add potency EFFICIENTLY

Be proactive in selecting quality – potency is the easiest thing to improve!

Hypothesis driven reduction of liabilities must include increased emphasis on physicochemical relationships

Potency, permeability and metabolic stability all correlated to physicochemical properties

Don’t improve solubility by adding a basic amine that has hERGaffinity - which has to be made less basic and then

increases metabolism,

reduces permeability,

decreases oral exposure,

increases molecular weight

results in metabolite issues that

requires EXHAUSTIVE preclinical profiling resource

Critical evaluation of early ADR

mitigation

• Technical considerations– Poorly optimized physicochemical properties, HT automation

might compromise data quality

– Exposure is not adequate

• Tactical considerations– Single target vs. pathway considerations

– Preference of management vs. mitigation

– Ignorance of integrative assessment

• Strategic considerations– Drug repositioning requires safety assessment adjusted to the

new indication

– Interpretation of complex data could lead to wrong conclusions

– Overestimation of predictive value – importance of follow-ups

Conclusions

• Early integrated risk assessment supports mitigation of

predicted clinical toxicities, ADRs

• Correct prediction depends on the knowledge of the

target associated with an ADR and the calculated TI

• Knowledge of the chemical structural and biological

target environment enables the development of in silico

technologies to support safety assessment

• Risk/Benefit evaluation is essential for clinical candidate

selection when off-target liabilities persist

• Medicinal chemistry can drive off-target mitigation by

linking physicochemical features or off-target SAR with

biological performance

Literature and links

Whitebread S, Hamon J, Bojanic D, Urban L (2005) In vitro Safety Pharmacology profiling: An

essential Tool for Drug Development. Drug Discovery Today; 10(21):1421-1433.

http://dx.doi.org/10.1016/S1359-6446(05)03632-9

Hamon J, Whitebread S (2009) In vitro safety pharmacology profiling: An important Tool to Decrease

Attrition. In: Faller B, Urban L (eds). Hit and Lead Profiling. Weinheim: Wiley VCH, p. 273-295.

http://share.nibr.novartis.intra/cpc/psp/Shared%20Documents/Literature/Hamon%20and%20Whitebre

ad%202009%20book%20chapter%20in%20Hit%20and%20Lead%20Profiling.PDF

Hamon J, Whitebread S, Techer-Etienne V, Le Coq H, Azzaoui K, Urban L (2009) In vitro safety

pharmacology profiling: What else beyond hERG? Future Med Chem; 1(4):645-665.

http://www.future-science.com/doi/full/10.4155/fmc.09.51. Erratum in: http://www.future-

science.com/doi/full/10.4155/fmc.09.128

Scheiber J, Jenkins JL, Sukuru SC, Bender A, Mikhailov D, Milik M, Azzaoui K, Whitebread S,

Hamon J, Urban L, Glick M, Davies JW (2009) Mapping Adverse Drug Reactions in Chemical

Space. J Med Chem; 52(9):3103-3107. http://dx.doi.org/10.1021/jm801546k

Azzaoui K, Hamon J, Faller B, Whitebread S, Jacoby E, Bender A, Jenkins J, Urban L (2007)

Modeling Promiscuity Based on in vitro Safety Pharmacology Profiling Data. ChemMedChem; 2(6):

874-880. http://dx.doi.org/10.1002/cmdc.200700036

Bender A, Jenkins J, Glick M, Davies J, Azzaoui K, Hamon J, Urban L, Whitebread S (2007)

Analysis of Pharmacology Data and the Prediction of Adverse Drug Reactions and Off-Target Effect

From Chemical Structure. ChemMedChem; 2(6): 861-873. http://dx.doi.org/10.1002/cmdc.200700026

28 | Off-Target Workshop | 2011 | Business Use Only

Steven Whitebread Dejan Bojanic

Jacques Hamon Michael Shultz

Dmitri Mikhailov Bob Pearlstein

Gul Erdemli Clayton Springer

Dejan Bojanic Bill Kluwe

Patrick Mueller PeterK Hoffmann

Doriano Fabbro

Kamal Azzaoui

Sepp Scheiber

Jeremy Jenkins

Acknowledgement