1 Integrating High Throughput Electrophysiology into the Drug ...

Integrating High Throughput
Electrophysiology into the Drug
Discovery Process
Peter Haddock, PhD.
Pfizer CNS Biology
PGRD Michigan
Automated Electrophysiology Users Meeting
Salt Lake City
February 16, 2006

Ion channel drug discovery

Pfizer Global Research and
Development
PGRD Ann Arbor, MI

Ion channels as drug targets
Marketed drugs
Excitable cells
Non-excitable cells
Others
Hypertension
Anxiety
Pain
Epilepsy
Insomnia
Incontinence
Stroke
Diabetes
Autoimmune
Cancer
COPD
Osteoporosis
Allergy/asthma
Ion channel
targets
• 30% of marketed drugs
• 15 of 100 bestselling drugs
• Sales $6-12 billion / year
• 7 superclasses
• ~400 ion channel genes
• Regulate an array of physiological
processes
• Role in numerous disease states
• Great opportunity for drug discovery
– Hampered by lack of high throughput
electrophysiology screening technologies

Historical approaches
Receptor binding
In vitro / in vivo efficacy
Attrition
Structure vs. activity
selectivity
safety
Manual patch
Candidate ion channel modulators

Realizing the full potential of ion
channels as a viable target class
selectivity
Ion channel
Discovery projects
CV safety
Requirement for quantum improvement in the throughput
of electrophysiological screening platforms
SAR
Focused ion channel
chemical libraries

Expanding throughput?

An Integrated Approach to Ion
Channel Drug Discovery
IDEA
Attrition / SAR
Characterization
DRUG
1000’s 100’s 10’s 1’s

Automated Electrophysiology Systems
in Pfizer Drug Discovery
• Assay development, data and experience
– PatchXpress
• hERG
• >18 months
– IonWorks Quattro
• Nav channels
• 4 months
– Manual patch clamp
– FLIPR/VIPR

PatchXpress
hERG
Nav
Cav

Key variables
• Cell type success
– RBL > CHO > HEK
• Cell husbandry and preparation
– Rigorous attention to detail – gentle approach to cell harvest
– 60-70% confluence
– Short trypsinization period
– 30 min rest period prior to use
– Cell tituration prior to chip loading
• Patch settings
– Suction, settling time, Ra optimization etc.
• Compound preparation
– Glass vials and compound plates
– Multiple drug additions
– Length of addition
• Dependent on compound class

Assay development
Manual system
Automated systems
Cell line and current
characterization
Voltage protocols
Signal/noise
Clonal analysis +
Data comparison
with manual system
Cell isolation/solutions
endpoints

PatchXpress validation
Transitioning hERG screening to an automated platform
1. Compound potency
– Manual patch ≡ PatchXpress?
2. Effect of temperature?
– Manual (35 o C) vs. PatchXpress (22 o C)
3. Site-to-site variation
– Global organization

Measuring hERG activity on PX
+30mV
-80mV
Pseudo cardiac action potential
200 pA
Control
dofetilide-sensitive (hERG) current
500 ms
Start
10 µM dofetilide
10µM dofetilide
baseline
A B C
Rising drug concentrations

hERG pharmacology on PX
1.5
100
peak hERG current (nA)
1.0
0.5
Control
[A]
[B]
[C]
% inhibition
80
60
40
20
0.0
[D]
10 µM dofetilide
0
1 10 100 1000
[Compound] (µM)

Compound Potency
manual vs. PatchXpress
Compound
Manual Rig IC 50
RT (nM)
PatchXpress IC 50
RT (nM)
Ratio IC 50
PatchXpress/Manual
Rig (RT)
Bepridil 222 648 2.9
Cisapride 40 57 1.4
Citalopram 2700 2700 1.0
E-4031 24 28 1.2
Flecainide 1600 1600 1.0
Fluoxetine 1100 1200 1.1
Haloperidol 40 40 1.0
Propranolol 14600 11200 0.8
Quinidine 1400 1300 0.9
Risperidone 375 522 1.4
dl-Sotalol 354000 421000 1.2
Verapamil 745 1040 1.4
Mean 1.3
SEM 0.2
n 12
PatchXpress hERG IC 50
(nM, RT)
1000000
100000
10000
1000
100
Line of Identity
10
10 100 1000 10000 100000 1000000
Manual hERG IC 50
(nM, RT)

Effect of temperature
manual vs. PatchXpress
Compound
Manual IC 50 (nM)
(35 o C)
PatchXpress IC 50
(nM) (RT)
Ratio
RT PatchXpress / 35 o C
manual IC 50
1000000
PF# 1 1600 996 0.62
PF# 2 130 158 1.22
PF# 3 240 238 0.99
PF# 4 1500 2301 1.53
PF# 5 1660 2217 1.34
PF# 6 1230 459 0.37
PF# 7 2570 2278 0.89
PF# 8 2200 2513 1.14
PF# 9 3700 1932 0.52
PF# 10 920 831 0.90
Bepridil 26 64 2.46
Cisapride 15.2 28 1.84
Risperidone 75 245 3.27
Quinidine 850 599 0.70
Manual hERG IC 50
(nM, 35 o C)
100000
10000
1000
100
10
Line of Identity
R = 0.97
Mean 1.3
SEM 0.2
10 100 1000 10000 100000 1000000
PX hERG IC 50
(nM, RT)

Site-to
to-site variation
Compound
Pfizer Site #1
PatchXpress IC 50
(nM)
Pfizer site #2
PatchXpress IC 50
(nM)
Cisapride 57 34
Citalopram 2700 1780
E-4031 28 33
Flecainide 1600 902
Quinidine 1300 1030
dl-Sotalol 421000 526000
Pfizer Site #2 hERG IC 50
(nM)
100000
10000
1000
100
line of identity
10
10 100 1000 10000 100000
Pfizer Site #1 hERG IC 50
(nM)

Minimizing Potency Shifts on PX
• Compound solvation
• Plastic vs. glass compound plates
• Single vs. double compound addition
Compound
PX Single Addition
(plastic compound plate)
PX Double Addition
(glass compound plate)
Manual
PX
Single addition / manual
PX
Double addition / manual
1 57 28 15 3.8 1.8
2 120 64 26 4.6 2.5
3 265 158 130 2.0 1.2
4 470 238 240 2.0 1.0
5 512 245 75 6.8 3.3
6 988 459 1230 0.8 0.4
7 1473 599 850 1.7 0.7
8 1865 831 920 2.0 0.9
9 2658 1932 3700 0.7 0.5
10 2704 2513 2200 1.2 1.1
11 2930 2278 2570 1.1 0.9
12 3397 2301 1500 2.3 1.5
13 4057 2217 1660 2.4 1.3
14 4802 996 1600 3.0 0.6
Mean 2.5 1.3
SEM 0.4 0.2

PX hERG Data Analysis
• Manual analysis vs. DataXpress script
• ± rundown correction
#
Excel Analysis
No RD Correction
DX Analysis
No RD Correction
DX Analysis
+ RD correction Excel / DX
No Correction
IC 50 Ratios
Excel / DX + RD
correction
DX no correction /
DX + RD correction
1 49 55.5 49 0.9 1.0 1.1
2 130 139 159 0.9 0.8 0.9
3 320 426 369 0.8 0.9 1.2
4 440 477 470 0.9 0.9 1.0
5 460 796 732 0.6 0.6 1.1
6 880 1026 981 0.9 0.9 1.1
7 1600 1847 1703 0.9 0.9 1.1
8 1700 1997 1865 0.9 0.9 1.1
9 2200 2323 2191 1.0 1.0 1.1
10 2300 2603 2658 0.9 0.9 1.0
11 2400 3560 2930 0.7 0.8 1.2
12 2500 3835 3354 0.7 0.7 1.1
13 2600 3068 2788 0.9 0.9 1.1
14 2700 3067 3166 0.8 0.9 1.0
15 2800 3623 3397 0.8 0.8 1.1
16 3100 3645 3452 0.9 0.9 1.1
17 3100 4092 3454 0.8 0.9 1.2
18 3500 4016 3910 0.9 0.9 1.0
19 3900 4538 4407 0.9 0.9 1.0
20 4500 5234 4802 0.9 0.9 1.1
21 4800 5846 5865 0.8 0.8 1.0
22 6300 8850 7515 0.7 0.8 1.2
23 9900 13624 10719 0.7 0.9 1.3
24 16500 19161 18386 0.9 0.9 1.0
25 20800 26379 23718 0.8 0.9 1.1
26 54000 83253 65869 0.7 0.8 1.3
27 55400 64085 62398 0.9 0.9 1.0
28 99200 103955 115277 1.0 0.9 0.9
Mean 0.8 0.9 1.1
SEM 0.02 0.01 0.02

PX Impact
• All pre-CAN nomination hERG screening
performed on PX
– Global standard
• Manual work reserved for post-CAN
package
• Progression of two ion channel discovery
programs to CAN
• FTE use
– 1x PX vs. 5x manual rigs

PatchXpress – beyond hERG screening
Cav channels
Ligand-gated channels
0
500 µM Ni 2+ [ligand] µM 0.3 1 3 10 30 100
100
300
500pA
25ms
30
control

PX Issues
• Compound addition can result in cell loss
• relatively rapid addition
• double addition protocol
• Maximum success rate < 60 %
• < 9 cells/chip
• Absence of rapid post-acquisition data
interrogation tools
• Not truly HTS/PMC compatible
– fills a niche

IonWorks Quattro
Nav
hERG

Characteristics of Nav IonWorks
experiments
350
3.5 MΩ
10
300
Count
250
200
150
100
4.1 MΩ
Peak current (nA)
1
0.1
50
0
3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
Hole resistance (MΩ)
0.01
0 100 200 300 400 500 600 700 800
Cell number

IonWorks protocol
• Single hole mode
• HEK-Nav cell line
• P13 / 50-60 % confluence
• T75 - ~10 6 cells / ml
• 50/50 Trypsin/Versene
• 10 min Amphotericin soak
• Pre / post compound read
• < 0.3 % DMSO
0
-90 mV
-100 20 ms

Seal Quality and Quantity
Seal resistance > 100 MΩ (~80 % success rate)
100
Inital read
100
Pre-compound
100
Post-compound
300
250
Cell count
80
60
40
20
80
60
40
20
80
60
40
20
# cells with seals >100 MΩ
200
150
100
50
0
0
0 100 200 300 400 500
0
0 100 200 300 400 500
0 100 200 300 400 500
0
initial
pre-compound
post-compound
Seal resistance (MΩ)
Suitable for transfer to PPC mode

Nav1 Pharmacology
• Single pulse
• Read pre/post compound
• Peak amplitude filter >0.2 nA
• Seal resistance filter >100 MΩ
• Experiment duration 55 min
0.1 nM
TTX
100 nM
100
0
80
2 ms
0.5 nA
30 nM TTX
% inhibition
60
40
IC 50
= 12.7 nM
control
20
0
0.01 0.1 1 10 100 1000
TTX (nM)

Nav - Drug Use-Dependency
10 Hz pulse train
Seal resistance > 100 MΩ (~80 % success rate)
• 35 s pre-hold at Vm before pulse train
• Metric to quantify amplitude of 25 th pulse
– Control vs. compound
% Inhibition
0
-25
-50
-75
-100
-125
-7 -6 -5 -4 -3
log [ ]

hERG - IonWorks
Seal resistance > 100 MΩ (~70 % success rate)
• HEK-hERG cell line
• Single hole mode
• 50-60 % confluence
• T75 - ~10 6 cells / ml
• 50/50 Trypsin/Versene
• 10 min Amphotericin soak
• < 0.3 % DMSO
500ms
0.2 nA

hERG - IonWorks
800
800
800
1
count
700
600
500
400
700
600
500
400
700
600
500
400
Peak hERG tail current (nA)
0.1
0.01
1E-3
300
300
300
200
100
200
100
200
100
1E-4
150
0 200 400 600 800 1000 1200 1400 1600 1800 2000
cell number
0
0
0
0 100 200 300 400 500 0 100 200 300 400 500
seal resistance (MΩ)
• Assay development
0 100 200 300 400 500
count
120
90
60
30
0
0 1 2 3
peak hERG tail current (nA)

IonWorks Utility
Advantages
• Rapid SAR analysis and compound attrition
– Ability to support high-speed chemistry approaches
• Simple to use acquisition interface
• Simple post-acquisition analysis tools
• Efficient FTE use
• Cost / data point
Limitations
• Single compound addition, no washout
• No families of currents
• Current – voltage relationships
• Steady-state activation / inactivation
• Discontinuous voltage-control
• Voltage-dependent inhibitors
• Not considered a hindrance relative to throughput and position in
screening funnel
• Other platforms more suited to measure additional endpoints

Implications of PX and IW
Quattro integration
• Expanded drug discovery support
– Target validation
– SAR
– increased colleague confidence in doability of
target class within portfolio
• Safety-related screening
– more
– earlier
– significant reduction in manual rig time
• reserved to address key kinetic issues

High throughput realized?
# compounds tested / week / FTE
2
25
250
Significant impact on content of the discovery portfolio

Integration into a drug discovery
screening funnel
Idea
IonWorks
Manual patch
- cell line selection
- cell line/assay optimization
- hit ID
- attrition
- leads
SAR
selectivity
safety
IonWorks
FLIPR
- lead optimization
- focus on throughput
Manual patch
PatchXpress
in vitro / in vivo efficacy
Candidate drugs
- extended electrophysiological
characterization
- candidate quality attributes

Use the Right Tool for the Job
x
• IonWorks and PatchXpress fill certain niches
– facilitate credible prosecution of ion channels as chemistrysupported
drug targets
• Manual patch is not redundant
– like any tool, use it for what it is designed for
• Like other target classes, ion channels need a
range of techniques to be effectively pursued

Summary
• New era in ion channel drug discovery
• Quantum improvement in ability to screen at
capacity compatible with drug discovery
• Positive impact on
– Perception of ion channel project doability
– New targets
– RIP targets
– CV safety of all target classes
• Toolbox of complementary technologies now
available

Acknowledgements
• Ben Wilenkin
• Mark Weber
• Chasta Khozouie
• Brandi Soldo
• Bernard Fermini
• David Caouette
• Gareth Waldron
• Kirk Schroeder
• Sohiel Memarsadeghi

Questions?