Cardiac Safety Testing Services

Why Cardiac Safety Testing?

� S7B Guidance

� Objective

− Identify potential of a test substance or its metabolites to delay ventricular

repolarization.

− Relate extent of delayed ventricular repolarization to concentrations of a test

substance and its metabolites.

� Components that contribute to an assessment of cardiac risk

− In Vitro IKr Assay

– effect on native or expressed IKr (such as hERG)

− In Vivo QT Assay

– effect on QT interval

– this assay can be designed to meet the objective of both the ICH S7A (cardiovascular

core battery) and S7B

Ion Channels that Contribute to Cardiac Action Potential

Normal AP

K+ Channel Block

Ca2+ Channel Block

Na+ Channel Block

time

voltage

Inhibition of cardiac ion currents can alter QT interval, cause arrhythmia or death

Discovery Safety Testing: Detect cardiac safety liabilities early.

4

Cardiac Safety Testing Services

� Radioligand Binding Assays (Pharmacology, Taiwan)

− Ideal for early hit selection profiling

� Cellular patch clamp assays (Pharmacology, Bothell)

− Automated Patch Clamp (APC) with PatchXpress

– cost-effective technology providing a medium-throughput method usually used in

discovery stage after hit selection

� Tissue Assays (Pharmacology, Taiwan)

− L-Type Calcium channel Atrial Inotropy

� In Vivo Assays – (Pharmacology, Taiwan and DSA, Lyon)

− - Cardiovascular, QTc Interval

InformationValue

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Ion Channel Testing Services

� Services at Bothell SiteNon-GLP (10 day TAT)

- Cardiac

– hERG (Kv11.1)

– hNav1.5

– hCav1.2

- Immuno-suppression and Obesity

– hKv1.3

Functional Ion Channel Services in Bothell

Discovery Safety Testing

Crucial attributes for discovery assays:

� Fast

� 10-day Turnaround time on Automated Patch Clamp (PatchXpress)

assays

� Medium- to High-throughput capacity

� Automated Patch Clamp (PatchXpress) assays are medium-throughput at

modest cost compared to conventional patch clamp

� Predictive of clinical outcome

� Over-expressed human cardiac ion channels that play central role in AP

hKv11.1 (hERG)

hNav1.5

hCav1.2

� Assays validated against drugs with known cardiac liabilities or effects;

compare favorably with conventional patch clamp

Fail Early

hERG Validation (PatchXpress)

hERG Validation (PatchXpress)

hERG validation studies withcontrol compounds

-9 -8 -7 -6 -5 -40

25

50

75

100

Cisapride

Haloperidol

Risperidone

Verapamil

Pimozide

Astemizole

Quinidine

Ketoconozole

E4031

Terfenadine

MoxifloxacineLog Compound [uM]

Percent of Control

Assay comparison

IC 50 (nM)

Patch Express Conventional Patch Clamp Radioligand Binding

Quinidine 760 + 91 300-1000 13155

Cisapride* 13 + 1 45 158

Haloperidol 90 + 21 93 250

Risperidone 379 + 59 394 5865

Verapamil 535 + 46 140-830 5600

Astemizole 13 + 2 26 15

Pimozide 12 + 4 18 53

Ketoconozole 2,340 + 250 1920-4700 19050

E4031 32 + 4 18-36 75

Terfenadine 38 + 7 28-56 127

Moxifloxacine 39,167 + 4,061 41,000-170,000 NA

* - IC50 curve for Cisapride is not representative of current data.

hERG Validation (PatchXpress)

Comparison of PatchXpress data to conventional patch clamp (Literature values)

Nav1.5 Validation (PatchXpress)

Voltage ClampVoltage ClampVoltage ClampVoltage ClampProtocolProtocolProtocolProtocol

-10 mV

-120 mV

0.5 nA

5 ms

-75 -50 -25 25 50 75

-3

-2

-1

1

B

1 nA

3 ms

A

Nav1.5 Validation (PatchXpress)

hNav 1.5 validation studies withcontrol compounds

41.4 + 6.5

18.8 + 4.6

7.6 + 1.7

7.58 + 1.3

6.6 + 1.3

0.996 + 0.22

0.676 + 0.061

0.149 + 0.054

IC50 (uM)Rank Order of Potency

Flecainide

Imipramine

Lidocaine

Quinidine

Terfenadine

TTX

Dibucaine

Veratridine

-8 -7 -6 -5 -4 -3

0

25

50

75

100

Log Compound

[uM]

Percent of Control

Nav1.5 Validation (PatchXpress)

PX pIC50 vs. RBA pIC50

Dibucaine

Terfenadine

Veratridine

Flecanide

Propranolol

Imipramine

Lidocaine

y = 0.9863x + 0.2804

R2 = 0.8216

3.00

4.00

5.00

6.00

7.00

8.00

3.00 4.00 5.00 6.00 7.00 8.00

Radioligand Binding Assay (RBA) pIC50 (nM)

PatchXpress (PX) pIC50 (nM)

Cav1.2 Validation (PatchXpress)

Cardiac Ca2+ channel hCav1.2

-40 mV

0.1 nA

10 ms

0.2 nA Normalized Current

Test pulse (mV)

1

.8

.6

.4

.2

-40 -20 0 20 40 60

-80 mV

+10 mV

2 nA

10 ms

Cav1.2 Validation (PatchXpress)

0.114

1.19

0.177

0.824

36.5

0.87

0.0535

0.138

hCav 1.2 validation studies withcontrol compounds

-9 -8 -7 -6 -5 -4 -30

25

50

75

100

Nitrendipine

Lacidipine

Nimodipine

Isradipine

Diltiazem

Nicardipine

Nifedipine

Bepridil

Log Compound [uM]

Percent of Control

IC50 (µµµµM)

Cav1.2 Validation (PatchXpress)

IC50 correlation between the PatchXpress 7000A vs. referenced

conventional patch-clamp results.

Conventional pIC50 vs. PX pIC50

Diltiazem

Nitrendipine

Bepridil

Nimodipine

Nicardipine

Nifedipine

Lacidipine

Isradipine

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5

Conventional log pIC50(nM)

PX log pIC50(nM)

Spearman r = 0.90, p <0.01