Upload
shiminwang
View
1.629
Download
1
Embed Size (px)
DESCRIPTION
Citation preview
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
5
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