NaV1.5 peak assays
Peak sodium ion channel (NaV1.5 peak)
The voltage gated cardiac sodium channel Nav1.5 peak, encoded by the SCN5A gene, is a key component for the initiation and transmission of this excitability throughout the heart. Nav1.5 peak activation induces the depolarization of the cell membrane and initiates the phase I of the action potential (dV/dtmax). When the Nav1.5 peak channel cannot adequately conduct the electrical current across the cell membrane, fatal ventricular disorder could occur and could trigger ventricular arrhyhthmias.
Nav1.5 peak assays & preclinical studies
Nav1.5 peak channel assays aim to identify potential blocking effects early in drug discovery phase. Numerous clinically approved drugs reported to increase the pro arrhythmogenic risk (including sudden death, fatal fibrillation) through excessive Nav1.5 peak channel blockade known to increase the threshold of arrhythmias (leading circle). As a result, a large number of drugs have been withdrawn during late stage of clinical trials or from the market.
What is the added value of such a study?
- Is a cardiac ion channel recommended by the CiPA
- Threshold concentration of hNav1.5 peak blockade
- Leading to the determination of the safety range versus efficacious concentration
- You obtain your safety margin
Automated Patch Clamp
Technique
- Human Embryonic Kidney HEK-293 cells
- Experiment conducted at room temperature or at physiological temperature (35°C)
- Up to 6 increasing cumulative concentrations of test compound or biologic.
Measured parameters
- Amplitude of the peak current upon depolarization to –30mV (pA)
- Amplitude of the base current at –110mV (pA)
- Ion current amplitude measurement
- Inhibition of NaV1.5 peak current amplitude (%)
- IC50 value (at least 4 concentrations required)
Main advantages
- High throughput screening
- Cost effective
- Fast process to delivery results
- Perfect at earliest stages
- Design protocol could be adapted
Manual Patch Clamp
Technique
- Human Embryonic Kidney HEK-293 cells
- Experiment conducted at room temperature or at physiological temperature (35°C)
- Up to 5 increasing concentrations of test compound tested independently
- At least 3 treated cells are recommended
Measured parameters
- Amplitude of the peak current upon depolarization to –30mV (pA)
- Amplitude of the base current at –110mV (pA)
- Ion current amplitude measurement
- Inhibition of NaV1.5 peak current amplitude (%)
- IC50 value (at least 4 concentrations required)
Main advantages
- Technically robust
- Highly informative (more accurate IC50 value)
- Strongly replicable
- Design protocol could be adapted
Manual Patch Clamp - GLP Conditions
Technique
- Human Embryonic Kidney HEK-293 cells
- Experiment conducted at room temperature or at physiological temperature (35°C)
- At least 4 increasing concentrations of test compound tested independently
- At least 5 treated cells is highly recommended for a regulatory application
- Vehicle group is also highly recommended
- Sampling pre- and post- perfusion for analytical quantification
Measured parameters
- Amplitude of the peak current upon depolarization to –30mV (pA)
- Amplitude of the base current at –110mV (pA)
- Ion current amplitude measurement
- Inhibition of NaV1.5 peak current amplitude (%)
- IC50 value
Main advantages
- Technically robust
- Highly informative (more accurate IC50 value)
- Strongly replicable
- Design protocol could be adapted
- Sampling
- Analytical quantification under GLP conditions (also mandatory) could be performed by the Sponsor or with PhysioStim
Stimulation protocol
Typical effects of flecainide on Nav1.5 peak current recorded from HEK-293 cells.
Reference compounds or positive controls
Reference CompoundsIC₅₀
Flecainide1.5 µM
Quinidine21.1 µM
