Authors :
Luis Williams, PhD – Quiver Bioscience
Hongkang Zhang, PhD – Quiver Bioscience
Joshua Meier, BS Candidate – Quiver Bioscience
Jennifer Grooms, BS – Quiver Bioscience
Steven Ryan, PhD – Quiver Bioscience
Benjamin Harwood, PhD – Quiver Bioscience
James Fink, PhD – Quiver Bioscience
Owen McManus, PhD – Quiver Bioscience
Graham Dempsey, PhD – Quiver Bioscience
Presenting Author: Steven Dworetzky, PhD – Biohaven Pharmaceuticals
Rationale:
Epilepsy is one of the most common chronic neurological conditions. BHV-7000 is a novel and selective activator of Kv7.2/Kv7.3, a key ion channel involved in neuronal signaling and regulating hyperexcitability in epilepsies, in clinical development for focal-onset seizures, generalized seizures, and mood disorders. In this study, an in vitro all-optical electrophysiology platform was used to further characterize the mechanistic activity of BHV-7000 under different treatment regimens and stimulus conditions.
Methods:
The acute and chronic pharmacological effects of BHV-7000 on the neuronal excitability of primary rat cortical neurons were evaluated using the all-optical electrophysiology platform Optopatch, which measures neuronal activity with single-cell and single action potential resolution, and uses machine learning to profile drug interactions for drug discovery optimization. Each experimental round used 3x384-well plates with early and chronic treatment regimens: Chronic Regimen A was 8 days, 5 days, 48 hrs and acute compound addition, and Regimen B used 48 hrs and acute treatment prior to optical physiology measurements. The stimulus protocol is composed of multiple ‘epochs’ of differing stimulus shapes, intensities, and durations.
Results:
Functional measurements were made from more than 200,000 neurons under two different treatment regimens and the results demonstrate a concentration-dependent dampening of neuronal excitability under gentle stimulus levels and increasing rheobase under ramp stimulus with an EC50 of ~100 nM, consistent with the brain exposures determined EC50 in the rat BHV-7000 MES studies. BHV-7000 induced a concentration-dependent firing rate reduction in both long duration stimulus with low amplitude depolarization and the beginning of the ramp stimulus. Both treatment regimens tended to give rise to similar EC50 values, although more features tend to be altered under the more chronic regimen A. EC50 values were based on common features with unique features providing additional mechanistic insight of compound interaction with the Kv7 channels. Additional analyses of the BHV-7000 data focused on select, shared Optopatch features that are impacted across several known anti-seizure medicines (ASMs) to provide additional translatability to in vivo data.
Conclusions:
Overall, BHV-7000 demonstrated potent in vitro effects to reduce neuronal activity impacting a diverse set of Optopatch functional features across the stimulus protocol, including spike timing and spike shape features in different stimulus periods, which indicates lower excitability near action potential threshold. Compound effects were more pronounced (additional altered features detected) under the more Chronic Regimen A compared to Regimen B, suggestive of neuronal remodeling with longer treatment.
Lastly, the fraction of neurons staying completely silenced during a long gentle blue stimulus period can effectively distinguish ASMs treated wells from DMSO wells in a parallel neuronal excitability assay using human iPS cell-derived cortical excitatory (“NGN2”) neurons, suggesting relevance of this measure for predicting an efficacious ASM.
Funding: Biohaven