Background & Aims

Chronic pain is highly prevalent and remains a significant unmet global medical need. As part of a search for modulatory genes that confer pain resilience, we have studied two family cohorts with inherited erythromelalgia (IEM) where one individual reported much less pain than other family members that share the same pathogenic gain-of-function Nav1.7 mutation that confers hyperexcitability in pain-signaling dorsal root ganglion (DRG) neurons. In each of these kindreds, the pain-resilient individual carried a gain-of-function variant in Kv7.2 or Kv7.3, two potassium channels that stabilize membrane potential and reduce excitability. Our findings show that these gain-of-function Kv7.2 and 7.3 variants reduce DRG neuron excitability suggesting that Kv7.2/7.3 activators may attenuate sensory neuron firing to alleviate pain.

In the present study, we assess the effects of BHV-7000, a selective Kv7.2/7.3 activator in clinical development for epilepsy, on IEM sensory neuron excitability

Methods

Standard patch-clamp methods were employed to record from human derived-iPSC sensory neurons (iPSC-SNs, Axol Bioscience). After drug application, three endpoints were assessed: changes in resting membrane potential, rheobase, and action potential firings at 3X rheobase. IEM-iPSCs were generated from the blood samples of family members previously identified (Mis et al., 2019; Yuan et al., 2021) to carry pathogenic mutations of Nav1.7 that cause inherited erythromelalgia (IEM) but with no Kv7 variants (IEM-iPSC-SNs). MEA recordings (Maestro, Axion Biosystems) were obtained to assess spontaneous firing as previously described (Mis et al., 2019). Current clamp recordings were obtained using an EPC-10 amplifier and the Patch Master program (HEKA Elecktronik). iPSC-SNs with a stable membrane potential were chosen for analysis. Resting membrane potential was determined immediately after switching into current-clamp mode as the mean membrane voltage in the absence of current stimulation.

Results

At 1 and 10 µM in iPSC-SNs not carrying IEM mutations of Nav1.7, BHV-7000 demonstrated a hyperpolarization of -3.5±0.4 and -6.5± 0.7 in resting membrane potential (mV) and 36±3.4 and 100±8.9 change in rheobase (pA) from baseline, respectively. At 3x rheobase, 1 and 10 µM reduced the number of APs by 52 and 87% compared to control. Results recorded from IEM-iPSC-SNs, obtained from a subject with IEM and carrying the Nav1.7-S241T mutation, displayed robust spontaneous activity measured by multi-electrode array recordings. In multiple independent differentiations, analysis of the total spikes showed that BHV-7000 reduced spontaneous activity with an average IC50 of about 75 nM and the average maximal inhibition was about 92%. In IEM-iPSC-SNs from a Nav1.7-F1449V mutation IEM patient, BHV-7000 had an IC50 value of about 70 nM and inhibited spontaneous activity by about 95%.

Conclusions

This study demonstrated that BHV-7000 hyperpolarized the resting membrane potential, increased the rheobase, and decreased the action potential firing rate at 3X rheobase from iPSC-SNs using standard patch-clamp recordings. In IEM-iPSC-SNs from 2 IEM patients, multi-electrode-array recordings demonstrated potent inhibition of spontaneous firing with BHV-7000. In addition, current-clamp recording from one IEM-iPSC cell line demonstrated that BHV-7000 hyperpolarized iPSC-SN resting membrane potential. These results implicate Kv7.2/Kv7.3 channels as effective modulators of sensory neuron excitability, with and without sodium channel mutations, and suggest that BHV-7000 can specifically target Kv7.2/Kv7.3 currents to reduce excitability in sensory neurons with the potential to be an effective treatment toward pain relief in IEM patients.

References

Dib-Hajj SD & Waxman SG. (2019). Resilience to Pain: A Peripheral Component Identified Using Induced Pluripotent Stem Cells and Dynamic Clamp. J Neurosci 39, 382-392. • DOI: 10.1523/JNEUROSCI.2433-18.2018

Yuan JH, Estacion M, Mis MA, Tanaka BS, Schulman BR, Chen L, Liu S, Dib-Hajj FB, Dib-Hajj SD & Waxman SG. (2021). KCNQ variants and pain modulation: a missense variant in Kv7.3 contributes to pain resilience. Brain Commun 3, fcab212. DOI: 10.1093/braincomms/fcab212

Presenting Author

Mark Estacion

Poster Authors

Mark Estacion

PhD

Yale University

Lead Author

Topics

  • Treatment/Management: Pharmacology: Novel Targets