Modulation of human dorsal root ganglion neuron excitability by Nav1.7 inhibition

Reviewer #1 (Public review):

Summary:

Fujita and colleagues investigated two selective peripheral nerve voltage-gated sodium channel inhibitors targeting either Nav1.7 or Nav1.8 on the excitability of human dorsal root ganglion neurons. The authors discovered that Nav1.8 inhibition is more effective at suppressing repetitive firing of DRG neurons, and this may explain the greater clinical efficacy observed for suzetrigine.

Strengths:

The study is interesting, and the findings are conceptually satisfying in that they may explain one aspect of Nav1.7 vs Nav1.8 targeting success.

Weaknesses:

(1) The use of postmortem human DRG neurons provides translational relevance, but the use of these cells is also a liability, given their high degree of variability. Of note are the 10 to 20-fold differences in baseline properties among cells, which dwarf the effects of the test compounds. The experiments may suffer from undersampling.

(2) A potential confounder when using post-mortem human DRG neurons is heterogeneity of cell types. The methods clearly state that the cells selected for recording were of 'generally' small size, but specific criteria for what constitutes 'small' or other unstated selection criteria were not provided. A table of individual cell capacitance and input resistance values, along with information about individual donors (age, sex, ethnicity), is important to include. Additionally, some discussion of how DRG neuron heterogeneity impacts the findings. This relates to concern #1 about sample size determination and how cell heterogeneity factored into this calculation.

Reviewer #2 (Public review):

Summary:

The authors examine the functional role of Nav1.7 voltage-gated sodium channels in human sensory neuron electrogenesis using a Nav1.7 selective inhibitor and human dorsal root ganglion neurons obtained from organ donors. Patch-clamp electrophysiology is used at physiological temperature to measure the impact of Nav1.7 inhibition on sensory neurons' action potential firing. This is an important topic as Nav1.7 and Nav1.8 have been identified as therapeutic targets for the treatment of pain, but there has been mixed success with isoform-specific inhibitors in clinical trials. The data suggest that Nav1.7 and Nav1.8 have overlapping yet complementary functions in nociceptor neurons and that targeting both may be most effective for reducing nociception.

Strengths:

The data are of high quality. Action potential properties are measured at 37 degrees Celsius. Threshold is measured using brief pulses. The Nav1.7 inhibitor has been reported to be highly selective for Nav1.7 over Nav1.8 and moderately selective for Nav1.7 over Nav1.1 and Nav1.6. Data are collected using identical conditions and protocols to a previous study on the role of Nav1.8 in similar neurons.

Weaknesses:

The study relies on a single Nav1.7 inhibitor that has not been extensively characterized. One prior study indicates that the IC50 is around 140 nM, thus the 600 nM concentration used in this study could be predicted to reduce Nav1.7 currents by 80%. However, there is no voltage-clamp data in the current study to confirm this, and therefore, it is unclear if the batch of AM-2099 is as potent as reported in the paper that initially described its selectivity. The impact of Nav1.7 inhibition is compared to data from a previous study by this lab, and this is a minor concern. It would have been interesting to see if the combined inhibition of Nav1.7 and Nav1.8 completely blocked action potential generation in the human DRG neurons.

Reviewer #3 (Public review):

Summary:

In this manuscript, Fujita/Jo/Stewart/Osorno et al. investigate the contribution of Nav1.7 in regulating the excitability and firing properties of human dorsal root ganglion (hDRG) neurons in vitro. The authors characterize the effects of a previously reported Nav1.7-selective blocker AM-2099 in cultured hDRG neurons from postmortem organ donors. The authors observed modest changes in many of the properties expected by inhibiting Nav channels, including decreased action potential upstroke rate and amplitude, while increasing the voltage and current thresholds for spike generation. However, AM-2099 did not change the maximum number of APs in response to suprathreshold stimulation, leading the authors to conclude that Nav1.7 inhibition alone has limited efficacy in reducing the firing properties of hDRG neurons and that Nav1.7 blockers may have limited efficacy as analgesics. This is surprising, given that patients with loss-of-function mutations in Nav1.7 suffer from congenital insensitivity to pain. While it may indeed be true that pharmacological inhibition of Nav1.7 is unlikely to produce analgesia, the present study was limited to a single concentration of AM-2099. The manuscript would be significantly strengthened by a more careful and thorough pharmacological characterization of this compound, which has not been widely used or validated in native human DRG neurons.

Strengths:

Experiments are well-designed and executed, and the results presented are convincing. The focus on voltage-gated sodium channels in native human DRG neurons is highly relevant to recent efforts to develop safer analgesic options for chronic pain in people.

Weaknesses:

Only a single concentration of AM-2099 was used for all experiments. This compound was reported to be selective for cloned human Nav1.7 channels in heterologous systems, but has not been validated in other studies after the original publication in 2016. Since the original study reported a substantial state-dependent block of recombinant Nav1.7 channels, more detailed pharmacological characterization of AM-2099 is needed in human DRG neurons to fully support these claims. This study would be significantly strengthened by the inclusion of dose-response curves to assess how much of the sodium current is inhibited at this concentration, confirming selectivity in hDRG, and whether maximal inhibition of Nav1.7 still has limited efficacy in reducing the firing of native human sensory neurons.