Background & Aims

The voltage-gated sodium (Nav) channel 1.8 isoform is predominantly expressed in C and A? peripheral sensory neurons, important in pain transmission, and has strong human biology support i.e. humans with gain of function mutation in Nav1.8 experience painful peripheral neuropathies. However, highly selective, and potent “drug-like” inhibitors for this channel have been difficult to develop and only recently have made their way to clinical testing. Our efforts to develop a small molecule inhibitor, yielded a series of molecules with favorable in vitro potency and selectivity in humans but limited or no activity in rodents plausibly due to differences in protein homology between the rodent and human Nav1.8 channels. To support drug discovery efforts, a transgenic rat expressing the human Nav1.8 channel was developed. Using these animals, we identified MSD199 as being efficacious in various inflammatory and neuropathic behavior pain assays.

Methods

Automated Patch Clamp (QUBE): HEK293 cell lines stably expressing human Nav isoforms (Kraus et al 2021) were used to establish an in vitro selectivity profile for MSD199.
Manual Patch Clamp: Human Nav1.8 HEK293 recombinant cells or acutely dissected rat or humanized rat dorsal root ganglion cells were used to determine in vitro potency of MSD199.
Capsaicin Nocifensive Behavior: Nocifensive behaviors (number of flinches in first 5 min) in response to intraplantar injection of 0.01% 25?L capsaicin were counted in MSD199 or vehicle-treated animals.
Complete Freund’s Adjuvant (CFA) model: Changes in the thermal latency (Hargreaves method) of rats treated with MSD199 or vehicle, 1 day after receiving intra-plantar injection of CFA (50?L, 1 mg/ml) were measured.
Spinal nerve ligation model: Baseline mechanosensitivity was assessed with von Frey filaments via Up/Down method. Thereafter, lumbar 5 spinal nerve ligation was performed and mechanical testing was performed 7 days post surgery.

Results

MSD 199 is a potent inhibitor of human Nav1.8 channels (IC50= 3.4nM) vs. rodent Nav1.8 (IC50=4826 nM). It is highly selective over other Nav isoforms (Nav1.2, Nav1.5, Nav1.6, or Nav1.7) with IC50= >33800nM and is ~2000-fold more potent versus Nav1.4 (IC=8370 ± 1429nM. Its potency measured in humanized Nav1.8 rat DRG neurons was 5.6 nM. In vivo, MSD199 had no efficacy in wild type rats but dose-dependently reduced the capsaicin-evoked nocifensive behaviors at doses ? 1 mg/kg compared to vehicle in humanized Nav1.8 animals. High dose (10mg/kg) MSD199 significantly increased response latency to Complete Freund’s Adjuvant-induced hyperalgesia in the Hargreaves noxious radiant heat assay, consistent with approved analgesics. Finally, 10 mpk, but not 1 mpk, significantly reversed mechanical allodynia in the spinal nerve ligation assay when compared to vehicle controls.

Conclusions

These results highlight the utility of humanized transgenic animals when cross-species potency shifts are observed within an otherwise promising chemical series. MSD199 is a promising Nav1.8 inhibitor with significant efficacy reported in CNB, CFA and SNL assays.

References

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Presenting Author

Dillon McDevitt

Poster Authors

Dillon McDevitt

PhD

Merck & Co.

Lead Author

E-mail

Topics

  • Mechanisms: Biological-Systems (Physiology/Anatomy)