SCN9A: A pivotal therapeutic target for analgesic drug development

• Gene Information: The sodium voltage-gated channel alpha subunit 9 (SCN9A) gene encodes the Nav1.7 sodium channel protein. Gain-of-function and loss-of-function mutations correspond to enhanced and diminished pain sensation, respectively.
• Protein Expression: Nav1.7 is predominantly expressed in peripheral sympathetic and sensory neurons. In response to membrane depolarization, it mediates the generation and repetitive firing of action potentials, playing a central role in electrical signal transduction within nociceptive neurons. Accordingly, it serves as a promising target for the development of effective and safe analgesics.
• Signaling Pathway: Nerve injury increases p38 MAPK and other kinases to upregulate Nav1.7 and induce abnormal neuronal discharges. Inflammation raises levels of Nav1.7-regulating phosphorylating kinases, increasing aberrant action potentials. Together, these pathways drive pain.
• Therapeutic Inhibition: Raxatrigine is a selective Nav1.7 inhibitor. It blocks voltage-gated sodium channel Nav1.7 in a state-dependent manner to suppress aberrant high-frequency discharges in nociceptive neurons, with weak inhibitory effects on other sodium channel subtypes, thereby alleviating neuropathic pain.

SCN9A

• The exons 2-27 of mouse Scn9a gene that encode the whole molecule (ATG to STOP codon) were replaced by human counterparts in B-hSCN9A mice. The promoter, 5’UTR and 3’UTR region of the mouse gene are retained.
• The human SCN9A expression is driven by endogenous mouse Scn9a promoter, while mouse Scn9a gene transcription and translation will be disrupted.

• (A) Human SCN9A mRNA is exclusively detectable in dorsal root ganglia (DRG)  of homozygous B-hSCN9A mice, but not in wild-type C57BL/6 mice.
• (B) Relative SCN9A mRNA expression levels in DRG were comparable between B-hSCN9A mice and wild-type C57BL/6JNifdc mice, regardless of gender.

Strain-specific SCN9A expression analysis in wild-type C57BL/6 mice and homozygous B-hSCN9A mice. (A) Dorsal root ganglia (DRG) RNA was isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hSCN9A mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human SCN9A primers. (B) Quantitative real-time PCR (qRT-PCR) analysis of relative SCN9A mRNA expression in the DRG of male and female wild-type C57BL/6JNifdc mice and homozygous B-hSCN9A mice. Expression levels were normalized to SCN9A expression in female B-hSCN9A mice. Values are expressed as mean ± SEM.

• SCN9A protein was detectable in DRG, brain, cerebellum and testis from homozygous B-hSCN9A mice and wild-type C57BL/6JNifdc.

Protein expression analysis of SCN9A in homozygous B-hSCN9A mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hSCN9A mice (H/H), and then analyzed by western blot with anti-SCN9A antibody. 40 μg total protein was loaded for western blotting analysis. SCN9A protein was detectable in DRG, brain, cerebellum and testis from homozygous B-hSCN9A mice and wild-type C57BL/6 mice, as the antibody was cross-reactive between human and mouse.

Experimental schedule for the CFA-induced Inflammatory Pain Model and in vivo efficacy of Raxatrigine analog in wild-type mice and B-hSCN9A mice. The mice underwent a 3-day acclimation period in test cages. Mice received subcutaneous CFA injection in the hind paws on Day 0 to establish an inflammatory pain model induced by CFA. Pain thresholds were measured with the Von Frey test 2 h before compound administration. Raxatrigine analog was orally administered, and follow-up Von Frey threshold measurements were performed at 1 h and 2 h after dosing.

CFA-induced inflammatory pain and analgesic effects of Raxatrigine analog in wild-type mice and B-hSCN9A mice. In wild-type mice and B-hSCN9A mice, intraplantar CFA injection markedly reduced mechanical pain thresholds. Raxatrigine analog significantly alleviated inflammatory pain with an analgesic effect lasting up to 2 h in wild-type mice and B-hSCN9A mice. B-hSCN9A mice provide a powerful preclinical model for in vivo evaluation of the efficacy of targeted human SCN9A analgesic drugs. *P < 0.05, **P < 0.01, ***P < 0.001.