Toward Brain NaV1.8 Imaging with [11C]Suzetrigine

<b>Background/Objective:</b> Acute and chronic pain affect millions of individuals, yet there are currently no molecular imaging tools to directly assess pain-related mechanisms in the central nervous system (CNS). The voltage-gated sodium channel Na_V1.8 plays a pivotal role in neuropathic pain by increasing the excitability of nociceptive neurons following nerve injury or inflammation. In this work, we aimed to develop a novel positron emission tomography (PET) imaging probe for Na_V1.8 to facilitate noninvasive quantification of this target in the CNS and thereby advance our understanding of pain neurobiology. <b>Methods:</b> We selected the compound suzetrigine, a U.S. FDA-approved, highly selective non-opioid Na_V1.8 inhibitor, as the first candidate for a Na_V1.8-targeted PET tracer. The compound was first assessed using in silico docking and CNS multiparameter optimization (MPO) analysis to evaluate target binding and predicted brain penetrability. Radiolabeling was accomplished by <i>O</i>-methylation with [¹¹C]methyl iodide to yield [¹¹C]suzetrigine without structural modification. The tracer was then evaluated using in vitro binding assays, including autoradiography and saturation binding on rat brain tissues, to determine binding parameters (K_D, B_max), and using in vivo PET imaging in rats to assess brain uptake, time-activity curves (TACs), and tracer behavior under baseline and pretreatment conditions. Pretreatment was performed with unlabeled suzetrigine, the P-glycoprotein (P-gp) inhibitor verapamil, and the heterologous Na_V1.8 inhibitor A-803467. <b>Results:</b> In silico docking demonstrated favorable binding of suzetrigine to the Na_V1.8 active site, and the calculated CNS MPO score (>3.5) suggested adequate brain penetration. Radiochemical synthesis of [¹¹C]suzetrigine via <i>O</i>-methylation yielded a high decay-corrected radiochemical yield (19.2 ± 2.7%, <i>n</i> = 3), excellent purity (>98%, <i>n</i> = 3), and moderate molar activity (62.9 ± 51.8 MBq/nmol, <i>n</i> = 3). Autoradiography on rat brain tissue confirmed saturable and selective binding of [¹¹C]suzetrigine to Na_V1.8. Saturation binding assays revealed a B_max = 93 fmol/mg and a K_D = 0.1 nM, supporting the imageability of Na_V1.8 in the brain using this tracer. In vivo PET imaging in rats demonstrated rapid and sufficient brain uptake but revealed unexpected tracer behavior: signal intensity markedly increased following pretreatment with either unlabeled suzetrigine or the P-gp inhibitor verapamil, and showed a slight increase after pretreatment with the heterologous Na_V1.8 inhibitor A-803467. Detailed analysis of PET images, TACs, and normalized area-under-curve (AUC) values indicated that these atypical uptake patterns were primarily attributable to P-gp-mediated effects, although additional factors may also contribute. <b>Conclusions:</b> [¹¹C]Suzetrigine exhibits high affinity, good brain uptake, and selective target engagement in vitro, supporting its potential as a first-in-class Na_V1.8-PET tracer. However, in vivo performance is confounded by P-gp-mediated efflux and possibly other mechanisms that limit accurate quantification of Na_V1.8 in the living brain. These findings underscore the critical role of efflux transporters in CNS radiotracer development and highlight the need for design strategies that mitigate P-gp interaction when targeting ion channels in the brain. Future studies will include imaging under constant P-gp inhibition, arterial blood sampling for radiometabolite analysis and full kinetic modeling, and evaluation in non-human primates to assess translational feasibility.