Contact PD/PI: Patwardhan, Amol M Project Summary: Epidural/spinal administration of analgesics such as opioids, ziconotide and local anesthetics have profound efficacy in some of the most intractable pain conditions such as severe neuropathic pain after failed back surgery, cancer pain and post-operative pain after major abdominal/thoracic surgeries. Despite their profound efficacy, their use is limited primarily because of the side effects such as tolerance, granuloma, psychosis and motor block. Discovery and validation of new spinal analgesic targets for development of therapeutics is urgently needed. Here we propose to validate a novel spinal analgesic target, neurotensin receptor 2 (NTSR2), based upon our mechanistic studies of Contulakin-G (CGX), that has shown preliminary efficacy in humans suffering from one of the hardest to treat neuropathic pain condition-spinal cord injury associated pain. CGX is a snail venom derived peptide that has homology with mammalian neurotensin and was shown to be safe in humans. A small, pilot Phase1A study demonstrated analgesic effect in some patients with spinal cord injury-associated pain. Although, CGX does not have favorable pharmacokinetic properties, these studies suggested a possibility of a novel, non-opioid, analgesic mechanism that is active in humans. Our preliminary studies suggest CGX produces its analgesic actions via activation of spinal neurotensin receptor 2 (NTSR2) and subsequent inhibition of voltage-gated calcium channels. NTSR2 is highly expressed in small/medium size sensory neurons in rodents and co-expressed with voltage gated calcium channels. Transcriptomics confirmed NTSR2 expression in human dorsal root ganglia sensory neurons. Importantly, our pilot studies show that NTSR2 activation by CGX produces profound analgesia and is not associated with unwarranted side effects such as rapid tolerance or motor blockade. Preliminary data thus support a role of spinal NTSR2 in pain modulation, but validation of this receptor as an analgesic target has not been done. In this project, we propose to perform a robust validation of spinal NTSR2 as an analgesic target utilizing three species of both sexes (rat, mice and human), two models (neuropathic pain and post-surgical pain), pharmacological (SA1) and state of the art genetic tools such as CRISPR-Cas9 editing (SA2) and assessment of both sensory and affective measures of pain. Moreover, we propose a rigorous, two-site parallel confirmation study (SA3) designed after multisite clinical trials to further authenticate spinal NTSR2 as an analgesic target. If successful, proposed studies could lead to a development of non-opioid spinal analgesic that has high translational potential. Page 6 Project Summary/Abstract