Excessive activation of NMDA receptor (NMDAR) signaling within the spinal dorsal horn contributes to central sensitization and the induction and maintenance of pathological pain states. However, direct antagonism of NMDARs produces undesirable side effects which limit their clinical use. NMDAR activation produces central sensitization, in part, by initiating a signaling cascade that activates the enzyme neuronal nitric oxide synthase (nNOS) and generates the signaling molecule nitric oxide (NO). NMDAR-mediated activation of nNOS requires a scaffolding protein, postsynaptic density protein 95 kDa (PSD95), which tethers nNOS to NMDARs. Thus, disrupting the protein-protein interaction between PSD95 and nNOS may inhibit pro-nociceptive signaling mechanisms downstream of NMDARs and suppress central sensitization while sparing unwanted side effects associated with NMDAR antagonists. We examined the impact of small molecule PSD95-nNOS protein-protein interaction inhibitors (ZL006, IC87201) on both nociceptive behavior and formalin-evoked Fos protein expression within lumbar spinal dorsal horn of rats. Comparisons were made with ZL007, an inactive analog of ZL006, and the NMDAR antagonist MK-801. IC87201 and ZL006, but not ZL007, suppressed phase 2 of formalin-evoked pain behavior and decreased the number of formalin-induced Fos-like immunoreactive cells in spinal dorsal horn regions associated with nociceptive processing. MK-801 suppressed Fos protein expression in both dorsal and ventral horns. MK-801 produced motor ataxia in the rotarod test whereas IC87201 and ZL006 failed to do so. ZL006 but not ZL007 suppressed paclitaxel-induced mechanical and cold allodynia in a model of chemotherapy-induced neuropathic pain. Co-immunoprecipitation experiments revealed the presence of the PSD95-nNOS complex in lumbar spinal cord of paclitaxel-treated rats, although ZL006 did not reliably disrupt the complex in all subjects. The present findings validate use of putative small molecule PSD95-nNOS protein-protein interaction inhibitors as novel analgesics and demonstrate, for the first time, that these inhibitors suppress inflammation-evoked neuronal activation at the level of the spinal dorsal horn.

The G-protein coupled receptor GPR55 has been postulated to serve as a novel cannabinoid receptor. A previous report indicated that GPR55 knockout mice fail to develop mechanical hyperalgesia, suggesting a pro-nociceptive role for GPR55 in the control of nociceptive responding. However, GPR55 knockout mice remain incompletely characterized in models of pathological pain. Here we provide a comprehensive assessment of responses of GPR55 knockout and wild-type mice to mechanical and thermal (heat, cold) stimulation in multiple, mechanistically distinct models of inflammatory and neuropathic pain. Inflammatory sensitization was produced by intraplantar administration of capsaicin, formalin or complete Freund’s adjuvant. No differences in responding were detected between GPR55 knockout and wild-type mice in any model of inflammatory nociception assessed. Neuropathic pain was induced by partial sciatic nerve ligation (which induces hypersensitivity to mechanical, cold and heat stimulation) or by treatment with the chemotherapeutic agent paclitaxel (which induces hypersensitivity to mechanical and cold stimulation only). No differences were observed between GPR55 knockout and wild type mice in either development or maintenance of neuropathic nociception in either neuropathic pain model. In conclusion, genetic deletion of GPR55 did not alter the development of pathological pain in adult mice in any chronic pain model evaluated.

Neuropathic pain impacts approximately 3–4.5% of the global population and remains an unresolved health problem. The management of neuropathic pain has two distinct goals—prevention of development and control of established neuropathic pain. We examined the impact of both prophylactic and therapeutic treatments with the tricyclic antidepressant desipramine on the development and maintenance of toxic neuropathic pain induced by the chemotherapeutic agent paclitaxel. We also investigated the involvement of endogenous analgesic (i.e., endogenous opioid and endocannabinoid) systems in the antinociceptive actions of desipramine in these two distinct phases of neuropathic pain. Chronic subcutaneous infusion of desipramine via osmotic pumps suppressed both the development and maintenance of paclitaxel-induced neuropathic pain. However, only prophylactic desipramine treatment blocked the development of neuropathic pain throughout the three month observation interval. The opioid receptor antagonist naloxone blocked the antinociceptive effects of both prophylactic and therapeutic desipramine treatments throughout the entire timecourse of desipramine-induced antinociception. By contrast, cannabinoid CB1 and CB2 receptor antagonists partially attenuated the antinociceptive actions of desipramine in a manner that was restricted to the development phase of paclitaxel-induced neuropathic pain only. Paclitaxel decreased cell viability in TMD231 tumor cells in an MTT assay in vitro. Notably, desipramine (1 nM to 1 μM) alone did not alter tumor cell viability and did not prevent the cytotoxic effects of paclitaxel under identical conditions. The highest concentration of desipramine (10 μM) reduced tumor cell viability alone and enhanced the cytotoxic effects of paclitaxel. Our study identifies a previously unrecognized preemptive analgesic strategy that prevents development of paclitaxel-induced neuropathic pain, and also dissects receptor mechanisms underlying desipramine-induced antinociceptive effects. This information may be applied to improve current therapeutic strategies with the goal of preventing and managing neuropathic pain induced by chemotherapeutic treatment.

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Elevated N-methyl-D-aspartate receptor activity contributes to central sensitization. Our laboratories and others recently reported that disrupting protein–protein interactions downstream of N-methyl-D-aspartate receptors suppresses pain. Specifically, disrupting binding between the enzyme neuronal nitric oxide synthase and either its upstream (postsynaptic density 95 kDa, PSD95) or downstream (e.g. nitric oxide synthase 1 adaptor protein, NOS1AP) protein partners suppressed inflammatory and/or neuropathic pain. However, the lack of a small-molecule neuronal nitric oxide synthase-NOS1AP inhibitor has hindered efforts to validate the therapeutic utility of disrupting the neuronal nitric oxide synthase-NOS1AP interface as an analgesic strategy. We, therefore, evaluated the ability of a putative small-molecule neuronal nitric oxide synthase-NOS1AP inhibitor ZLc002 to disrupt binding between neuronal nitric oxide synthase and NOS1AP using ex vivo, in vitro, and purified recombinant systems and asked whether ZLc002 would suppress inflammatory and neuropathic pain in vivo. In vitro, ZLc002 reduced co-immunoprecipitation of full-length NOS1AP and neuronal nitric oxide synthase in cultured neurons and in HEK293T cells co-expressing full-length neuronal nitric oxide synthase and NOS1AP. However, using a cell-free biochemical binding assay, ZLc002 failed to disrupt the in vitro binding between His-neuronal nitric oxide synthase1-299 and glutathione S-transferase-NOS1AP400-506, protein sequences containing the required binding domains for this protein–protein interaction, suggesting an indirect mode of action in intact cells. ZLc002 (4–10 mg/kg i.p.) suppressed formalin-evoked inflammatory pain in rats and reduced Fos protein-like immunoreactivity in the lumbar spinal dorsal horn. ZLc002 also suppressed mechanical and cold allodynia in a mouse model of paclitaxel-induced neuropathic pain. Anti-allodynic efficacy was sustained for at least four days of once daily repeated dosing. ZLc002 also synergized with paclitaxel when administered in combination to reduce breast (4T1) or ovarian (HeyA8) tumor cell line viability but did not alter tumor cell viability without paclitaxel. Our results verify that ZLc002 disrupts neuronal nitric oxide synthase-NOS1AP interaction in intact cells and demonstrate, for the first time, that systemic administration of a putative small-molecule inhibitor of neuronal nitric oxide synthase-NOS1AP suppresses inflammatory and neuropathic pain.