Cellular prion protein protects from inflammatory and neuropathic pain

Cellular prion protein (PrPC) inhibits N-Methyl-D-Aspartate (NMDA) receptors. Since NMDA receptors play an important role in the transmission of pain signals in the dorsal horn of spinal cord, we thus wanted to determine if PrPC null mice show a reduced threshold for various pain behaviours

NMP-7 inhibits chronic inflammatory and neuropathic pain via block of Cab3.2 T-type calcium channels and activation of CB2 receptors

Background: T-type calcium channels and cannabinoid receptors are known to play important roles in chronic pain, making them attractive therapeutic targets. We recently reported on the design, synthesis and analgesic properties of a novel T-type channel inhibitor (NMP-7), which also shows mixed agonist activity on CB1 and CB2 receptors in vitro. Here, we analyzed the analgesic effect of systemically delivered NMP-7 (intraperitoneal (i.p.) or intragstric (i.g.) routes) on mechanical hypersensitivity in inflammatory pain induced by Complete Freund’s Adjuvant (CFA) and neuropathic pain induced by sciatic nerve injury. Results: NMP-7 delivered by either i.p. or i.g. routes produced dose-dependent inhibition of mechanical hyperalgesia in mouse models of inflammatory and neuropathic pain, without altering spontaneous locomotor activity in the open-field test at the highest active dose. Neither i.p. nor i.g. treatment reduced peripheral inflammation per se, as evaluated by examining paw edema and myeloperoxidase activity. The antinociception produced by NMP-7 in the CFA test was completely abolished in CaV3.2-null mice, confirming CaV3.2 as a key target. The analgesic action of intraperitoneally delivered NMP-7 was not affected by pretreatment of mice with the CB1 antagonist AM281, but was significantly attenuated by pretreatment with the CB2 antagonist AM630, suggesting that CB2 receptors, but not CB1 receptors are involved in the action of NMP-7 in vivo. Conclusions: Overall, our work shows that NMP-7 mediates a significant analgesic effect in a model of persistent inflammatory and chronic neuropathic pain by way of T-type channel modulation and CB2 receptor activation. Thus, this study provides a novel therapeutic avenue for managing chronic pain conditions via mixed CB ligands/ T-type channel blockers

Analgesic Effect of a Mixed T-Type Channel Inhibitor/CB2 Receptor Agonist

Background: Cannabinoid receptors and T-type calcium channels are potential targets for treating pain. Here we report on the design, synthesis and analgesic properties of a new mixed cannabinoid/T-type channel ligand, NMP-181. Results: NMP-181 action on CB1 and CB2 receptors was characterized in radioligand binding and in vitro GTP gamma[S-35] functional assays, and block of transiently expressed human Cav3.2 T-type channels by NMP-181 was analyzed by patch clamp. The analgesic effects and in vivo mechanism of action of NMP-181 delivered spinally or systemically were analyzed in formalin and CFA mouse models of pain. NMP-181 inhibited peak Ca(V)3.2 currents with IC50 values in the low micromolar range and acted as a CB2 agonist. Inactivated state dependence further augmented the inhibitory action of NMP-181. NMP-181 produced a dose-dependent antinociceptive effect when administered either spinally or systemically in both phases of the formalin test. Both i.t. and i.p. treatment of mice with NMP-181 reversed the mechanical hyperalgesia induced by CFA injection. NMP-181 showed no antinocieptive effect in Ca(V)3.2 null mice. The antinociceptive effect of intrathecally delivered NMP-181 in the formalin test was reversed by i.t. treatment of mice with AM-630 (CB2 antagonist). In contrast, the NMP-181-induced antinociception was not affected by treatment of mice with AM-281 (CB1 antagonist). Conclusions: Our work shows that both T-type channels as well as CB2 receptors play a role in the antinociceptive action of NMP-181, and also provides a novel avenue for suppressing chronic pain through novel mixed T-type/cannabinoid receptor ligands

Functional characterization and analgesic effects of mixed cannabinoid receptor/T-type channel ligands

<p>Abstract</p> <p>Background</p> <p>Both T-type calcium channels and cannabinoid receptors modulate signalling in the primary afferent pain pathway. Here, we investigate the analgesics activities of a series of novel cannabinoid receptor ligands with T-type calcium channel blocking activity.</p> <p>Results</p> <p>Novel compounds were characterized in radioligand binding assays and <it>in vitro </it>functional assays at human and rat CB1 and CB2 receptors. The inhibitory effects of these compounds on transient expressed human T-type calcium channels were examined in tsA-201 cells using standard whole-cell voltage clamp techniques, and their analgesic effects in response to various administration routes (intrathecally, intraplantarly, intraperitoneally) assessed in the formalin model. A series of compounds were synthesized and evaluated for channel and receptor activity. Compound NMP-7 acted as non-selective CB1/CB2 agonist while NMP4 was found to be a CB1 partial agonist and CB2 inverse agonist. Furthermore, NMP-144 behaved as a selective CB2 inverse agonist. All of these three compounds completely inhibited peak Cav3.2 currents with IC₅₀values in the low micromolar range. All compounds mediated analgesic effects in the formalin model, but depending on the route of administration, could differentially affect phase 1 and phase 2 of the formalin response.</p> <p>Conclusions</p> <p>Our results reveal that a set of novel cannabinioid receptor ligands potently inhibit T-type calcium channels and show analgesic effects <it>in vivo</it>. Our findings suggest possible novel means of mediating pain relief through mixed T-type/cannabinoid receptor ligands.</p

Anxiolytic effects of the flavonoid luteolin in a mouse model of acute colitis

Abstract Anxiety related disorders commonly occur in association with major depressive disorder (MDD) in individuals suffering from peripheral inflammation, with a higher prevalence among IBS patients. We have previously shown that the bioflavonoid luteolin has pronounced analgesic and antidepressant-like effects in mice with dextran sodium sulfate (DSS)-induced colitis. Here, we further evaluate the biological effect of luteolin as a possible anxiolytic agent in DSS treated mice. Anxiolytic action was evaluated using the open field test (OF), the novelty suppressed feeding test (NSFT) and the elevated plus maze test (EPM). Luteolin increased the number of crossings in the center of the OF apparatus, reduced the latency to interact with the food pellet in the NSFT, and increased the time spent in the open arms in the EPM. These results suggest luteolin as a possible natural anxiolytic molecule without sedative effects, thus reinforcing its therapeutic potential for the comorbidities involving peripheral inflammation, pain, mood and anxiety-related disorders

Ethosuximide inhibits acute histamine- and chloroquine-induced scratching behavior in mice

Abstract We have recently reported that the Cav3.2 T-type calcium channel which is well known for its key role in pain signalling, also mediates a critical function in the transmission of itch/pruritus. Here, we evaluated the effect of the clinically used anti-seizure medication ethosuximide, a well known inhibitor of T-type calcium channels, on male and female mice subjected to histaminergic- and non-histaminergic itch. When delivered intraperitoneally ethosuximide significantly reduced scratching behavior of mice of both sexes in response to subcutaneous injection of either histamine or chloroquine. When co-delivered subcutaneously together with either pruritogenic agent ethosuximide was also effective in inhibiting scratching responses in both male and female animals. Overall, our results are consistent with an important role of Cav3.2 T-type calcium channels in modulating histamine-dependent and histamine-independent itch transmission in the primary sensory pathway. Our findings also suggest that ethosuximide could be explored further as a possible therapeutic for the treatment of itch

Disrupting USP5/Cav3.2 interactions protects female mice from mechanical hypersensitivity during peripheral inflammation

Abstract Cav3.2 T-type calcium channels are important for the signaling of nociceptive information in the primary afferent pain pathway. During neuropathy and peripheral inflammation, Cav3.2 channels are upregulated due to an increased association with the deubiquitinase USP5. Disrupting these interactions in male mice by the use of cell permeant peptides reverses mechanical and thermal hypersensitivity. Here we explore the effects of interfering with USP5 binding to the channel in female mice with synchronized estrous cycle. We show that intrathecal delivery of a cell-penetrating TAT peptide corresponding to the UBPc domain of USP5 fully reverses mechanical hypersensitivity in mice intraplantarly injected with Complete Freund’s Adjuvant. Hence, the USP5 mediated dysregulation of Cav3.2 channel activity does not exhibit sex differences, and potential therapeutics targeting this interaction should be effective in both male and female subjects

The terpenes camphene and alpha-bisabolol inhibit inflammatory and neuropathic pain via Cav3.2 T-type calcium channels

Abstract T-type calcium channels are known molecular targets of certain phytocannabinoids and endocannabinoids. Here we explored the modulation of Cav3.2 T-type calcium channels by terpenes derived from cannabis plants. A screen of eight commercially available terpenes revealed that camphene and alpha-bisabolol mediated partial, but significant inhibition of Cav3.2 channels expressed in tsA-201 cells, as well as native T-type channels in mouse dorsal root ganglion neurons. Both compounds inhibited peak current amplitude with IC50s in the low micromolar range, and mediated an additional small hyperpolarizing shift in half-inactivation voltage. When delivered intrathecally, both terpenes inhibited nocifensive responses in mice that had received an intraplantar injection of formalin, with alpha-bisabolol showing greater efficacy. Both terpenes reduced thermal hyperalgesia in mice injected with Complete Freund’s adjuvant. This effect was independent of sex, and absent in Cav3.2 null mice, indicating that these compounds mediate their analgesic properties by acting on Cav3.2 channels. Both compounds also inhibited mechanical hypersensitivity in a mouse model of neuropathic pain. Hence, camphene and alpha-bisabolol have a wide spectrum of analgesic action by virtue of inhibiting Cav3.2 T-type calcium channels

Analgesic effects of optogenetic inhibition of basolateral amygdala inputs into the prefrontal cortex in nerve injured female mice

Abstract Peripheral nerve injury can lead to remodeling of brain circuits, and this can cause chronification of pain. We have recently reported that male mice subjected to spared injury of the sciatic nerve undergo changes in the function of the medial prefrontal cortex (mPFC) that culminate in reduced output of layer 5 pyramidal cells. More recently, we have shown that this is mediated by alterations in synaptic inputs from the basolateral amygdala (BLA) into GABAergic interneurons in the mPFC. Optogenetic inhibition of these inputs reversed mechanical allodynia and thermal hyperalgesia in male mice. It is known that the processing of pain signals can exhibit marked sex differences. We therefore tested whether the dysregulation of BLA to mPFC signaling is equally altered in female mice. Injection of AAV-Arch3.0 constructs into the BLA followed by implantation of a fiberoptic cannula into the mPFC in sham and SNI operated female mice was carried out, and pain behavioral responses were measured in response to yellow light mediated activation of this inhibitory opsin. Our data reveal that Arch3.0 activation leads to a marked increase in paw withdrawal thresholds and latencies in response to mechanical and thermal stimuli, respectively. However, we did not observe nerve injury-induced changes in mPFC layer 5 pyramidal cell output in female mice. Hence, the observed light-induced analgesic effects may be due to compensation for dysregulated neuronal circuits downstream of the mPFC