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

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

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

Mastering tricyclic ring systems for desirable functional cannabinoid activity

There is growing interest in using cannabinoid receptor 2 (CB2) agonists for the treatment of neuropathic pain and other indications. In continuation of our ongoing program aiming for the development of new small molecule cannabinoid ligands, we have synthesized a novel series of carbazole and γ-carboline derivatives. The affinities of the newly synthesized compounds were determined by a competitive radioligand displacement assay for human CB2 cannabinoid receptor and rat CB1 cannabinoid receptor. Functional activity and selectivity at human CB1 and CB2 receptors were characterized using receptor internalization and [35S]GTP-γ-S assays. The structure–activity relationship and optimization studies of the carbazole series have led to the discovery of a non-selective CB1 and CB2 agonist, compound 4. Our subsequent research efforts to increase CB2 selectivity of this lead compound have led to the discovery of CB2 selective compound 64, which robustly internalized CB2 receptors. Compound 64 had potent inhibitory effects on pain hypersensitivity in a rat model of neuropathic pain. Other potent and CB2 receptor–selective compounds, including compounds 63 and 68, and a selective CB1 agonist, compound 74 were also discovered. In addition, we identified the CB2 ligand 35 which failed to promote CB2 receptor internalization and inhibited compound CP55,940-induced CB2 internalization despite a high CB2 receptor affinity. The present study provides novel tricyclic series as a starting point for further investigations of CB2 pharmacology and pain treatment.Fil: Petrov, Ravil R.. University Of Montana; Estados UnidosFil: Knight, Lindsay. Indiana University; Estados UnidosFil: Chen, Shao Rui. University Of Texas; Estados UnidosFil: Wager Miller, Jim. Indiana University; Estados UnidosFil: McDaniel, Steven W.. University Of Montana; Estados UnidosFil: Diaz, Fanny. University Of Montana; Estados UnidosFil: Barth, Francis. Sanofi-aventis R&D; FranciaFil: Pan, Hui Lin. University Of Texas; Estados UnidosFil: Mackie, Ken. Indiana University; Estados UnidosFil: Cavasotto, Claudio Norberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires; ArgentinaFil: Diaz, Philippe. University Of Montana; Estados Unido