Effect of ABT-639 on Cav3.2 channel activity and its analgesic actions in mouse models of inflammatory and neuropathic pain.

Cav3.2 T-type calcium channels are important targets for pain relief in rodent models of inflammatory and neuropathic pain. Even though many T-type channel blockers have been tested in mice, only one molecule, ABT-639, has been tested in phase II clinical studies and did not produce analgesic effects over placebo. Here we examined the effects of ABT-639 on Cav3.2 channel activity in tsA-201 cells and dorsal root ganglion (DRG) neurons, in comparison with another established Cav3.2 inhibitor Z944. These experiments revealed that Z944 mediated ∼100-fold more potent inhibition of Cav3.2 currents than ABT-639, with the latter blocking channel activity by less than 15 percent when applied at a concentration of 30 µM. A slight increase in ABT-639 potency was observed at more depolarized holding potentials, suggesting that this compound may act preferentially on inactivated channels. We tested the effects of both compounds in the Complete Freund's Adjuvant (CFA) model of chronic inflammatory pain, and in partial sciatic nerve injury model of neuropathic pain in mice. In the neuropathic pain model, both Z944 and ABT-639 reversed mechanical hypersensitivity to similar degrees when delivered systemically, but remarkably, when delivered intrathecally, only Z944 was effective. In the CFA model, both compounds reversed thermal hyperalgesia upon systemic delivery, but only Z944 mediated pain relief upon intrathecal delivery, indicating that ABT-639 acts primarily at peripheral sites. ABT-639 lost its analgesic effects in CFA treated Cav3.2 null mice, indicating that these channels are essential for ABT-639-mediated pain relief despite its poor inhibition of Cav3.2 currents.

Icariside II, a Prenyl-Flavonol, Alleviates Inflammatory and Neuropathic Pain by Inhibiting T-Type Calcium Channels and USP5-Cav3.2 Interactions.

Cav3.2 channels play an important role in the afferent nociceptive pathway, which is responsible for both physiological and pathological pain transmission. Cav3.2 channels are upregulated during neuropathic pain or peripheral inflammation in part due to an increased association with the deubiquitinase USP5. In this study, we investigated nine naturally occurring flavonoid derivatives which we tested for their abilities to inhibit transiently expressed Cav3.2 channels and their interactions with USP5. Icariside II (ICA-II), one of the flavonols studied, inhibited the biochemical interactions between USP5 and Cav3.2 and concomitantly and effectively blocked Cav3.2 channels. Molecular docking analysis predicts that ICA-II binds to the cUBP domain and the Cav3.2 interaction region. In addition, ICA-II was predicted to interact with residues in close proximity to the Cav3.2 channel's fenestrations, thus accounting for the observed blocking activity. In mice with inflammatory and neuropathic pain, ICA-II inhibited both phases of the formalin-induced nocifensive responses and abolished thermal hyperalgesia induced by injection of complete Freund's adjuvant (CFA) into the hind paw. Furthermore, ICA-II produced significant and long-lasting thermal anti-hyperalgesia in female mice, whereas Cav3.2 null mice were resistant to the action of ICA-II. Altogether, our data show that ICA-II has analgesic activity via an action on Cav3.2 channels.

The natural product argentatin C attenuates postoperative pain via inhibition of voltage-gated sodium and T-type voltage-gated calcium channels.

BACKGROUND AND PURPOSE: Postoperative pain occurs in as many as 70% of surgeries performed worldwide. Postoperative pain management still relies on opioids despite their negative consequences, resulting in a public health crisis. Therefore, it is important to develop alternative therapies to treat chronic pain. Natural products derived from medicinal plants are potential sources of novel biologically active compounds for development of safe analgesics. In this study, we screened a library of natural products to identify small molecules that target the activity of voltage-gated sodium and calcium channels that have important roles in nociceptive sensory processing. EXPERIMENTAL APPROACH: Fractions derived from the Native American medicinal plant, Parthenium incanum, were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion (DRG) neurons. Further separation of these fractions yielded a cycloartane-type triterpene identified as argentatin C, which was additionally evaluated using whole-cell voltage and current-clamp electrophysiology, and behavioural analysis in a mouse model of postsurgical pain. KEY RESULTS: Argentatin C blocked the activity of both voltage-gated sodium and low-voltage-activated (LVA) calcium channels in calcium imaging assays. Docking analysis predicted that argentatin C may bind to NaV 1.7-1.9 and CaV 3.1-3.3 channels. Furthermore, argentatin C decreased Na+ and T-type Ca2+ currents as well as excitability in rat and macaque DRG neurons, and reversed mechanical allodynia in a mouse model of postsurgical pain. CONCLUSION AND IMPLICATIONS: These results suggest that the dual effect of argentatin C on voltage-gated sodium and calcium channels supports its potential as a novel treatment for painful conditions.

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

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.

Protective effects of cerebrolysin in a rat model of optic nerve crush.

To investigate the effects of cerebrolysin (Cbl) on optic nerves (ON) and retinal ganglion cells (RGC) in a rat model of ON crush. Rats received intravitreal injection of Cbl (n = 20), intra-ON injection of Cbl (n = 20), intraperitoneal injection (IPI) of Cbl (n = 20), or phosphate buffered saline (PBS; n = 20) every day for 2 weeks after ON crush injury. At 3 weeks post-trauma, RGC density was counted by retrograde labeling with FluoroGold and visual function was assessed by flash visual-evoked potentials. Activities of microglia after insults were quantified by immunohistochemical analysis of the presence of ED1 in the optic nerve. At 3 weeks postcrush, the densities of RGCs in the Cbl-IVI group (1125 ± 166/mm(2)) and in the Cbl-IPI treatment group (1328 ± 119/mm(2)) were significantly higher than those in the PBS group (641 ± 214/mm(2)). The flash visual-evoked potential measurements showed that latency of the P1 wave was significantly shorter in the Cbl-IVI- and Cbl-IPI-treated groups (105 ± 4 ms and 118 ± 26 ms, respectively) than in the PBS-treated group (170 ± 20 ms). However, only Cbl IPI treatment resulted in a significant decrease in the number of ED1-positive cells at the lesion sites of the ON (5 ± 2 cells/vs. 30 ± 4 cells/high-power field in control eyes). Treatment with intra-ON injection of Cbl was harmful to the optic nerve in the crush model. Systemic administration of Cbl had neuroprotective effects on RGC survival and visual function in the optic nerve crush model.