Figure 7. Sensory neuron excitability is decreased by CBD3063.(A) Representative traces in response to the indicated current injection steps from rat DRG neurons treated 0.1% DMSO (control; black circles) or 20 µM CBD3063 (cyan squares). (B) Quantification of resting membrane potential in millivolts (mV) in the two conditions. (C) Quantification of the rheobase in the presence of DMSO or 20 µM CBD3063. (D) Summary of the number of evoked action potentials in response to current injection between 0-120 pA. N=6 cells; p value as indicated; Mann-Whitney test (B and C) and Multiple Mann-Whitney test. Error bars indicate mean ± SEM.
Disruption of Cav2.2CRMP2 binding confers antinociception
In humans, targeting auxiliary subunits that regulate Cav2.2 channels is effective against chronic pain. For example, targeting α2δ-1 subunit with Gabapentin and Pregabalin alleviates pain by interfering with Cav2.2–α2δ-1 interaction (Bauer, Rahman, Tran-van-Minh, Lujan, Dickenson & Dolphin, 2010; Hendrich et al., 2008; Sutton, Martin, Pinnock, Lee & Scott, 2002). Consequently, we tested if interrupting CRMP2 binding to Cav2.2 with CBD3063 could be antinociceptive in a rat model of spared nerve injury (SNI). This model of neuropathic pain results in long-lasting thermal hyperalgesia and mechanical allodynia on the affected hind paw (Decosterd & Woolf, 2000). We used male rats for these studies since interrupting the Cav2.2–CRMP2 interaction in vivo does not display sex dimorphism with equivalent effects observed in females (Ju, Li, Allette, Ripsch, White & Khanna, 2013; Ripsch, Ballard, Khanna, Hurley, White & Khanna, 2012) and males (Fischer, Pan, Vilceanu, Hogan & Yu, 2014; Francois-Moutal et al., 2015; Moutal et al., 2018).
Ligation of the tibial and peroneal nerves in male rats resulted in the development of mechanical hypersensitivity (Figure 8 ). We chose to administer CBD3063 intrathecally (0.3 μg/kg) to directly reach the site of action of Cav2.2 in the SDH, where the central termini of primary afferent fibers synapse with second order neurons in the SDH. A single intrathecal administration of CBD3063, 7 days following SNI, resulted in a significant reversal of mechanical allodynia that started 60 minutes post-injection and lasted up to 36 hours (Figure 8A ). A significant increase in the area under the curve was observed after injection of CBD3063 compared to the control group (DMSO: 7.650 ± 0.84; CBD3063: 57.05 ± 3.84; Figure 8B ).
To assess the long-term antinociceptive effect of CBD3063, rats were injected with CBD3063 (0.3 µg/kg) starting 7 days after SNI surgery and once a day for 14 days. On days 1, 3, 5, 7, 10 and 14, paw withdrawal thresholds were determined at six hours following each injection (Figure 8C ). Repeated administration of CBD3063 resulted in a prolonged reversal of SNI-induced mechanical allodynia (Figure 8C ). A significant increase in the area under the curve was observed following treatment with CBD3063 for 14 days (DMSO: 3.37 ± 0.86; CBD3063: 47.26 ± 8.22; Figure 8D ). These results demonstrate that targeting the Cav2.2–CRMP2 interaction results in a long-lasting reversal of mechanical hypersensitivity, without any tolerance (loss of antinociception), in a nerve injury model of persistent pain.