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 Cav2.2 –CRMP2 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.