Spinal α3 GlyRs have been proposed as an important target for pain treatment. However, the α3 GlyR-based therapeutic agents in the treatment of chronic pain or other diseases are not yet available. The current study has provided several lines of evidence to suggest that CBD and DH-CBD suppress persistent inflammatory and neuropathic pain by targeting the α3 GlyRs in rodents. Consistent with in vitro observation that DH-CBD was more efficacious than CBD in potentiating IGly, DH-CBD was more potent than CBD in reducing chronic pain. DH-CBD also attenuated i.t. PGE2-induced persistent pain hypersensitivity in mice. Several lines of evidence suggest that cannabinoid-induced analgesia is mediated through the α3 GlyR-dependent pathway. First, both CBD and DH-CBD–induced analgesic effects were significantly reduced in mice lacking the α3 GlyRs but not in mice lacking the CB1 and CB2 receptors. Second, DD-CBD inhibited DH-CBD–induced potentiation of the α3 GlyRs and analgesic effect in chronic pain. Third, structural and functional analysis reveals that the magnitude of the cannabinoid-induced analgesic effect was correlated with cannabinoid potentiation of the α3 GlyRs but not with the cannabinoid binding affinity for CB1 and CB2 receptors. Because of substantially reduced CB1 binding affinity, DH-CBD, even at high concentrations (50 mg/kg i.p.), did not produce the psychoactive effects commonly associated with cannabinoid activation of CB1 receptors. Collectively, glycinergic cannabinoids represent a new class of therapeutic agents that selectively relieve pathological pain by targeting the α3 GlyRs.
The data from NMR titration and NOESY experiments strongly indicates the direct interaction of CBD with residue S296. The change in NMR signal intensity upon CBD titration suggests that the protein motion at S296 is sensitive to CBD binding. The molecular model of the α3 GlyR TM domains reveals that S296 is located near the intracellular end of the TM3 helix, with its side chain facing the lipids. Direct interaction of CBD with α3 GlyR-TM protein was confirmed by the intermolecular NOESY cross peaks between CBD and the protein. There is a transition from the free to the CBD-bound state as indicated by the observation that a free S296 resonance and a bound resonance appeared sequentially with an intermediate coexistence of both peaks. This finding also favors a protein conformational change at S296 in the presence of CBD.
Our molecular dynamics simulations suggest that CBD-α3 GlyR binding interactions involve the S296 residue of the α3 GlyR TM domain on the principal side, and the lipid molecules on the complementary side. The molecular docking analyses reveal that the binding free energies of the potentiator CBD and the inhibitor DD-CBD at this protein-lipid interfacial site are very similar, suggesting that the binding affinities are within the same order of magnitude. This finding favors the idea that the DD-CBD inhibition of CBD potentiation of IGly is through a competitive mechanism by acting near or at the same site involving S296. It should be pointed out that both DH-CBD and DD-CBD are modulators of GlyRs. Unlike orthosteric ligands, these modulators bind to allosteric sites. The molecular nature of competitiveness of these modulators remains unknown. Although the data from the in vitro study together with the result of molecular dynamics simulations suggest that DD-CBD may compete for the same site with CBD, our experimental data alone are not sufficient to conclude that DD-CBD acts as a competitive antagonist of CBD. Future studies should be performed to examine the effect of DD-CBD on the purified α1 or α3 GlyR-TM proteins using NMR analysis.
The kinetic analysis suggests that DH-CBD increases the agonist binding affinity of GlyRs. DH-CBD accelerated receptor activation rate and, on the other side, slowed receptor deactivation rate. Although receptor activation time represents agonist binding and/or channel gating, receptor deactivation time reflects the kinetics of agonist unbinding/channel closing or combination of two. The DH-CBD–induced changes in GlyR kinetics appears relevant to DH-CBD–induced potentiation of IGly because both S296A and DD-CBD, which inhibited DH-CBD potentiation of IGly, abolished DH-CBD alteration of receptor gating kinetics. DH-CBD shifted in a parallel manner the glycine-concentration response curve to the left, favoring a hypothesis that DH-CBD allosterically increases the agonist binding affinity of GlyRs. However, this notion should be made with caution, as slow deactivation time of GlyRs could reflect a slow channel closing, unbinding rate, or both in the presence of DH-CBD. In this scenario, slow deactivation time could be a result of slow wash time of DH-CBD because of its hydrophobic nature. One can also argue that the deactivation rate could be contaminated with desensitization rate. However, it is unlikely to be the case in our study because DH-CBD did not significantly affect GlyR desensitization rate while slowing deactivation.
Several preclinical persistent and chronic pain models were tested in this study. Intraplantar CFA injection has been widely used as an inflammatory pain model. Both systemic and i.t. injection of DH-CBD significantly reduced mechanical pain hypersensitivity induced by CFA. PGE2
is one of the major proinflammatory substances that promote nociceptive processing in the spinal cord and peripheral tissues upon various noxious stimuli such as CFA (Vanegas and Schaible, 2001
; Harvey et al., 2004
; Zeilhofer et al., 2012
). Consistent with the observations in CFA-induced inflammatory pain models, DH-CBD also produced an analgesic effect in i.t. injection of PGE2
-induced nociception in mice. More importantly, i.t. application of DH-CBD exerted potent inhibition of chronic neuropathic pain in rats. Neuropathic pain is a substantial health issue because currently available therapies are far from satisfactory. Although neuropathic pain and inflammatory pain differ in pathogenesis, molecular mechanisms, and treatments, these two types of persistent pain may be modulated by similar synaptic mechanism at the spinal level (Zeilhofer et al., 2012
). The data presented in our study suggest that the α3 GlyR contributes to the mechanisms that modulate both types of pain. Yet, we cannot exclude the potential involvement of other subtypes of GlyRs in the pain modulation. In addition to reducing chronic pain, DH-CBD can also attenuate acute pain. DH-CBD increased the time latency in the tail flick reflex in mice (Xiong et al., 2011
). This analgesic effect induced by DH-CBD was abolished in mice depleted with the α3 GlyRs but remained intact in mice depleted with the CB1 receptors. Consistent with this idea, i.t. application of DH-CBD at higher doses also significantly increased the contralateral PWL from baseline in both inflammatory and neuropathic pain in rats.
i.t. application of DH-CBD seems the most efficacious way to suppress mechanical and thermal pain hypersensitivity in both inflammatory and neuropathic pain conditions. This idea is consistent with the distinct distribution pattern of the α3 GlyRs in lamina II of the spinal dorsal horn (Harvey et al., 2004
). Moreover, the α3 GlyRs are either absent or less expressed in primary sensory neurons such as dorsal root ganglion neurons (Lynch, 2004
). Oral administration of cannabinoids is not an ideal route for drug delivery because primary cannabinoids are largely metabolized by the liver (Huestis and Pertwee, 2005
). It is worth mentioning that one of the common practices to deliver medical cannabis to humans is via sublingual spray, which bypasses the liver and delivers drugs directly into the blood stream (Nurmikko et al., 2007
). Collectively, we propose that i.t. injection of cannabinoids should be the most efficacious route to treat patients with chronic neuropathic pain.
Among 11 cannabinoid analogues evaluated in this study, DH-CBD has emerged as an ideal glycinergic cannabinoid that can be used to treat chronic pain without causing aversive effects. Unlike some of the analogues that not only showed relatively high efficacy in potentiating IGly but also demonstrated relatively high affinity to bind to CB1 receptors, DH-CBD displayed a low affinity for CB1 receptors and at the same time is one of the most efficacious positive modulators of GlyRs. It has been shown consistently in our correlation analysis that most psychoactive effects induced by cannabinoids are associated with CB1 receptor binding affinity but not cannabinoid-induced potentiation of GlyRs. Conversely, the cannabinoid-induced analgesic effect in chronic pain is correlated with cannabinoid potentiation of GlyRs but not with cannabinoid binding affinity to CB1 receptors. These principles may apply to future studies in developing a new generation of glycinergic cannabinoids in the treatment of chronic pain. In addition to lacking a psychoactive side effect, glycinergic cannabinoids are unlikely to develop drug tachyphylaxis or tolerance, one of the major barriers for long-term pain management with currently available clinical agents. Repeated application of DH-CBD either i.p. or i.t. exhibited similar analgesic potency in both inflammatory and neuropathic pain. This finding is not unexpected because glycinergic cannabinoids act on the GlyRs as allosteric modulators instead of agonists or antagonists.
Collectively, we have provided evidence to suggest that glycinergic cannabinoids are ideal therapeutic agents in the treatment of inflammatory and neuropathic pain. They can effectively attenuate pathological pain without significantly causing major psychoactive side effect and analgesic tolerance. The mechanistic details of drug–receptor interaction could help to develop novel agents for the treatment of painful conditions and other diseases involving GlyR impairment.