Search

Uma Anand,1 Barbara Pacchetti,2 Praveen Anand,3 Mikael Hans Sodergren1,2 1Medical Cannabis Research Group, Department of Surgery and Cancer, Imperial College London, London, W12 0HS, UK; 2Curaleaf International Limited, London, EC2A 2EW, UK; 3Professor of Clinical Neurology, Department of Brain Sciences, Imperial College London, London, W12 0HS, UKCorrespondence: Uma Anand, Medical Cannabis Research Group, Department of Surgery and Cancer, Imperial College London, Hammersmith Campus, Du Cane Road, London, W12 0HS, UK, Tel +44 020 3313 2362, Fax +44 020 3313 3363, Email u.anand@imperial.ac.ukBackground: The endocannabinoid 2-Arachidonyl glycerol (2-AG) exerts dose-related anti-nociceptive effects, which are potentiated by the related but inactive 2-palmitoyl glycerol (2-PG) and 2-linoleoyl glycerol (2-LG). This potentiation of analgesia and other in vivo measures was described as the “entourage effect”. We investigated this effect on TRPV1 signalling in cultured dorsal root ganglion (DRG) nociceptors.Methods: Adult rat DRG neurons were cultured in medium containing NGF and GDNF at 37°C. 48 h later cultures were loaded with 2 μM Fura2AM for calcium imaging, and treated with 2-AG, 2-PG and 2-LG, individually or combined, for 5 min, followed by 1 μMol capsaicin. The amplitude and latency of capsaicin responses were measured (N=3– 7 rats, controls N=16), and analysed.Results: In controls, 1 μMol capsaicin elicited immediate calcium influx in a subset of neurons, with average latency of 1.27 ± 0.2 s and amplitude of 0.15 ± 0.01 Units. 2-AG (10– 100 μMol) elicited calcium influx in some neurons. In the presence of 2-AG (0.001– 100 μMol), capsaicin responses were markedly delayed in 64% neurons by up to 320 s (P< 0.001). 2-PG increased capsaicin response latency at 0.1 nMol-100 μMol (P< 0.001), in 60% neurons, as did 2-LG at 0.1– 100 μMol (P< 0.001), in 76% neurons. Increased capsaicin response latency due to 2-AG and 2-PG was sensitive to the CB2 but not to the CB1 receptor antagonist. Combined application of 1 μMol 2-AG, 5 μMol 2-PG and 10 μMol 2-LG, also resulted in significantly increased capsaicin response latency up to 281.5 ± 41.5 s (P< 0.001), in 96% neurons, that was partially restored by the CB2, but not the CB1 antagonist.Conclusion: 2-AG, 2-LG and 2-PG significantly delayed TRPV1 signalling in the majority of capsaicin-sensitive DRG neurons, that was markedly increased following combined application. Further studies of these endocannabinoids are required to identify the underlying mechanisms.Keywords: entourage effect, endocannabinoids, DRG neurons, nociception, analgesia

Uma Anand,1 Christian Oldfield,1 Barbara Pacchetti,2 Praveen Anand,1 Mikael H Sodergren1,2 1Faculty of Medicine, Imperial College London, Hammersmith Hospital, London, W12 ONN, UK; 2EMMAC Life Sciences Ltd, London, UKCorrespondence: Uma AnandFaculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London, W12 ONN, UKEmail u.anand@imperial.ac.ukBackground: The analgesic effects of Cannabis sativa are mediated by ∆9 tetrahydrocannabinol (THC), but the contributions of other bioactive complex components, including cannabigerol (CBG) and cannabidiol (CBD), are unclear. We describe the individual and combined effects of CBG, CBD and THC, on blocking capsaicin responses in dorsal root ganglion (DRG) neurons, in an in vitro model of nociceptor hypersensitivity.Materials and Methods: Adult rat DRG were dissected and enzyme digested to obtain a neuronal suspension in BSF2 medium containing 2% fetal calf serum, and the neurotrophic factors NGF and GDNF. After 48 h, cultured neurons were loaded with Fura-2 AM, to determine the effects of cannabinoids on capsaicin responses using calcium imaging. In control experiments, neurons were treated with vehicle, followed by 1 μM capsaicin. In cannabinoid treated cultures, CBG, CBD or THC were applied individually, or combined (1:1:1 ratio), followed by 1 μM capsaicin. Data from n = 6 experiments were analysed with Student’s t-test and Pearson’s correlation coefficient.Results: CBG, CBD and THC, applied individually, elicited dose-related calcium influx in a subset of DRG neurons, and a corresponding dose-related reduction of subsequent responses to capsaicin. Maximum inhibition of capsaicin responses was observed at 30 μM CBG, 100 μM CBD, and 100 μM THC individually, and with combined CBD+CBG+THC (1:1:1) at 90 μM. THC+CBD+CBG combined in a 1:1:1 proportion has the potential to enhance the potency of these compounds applied individually. There was a high correlation between cannabinoid-mediated calcium influx and reduction of capsaicin responses: CBG = − 0.88, THC = − 0.97, CBD = − 0.99 and combined CBG + THC + CBD = − 1.00.Conclusion: CBG, CBD and THC demonstrated potent dose-related inhibition of capsaicin responses in DRG neurons when applied individually in vitro, and enhanced when applied in combination, being most effective at 90 μM. Thus, efficacy and tolerability of THC could be improved in combination with CBG and CBD at optimal concentrations, which deserve further studies in vivo.Keywords: cannabinoid, CBG, CBD, THC, TRPV1, DRG neurons, pain

Uma Anand,1 Ben Jones,2 Yuri Korchev,3 Stephen R Bloom,2 Barbara Pacchetti,4 Praveen Anand,5 Mikael Hans Sodergren1 1Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital, London W12 ONN, UK; 2Department of Metabolism, Digestion and Reproduction, Imperial College London, Hammersmith Hospital, London W12 ONN, UK; 3Nanomedicine Laboratory, BN5 Commonwealth Building, Imperial College London, Hammersmith Hospital, London W12 ONN, UK; 4EMMAC Life Sciences Ltd, London EC2A 2EW, UK; 5Peripheral Neuropathy Unit, Department of Brain Sciences, Imperial College London, Hammersmith Hospital, London W12 ONN, UKCorrespondence: Uma Anand Email u.anand@imperial.ac.ukIntroduction: Cannabidiol (CBD) is reported to produce pain relief, but the clinically relevant cellular and molecular mechanisms remain uncertain. The TRPV1 receptor integrates noxious stimuli and plays a key role in pain signaling. Hence, we conducted in vitro studies, to elucidate the efficacy and mechanisms of CBD for inhibiting neuronal hypersensitivity in cultured rat sensory neurons, following activation of TRPV1.Methods: Adult rat dorsal root ganglion (DRG) neurons were cultured and supplemented with the neurotrophic factors NGF and GDNF, in an established model of neuronal hypersensitivity. Neurons were stimulated with CBD (Adven 150, EMMAC Life Sciences) at 1, 10, 100 nMol/L and 1, 10 and 50 μMol/L, 48 h after plating. In separate experiments, DRG neurons were also stimulated with capsaicin with or without CBD (1 nMol/L to10 μMol/L), in a functional calcium imaging assay. The effects of the adenylyl cyclase activator forskolin and the calcineurin inhibitor cyclosporin were determined. We also measured forskolin-stimulated cAMP levels, without and after treatment with CBD, using a homogenous time-resolved fluorescence (HTRF) assay. The results were analysed using Mann-Whitney test.Results: DRG neurons treated with 10 and 50 μMol/L CBD showed calcium influx, but not at lower doses. Neurons treated with capsaicin demonstrated robust calcium influx, which was dose-dependently reduced in the presence of low dose CBD (IC50 = 100 nMol/L). The inhibition or desensitization by CBD was reversed in the presence of forskolin and cyclosporin. Forskolin-stimulated cAMP levels were significantly reduced in CBD treated neurons.Conclusion: CBD at low doses corresponding to plasma concentrations observed physiologically inhibits or desensitizes neuronal TRPV1 signalling by inhibiting the adenylyl cyclase – cAMP pathway, which is essential for maintaining TRPV1 phosphorylation and sensitization. CBD also facilitated calcineurin-mediated TRPV1 inhibition. These mechanisms may underlie nociceptor desensitization and the therapeutic effect of CBD in animal models and patients with acute and chronic pain.Keywords: cannabidiol, CBD, chronic pain, DRG neurons, cAMP, calcium imaging, desensitization