Modulatory Effects of “Minor” Cannabinoids in an in vitro Model of Neuronal Hypersensitivity

Uma Anand,1 Praveen Anand,2 Mikael H Sodergren1,3 1Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Hammersmith Hospital, London, W12 0HS, UK; 2Department of Brain Sciences, Faculty of Medicine, Imperial College London, Hammersmith Hospital, London, W12 0HS, UK; 3Curaleaf International, Guernsey, UKCorrespondence: Uma Anand, Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London, W12 0HS, UK, Tel +44 20 3313 3319, Email [email protected]: Effective treatment for neuropathic pain remains an unmet clinical need. The therapeutic benefits of the Cannabis plant are well known, especially for pain relief. Here, we have assessed ten “minor” cannabinoids for their analgesic effects in an established model of neuronal hypersensitivity, a key mechanism which underlies neuropathic pain.Methods: Adult rat DRG neurons were cultured in medium containing 100 ng/mL nerve growth factor (NGF) and 50 ng/mL glial cell-line derived neurotrophic factor (GDNF) for 48 hours to sensitize the neurons. Ca2+ imaging was used to measure the responses to pain stimulation using capsaicin, and to determine the modulatory effects of the cannabinoids, in individual neurons.Results: Control neurons (nociceptors) showed robust responses of Ca2+ influx to capsaicin application, while neurons treated with ten minor cannabinoids tetrahydrocannabiorcol (THCC), cannabitriol (CBT), cannabidivarin (CBDV), cannabinol (CBN), cannabichromene (CBC), cannabichromevarin (CBCV), cannabicitran (CBCT), cannabigerol monomethyl ether (CBGM), tetrahydrocannabutol (THCB) or tetrahydrocannabiphorol (THCP), at concentrations of 0.001– 100 μM, showed differential dose-related effects on the responses to capsaicin. Ca2+ influx in response to capsaicin application was completely inhibited for each compound in 35– 78% capsaicin-sensitive neurons, while other neurons showed reduced responses. The opioid receptor agonist morphine and α 2δ 1- Ca2+ channel inhibitor gabapentin were also tested for comparison and showed similar results. All the cannabinoids tested here inhibited calcium influx in response to capsaicin, and two, namely, CBN and THCC elicited calcium influx at higher doses. Inhibition of Ca2+ influx due to cannabichromene (CBC) was reversed by the potassium channel inhibitor Tertiapin Q.Conclusion: All the cannabinoids tested here inhibited TRPV1 signalling. CBC targeted K+ channels to block TRPV1 mediated Ca2+ influx, demonstrating potential analgesic effects in vitro.Plain Language Summary: Nerve damage due to disease or injury causes neuropathic pain which is very difficult to treat and affects the quality of life of affected individuals. The cannabis plant is known for its medicinal properties for millennia, especially for treating pain. This is due to its constituents such as THC which is psychoactive, limiting its usefulness. There are several other constituents also known as cannabinoids, which are known not to be psychoactive, and whose medicinal effects are unknown. In this study, we examined ten different cannabis constituents for their ability to block pain signals in nerve cells which sense pain in the body. This was done by growing nerve cells or sensory neurons that sense pain, in a dish and treating them with NGF and GDNF that increase the nerve signals which lead to neuropathic (nerve) pain. We then used capsaicin, the hot ingredient of chilli peppers, to stimulate these neurons (nerve cells) to generate pain signalling. These signals were measured by using a calcium indicator dye to identify which neurons were responding and how large their responses were to capsaicin. We observed that treating the neurons with individual cannabinoids either completely blocked or reduced the capsaicin responses to a very large extent, in most neurons. We have also shown that the cannabinoid cannabichromene blocked responses to capsaicin by activating the potassium channels in the neuron membrane, by using bee venom, which blocks potassium channels. As different cannabinoids inhibited capsaicin responses to a greater or lesser extent, we can identify which cannabinoids are likely to be most useful for blocking pain signalling to develop new drugs for providing pain relief without side effects.Keywords: chronic pain, analgesia, cannabinoids, sensory neurons, tertiapin Q