Differences in receptor binding affinity of several phytocannabinoids do not explain their effects on neural cell cultures

Highlights

• We determined CB1 and CB2 receptor affinity of several phytocannabinoids.

• We evaluated cell toxicity of several phytocannabinoids in neuroblastoma cells.

• We evaluated cell toxicity of several phytocannabinoids in murine dopaminergic neurons.

• Toxic effects of phytocannabinoids do not correspond to binding to the CB1 receptor.

Abstract

Phytocannabinoids are potential candidates for neurodegenerative disease treatment. Nonetheless, the exact mode of action of major phytocannabinoids has to be elucidated, but both, receptor and non-receptor mediated effects are discussed. Focusing on the often presumed structure–affinity-relationship, Ki values of phytocannabinoids cannabidiol (CBD), cannabidivarin (CBDV), cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), THC acid (THCA) and THC to human CB1 and CB2 receptors were detected by using competitive inhibition between radioligand [³H]CP-55,940 and the phytocannabinoids. The resulting Ki values to CB1 range from 23.5 μM (THCA) to 14,711 μM (CBDV), whereas Ki values to CB2 range from 8.5 μM (THCA) to 574.2 μM (CBDV). To study the relationship between binding affinity and effects on neurons, we investigated possible CB1 related cytotoxic properties in murine mesencephalic primary cell cultures and N18TG2 neuroblastoma cell line. Most of the phytocannabinoids did not affect the number of dopaminergic neurons in primary cultures, whereas propidium iodide and resazurin formation assays revealed cytotoxic properties of CBN, CBDV and CBG. However, THC showed positive effects on N18TG2 cell viability at a concentration of 10 μM, whereas CBC and THCA also displayed slightly positive activities. These findings are neither linked to structural characteristics nor to the receptor binding affinity therewith pointing to another mechanism than a receptor mediated one.

Introduction

In recent years, phytocannabinoids as pharmacological active compounds isolated of the plant Cannabis sativa are discussed for neurodegenerative disease treatment, especially in Parkinson's or Alzheimer's disease research. It is known that tetrahydrocannabinol (THC) participates e.g. in cascades of inflammatory processes, and it is already used as a therapeutic drug because of its antiemetic and analgesic properties (Borgelt et al., 2013, Saito et al., 2012).

According to recent literature, the main cannabinoid receptors, CB1 and CB2, contain seven transmembrane spanning domains. They are both coupled by G_i/o proteins to adenylyl cyclase in a negative way, and also to the mitogen-activating protein (MAP) kinase in an activating manner (Howlett et al., 2002). However, CB1 can also act through G_S proteins to activate adenylate cyclase (Pertwee, 2006) and affects ion fluxes of certain potassium and calcium channels (Howlett, 2005). In general, the mammalian CB1 is densely expressed in mesencephalon, basal ganglia, the cerebellum and the hippocampus (Maldonado et al., 2011) and is located presynaptically on neuronal membranes. Further on, it is described that CB1 receptor distribution on fetal brain cells differs from those observed in adult brains due to brain formation development (Hernández et al., 2000). Its detailed three-dimensional structure still has to be determined as no computer model for prediction of phytocannabinoid receptor binding is yet available (Fig. 1). When receptors are activated by phytocannabinoids, the cyclic adenosine monophosphate (cAMP) level is decreased by inhibiting adenylate cyclase and stimulating MAP kinase. This process shortens the duration of presynaptic action potential thereby limiting the Ca^2 + entry as well as the activation of phospholipases A and C (Howlett, 2005). This leads to a reduction in excitatory and inhibitory transmitter release (Elphick and Egertová, 2001, Pertwee, 2006). In the brain, the CB2 receptor which possesses a protein sequence similarity of 48% to CB1 (Howlett et al., 2002), is especially expressed in microglia depending on the activated state of the cell (Carlisle et al., 2002). This receptor is associated with the immune system and neuron-inflammation response modulation (Stella, 2010, Galiègue et al., 1995). It is assumed that CB2 activation leads to a suppression of microglial phagocytosis activation, which might be essential for Alzheimer's disease treatment since this disease is accompanied by chronic inflammation and loss of neurons (Ehrhart et al., 2005). CB2 agonists are supposed to serve as alternatives for conventional therapeutic drugs for inflammation of the central nervous system and neuropathic pain treatment (Cheng and Hitchcock, 2007).

THC is described as an agonist to CB1 and CB2 and, besides the generally known psychoactive effects, its derivatives or even itself is used to treat anorexia in AIDS patients or diminish nausea or vomiting in cancer patients undergoing chemotherapy (Pertwee, 2009). During the last years, phytocannabinoid studies revealed various beneficial properties of THC. Compared to these findings, the precursor of THC, THCA, as well as the metabolite cannabinol (CBN) do not seem to have psychoactive properties, but analgesic, anti-inflammatory and neuroprotective effects are described (Ruhaak et al., 2001, Formukong et al., 1988). The second main compound of Cannabis, cannabidiol (CBD) has lower psychoactive properties and is therefore considered as a promising candidate for medical purposes. Due to its several benefits, CBD is discussed as a therapeutic agent for diabetes, ischemia and cancer treatment. Preclinical studies already revealed positive effects of CBD on these diseases (Izzo et al., 2009). Also its homologue, cannabidivarin (CBDV) revealed anticonvulsant activity in rodents (Hill et al., 2012). Further on, cannabigerol (CBG) and cannabichromene (CBC) are both described as non-psychoactive and anti-inflammatory substances (Colasanti, 1990, Borrelli et al., 2013, Izzo et al., 2012), but additional studies are required to verify definite medical effects.

Phytocannabinoids are promising therapeutic agents especially for neurodegenerative disease treatment, but their exact mode of action has to be elucidated to prevent severe side-effects and damages to the brain. Therefore, we determined the binding affinity of the main compounds of Cannabis to CB1 and CB2 and their influences on the cell viability of primary dopaminergic as well as N18TG2 neuroblastoma cells, both of which are known to express CB1 receptors (Howlett et al., 2009).