Big conductance calcium-activated potassium channel openers control spasticity without sedation.

BACKGROUND AND PURPOSE: Our initial aim was to generate cannabinoid agents that control spasticity, occurring as a consequence of multiple sclerosis (MS), whilst avoiding the sedative side effects associated with cannabis. VSN16R was synthesized as an anandamide (endocannabinoid) analogue in an anti-metabolite approach to identify drugs that target spasticity. EXPERIMENTAL APPROACH: Following the initial chemistry, a variety of biochemical, pharmacological and electrophysiological approaches, using isolated cells, tissue-based assays and in vivo animal models, were used to demonstrate the activity, efficacy, pharmacokinetics and mechanism of action of VSN16R. Toxicological and safety studies were performed in animals and humans. KEY RESULTS: VSN16R had nanomolar activity in tissue-based, functional assays and dose-dependently inhibited spasticity in a mouse experimental encephalomyelitis model of MS. This effect occurred with over 1000-fold therapeutic window, without affecting normal muscle tone. Efficacy was achieved at plasma levels that are feasible and safe in humans. VSN16R did not bind to known CB1 /CB2 /GPPR55 cannabinoid-related receptors in receptor-based assays but acted on a vascular cannabinoid target. This was identified as the major neuronal form of the big conductance, calcium-activated potassium (BKCa ) channel. Drug-induced opening of neuronal BKCa channels induced membrane hyperpolarization, limiting excessive neural-excitability and controlling spasticity. CONCLUSIONS AND IMPLICATIONS: We identified the neuronal form of the BKCa channel as the target for VSN16R and demonstrated that its activation alleviates neuronal excitability and spasticity in an experimental model of MS, revealing a novel mechanism to control spasticity. VSN16R is a potential, safe and selective ligand for controlling neural hyper-excitability in spasticity.

Genetic background can result in a marked or minimal effect of gene knockout (GPR55 and CB2 receptor) in experimental autoimmune encephalomyelitis models of multiple sclerosis.

Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 (tm1Zim)) CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 (Dgen)) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 (tm1Zim) mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some transgenic/gene knockout and other studies on low-EAE susceptibility backgrounds with inconsistent disease course and susceptibility.

Inhibition of human neutrophil chemotaxis by endogenous cannabinoids and phytocannabinoids: evidence for a site distinct from CB1 and CB2.

Here, we show a novel pharmacology for inhibition of human neutrophil migration by endocannabinoids, phytocannabinoids, and related compounds. The endocannabinoids virodhamine and N-arachidonoyl dopamine are potent inhibitors of N-formyl-l-methionyl-l-leucyl-l-phenylalanine-induced migration of human neutrophils, with IC(50) values of 0.2 and 8.80 nM, respectively. The endocannabinoid anandamide inhibits human neutrophil migration at nanomolar concentrations in a biphasic manner. The phytocannabinoid (-)-cannabidiol is a partial agonist, being approximately 40 fold more potent than (+)-cannabidiol; abnormal-cannabidiol is a full agonist. Furthermore, the abnormal-cannabidiol (CBD) analog trans-4-[3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-methyl-1,3-benzenediol (O-1602) inhibits migration, with an IC(50) value of 33 nM. This reported profile of agonist efficacy and potency parallels with the pharmacology of the novel "abnormal-cannabidiol" receptor or a related orphan G protein-coupled receptor, which are already known to modulate cell migration. Although having no effect alone, N-arachidonoyl l-serine attenuated inhibition of human neutrophil migration induced by anandamide, virodhamine, and abnormal-CBD. Our data also suggest that there is cross-talk/negative co-operativity between the cannabinoid CB(2) receptor and this novel target: CB(2) receptor antagonists significantly enhance the inhibition observed with anandamide and virodhamine. This study reveals that certain endogenous lipids, phytocannabinoids, and related ligands are potent inhibitors of human neutrophil migration, and it implicates a novel pharmacological target distinct from cannabinoid CB(1) and CB(2) receptors; this target is antagonized by the endogenous compound N-arachidonoyl l-serine. Furthermore, our findings have implications for the potential pharmacological manipulation of elements of the endocannabinoid system for the treatment of various inflammatory conditions.

An in situ model to study the toothpaste abrasion of enamel.

OBJECTIVES: In order to understand the clinical relevance of dentifrice abrasivity on the dentition in vivo, an in situ enamel wear model has been developed. MATERIALS AND METHODS: Polished human enamel blocks were indented with a Knoop diamond, attached to dentures and worn by adult volunteers for 24 h per day. The blocks were brushed for 30 s, twice per day with dentifrices of known relative dentine abrasivity (RDA) and relative enamel abrasivity (REA). The dentifrices used were either dentifrice A (RDA=85, REA=3.4), dentifrice B (RDA=189, REA=2.0) or dentifrice C (RDA=132, REA=42.7). After 28 days, the blocks were removed and the geometry of each Knoop indent was remeasured. From the baseline and post-treatment values of indent length, the amount of enamel wear was calculated from the change in the indent depth. RESULTS: The median values for enamel wear of dentifrices A, B and C were -0.02, 0.01 and -0.48 microm, respectively. The differences between dentifrice C and dentifrices A and B were of statistical significance. CONCLUSION: This study has demonstrated the usefulness of an in situ technique for investigating the relationship between the abrasivity of a dentifrice in vitro and the wear of enamel in situ.