Assessment of the in vitro acaricidal activity of Bravecto® (fluralaner) and a proposed orange oil-based formulation vehicle for the treatment of Sarcoptes scabiei.

BACKGROUND: Sarcoptic mange is a serious animal welfare concern in bare-nosed wombats (Vombatus ursinus). Fluralaner (Bravecto®) is a novel acaricide that has recently been utilised for treating mange in wombats. The topical 'spot-on' formulation of fluralaner can limit treatment delivery options in situ, but dilution to a volume for 'pour-on' delivery is one practicable solution. This study investigated the in vitro acaricidal activity of Bravecto, a proposed essential oil-based diluent (Orange Power®), and two of its active constituents, limonene and citral, against Sarcoptes scabiei. METHODS: Sarcoptes scabiei were sourced from experimentally infested pigs. In vitro assays were performed to determine the lethal concentration (LC50) and survival time of the mites when exposed to varying concentrations of the test solutions. RESULTS: All compounds were highly effective at killing mites in vitro. The LC50 values of Bravecto, Orange Power, limonene and citral at 1 h were 14.61 mg/ml, 4.50%, 26.53% and 0.76%, respectively. The median survival times of mites exposed to undiluted Bravecto, Orange Power and their combination were 15, 5 and 10 min, respectively. A pilot survival assay of mites collected from a mange-affected wombat showed survival times of < 10 min when exposed to Bravecto and Orange Power and 20 min when exposed to moxidectin. CONCLUSIONS: These results confirm the acaricidal properties of Bravecto, demonstrate acaricidal properties of Orange Power and support the potential suitability of Orange Power and its active constituents as a diluent for Bravecto. As well as killing mites via direct exposure, Orange Power could potentially enhance the topical delivery of Bravecto to wombats by increasing drug penetration in hyperkeratotic crusts. Further research evaluating the physiochemical properties and modes of action of Orange Power and its constituents as a formulation vehicle would be of value.

Subunit-specific modulation of glycine receptors by cannabinoids and N-arachidonyl-glycine.

Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord motor and pain sensory neurons. Recent studies demonstrated apparently contradictory (potentiating versus inhibitory) effects of the endocannabinoid anandamide on these receptors. The present study characterised the effects of cannabinoid agonists on alpha1, alpha1beta, alpha2 and alpha3 GlyRs recombinantly expressed in HEK293 cells with the aims of reconciling effects of cannabinoids on these receptor subtypes and to establish the potential of different GlyR isoforms as novel physiological or analgesic targets for cannabinoids. The compounds investigated were anandamide, HU-210, HU-308, WIN55,212-2 and the endogenous non-cannabinoid, N-arachidonyl-glycine. The latter compound was chosen due to the structural similarity with anandamide and known analgesic actions in the spinal cord. Recombinant alpha1 and alpha1beta GlyRs were potentiated by anandamide and HU-210 at submicromolar concentrations, whereas WIN55,212-2 had no effect and HU-308 produced only weak inhibition. By contrast, N-arachidonyl-glycine exerted complex effects including both potentiation and inhibition. Anandamide had no effect at alpha2 or alpha3 GlyRs although the other cannabinoids produced potent inhibition. On alpha2 GlyRs, the inhibitory potency sequence was HU-210=WIN55,212-2>HU-308>N-arachidonyl-glycine but on alpha3 GlyRs it was HU-210=WIN55212=HU-308>N-arachidonyl-glycine. These results suggest that alpha1, alpha2 and alpha3 containing GlyRs exhibit distinct pharmacological profiles for cannabinoids. We conclude that cannabinoid agonists may be useful as pharmacological tools for selectively inhibiting alpha2 and alpha3 GlyRs. Our results also establish GlyRs as potential novel targets for endogenous and exogenous cannabinoids.

A proposed structural basis for picrotoxinin and picrotin binding in the glycine receptor pore.

Picrotoxin, an antagonist of structurally-rated GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs), is an equimolar mixture of picrotoxinin (PTXININ) and picrotin (PTN). These compounds share a common structure except that PTN contains a slightly larger dimethylmethanol in place of the PTXININ isopropenyl group. Although the homomeric alpha1 GlyR is equally sensitive to both compounds, we show here that homomeric alpha2 and alpha3 GlyRs, like most GABA(A)Rs, are selectively inhibited by PTXININ. As conservative mutations to pore-lining 6' threonines equally affect the sensitivity of the alpha1 GlyR to both compounds, we conclude that PTXININ and PTN bind to 6' threonines by hydrogen bonding with exocyclic oxygens common to both molecules. In contrast, substitution of the 2' pore-lining glycine by serine selectively reduces PTN sensitivity, whereas the introduction of 2' alanines selectively increases PTXININ sensitivity. These results define the orientation of PTXININ and PTN binding in the alpha1 GlyR pore and allow us to conclude that the relatively reduced sensitivity of PTN at GABA(A)Rs and alpha2 and alpha3 GlyRs is due predominantly to its larger size and reduced ability to form hydrophobic interactions with 2' alanines.

GlyR alpha3: an essential target for spinal PGE2-mediated inflammatory pain sensitization.

Prostaglandin E2 (PGE2) is a crucial mediator of inflammatory pain sensitization. Here, we demonstrate that inhibition of a specific glycine receptor subtype (GlyR alpha3) by PGE2-induced receptor phosphorylation underlies central inflammatory pain sensitization. We show that GlyR alpha3 is distinctly expressed in superficial layers of the spinal cord dorsal horn. Mice deficient in GlyR alpha3 not only lack the inhibition of glycinergic neurotransmission by PGE2 seen in wild-type mice but also show a reduction in pain sensitization induced by spinal PGE2 injection or peripheral inflammation. Thus, GlyR alpha3 may provide a previously unrecognized molecular target in pain therapy.