The synergistic action of imidacloprid and flumethrin and their release kinetics from collars applied for ectoparasite control in dogs and cats

<p>Abstract</p> <p>Background</p> <p>The control of tick and flea burdens in dogs and cats has become essential to the control of important and emerging vector borne diseases, some of which are zoonoses. Flea worry and flea bite hypersensitivity are additionally a significant disease entity in dogs and cats. Owner compliance in maintaining the pressure of control measures has been shown to be poor. For these reasons efforts are continuously being made to develop ectoparasiticides and application methods that are safe, effective and easy to apply for pet owners. A new polymer matrix collar has recently been developed which is registered for 8 months use in cats and dogs. The basic properties of this collar have been investigated in several <it>in vitro </it>and <it>in vivo </it>studies.</p> <p>Methods</p> <p>The effects of imidacloprid, flumethrin and the combination were evaluated in vitro by means of whole cell voltage clamp measurement experiments conducted on isolated neuron cells from <it>Spodoptera frugiperda</it>. The in vitro efficacy of the two compounds and the combination against three species of ticks and their life stages and fleas were evaluated in a dry surface glass vial assay. The kinetics of the compounds over time in the collar were evaluated by the change in mass of the collar and measurement of the surface concentrations and concentrations of the actives in the collar matrix by HPLC. Hair clipped from collar treated dogs and cats, collected at various time points, was used to assess the acaricidal efficacy of the actives ex vivo.</p> <p>Results</p> <p>An <it>in vitro </it>isolated insect nerve model demonstrated the synergistic neurotoxic effects of the pyrethroid flumethrin and the neonicotinoid imidacloprid. An <it>in vitro </it>glass vial efficacy and mortality study against various life stages of the ticks <it>Ixodes ricinus, Rhipicephalus sanguineus </it>and <it>Dermacentor reticulatus </it>and against the flea (<it>Ctenocephalides felis</it>) demonstrated that the combination of these products was highly effective against these parasites. The release kinetics of these actives from a neck collar (compounded with 10% imidacloprid and 4.5% flumethrin) was extensively studied in dogs and cats under laboratory and field conditions. Acaricidal concentrations of the actives were found to be consistently released from the collar matrix for 8 months. None of the collar studies in dogs or cats were associated with any significant collar related adverse event.</p> <p>Conclusion</p> <p>Here we demonstrated the synergism between the pyrethroid flumethrin and the neonicotinoid imidacloprid, both provided in therapeutically relevant doses by a slow release collar matrix system over 8 months. This collar is therefore a convenient and safe tool for a long-term protection against ectoparasites.</p

Ce SLO-1 and hum KCNMA1 currents are blocked by the BK channel antagonist penitrem A.

<p><b>A</b>. Representative whole cell currents recorded from HEK293 cells expressing either Ce SLO-1 or hum KCNMA1 before and after penitrem A (1μM) application. Membrane potential was held at -60mV and stepped to between -100 and +90mV in 10mV increments for 50ms. <b>B</b>. A time-course of steady state Ce SLO-1 and hum KCNMA1 currents at +70mV (stepped every 10s for 40ms from -60mV holding potential) before and during application of DMSO (0.01%) or penitrem A (1μM). The current response is shown as a percentage change from the mean of pre-drug current amplitude during the 0 to 2 min time period. An arrow indicates the time point at which the drug solution reached the chamber with the cells. Free intracellular [Ca²⁺] was 100μM for Ce SLO-1 and 300nM for hum KCNMA1. Data points are the mean ± s.e.mean. p<0.0001 for penitrem A inhibited current compared to DMSO treatment, two-way ANOVA with Bonferroni post-hoc tests.</p

The biphasic effect of emodepside on hum KCNMA1 currents.

<p>Time-course analysis of steady state hum KCNMA1 currents expressed in HEK293 cells at +70mV (stepped every 10s for 40ms from -60mV holding potential) before and during application of 0.01% DMSO or emodepside. The current response is shown as a percentage change from the mean of pre-drug current amplitude during the 0 to 2 min time period. Arrow indicates the time of emodepside application at <b>A</b>. 1nM <b>B</b>. 10nM and <b>C</b>. 100nM. Data points are the mean ± s.e.mean. 100nM emodepside had a biphasic effect on hum KCNMA1 currents eliciting a significant increase between 4 and 8 min application (p<0.05) and a significant decrease after 20 min application (p<0.001); two-way ANOVA with Bonferroni post-hoc tests.</p

Dm Slo currents are blocked by the BK channel antagonists verruculogen and penitrem A.

<p>Top panel shows representative traces of Dm Slo whole cell currents stable expressed in CHO cells before and after verruculogen (3μM) application. Membrane potential was held at -70mV and stepped to between -120 and +60mV in 10mV increments for 100ms. Bottom panel shows dose-response curves for verrucologen (circles) and penitrem A (squares) on Dm Slo. Membrane potential was held at -70mV. Test pulses to + 60mV for 100ms. Each data points represent the mean ± s.e. of n = 3 cells.</p

Intracellular Ca²⁺ ions are required for emodepside activation of Dm Slo.

<p><b>A</b>. Representative traces of Dm Slo whole cell currents stable expressed in CHO cells before (left hand traces) and after (right hand traces) emodepside (10 μM) application at different free intracellular [Ca²⁺] (“0 nM”, 52 nM and 290 nM as indicated). Emodepside was applied for 10 min for experiments with low free intracellular [Ca²⁺] (“0 nM” and 52 nM). Membrane potential was held at -70 mV and stepped to between -120 and +60 mV in 10 mV increments for 100 ms. <b>B</b>. Current-voltage relationship of Dm Slo whole cell currents after emodepside application at two different free intracellular Ca²⁺ concentrations, as indicated. Membrane potential was held at -70 mV and stepped to between -120 and +60 mV in 10 mV increments for 100 ms. Data points are the mean ± s.e.mean of n cell recordings.</p

A comparison of the voltage and Ca²⁺ -dependent activation of human KCNMA1 and <i>C</i>. <i>elegans</i> SLO-1a.

<p><b>A</b>. Representative traces of currents recorded from HEK293 cells expressing eGFP alone (control), Ce SLO-1 or hum KCNMA1. Membrane potential was held at -60mV and stepped to between -100 and +90mV in 10mV increments for 50ms. Free [Ca²⁺] in the internal solution was 300nM or 100μM as indicated. <b>B</b>. Current-voltage relationship for whole cell currents recorded from cells expressing eGFP alone (control), Ce SLO-1 or hum KCNMA1 at 300nM or 100μM free intracellular [Ca²⁺]. Membrane potential was held at -60mV and stepped to between -100 and +90mV in 10mV increments for 50ms. Data points are the mean ± s.e.mean of ‘n’ cell recordings (shown in brackets). Currents recorded from Ce SLO-1 in the presence of 300nM Ca²⁺, Ce SLO-1 in the presence of 100μM Ca²⁺ and hum KCNMA1 in the presence of 300nM Ca²⁺ were significantly different from eGFP recorded in 300nM and 100μM Ca²⁺; p<0.001, two-way ANOVA with Bonferroni post-hoc test.</p

Emodepside does not increase cytosolic Ca²⁺ in HEK293 cells.

<p>Ca²⁺ fluorescence measured in HEK293 cells loaded with Fluo-3, AM dye after treatment with 1% DMSO (control), 100μM, 10μM, 1μM and 100nM emodepside (1% DMSO) or ionomycin (1% DMSO). The Ca²⁺ fluorescence was measured at excitation of 480p and emission of 525p in the FLUO Star OPTIMA machine. The readings for each treatment were taken in triplicate. Data were pooled from at least three independent experiments and are shown as mean ± s.e.mean.</p