For Dose 1 and Dose 2, early blood samples were taken at 2, 6 and 12 h after treatment,

for the remaining doses the 2 and 12 h plasma collections were eliminated. The highest plasma concentration of 0.82 μg/ml was measured at the 6 h time point after dose 1 (Fig. 3). Pharmacokinetic profiles for afoxolaner were observed to be predictable and reproducible following multiple dosing (Fig. 3). Mean afoxolaner plasma concentrations at 6 h were 0.82, 0.81, 0.97, 0.91, and 0.80 μg/ml for Doses 1 through 5, respectively. There was no apparent difference in the trough concentrations as drug discovery mean minimum afoxolaner plasma concentrations (Cmin) collected at 30 days post-dose were 0.09, 0.09, 0.12, 0.10 and 0.15 μg/ml for Doses 1 through 5, respectively ( Fig. 3). These data indicate that steady state had been reached by the 2nd dose. No adverse clinical signs were observed during the study. A KD50 (50% knockdown concentration) value of 0.35 μg per cockroach was determined. At the higher injected dose,

symptoms were observed within 10 min, initially appearing as brief periodic LY2835219 datasheet wing fluttering which progressed over time until the insects became uncoordinated and had difficulty remaining upright. Once prostrate, cockroaches displayed periodic volleys of leg tremors. The rapid onset and excitatory nature of toxicity suggested involvement of a neuronal target. By doing extracellular recordings on nerve 5 (N5) of the metathoracic Carnitine palmitoyltransferase II ganglion of American cockroaches, under control conditions, a single air puff to the cerci produced a rapidly adapting volley of action potentials with a spike frequency between 75 and 175 Hz. Injection of CPD I (10 μg) into the body cavity produced no significant effect on spontaneous action potential frequency. However, adaptation of the air puff-induced N5 activity was inhibited by CPD I, resulting in a strong increase of spike frequency (Fig. 4b). Similarly, bath perfusion of CPD I (10 mM) induced a strong increase in the air puff-induced spike frequency indicating increased excitability

due to blocking of inhibitory neuronal activity (Fig. 4c). The fact that the spontaneous action potential frequency remained unaffected suggested that action at the neurotransmitter receptors was a more likely target than action at voltage-gated ion channels. As the neurotransmitters involved in the cercal reflex include both excitatory nicotinic acetylcholine receptors (nAChRs) and inhibitory GABA receptors (GABARs), action of CPD I was investigated on both neurotransmitter receptors. Although no effect was observed on nAChRs (data not shown), the compound potently inhibited GABA-induced currents in American cockroach thoracic neurons. CPD I inhibited GABA-induced currents with an IC50 value of 10.8 nM (Fig. 5) with prolonged saline rinse (>15 min) resulting in partial recovery of the GABA response.