Study 3 provided a dosage titration of afoxolaner in dogs (n = 3) using oral administration. Results supported the previously observed flea and tick effectiveness for one month. Moreover, plasma ABT-263 ic50 level data and efficacy results indicated that there was no apparent prandial effect on the systemic absorption and effectiveness of afoxolaner. The compound was well absorbed with concentrations above those needed for effectiveness

achieved at the first sampling time on the first day of the study and the plasma concentrations remained high enough to support effective flea and tick kill for the entire 33 days of the study. The pharmacokinetic profiles suggested dose proportionality over the range of doses tested, providing further indication of a good safety profile, and

no dog showed an adverse event attributable to the drug at any time during this study. Study 4 was conducted to explore the effects of repeated dosing and dogs (n = 6) received 5 monthly doses of afoxolaner in the experimental oral solution. Effectiveness results showed nothing less than 99% against fleas during the 26 challenges. Effectiveness against ticks at the end of each month (immediately before the next monthly dose) was also very good with values of 100, 99, 82, 99 and 92% for months 1 through 5, respectively. The pharmacokinetic profiles observed in this study were remarkably consistent with similar Cmax and Cmin for each monthly dose, and minimal accumulation of afoxolaner recorded over repeated dosages. Veterinary clinical examinations Bay 11-7085 conducted throughout the study showed no LY294002 in vitro indication of adverse effects. The lack of accumulation following multiple dosing, the observed safety, and the sustained effectiveness confirmed the potential for afoxolaner to become a convenient and safe ectoparasiticide for use in dogs. The mode of action studies demonstrated that afoxolaner and compounds of the isoxazoline class control insects by inhibition of GABA-gated chloride ion channels (Ozoe et al., 2010 and Gassel et al., 2014). It can be expected

that afoxolaner will act on fleas and ticks in a similar way. GABA-gated chloride ion channels are the target of ivermectin, fipronil, and cyclodienes; however ivermectin binds to a distinct site and activates rather than blocks GABA-gated chloride channels (Garcia-Reynaga et al., 2013 and Gassel et al., 2014). It has been well established that replacement of alanine with serine at position 302 of the rdl gene confers strong resistance to cyclodienes and moderate resistance to fipronil in some arthropods ( Ffrench-Constant et al., 2000, Bloomquist et al., 1992 and Bloomquist, 1993). It is particularly noteworthy that no significant difference in afoxolaner sensitivity was observed in Drosophila toxicity or receptor studies for wild type versus A302S mutants. These findings indicate that afoxolaner binds to the target in a manner distinct from cyclodienes and phenylpyrazoles.