Comb counts were performed following the method described by Zaks

Comb counts were performed following the method described by Zakson et al. (1995). All tick challenges were performed by placing 50 live, unfed adult American dog ticks, Dermacentor variabilis, onto the dorsal midline of dogs at various times throughout the studies. Ticks were removed,

categorized as dead or alive, and counted by hand and comb following 48 h of drug exposure. The initial day of the flea or tick challenge is recorded in the specific table for each study (Table 2, Table 3, Table 4, Table 5 and Table 6) whereas the removal of fleas and ticks took place 24 and 48 h later, respectively. All flea and tick evaluations were blinded. Effectiveness was calculated at given time points, by the classic formula (1 − [geo mean flea/tick count treated dogs − geo SNS-032 ic50 mean flea/tick count control dogs]/[geo mean flea/tick count on control dogs]) × 100. At a minimum, clinical observations for adverse reactions were performed post-treatment at 30 min, 2 and 4 h, and then daily for the duration of each study. Additional details were noted in Study 4 (see below). Afoxolaner plasma levels were determined in each animal study. At various times during the studies, blood samples were collected from the dogs and placed in heparinized tubes. The blood samples were centrifuged and the plasma was

separated and immediately frozen. Plasma was thawed and afoxolaner was extracted from Akt targets a 50-μL aliquot diluted with 450 μL of HPLC grade water in a 2-ml plastic centrifuge tube. The sample was further

diluted with 500 μL of HPLC grade acetonitrile. Samples were centrifuged for 5 min at 14,000 rpm in a microcentrifuge science tube after vigorous mixing at each step. A 10-μL aliquot was then analyzed for afoxolaner by LC/MS/MS. The LC/MS/MS system consisted of a Waters Quattro Micro™ Mass Spectrometer interfaced to a Waters Alliance HT 2795 HPLC and equipped with a 2.1 mm × 50 mm, 5 μm Zorbax SB C18 column. Afoxolaner was eluted from the column using a gradient of water/acetonitrile, each containing 0.1% formic acid. Food and water were provided for all dogs following USDA guidelines and access to veterinary care was available throughout the studies. All testing was performed under the guidelines set forth by the Institutional Animal Care and Use Committee of DuPont (USDA, 2008). Afoxolaner was prepared for a dosage of 2.5 mg/kg to be administered once, orally, at the rate of 0.2 ml/kg. The vehicle was administered in the same manner. Two beagle dogs were used for the study with one randomly assigned to treatment and the other assigned to vehicle. Flea and tick challenges were made at periodic intervals over the course of 46 days and counts were performed approximately 24 h (fleas) or 48 h (ticks) after challenge. Blood samples were taken at least weekly for the duration of the study. Afoxolaner was prepared for administration at dosages of 1.5, 2.5 or 3.5 mg/kg to be delivered once, orally at the rate of 0.

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