Blood was collected at time points between the bile and urine collection periods (e.g. at 1.5 h, this website the midpoint of the 0–3 h collection period). Afoxolaner urine and bile concentrations
were determined quantitatively using an LC–MS method (Wenkui et al., 2013). Additionally, plasma, urine and bile samples were scanned for metabolites of afoxolaner. Biliary and renal clearances were calculated for each dosing interval (Rowland and Tozer, 1995) as in the following: biliary clearance = bile flow × concentration in bile/concentration in plasma and urinary clearance = urine flow × concentration in urine/concentration in plasma. The dosing intervals for each animal were averaged for each matrix. The bile and urine flow parameter for dogs were based on the data published by Davies and Morris (1993). To estimate the percentage of the total clearance attributed to biliary and renal clearance, the average total clearance (5.0 ± 1.2 mL/h/kg) from the IV Treatment Group in PK Study 2 was used. Metabolite identification was performed on plasma samples taken 4 h and 1, 2, 4 and 6 days after administration of 25 and 100 mg/kg given orally as a solution
in dog studies. Sixteen male and female Beagle dogs (4 dogs/group; 3 oral dose groups/study and 1 control group/study) were studied to determine the effectiveness of afoxolaner when administered once as an oral soft chewable to dogs against induced infestations with 50 D. variabilis ticks and 100 C. felis fleas (Study 1) and 50 R. sanguineus
sensu lato and 100 C. felis (Study 2). For each Farnesyltransferase study, Treatment Group 1 included Selleckchem Luminespib 4 untreated control dogs and Treatment Groups 2, 3 and 4 were treated with soft chewable formulations administered orally at approximately 1.5, 2.5 and 3.5 mg/kg body weight, respectively. All dogs were infested with 100 ± 5 adult C. felis and 50 ± 5 adult D. variabilis or R. sanguineus sensu lato. Study 1 dogs were infested with C. felis on days −1, 8, 15, 22, 29, 35, 43 and 57. Study 1 dogs were infested with D. variabilis on days −1, 7, 14, 21, 28, 34 and 42. Study 2 dogs were infested with C. felis on days −1, 8, 15, 22, 29, 36, and 43. Study 2 dogs were also infested with R. sanguineus sensu lato on days −1, 7, 14, 21, 28, 35 and 42. All ticks and fleas were counted upon removal on Days 2, 9, 16, 23, 30, 37 and 44 for both studies, they were then categorized as live or dead (and for ticks also attached or free). Additionally, fleas were counted on Day 58 in Study 1. Blood samples were collected prior to treatment, on Day 0 at 4 and 12 h and on Days 1, 2, 9, 16, 23, 30, 36 (Day 37 for Study 2), and 44 in Study 1 and 2 and additionally on Days 51, 58, 64, 72, 79, 86 in Study 1. The later sampling times in study 2 were taken to determine the full pharmacokinetic profile of afoxolaner in dogs.