, 2008), which makes the duration of its action too short for convenient and compliant use as an oral monthly product. Spinosad, on the other hand, has a long plasma half-life of 7–10 days (Holmstrom et al., 2012) therefore preventing and treating flea infestations for one month. In contrast to the currently available oral antiparasitic agents for dogs, the ideal pharmacokinetic and efficacy profile would demonstrate both a rapid onset of action and monthly duration of efficacy against fleas and multiple tick species. These attributes are achieved with an easy-to-administer oral soft chewable formulation for dogs, as demonstrated by the afoxolaner pharmacokinetic profile and PK/PD correlation

SB203580 discussed herein and supported by both efficacy and safety data. Protein binding was determined via equilibrium dialysis using a Dianorm Multi-Equilibrium DIALYZER at 200, 500, 1000, 2000 and 10,000 ng/mL of afoxolaner in dog plasma Talazoparib cell line with buffer and plasma chambers separated by a semi-permeable cellulose membrane with a molecular weight cutoff of 10,000 Dalton. Incubations were performed for 2.5 h (37 °C), after which the afoxolaner concentrations were measured in the buffer and plasma compartments using LC–MS analysis (see below). For all in vivo studies, animals were treated with the final formulation of afoxolaner except when noted. All dogs were purpose-bred, ≥6 months of age, and weighed approximately 5–19 kg.

The number of dogs in each study and other study details are described in Table 1 and in the text below. All animal procedures in these studies were reviewed and approved by Merial Institutional Tryptophan synthase Animal Care and Use Committee (IACUC) and dogs were handled with due regard for their welfare (USDA, 2008, 9 CFR). Blood samples

were taken prior to treatment, and periodically until the end of each study. All blood samples were processed into plasma and stored frozen until analyzed. Afoxolaner plasma concentrations were determined using a validated LC–MS method (Nguyen, unpublished data) and pharmacokinetic analysis via noncompartmental and/or compartmental analysis was performed. All study times are relative to Day 0, the day of dosing. Six pharmacokinetic studies were performed. The purpose and study design details are given in Table 1. Some details of PK Study 6 and two efficacy studies are given below. Six male Beagles were studied to determine the renal and biliary clearance of afoxolaner when administered as a soft chewable formulation once, orally, at 30–45 mg/kg (PK Study 6). Four of the dogs underwent bile duct cannulation prior to study start to allow bile collection throughout the study. Hematology and serum chemistry samples from all dogs were evaluated prior to treatment to assure normal liver functioning. Dogs were fasted prior to treatment. Plasma, urine and bile samples were collected and stored frozen (−20 °C) until analysis.