Chewable tablets containing 13.64% (w/w) fluralaner were formulated for oral administration to dogs. Test material for i.v. administration was formulated as solution with 2.5 mg fluralaner /mL in a Polyethylene glycol (PEG) 200 (90% v/v) vehicle containing 10% v/v water for injection.
Healthy male and female Beagle dogs were kept indoors in pens with sealed floors and individually housed until 5 or 6 weeks after fluralaner administration (oral or i.v.) to avoid potential cross contamination between animals. Thereafter, dogs were housed in groups of 3 of the same treatment group and sex. Room environment was monitored continuously, with a temperature of 15-21°C, relative humidity of 40-70%, 10–20 air changes per hour and a 12-hour fluorescent light/12-hour dark cycle. Dogs were fed once daily in the morning with a standard dog diet and had ad libitum access to water. On the day of oral fluralaner administration, the dogs received one-half of their daily food ration shortly before treatment and the remainder immediately after treatment, because administration of chewable tablets containing fluralaner with food increases its bioavailability.
To determine the rate and extent of systemic exposure after oral administration and oral dose proportionality, a parallel-group study was conducted with 3 treatment groups. Dogs received either 12.5, 25, or 50 mg (target dose) fluralaner/kg BW orally, with the mid dose (25 mg/kg BW) based on the minimum recommended treatment dose. Additional pharmacokinetic parameters such as total body clearance and volume of distribution were determined in a separate study involving 6 Beagles administered 12.5 mg fluralaner/kg BW by slow i.v. infusion. All dose rates in the following sections are expressed in mg fluralaner per kg BW.
In both studies, dogs were randomized to treatment groups (3 dogs per sex per group), within sex, and blocked by body weight to ensure a balanced distribution. Both studies were compliant with the principles of Good Laboratory Practice. The animal work was conducted in compliance with respective national legislation and approved by Animal Experimentation Ethics Committee at Harlan Laboratories S.A.
Individual oral doses were determined on the basis of individual body weights and the nominal content of fluralaner in the tablets. Dogs received whole tablets using either individual 112.5 mg, 250 mg or 500 mg fluralaner tablets, or a combination of tablets to achieve a dose close to the calculated target dose. Oral dosing was facilitated by placing the calculated target dose on the back of the dog’s tongue. Intravenous injection dose volumes were determined on the basis of individual body weights and were administered as a constant rate infusion over 5 minutes using an automatic injection system (KDS Model 200, KD Scientific Inc., Holliston, USA). The injection rate per hour was approximately 12 times the respective dose volume to ensure complete administration within 5 minutes.
Blood samples were collected from the jugular vein into sodium-citrate tubes before and at 2, 4, and 8 hours and 1, 2, 3, 4, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, and 112 days after oral dosing and 15 min, 2, 4 and 8 hours, and 1, 2, 3, 4, 7, 14, 21, 28, 35, 49, 63, 77, 91, and 112 days after i.v. dosing. Plasma was harvested by centrifugation and stored frozen in sterile plastic vials until analysis. The dogs were closely observed for 1 hour after dosing and once daily thereafter.
Plasma samples were extracted by protein precipitation with acetonitrile and diluted with 0.1% formic acid. The resultant solution was analyzed quantitatively using automated solid phase extraction coupled to liquid chromatography with mass spectrometric detection (online SPE-HPLC-MS/MS). The linear range of the method for determination of fluralaner was 10.0 to 2500 ng/mL, with a lower limit of quantification (LLOQ) of 10.0 ng/mL. Pharmacokinetic parameters for fluralaner were calculated using non-compartmental methods with the validated software WinNonlin® Professional Version 5.3 (Pharsight Corporation, California, USA). The peak plasma concentration (Cmax) and time to peak concentration (tmax) were observed values. The elimination half-life (t1/2) was calculated by linear regression using the slope of the terminal segment of the semilogarithmic plasma concentration versus time curve. The area under the concentration versus time curve (AUC) from time 0 to the last measurable concentration (AUC(0→t)) was calculated using the linear trapezoidal rule. The AUC from time 0 extrapolated to infinity (AUC(0→∞)) was determined as AUC
, where Ct is the plasma concentration at time t and λz is the first order rate constant associated with the terminal (log-linear) portion of the curve. The apparent volume of distribution (Vz) after i.v. administration, based on the terminal phase was calculated as Dose/λ
× AUC. Total body clearance (Cl) after i.v. administration was calculated as Dose/AUC. Bioavailability (F%) via the oral route was calculated using mean AUC(0→∞) as (AUC
) × (Dose
) × 100. Mean residence time (MRT) extrapolated to infinity was calculated as the ratio of AUMC/AUC; where AUMC is the area under the first moment curve. Dose proportionality was tested for exposure parameters Cmax and AUC(0→t). For this purpose dose-normalized (nominal dose) values were analyzed using an appropriate analysis of variance (ANOVA) regression model. The tests were two-sided at the 0.05 level of significance. All data are expressed as arithmetic mean ± SD unless otherwise stated.