Purpose-bred mixed sex adult Beagle dogs and weighing approximately 9 to 16 kg were used. Each animal was uniquely identified and acclimatized to the study conditions for at least 1 week. Only healthy animals were included and suitability was evaluated by physical examination and clinical pathology. Dogs were housed indoors, in climate-controlled facilities in accordance with accepted laboratory animal care and use guidelines. They were kept in small groups except for the days around treatment administration where dogs were housed individually for at least 1 day, to avoid potential cross contamination between animals. Dogs were allowed the daily opportunity for outdoor exercise and social interaction. They were fed once daily with an appropriate ration of a commercial canine feed and water was available ad libitum. Dogs were observed for general health, behaviour and appetite at least once daily throughout the studies. All animals returned to their normal housing facilities on completion of the studies.
In the first study, designed to investigate the effect of feeding on lotilaner pharmacokinetics after oral administration, 25 mixed sex adult dogs were allocated to five treatment groups with five dogs in each group. Each dog received a single oral administration of a close-to-final tablet formulation, at the target dose of 15 mg/kg lotilaner (the initially intended therapeutic dose). Dogs were fasted overnight and five different feeding regimes were tested as follows: dogs received their full daily food allowance (i) 30 min prior to; (ii) at the same time; (iii) 30 min after; (iv) 5 h after treatment administration; or (v) only one-third of their full daily food allowance at the same time of treatment administration. Blood specimens were collected from the jugular vein in K3-EDTA tubes at pre-dose and at 30 min, at 1, 2, 4, 8, 24, 48 and 72 h and at 7, 14 and 21 days post-treatment.
In the second study, intended to determine the pharmacokinetic profile of lotilaner after intravenous and oral administration, 26 mixed sex adult dogs were allocated to three treatment groups as follows: one intravenous group of eight dogs, one oral group of 12 dogs treated 30 (± 5) min after feeding, and one oral fasted group of six dogs. Each dog from the oral groups received a single administration of the final tablet formulation (containing 35% w/w lotilaner), at the target minimum dose of 20 mg/kg lotilaner, which was the final therapeutic dose. Each dog from the intravenous group received a single administration of lotilaner in a solution consisting of 23% w/v physiological saline and tetraglycol ad 100% w/v, at the target dose of 3 mg/kg lotilaner, which was a dose known to be well tolerated intravenously. Blood specimens were collected from the jugular vein in K3-EDTA tubes at pre-dose and at 5 min (intravenous only), 30 min, at 1, 2, 4, 8, 24, 48 and 72 h and 7, 14, 21, 28 and 35 days post-treatment.
For both studies, whole blood specimens were stored frozen (for a maximum of 5 months) at approximately −20 °C until analysis with a validated LC-MS/MS method. During validation, stability at storage conditions was demonstrated for at least 9 months.
Analysis of lotilaner in blood
Lotilaner was quantitatively analysed in blood using an analytical method involving liquid chromatography with tandem mass spectrometry detection (LC-MS/MS). Whole dog blood samples (80 μl) were extracted by precipitation with acetonitrile and further diluted with acetonitrile. A proprietary closely related chemical analogue was used as the internal standard. Ten microliters of each diluted supernatant were chromatographed by HPLC on a reversed-phase column [Thermo Betasil C18, 5 μm (50 × 4.6 mm)] with an isocratic mobile phase consisting of 0.1% formic acid and acetonitrile (15:85 v:v) using a flow rate of 0.8 ml/min and quantitatively analyzed on an AB Sciex API 5000 or API 5500 triple quadrupole mass spectrometer system using the negative Turbo IonSpray ionization mode and multiple reaction monitoring (MRM) of the transition m/z 596 to 181 for lotilaner.
The method was validated over a linear range of 6.8 to 6800 ng/ml, with a lower limit of quantification (LLOQ) of 6.8 ng/ml, according to FDA and EMA guidelines [17, 18]. Mean inter-day precision was 14.9% at LLOQ and ranged between 3.4 and 7.8% at the other levels and the mean inter-day accuracy ranged between 100.3 and 103.6%. In addition, specificity, dilution integrity, recovery and matrix effect, carryover, and stability in matrix and solutions were established. Long-term stability in frozen blood at −20 °C was demonstrated over 9 months.
The in vivo enantiomeric stability of lotilaner was investigated in an analytical study. Blood specimens from 16 adult dogs which had received a single oral administration of the pure enantiomer drug at 15 mg/kg (tablet or chewy formulation, during an efficacy study) were analysed at four time points (4 h and 28, 56 and 84 days post-dosing) using an enantioselective analytical method. This method involved precipitation of 200 μl whole blood with acetonitrile and subsequent solid phase extraction (SPE) on C18 cartridges, evaporation to dryness and reconstitution in heptane/ethanol 4:6, v/v. Enantiospecific analysis was carried out by chiral normal phase HPLC using a Daicel Chiralpak IA-3 column (150 × 4.6 mm) and a mobile phase consisting primarily of heptane and isopropanol. Mass spectrometric detection was performed on an AB Sciex API 4000 Qtrap triple quadrupole instrument using the negative Turbo IonSpray ionisation mode and multiple reaction monitoring (MRM).
Pharmacokinetic and statistical analysis
Pharmacokinetic parameters were calculated for individual animals using non-compartmental analysis. The validated statistical software SAS®, Version 9.2.2 was used for all calculations. The peak blood concentration (Cmax) and time to peak concentration (Tmax) were observed values, for the oral groups. The terminal half-life (T1/2z) was calculated by log-linear regression over a suitable time interval. The area under the concentration curve (AUC) between 0 and the last time point where the blood concentration was above the limit of quantitation (AUClast), was calculated by the linear trapezoidal rule and values below the limit of quantitation at the beginning of the profile were treated as zero. The area under the concentration curve from zero to infinity (AUCinf) was the sum of AUClast and the extrapolation after the last observed timepoint; the second term was calculated by log-linear extrapolation from the last observed time point to infinity, using the half-life. The mean residence time (MRT) was calculated as the ratio of AUMC/AUC; where AUMC is the area under the first moment curve.
The clearance per kilogram of body weight (CL), defined as dose per kilogram of body weight/AUC, the volume of distribution at steady-state per kilogram of body weight (Vss), which is CL × MRT and the apparent volume of distribution per kilogram of body weight (Vz), which is CL × T1/2z/ln(2), were determined for the intravenous group only.
Bioavailability (F%) in the oral groups was determined as (geometric mean of dose-normalized AUClast in the oral group) / (geometric mean of dose-normalized AUClast in the intravenous group). In this study, AUClast was also equal to AUC from 0 to 35 days (AUC0-35d). AUCinf was found to be an unsuitable parameter for the evaluation of bioavailability because it was not accurate due to the high percentage extrapolated beyond the last measured data point.
A one-way analysis of variance (ANOVA) was performed on log-transformed dose-normalized Cmax and AUC parameters, with treatment as fixed effect. The mean and the 90% confidence interval (CI) for the difference between two treatment groups was calculated on the log scale and then back-transformed to the original scale, leading to the ratio between the two groups of Cmax or AUC. The difference (on the log scale) between two treatment groups can be tested versus zero in a t-test (degrees of freedom given in subscripted parentheses after the symbol t in the tables; e.g. t
(21) meaning a t-value with 21 degrees of freedom).
Spanish translation of the article is available in Additional file 1. French translation of the Abstract is available in Additional file 2.