Animal management

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.

Experimental designs

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.

Enantiomeric stability

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 (C_max) and time to peak concentration (T_max) were observed values, for the oral groups. The terminal half-life (T_1/2z) 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 (AUC_last), 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 (AUC_inf) was the sum of AUC_last 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 (V_ss), which is CL × MRT and the apparent volume of distribution per kilogram of body weight (V_z), which is CL × T_1/2z/ln(2), were determined for the intravenous group only.

Bioavailability (F%) in the oral groups was determined as (geometric mean of dose-normalized AUC_last in the oral group) / (geometric mean of dose-normalized AUC_last in the intravenous group). In this study, AUC_last was also equal to AUC from 0 to 35 days (AUC_0-35d). AUC_inf 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 C_max 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 C_max 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).

Translations

Spanish translation of the article is available in Additional file 1. French translation of the Abstract is available in Additional file 2.

Enantiomeric stability in vivo

In 13 out of 16 adult dogs, no in vivo racemization was observed. In three dogs out of 16 animals, it was detectable only on day 84 but was negligible (less than 3%) and is believed to be of no clinical relevance for safety or efficacy. The absence of in vivo racemization in dogs after administration of pure enantiomer of lotilaner was clearly demonstrated. The absence of in vivo racemization is a prerequisite for a pure enantiomeric drug to make sense and for the investigation of the pharmacokinetics and safety of the opposite enantiomer to be omitted.

Effect of feeding in dogs

Since food may influence the pharmacokinetics and as feeding may facilitate treatment administration by the dog owner, the effect of feeding (time and amount) was evaluated in detail. Drug concentrations versus time profiles under the five tested feeding regimes are shown in Fig. 2. A pronounced feeding effect was found for lotilaner, however the exact time of feeding with respect to dosing (fed 30 min prior, fed at dosing, fed 30 min post-dosing) did not have a significant impact on bioavailability (see Table 1 for detailed test statistic and exact P-values). In addition, the reduction of the food ration to one-third of the daily ration also did not impact the bioavailability (Table 1). These findings offer a high degree of treatment flexibility to the dog owner, i.e. one-third of the daily ration is enough to provide adequate bioavailability and dosing can be performed at or around (± 30 min) the time of feeding. Hence, high bioavailability was found to be robust irrespective of changes in food amount and exact timing; moreover, it was achievable with both dry and wet food (unpublished data). However, fasted conditions (fed 5 h post-dose) yielded significantly lower bioavailability (Table 1), similarly to what was observed for fluralaner [19]. High bioavailability together with low between-animal variability is crucial in order to ensure reliable and robust efficacy, as lotilaner is a systemically acting ectoparasiticide and consequently blood concentrations are expected to be directly correlated with efficacy. Any individual case of low bioavailability would be expected to translate into the lower duration of efficacy.

Treatment groups		Ratioa (first/second)	90% CI	t-value	P-valueb
First	Second
Fed 30 min prior	Fed at dose	1.137	0.820–1.576	t (20) = 0.68	0.5052
Fed 30 min prior	Fed 1/3 of ration	1.252	0.904–1.735	t (20) = 1.19	0.2484
Fed 30 min prior	Fed 30 min after	1.029	0.743–1.426	t (20) = 0.15	0.8799
Fed 30 min prior	Fasted	2.250	1.624–3.118	t (20) = 4.29	0.0004
Fed at dose	Fed 1/3 of ration	1.101	0.795–1.526	t (20) = 0.51	0.6154
Fed at dose	Fed 30 min after	0.905	0.653–1.255	t (20) = -0.53	0.6050
Fed at dose	Fasted	1.979	1.428–2.742	t (20) = 3.61	0.0018
Fed 1/3 of ration	Fed 30 min after	0.822	0.593–1.139	t (20) = -1.04	0.3126
Fed 1/3 of ration	Fasted	1.797	1.297–2.490	t (20) = 3.10	0.0057
Fed 30 min after	Fasted	2.186	1.577–3.029	t (20) = 4.13	0.0005

^aRatio of bioavailabilities i.e. ratio of AUC_last

^b P-values in bold denote significance

Pharmacokinetic profile of lotilaner in dogs

The pharmacokinetic parameters of lotilaner are summarised in Table 2 and drug concentrations versus time profiles after intravenous and oral administration under fed or fasted conditions are shown in Fig. 3. The actual dose in the intravenous group ranged from 3.08 to 3.24 mg/kg, in the oral-fed group from 20.09 to 24.67 mg/kg, and in the oral-fasted group from 20.16 to 24.62 mg/kg. All pharmacokinetic parameters presented below are based on geometric means (considered as most appropriate, assuming that these parameters follow a log-normal distribution), except for T_max which can only take discrete values and is therefore based on the median.

Parameter	Intravenous, 3 mg/kg (n = 8)	Oral, fed 20 mg/kg (n = 12)	Oral, fasted 20 mg/kg (n = 6)
Actual doses (mg/kg)	3.08–3.24	20.09–24.67	20.16–24.62
Tmax (hours)	na	2 (range: 1–24)	4 (range: 2–24)
Cmax (ng/ml)	na	4011 ± 990	1454 ± 849
AUClast (day*ng/ml)	10,976 ± 1381	62,840 ± 17,346	18,592 ± 16,632
AUCinf (day*ng/ml)	17,844 ± 1937	118,600 ± 42,287	40,886 ± 31,915
Cmax (dose normalizeda) (ng/ml)	na	179.2 ± 43.5	65.4 ± 35.7
AUClast (dose normalizeda) (day*ng/ml)	3436 ± 430	2806 ± 801	837 ± 731
AUCinf (dose normalizeda) (day*ng/ml)	5586 ± 666	5297 ± 1921	1840 ± 1406
T1/2z (day)	24.6 ± 5.9	30.7 ± 10.0	38.8 ± 11.2
MRT (day)	36.0 ± 8.5	45.3 ± 13.9	56.9 ± 14.8
CL (l/kg/day)	0.18 ± 0.02	na	na
Vz (l/kg)	6.35 ± 1.27	na	na
Vss (l/kg)	6.45 ± 1.26	na	na
Bioavailability (F%)	na	81.7	24.3

^aDose-normalized to 1 mg/kg

All values (Mean and standard deviation) are based on geometric summary statistics (corresponding to summary statistics of log transformed values and then back-transformed), except for T_max which is based on the median

After intravenous administration at 3 mg/kg, a visual inspection of the profiles showed that lotilaner blood concentrations decreased bi-exponentially with a rapid distribution phase and a long elimination phase. The terminal half-life of lotilaner was 24.6 days and MRT was 36.02 days. Total blood clearance was 0.18 l/kg/day and the volumes of distribution V_z and V_ss were 6.35 and 6.45 l/kg, respectively. Mean dose-normalized AUC_last was 3436 day*ng/ml.

After oral administration at 20 mg/kg, a visual inspection of the profiles showed that lotilaner blood concentrations decreased bi-exponentially after T_max, with a rapid distribution phase within the first day of administration and a long elimination phase. After oral administration in fed conditions, detectable blood levels were identified in most treated dogs within 30 min and concentrations peaked quickly (mean dose-normalized C_max of 179 ng/ml) with a T_max at 2 h, indicating rapid dissolution and absorption of the chewable tablet. The terminal half-life was 30.7 days and MRT was 45.3 days. Mean dose-normalized AUC_last (= AUC_0-35d) was 2806 day*ng/ml. After oral administration in fasted conditions, lower blood concentrations of lotilaner were observed with a mean dose-normalized C_max of 65 ng/ml. T_max was observed later, at 4 h. The terminal half-life was 38.7 days and MRT was 56.9 days. Mean dose-normalized AUC_last (= AUC_0-35d) was 837 day*ng/ml. The mean terminal half-life after oral administration was in the same range as the one determined after intravenous administration, indicating that the terminal phase represented the true elimination phase.

In order to interpret the clearance, the overall body extraction ratio (which can be regarded as the percentage of drug being cleared by the entire body during a single passage through the clearing organs) was computed by the body clearance (0.18 l/kg/day) divided by the cardiac output (approximately 167 l/kg/day for a 10 kg dog) [21]. Hence, the total blood clearance corresponded to an overall extraction ratio of 0.1% and is therefore considered as very low. In addition, lotilaner had high volumes of distribution (> 6 l/kg), as expected for a lipophilic drug that would distribute in the fatty tissue. The low clearance combined with the large volume of distribution explains the long half-life of lotilaner in the dog [22, 23]. As compared to the other isoxazolines developed for dogs (afoxolaner, fluralaner and sarolaner), lotilaner in the present study had the longest half-life (approximately 4 weeks versus 2 weeks for the other compounds). This difference was mainly explained by the largest volume of distribution (approximately 6 l/kg for lotilaner versus 3 l/kg for the other compounds), whereas the clearance was in the same range (from 0.12 to 0.18 l/kg/day) [6, 8, 24]. This long terminal half-life and mean residence time explains the persistent systemic availability of lotilaner and provides effective blood concentrations for the entire duration of the inter-dosing interval of 1 month. Variability in half-life between individuals or between studies and populations was observed in the numerous studies performed during the development program, however, care was taken during dose characterization to select a robust dose high enough to provide 1 month duration of efficacy even in individuals with a shorter half-life and at the lowest possible therapeutic dose within the dose band.

The feeding effect on pharmacokinetics was multiple, not only did administration in fasted dogs lead to much lower bioavailability, but also to a delayed T_max and to an increase between-animal variability. Consequently, administration of lotilaner to fasted dogs is not recommended. Achievement of the maximum blood concentration within 2 h following lotilaner administration to dogs in the fed state aligns with the demonstrated rapid onset of adulticidal (flea and tick) activity [12–16]. Similarly, the demonstrated long half-life of lotilaner (30.7 days in the oral-fed group) and the sustained concentration levels above the estimated flea- and tick-lethal breakpoints through at least 1 month align with the prolonged effectiveness observed in multiple studies in which challenge with these parasites was extended through 35 days after lotilaner treatment.