Animals
A total of 24 purpose-bred laboratory Beagle dogs, including 12 males and 12 females approximately 10 months of age, were enrolled in the study. Dogs were housed individually in environmentally controlled indoor runs with ad libitum access to food and water.
Approximately 4 months prior to study initiation, dogs were surgically transplanted with 10 male and 10 female D. immitis worms (ZoeKY isolate) a known ML susceptible isolate (Rawlings et al. [8]). Prior to the study initiation, infection status was verified by a D. immitis antigen test (DiroCHEK® Heartworm Antigen Test Kit, Zoetis, Parsippany,NJ) and blood samples were verified to contain > 500 circulating microfilariae (MF) per mL by modified Knott’s method. No animal had previous exposure to a macrocyclic lactone.
Study Design
This study was conducted in compliance with requirements of US FDA GLP regulations (21CFR Part 58) [9]. The study aligned with recommendations of the “VICH GL 43 (Target Animal Safety) – Pharmaceuticals [10]”. To control bias, treatment allocation was not disclosed to any person responsible for conducting subjective observations. This study was also conducted in compliance with the Animal Welfare Act of 1966 (and subsequent amendments including the Animal Welfare Acts of 1970, 1976, and 1985) for the care of animals. The study procedures, and care and use of animals were reviewed and approved in advance of the study by test facility Institutional Animal Care and Use Committee.
The 24 dogs were randomized to treatments (8 per group) according to split-plot design within room, with sex as the whole plot factor and treatment as the split-plot factor. Animals were blocked by sex and microfilariae (MF) count and randomized within blocks of size 3 and assigned to treatment groups: negative control (empty capsule), 1X and 3X of the maximum recommended label dose of Simparica Trio. Dogs in the treated groups received Simparica Trio on days 0, 28 and 56 administered as whole tablets (Table 1). The recommended minimum dosage for the product is 1.2 mg/kg sarolaner + 5 mg/kg pyrantel + 24 µg/kg (0.024 mg/kg) moxidectin. However, per VICH GL 43, the margin of safety is assessed based on multiples of the maximum recommended therapeutic dose (MRTD) which is the dose intended for administration to the lightest-weight dog in the widest dose band. For Simparica Trio, the MRTD is 2.4 mg/kg of sarolaner + 10 mg/kg pyrantel + 48 µg/kg (0.048 mg/kg) moxidectin. The doses were based on the most recent individual body weight. Because point dosing of the calculated MRTD is not possible with whole tablets, doses were rounded up using the smallest tablet size.
In-life assessments included weekly body weight measurements, veterinary physical examinations, veterinary clinical observations, technician-led daily general health observations, and a daily quantitative estimate of food consumption. Blood collections for pharmacokinetic (PK) analysis were performed pre-dose, and 2, 8, 24, 72, 168, 336, 504, and 672 hours after each dose. Moxidectin, sarolaner and pyrantel plasma concentrations were measured using validated LC-MS/MS methods. Blood was also collected for heartworm testing throughout the study as follows: MF counts on Days − 14, -8, 0 (pre-dose), 1, 3, 7, 14, 21, 28 (pre-dose), 35, 56 (pre-dose), 63, and 83; and D. immitis antigen testing Day − 14 and pre-dose on days 28, 56, 83.
Table 1
Margin of safety study design of Simparica Trio in heartworm-infected (Dirofilaria immitis) Beagle dogs
Treatment Group | Treatment | Dose Level | # Dogs (M/F) | Dosing Days | Necropsy Day |
Control a | Placebo | 0 | 8 (4/4) | 0, 28, and 56 | 84 |
1X b | Sarolaner | 2.4 mg/kg | 8 (4/4) |
Pyrantel | 10 mg/kg |
Moxidectin | 48 µg/kg |
3X b | Sarolaner | 7.2 mg/kg | 8 (4/4) |
Pyrantel | 30 mg/kg |
Moxidectin | 144 µg/kg |
a Empty HPMC capsules for oral administration. |
b Maximum dose from the dose banding. |
Insert Table 1
Dogs were humanely euthanized after an intravenous injection of heparin, to facilitate parasite recovery, via an approved pentobarbital euthanasia solution on Day 84. After euthanasia, the pleural and peritoneal cavities were examined for adult D. immitis worms, and the posterior and anterior venae cavae were clamped before removal of the heart and lungs. The pre-cava, right atrium, right ventricle and pulmonary arteries (including those coursing through the lungs) were dissected and examined for worms. The worms from each dog were counted, identified as adult male or female, and classified as either dead or alive based on motility and appearance (Holmes et al. [11]).
Data Analysis
Observational data (general health observations, clinical observations, and physical exams) were summarized with frequency distributions by treatment and timepoint. Body temperature, body weight, weekly average food consumption, adult heartworm counts, and MF counts were summarized with descriptive statistics. Body weight, weekly average food consumption, and MF counts were statistically analyzed using a general linear mixed model for repeated measures including fixed effects of treatment, time, sex, and all interactions among those effects. The random effects included room, block within room and sex, the interaction of block and treatment within room and sex (animal term), and error. MF counts were natural log transformed prior to modeling and back-transformed least squares means and corresponding confidence intervals were reported by treatment and timepoint.
For sarolaner and moxidectin, plasma concentration means and 90% confidence intervals were calculated for each treatment and timepoint using the statistical model described above. A natural log transformation was applied to plasma concentration data prior to modeling and back-transformed least squares means and corresponding confidence intervals were reported by treatment and timepoint. For sarolaner and moxidectin estimates of the pharmacokinetic (PK) parameters Cmax, tmax, AUC0 − 672h, and t1/2 were made using non-compartmental methodology. For pyrantel Cmax, tmax and AUC0 − 24h were estimated. The means and confidence intervals for the PK parameters were calculated using a similar general linear mixed model for repeated measures as described above with dose period replacing time in the statistical model. AUC and Cmax were natural log transformed prior to modeling and back-transformed least squares means and corresponding confidence intervals were reported by treatment.
For the repeated measures models described above, if the treatment by sex by time interaction was significant at the 5% level of significance, then no further testing was done. If the sex by treatment by time interaction was not significant and the treatment by sex or time interaction was significant at the 10% level of significance, then treatment LS means were calculated and pairwise comparisons between control and treatments were performed at the unadjusted 10% level of significance, within each sex if the treatment by sex interactions was significant (P ≤ 0.10), or within timepoint if the treatment by time interaction was significant (P ≤ 0.10) or timepoint if the treatment by time interaction was significant (P ≤ 0.10). If none of the interactions involving treatment were significant and the treatment main effect was significant at the 10% level of significance, then the treatment LS means were calculated and pairwise comparisons between control and treatments were performed at the unadjusted 10% level of significance. If no treatment related effects were significant, no treatment comparisons were performed.