The novel isoxazoline ectoparasiticide, sarolaner, was identified during a lead optimization program for an orally-active compound with efficacy against fleas and ticks on dogs. The aim of the discovery program was to identify a novel isoxazoline specifically for use in companion animals, beginning with de novo synthesis in the Zoetis research laboratories. The sarolaner molecule has unique structural features important for its potency and pharmacokinetic (PK) properties, including spiroazetidine and sulfone moieties. The flea and tick activity resides in the chirally pure S-enantiomer, which was purified to alleviate potential off-target effects from the inactive enantiomer. The mechanism of action was established in electrophysiology assays using CHO-K1 cell lines stably expressing cat flea (Ctenocephalides felis) RDL (resistance-to-dieldrin) genes for assessment of GABA-gated chloride channel (GABACls) pharmacology. As expected, sarolaner inhibited GABA-elicited currents at both susceptible (CfRDL-A285) and resistant (CfRDL-S285) flea GABACls with similar potency. Initial whole organism screening was conducted in vitro using a blood feeding assay against C. felis. Compounds which demonstrated robust activity in the flea feed assay were subsequently tested in an in vitro ingestion assay against the soft tick, Ornithodoros turicata. Efficacious compounds which were confirmed safe in rodents at doses up to 30mg/kg were progressed to safety, PK and efficacy studies in dogs. In vitro sarolaner demonstrated an LC80 of 0.3μg/mL against C. felis and an LC100 of 0.003μg/mL against O. turicata. In a head-to-head comparative in vitro assay with both afoxolaner and fluralaner, sarolaner demonstrated superior flea and tick potency. In exploratory safety studies in dogs, sarolaner demonstrated safety in dogs≥8 weeks of age upon repeated monthly dosing at up to 20mg/kg. Sarolaner was rapidly and well absorbed following oral dosing. Time to maximum plasma concentration occurred within the first day post-dose. Bioavailability for sarolaner was calculated at >85% and the compound was highly protein bound (>99.9%). The half-life for sarolaner was calculated at 11-12 days. Sarolaner plasma concentrations indicated dose proportionality over the range 1.25-5mg/kg, and these same doses provided robust efficacy (>99%) for ≥35days against both fleas (C. felis) and multiple species of ticks (Rhipicephalus sanguineus, Ixodes ricinus and Dermacentor reticulatus) after oral administration to dogs. As a result of these exploratory investigations, sarolaner was progressed for development as an oral monthly dose for treatment and control of fleas and ticks on dogs.

Background: Fecal examination is an important component of routine companion animal wellness exams. Sensitivity and specificity of fecal examinations, however, are influenced by sample preparation methodologies and the level of training and experience of personnel who read fecal slides. The VETSCAN IMAGYST system consists of three components: a sample preparation device, a commercially available scanner, and an analysis software. The VETSCAN IMAGYST automated scanner and cloud-based, deep learning algorithm, locates, classifies, and identifies parasite eggs found on fecal microscopic slides. The main study objectives were (i) to qualitatively evaluate the capabilities of the VETSCAN IMAGYST screening system and (ii) to assess and compare the performance of the VETSCAN IMAGYST fecal preparation methods to conventional fecal flotation techniques. Methods: To assess the capabilities of VETSCAN IMAGYST screening components, fecal slides were prepared by the VETSCAN IMAGYST centrifugal and passive flotation techniques with 100 pre-screened fecal samples collected from dogs and cats and examined by both the algorithm and parasitologists. To determine the diagnostic sensitivity and specificity of the VETSCAN IMAGYST sample preparation techniques, fecal flotation slides were prepared by four different techniques (VETSCAN IMAGYST centrifugal and passive flotations, conventional centrifugal flotation, and passive flotation using OVASSAY ® Plus) and examined by parasitologists. Additionally, required sample preparation and scanning times were estimated on a subset of samples to evaluate VETSCAN IMAGYST ease-of-use. Results: The algorithm performance of the VETSCAN IMAGYST closely matched that of the parasitologists, with Pearsonʼs correlation coefficient (r) ranging from 0.83-0.99 across four taxa of parasites, Ancylostoma, Toxocara, Trichuris and Taeniidae. Both VETSCAN IMAGYST centrifugal and passive flotation methods correlated well with conventional preparation methods on all targeted parasites (diagnostic sensitivity of 75.8-100%, specificity of 91.8-100%, qualitative agreement between methods of 93.8-94.5%). Sample preparation, slide scan and image analysis were completed within 10-14 min by VETSCAN IMAGYST centrifugal and passive flotations, respectively. Conclusions: The VETSCAN IMAGYST scanning system with the VETSCAN IMAGYST sample preparation methods demonstrated a qualitative match in comparison to the results of parasitologists' examinations with conventional fecal

Background Moxidectin has previously shown limited efficacy (≤ 44.4%) against confirmed macrocyclic lactone (ML)-resistant Dirofilaria immitis strains at 3 µg/kg after single and multiple oral dosages. Three studies were conducted to evaluate higher oral moxidectin doses for efficacy against confirmed ML-resistant D. immitis strains. Methods Dogs were inoculated with 50 D. immitis L3 and randomly allocated to treatments. Study 1: 6 groups of dogs (n = 8) were inoculated with JYD-34 (Day − 30) and treated as follows: T01, negative control; T02–T05, moxidectin at 3, 6, 12 or 24 µg/kg, respectively, on Day 0 only; T06, moxidectin at 3 µg/kg on Days 0, 30 and 60. Study 2: 10 groups of dogs (n = 5) were inoculated (Day − 30) with either JYD-34 (T01, T03–05) or ZoeLA (T02, T06–T10) and treated as follows: T01 and T02, negative controls; T03–T05, moxidectin at 24, 40 or 60 µg/kg, respectively, on Days 0, 28 and 56; T06 and T09, moxidectin at 3 or 60 µg/kg on Day 0 only; T07, T08 and T10, moxidectin at 24, 40 or 60 µg/kg, respectively, on Days 0, 28 and 56. Study 3: 5 groups of dogs (n = 5) were inoculated with ZoeMO (Day − 28) and treated as follows: T01, negative control; T02, moxidectin at 3 µg/kg moxidectin on Day 0 only; T03–T05, moxidectin at 24, 40 or 60 µg/kg, respectively, on Days 0, 28 and 56. All dogs were necropsied for adult heartworm recovery ~ 4–5 months post-inoculation. Results All moxidectin-treated dogs showed significantly lower worm counts than controls. The efficacy of moxidectin administered once at 3 µg/kg was 19% (JYD-34), 44.4% (ZoeLA) and 82.1% (ZoeMO). Increasing both the dose and the number of dosages of moxidectin improved efficacy, with 100% protection obtained using three dosages of moxidectin at either 40 µg/kg (JYD-34, ZoeMO) or 60 µg/kg (ZoeLA). Three dosages of 24 µg/kg were also highly effective, providing ≥ 98.8% efficacy for all three strains. Conclusions Increasing both the dose and number of consecutive monthly dosages of moxidectin improved the efficacy against ML-resistant heartworms. Based on these data and other technical considerations, the 24 µg/kg dose was considered the optimal dose for further commercial development.

BackgroundMonthly topical and sustained-release injectable formulations of moxidectin are currently marketed; however, an oral formulation, while approved at a dose of 3 μg/kg, is not currently marketed in the United States. Although resistance of heartworms to all macrocyclic lactone (ML) heartworm preventives (ivermectin, milbemycin, selamectin and moxidectin) has been demonstrated, to date no data have been reported on the effectiveness of oral moxidectin against recent isolates of Dirofilaria immitis.MethodsA total of nine studies were conducted to determine the efficacy of moxidectin against a range of older and recently sourced heartworm isolates. Dogs (groups of three to eight) were inoculated with 50 D. immitis infective larvae (L3) from nine different isolates (MP3, Michigan, JYD-34, ZoeMO-2012, ZoeKy-2013, ZoeLA-2013, GCFL-2014, AMAL-2014 and ZoeAL-2015) and treated 28–30 days later with single oral doses of 3 μg/kg of moxidectin. Additionally, one group of dogs that was inoculated with JYD-34 was treated monthly for 3 consecutive months beginning 30 days post inoculation. Dogs were held for approximately 4 months after the initial (or only) treatment and then necropsied for recovery of adult heartworms.ResultsA single dose of 3 μg/kg of moxidectin was 100% effective in preventing the development of five of nine heartworm isolates (MP3, Michigan, ZoeKy, GCFL and ZoeAL isolates), confirming their susceptibility to oral moxidectin at this dose. MP3 and Michigan are isolates sourced from the field more than 9 years ago, while ZoeKy, ZoeAL and GCFL were isolated from the field within the past 2 to 3 years. Against JYD-34, ZoeMO, ZoeLA and AMAL isolates, a single dose of 3 μg/kg of moxidectin was not completely effective, with efficacies of 19%, 82%, 54% and 62%, respectively, demonstrating resistance of these heartworm isolates to oral moxidectin at this dosage. Three consecutive monthly doses of 3 μg/kg of moxidectin were also incompletely effective against the JYD-34 isolate, with an efficacy of 44%. JYD-34 was originally isolated in 2010, while ZoeMO, ZoeLA and AMAL were isolated within the past 2 to 3 years.ConclusionsA single oral dose (3 μg/mg) of moxidectin was 100% effective in preventing the development of ML-susceptible heartworm isolates while being incompletely effective against ML-resistant isolates.

Background Fecal examinations in pet cats and dogs are key components of routine veterinary practice; however, their accuracy is influenced by diagnostic methodologies and the experience level of personnel performing the tests. The VETSCAN IMAGYST system was developed to provide simpler and easier fecal examinations which are less influenced by examiners’ skills. This system consists of three components: a sample preparation device, an automated microscope scanner, and analysis software. The objectives of this study were to qualitatively evaluate the performance of the VETSCAN IMAGYST system on feline parasites (Ancylostoma and Toxocara cati) and protozoan parasites (Cystoisospora and Giardia) and to assess and compare the performance of the VETSCAN IMAGYST centrifugal flotation method to reference centrifugal and passive flotation methods. Methods To evaluate the diagnostic performance of the scanning and algorithmic components of the VETSCAN IMAGYST system, fecal slides were prepared by the VETSCAN IMAGYST centrifugal flotation technique with pre-screened fecal samples collected from dogs and cats and examined by both an algorithm and parasitologists. To assess the performance of the VETSCAN IMAGYST centrifugal flotation technique, diagnostic sensitivity and specificity were calculated and compared to those of conventional flotation techniques. Results The performance of the VETSCAN IMAGYST algorithm closely correlated with evaluations by parasitologists, with sensitivity of 75.8–100% and specificity of 93.1-100% across the targeted parasites. For samples with 50 eggs or less per slide, Lin’s concordance correlation coefficients ranged from 0.70 to 0.95 across the targeted parasites. The results of the VETSCAN IMAGYST centrifugal flotation method correlated well with those of the conventional centrifugal flotation method across the targeted parasites: sensitivity of 65.7–100% and specificity of 97.6–100%. Similar results were observed for the conventional passive flotation method compared to the conventional centrifugal flotation method: sensitivity of 56.4–91.7% and specificity of 99.4–100%. Conclusions The VETSCAN IMAGYST scanning and algorithmic systems with the VETSCAN IMAGYST fecal preparation technique demonstrated a similar qualitative performance to the parasitologists’ examinations with conventional fecal flotation techniques. Given the deep learning nature of the VETSCAN IMAGYST system, its performance is expected to improve over time, enabling it to be utilized in veterinary clinics to perform fecal examinations accurately and efficiently.