Safety of an oral combination of moxidectin, afoxolaner, and pyrantel pamoate in dogs.

NexGard®PLUS (moxidectin, afoxolaner, and pyrantel pamoate), is an oral combination product for dogs indicated for the prevention of heartworm disease, the treatment and prevention of flea and tick infestations, and the treatment of gastro-intestinal nematode infections. The safety of this product in dogs was evaluated in three studies. Study #1 was a margin-of-safety study conducted in puppies, dosed six times at 28-day intervals at 1X, 3X, or 5X multiples of the maximum exposure dose (equivalent to 24 µg/kg moxidectin, 5 mg/kg afoxolaner, and 10 mg/kg pyrantel). In Study #2, the product was administered to ABCB1-deficient collie dogs at a 1X dose twice at a 28-day interval, and at a 3X or 5X dose once. Study #3 evaluated the safety of the product at 1X and 3X doses administered three times at 4-week intervals, to dogs harboring adult Dirofilaria immitis. In the three studies, the safety was evaluated on the basis of multiple clinical observations and physical examinations, including a complete assessment of toxicity to macrocyclic lactones, and on comprehensive clinical and anatomical pathology evaluations in Study #1. No clinically significant combination product-related effects were observed in any of the three studies. No signs of macrocyclic lactone toxicity were observed in the ABCB1-deficient collie dogs. Some mild and self-resolving instances of emesis or diarrhea were occasionally observed in the 3X and 5X dosed dogs. NexGard® PLUS was demonstrated to be safe following multiple administrations in puppies, in ABCB1-deficient collie dogs, and in microfilaremic dogs infected with adult D. immitis.

Concurrent Validation of MI-CAT(V), a Clinical Metrology Instrument for Veterinarians Assessing Osteoarthritis Pain in Cats, through Testing for Firocoxib Analgesic Efficacy in a Prospective, Randomized, Controlled, and Blinded Study.

Veterinarians face the lack of a rapid, reliable, inexpensive, and treatment-sensitive metrological instrument reflecting feline osteoarthritis (OA) pain. The Montreal Instrument for Cat Arthritis Testing, for Use by Veterinarians (MI-CAT(V)) has been refined in 4 sub-sections, and we proposed its concurrent validation. Cats naturally affected by OA (n = 32) were randomly distributed into 4 groups of firocoxib analgesic (Gr. A: 0.40; B: 0.25; C: 0.15, and P: 0.00 mg/kg bodyweight). They were assessed during Baseline, Treatment, and Recovery periods using MI-CAT(V) and objective outcomes (effort path, stairs assay compliance, and actimetry). The MI-CAT(V) total score correlated to the effort path and actimetry (RhoS = -0.501 to -0.453; p < 0.001), also being sensitive to treatment responsiveness. The pooled treatment group improved its total, gait, and body posture scores during Treatment compared to the Baseline, Recovery, and placebo group (p < 0.05). The MI-CAT(V) suggested a dose-(especially for Gr. B) and cluster-response. Cats in the moderate and severe MI-CAT(V) clusters responded to firocoxib with a remaining analgesic effect, while the mild cluster seemed less responsive and experienced a negative rebound effect. The MI-CAT(V) was validated for its OA pain severity discriminatory abilities and sensitivity to firocoxib treatment, providing a new perspective for individualized care.

Plasma chemistry reference intervals for adult multi-drug-resistance gene deficient Collies.

BACKGROUND: Differences in the reference intervals between dog breeds have been recognized for many years. Due to the importance of multi-drug-resistance 1-deficient (MDR1) Collies in veterinary medicine, it is important to determine whether breed-specific reference intervals are needed for this group. OBJECTIVES: The goal of this study was to establish plasma chemistry reference intervals for adult MDR1-deficient Collies. METHODS: Plasma samples collected from 110 healthy male and female adult MDR1-deficient Collies were analyzed for 21 analytes on a Beckman AU480 clinical chemistry analyzer. Reference intervals were established using a nonparametric statistical method. RESULTS: Reference intervals were established for 21 biochemical measurands in healthy adult MDR1-deficient Collies. CONCLUSION: Plasma chemistry reference intervals for MDR1-deficient Collies were clinically similar to intervals earlier created for Beagles. Intervals for male and female MDR1-deficient Collies were very similar to each other.

Development of Two Innovative Performance-Based Objective Measures in Feline Osteoarthritis: Their Reliability and Responsiveness to Firocoxib Analgesic Treatment.

The metrological properties of two performance-based outcome measures of feline osteoarthritis (OA), namely Effort Path (Path) and Stairs Assay Compliance (Stairs), were tested. Cats naturally affected by OA (n = 32) were randomly distributed into four groups (A: 0.40, B: 0.25, C: 0.15, or D: 0.00 mg firocoxib/kg bodyweight) and assessed during baseline, treatment, and recovery periods. For Path, from an elevated walking platform, the cats landed on a pressure-sensitive mattress and jumped up onto a second elevated platform. Analysis included velocity, time to completion, peak vertical force (PVF), and vertical impulse. For Stairs, the number of steps and time to completion were recorded for 16 steps up and down in a 4 min period. Reliability was moderate to very good for Path and poor to good for Stairs. Different normalization methods are described in the manuscript. The placebo group remained stable within-time in Path, whereas treated cats trotted faster on the ramp (p < 0.0001), improved their PVF (p < 0.018) and completed the task quicker (p = 0.003). The percentage of cats completing the Stairs finish line was higher under treatment (p < 0.036), with huge effect size, the placebo group results being stable within-time. Both are promising performance-based outcome measures to better diagnose and manage feline OA pain.

Safety of oral afoxolaner formulated with or without milbemycin oxime in homozygous MDR1-deficient collie dogs.

Afoxolaner, an insecticide and acaricide compound of the isoxazoline class, is available for dogs as an oral ectoparasiticide medicine (NexGard®) and as an oral endectoparasiticide medicine in combination with milbemycin oxime (MO), a macrocyclic lactone (NexGard® Spectra). The safety of these two compounds, alone or in combination, was investigated in homozygous MDR1-deficient collie dogs, in two studies. Overall, 30 adult collie dogs were treated once orally, 9 with a placebo, 9 with afoxolaner, 6 with MO, and 6 with a combination of afoxolaner and MO. For afoxolaner, the mean investigated dosage corresponded to 3.8 and 4.7 multiples of the maximum recommended therapeutic doses (RTD) in NexGard® and NexGard® Spectra, respectively. For MO, the mean investigated dosage corresponded to 4.7 multiples of the maximum RTD in NexGard® Spectra. Dogs were closely monitored for adverse reactions on the day of treatment and for the following two days. No significant adverse reaction was observed in any dog from the afoxolaner or the afoxolaner + MO groups; in the MO-only treated group, mild and transient neurological signs were observed during the 4-8 h post-treatment window. These studies demonstrated a high level of safety of oral afoxolaner, alone or in combination with milbemycin oxime, in homozygous MDR1-deficient dogs.

The intravenous and oral pharmacokinetics of afoxolaner used as a monthly chewable antiparasitic for dogs.

The pharmacokinetics of afoxolaner in dogs was evaluated following either intravenous or after oral administration of NEXGARD(®), a soft chewable formulation. Afoxolaner is a member of one of the newest classes of antiparasitic agents, known as antiparasitic isoxazolines. The soft chewable formulation underwent rapid dissolution, and afoxolaner was absorbed quickly following oral administration of the minimum effective dose of 2.5mg/kg, with maximum plasma concentrations (Cmax) of 1,655 ± 332 ng/mL observed 2-6h (Tmax) after treatment. The terminal plasma half-life was 15.5 ± 7.8 days, and oral bioavailability was 73.9%. Plasma concentration-versus-time curves fit a 2-compartment model and increased proportionally with dose over the oral dose range of 1.0-4.0mg/kg, and over the oral dose range from 1.0 to 40 mg/kg. Following an intravenous dose of 1mg/kg, the volume of distribution (Vd) was 2.68 ± 0.55 L/kg, and the systemic clearance was 4.95 ± 1.20 mL/h/kg. Afoxolaner plasma protein binding was >99.9% in dogs. One major metabolite, formed following hydroxylation of afoxolaner, was identified in dog plasma, urine and bile. When afoxolaner is administered orally, there is a strong correlation between afoxolaner plasma concentration and efficacy with EC90 values of 23 ng/mL for Ctenocephalides felis and ≥ 100 ng/mL for Rhipicephalus sanguineus sensu lato and Dermacentor variabilis. The pharmacokinetic properties of afoxolaner are suited for a monthly administration product because the fast absorption and long terminal half-life support a rapid onset of action while ensuring month-long efficacy.

Assessment of the onset of action of afoxolaner against existing adult flea (Ctenocephalides felis) infestations on dogs.

The speed of kill of afoxolaner against experimental infestations by Ctenocephalides felis was evaluated after oral administration of afoxolaner in a soft chew (NEXGARD(®)) at a dose to achieve 2.5mg/kg bodyweight. Forty beagles were allocated to two treatment groups. Dogs in Treatment Group 1 were untreated controls. Dogs in Treatment Group 2 were treated on Day-0 with afoxolaner, according to their pre-treatment bodyweight. All dogs were infested with approximately 100 C. felis on Day-1. Live fleas were counted upon removal at 5 time points after treatment (i.e., 2, 4, 8, 12 and 24h after treatment). For each time point, counts were performed on 4 dogs from each of the treated and the untreated groups. Early curative flea killing efficacy was evaluated with respect to the untreated control group. The afoxolaner treated group had significantly fewer fleas than the untreated control group at 8, 12, and 24h (p<0.001). The percent efficacies of orally administered afoxolaner were 15.0%, 87.8%, 99.5%, 100.0%, and 100.0% at 2, 4, 8, 12, and 24h, respectively. In this study, afoxolaner began killing fleas by 2h after treatment with increasing efficacy at subsequent time points and had >99.5% efficacy at 8, 12, and 24h after treatment demonstrating an early onset of action.

Assessment of the efficacy of orally administered afoxolaner against Rhipicephalus sanguineus sensu lato.

Two studies were conducted to confirm that a single oral dose of the novel insecticide/acaricide afoxolaner is efficacious against existing infestations of Rhipicephalus sanguineus sensu lato in dogs and can control re-infestation for up to 35 days. Each study utilized 16 purpose bred adult dogs using a controlled randomized block design. One or two days prior to treatment, all dogs were infested with 50 unfed adult ticks. On Day 0 one group was treated with an oral chewable formulation of afoxolaner at a dose as close as possible to the minimum dose of 2.5mg/kg. Weekly re-infestations with 50 adult unfed ticks were repeated for five weeks. Forty-eight hours after treatment and after each re-infestation, the number of remaining live ticks on each dog was counted. Treatment with afoxolaner resulted in efficacies of 98.8-100% within 48 h on existing tick infestations, while the efficacy against new tick infestations was >95.7% over five weeks.

Evaluation of the efficacy of afoxolaner against Haemaphysalis longicornis on dogs.

A controlled study to assess the acaricidal efficacy of afoxolaner in dogs after a single oral administration was conducted against Haemaphysalis longicornis ticks. The study was characterized by a negative controlled randomized block design and included sixteen beagle dogs of both sexes. Starting two days before treatment, each dog was infested weekly with 50 ticks over 4 weeks. The number of live ticks was determined 48 h after treatment and then 48 h after each infestation. The mean dose of afoxolaner received by dogs was 3.0mg/kg (range: 2.5-3.1mg/kg). Afoxolaner rapidly eliminated pre-existing tick infestations (100% ticks killed within 48 h of treatment) and controlled weekly re-infestations (91.9% prophylactic efficacy at Day 30).

Safety evaluation of orally administered afoxolaner in 8-week-old dogs.

The safety profile of afoxolaner, a new isoxazoline molecule, was evaluated following the regulatory requirements when administered six times orally in a soft chewable formulation at a dose of at least 1×, 3× or 5× the maximum exposure dose (6.3mg/kg) in 8-week-old Beagle dogs. Thirty-two healthy puppies (16 males and 16 females) were enrolled and allocated randomly to one of four treatment groups. Treatments were administered at three, one-month dose intervals (Days 0, 28 and 56) followed by three, 2-week dose intervals (Days 84, 98 and 112). The study ended at Day 126. The groups were: Group 1: non-treated control; Group 2: afoxolaner chews administered at a dosage of at least 6.3mg/kg (1×); Group 3: afoxolaner chews administered at a dosage of at least 18.9 mg/kg (3×); and Group 4: afoxolaner chews administered at a dosage of at least 31.5mg/kg (5×). All dogs were examined for general health twice a day beginning on at least Day-14. Physical examinations, and blood collections for clinical pathology analysis and afoxolaner plasma concentrations, were performed throughout the study. On Day 126, 2 weeks following the last treatment, all dogs were humanely euthanized prior to the conduction of a full necropsy with tissue collection. No afoxolaner-related changes were observed in growth, physical variables, clinical pathology variables, or tissues examined histologically. No clinically or statistically significant health abnormalities related to the administration of afoxolaner were observed. Vomiting and diarrhea were observed sporadically across all groups including the controls. The kinetics of afoxolaner plasma concentrations was linear following 6 doses of 6.3, 18.9 and 31.5mg/kg and dose proportionality was demonstrated. There were no statistical differences (p<0.05) between samples taken on Days 55 and 83 when compared to Day 27. Based upon the results of this study, afoxolaner was shown to be safe when administered repeatedly in a soft chewable formulation at up to 5× the maximum exposure dose in dogs as young as 8 weeks of age.

Discovery of the development candidate N-tert-butyl nodulisporamide: a safe and efficacious once monthly oral agent for the control of fleas and ticks on companion animals.

Nodulisporic acid A (1) is a structurally complex fungal metabolite that exhibits systemic efficacy against fleas via modulation of an invertebrate specific glutamate-gated ion channel. In order to identify a nodulisporamide suitable for monthly oral dosing in dogs, a library of 335 nodulisporamides was examined in an artificial flea feeding system for intrinsic systemic potency as well as in a mouse/bedbug assay for systemic efficacy and safety. A cohort of 66 nodulisporamides were selected for evaluation in a dog/flea model; pharmacokinetic analysis correlated plasma levels with flea efficacy. These efforts resulted in the identification of the development candidate N-tert-butyl nodulisporamide (3) as a potent and efficacious once monthly oral agent for the control of fleas and ticks on dogs and cats which was directly compared to the topical agents fipronil and imidacloprid, with favorable results obtained. Multidose studies over 3 months confirmed the in vivo ectoparasiticidal efficacy and established that 3 lacked overt mammalian toxicity. Tissue distribution studies in mice using [(14)C]-labeled 3 indicate that adipose beds serve as ligand depots, contributing to the long terminal half-lives of these compounds.

Firocoxib efficacy preventing urate-induced synovitis, pain, and inflammation in dogs.

This positive-control study evaluated the efficacy of firocoxib versus carprofen, deracoxib, and meloxicam for the prevention of pain and inflammation in a urate crystal synovitis model of lameness. Lameness scoring and force plate gait analysis were used to assess efficacy. The resulting lameness scores and force plate ground reaction forces after urate crystal injection were not significantly different among the groups. Relative to each group's baseline (nonlame) score, only the firocoxib group was not significantly lame, based on lameness score, at the model's peak effect.

Efficacy and safety of firocoxib in the management of canine osteoarthritis under field conditions.

A total of 249 client-owned dogs with osteoarthritis were treated with firocoxib (5 mg/kg/day) or a positive control, etodolac (10-15 mg/kg/day), for 30 days. Veterinary examinations were performed on approximately days 0 (visit 1), 14 (visit 2), and 29 (visit 3). Based on defined noninferiority criteria, firocoxib and etodolac were comparable. Based on the magnitude of the change from baseline, improvement with firocoxib was significantly greater than with etodolac for lameness at a trot (visits 2 and 3) and for lameness at a walk, pain on manipulation, and range of motion (visit 3) (P < .05). In weekly owner evaluations, firocoxib provided significantly greater improvement than etodolac (P < .05) at each scoring.

MDR1-deficient genotype in Collie dogs hypersensitive to the P-glycoprotein substrate ivermectin.

Multidrug resistance (MDR) phenotypes in cancer cells are associated with overexpression of the drug carrier P-glycoprotein. The antiparasitic drug ivermectin, one of its substrates, abnormally accumulates in the brain of transgenic mice lacking the P-glycoprotein, resulting in neurotoxicity. Similarly, an enhanced sensitivity to ivermectin has been reported in certain dogs of the Collie breed. To explore the basis of this phenotype, we analyzed the canine P-glycoprotein-encoding MDR1 gene, and we report the first characterization of the cDNA for wild-type (Beagle) P-glycoprotein. The corresponding transcripts from ivermectin-sensitive Collies revealed a homozygous 4-bp exonic deletion. We established, by genetic testings, that the MDR1 frame shift is predictable. Accordingly, no P-glycoprotein was detected in the homozygote-deficient dogs. In conclusion, we characterized a unique case of naturally occurring gene invalidation. This provides a putative novel model that remains to be exploited in the field of human therapeutics and that might significantly affect tissue distribution and drug bioavailability studies.