Pharmacokinetics of orally administered single‐dose ponazuril in cats

Abstract Cats and kittens in animal shelters and catteries regularly suffer from severe gastrointestinal coccidiosis, which can be fatal, and there are no drugs labeled for feline coccidiosis in the United States. Ponazuril, a triazine‐class drug, is increasingly used at a dose of 50 mg/kg/d, orally, for three to five days in shelter environments for coccidiosis. A single oral dose of ponazuril paste 15% (Marquis®; Merial) at 50 mg/kg was administered to six healthy adult cats. Sample analysis was completed via high‐performance liquid chromatography. Plasma concentrations peaked at 7.49 ± 2.06 µg/ml at 14.67 ± 7.45 hr post‐administration. This study shows that ponazuril achieved a plasma concentration that inhibits growth of similar organisms after a single oral dose in cats. Further studies are necessary to optimize dosing for the treatment of clinical coccidiosis in cats.

Intestinal coccidiosis is difficult for shelters to manage as individuals most susceptible to clinical disease also are vulnerable to other life-threatening illnesses. The only Food and Drug Administration (FDA)-approved drug for treatment of coccidiosis in a small animal is the coccidiostatic drug sulfadimethoxine, it is administered for five to 21 days for coccidia, which makes it intensive from a time and economic standpoint. Lloyd and O'Brien reported in the early 2000s that toltrazuril reduces coccidia oocyst shedding and lessens diarrhea in kittens. (Lloyd & Smith, 2001;O'Brien et al., 2002) Dosing recommendations have ranged from only one dose, one dose followed by a second dose five days later, to daily doses for three or five days.
Unfortunately, toltrazuril is not available in the United States of America (US). Ponazuril, a metabolite of toltrazuril, is available in the United States, which is commonly used even though it can be harder to accurately dose due to the concentration of the commercially available product. A recent study by Litster demonstrated the highest reduction of fecal oocysts when naturally infected shelter cats were dosed with 50 mg/kg of ponazuril for three consecutive days. This is a much higher dosage than recommended for other species.
Of the cats, 12.5% had detectable oocysts at days three and four post-treatment. (Litster et al., 2014) It is unknown whether persistent shedding was due to inadequate absorption and, thus, subtherapeutic blood concentrations in some cats. If so, a higher dosage might be more efficacious in treating gastrointestinal coccidiosis. The objective of this study was to obtain pharmacokinetic parameters for a single oral administration of ponazuril (50 mg/kg) in healthy adult cats.

| Experimental subjects
Six healthy, female adult, purpose-bred domestic short hair cats from a research colony at the University of Tennessee, Knoxville, were used. Cats were excluded if abnormalities were present on laboratory diagnostics performed two months prior to the study (CBC, serum biochemistry profile, and urinalysis), or if there were any physical examination findings suggestive of systemic or gastrointestinal disease including poor hair coat, low body or muscle condition score (<5/9 or <3/3, respectively), or abnormalities identified on abdominal palpation or thoracic auscultation. The cats weighed 3.58-5.08 kg with a mean of 4.53kg. The study was approved by the Institutional Animal Care and Use Committee at the University of Tennessee, Knoxville (Protocol #2783-0920).
Studies using six animals allow the acquisition of precise preliminary estimates of the average pharmacokinetic parameters. (Riviere, 2011).
Twenty-four hours prior to ponazuril administration, 24G long line sampling catheters (MILA International) were placed after intramuscular sedation with 40 μg/kg dexmedetomidine (Dexdomitor ® ; Orion Co.) and 5 mg/kg ketamine (Zetamine ® ; Vet One). The following day (24 hr later), the cats were administered 50 mg/kg of ponazuril paste 15% (Marquis ® ; Merial), orally and monitored for complete ingestion. Ponazuril was weighed to the nearest microgram to ensure dose accuracy. Sampling catheters were removed within two days of placement due to decreased blood sampling frequency or lack of patency. Blood was collected at time 0, 15, and 30 minutes, then 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, and 336 hr. These time points were chosen based on clearance times observed in other species. (Dirikolu et al., 2009;Prado et al., 2011;Zou et al., 2014) Most samples consisted of 1ml of blood; no more than 24 ml of blood was taken from one cat over the 2 week period. For samples taken via catheter, a 3 ml sample was withdrawn prior to collection of the sample for testing, which was returned prior to flushing the catheter to limit the total volume of blood removed from each cat. The location of venipuncture for samples taken after catheters were removed alternated on the right and left sides from lateral saphenous, medial saphenous, cephalic, and jugular veins. Plasma was spun at 3,000 g for 15 min and frozen at −80ºC and assayed within 2 weeks of collection. Cats were monitored for lethargy, restlessness, change in appetite, vomiting, and diarrhea twice daily throughout the study.

| Extraction method
Ponazuril was extracted from plasma samples using a liquid-liquid extraction method. (Cox et al., 2021) Previously frozen plasma samples were thawed, vortex-mixed, and 100 µl of plasma was transferred to a 13 × 100 mm screw top tube followed by 10 µl of diclazuril (internal standard, 100 µg/ml) and 2 ml chloroform. The tubes were rocked for 15 min and then centrifuged for 20 min at 1000 g. The organic layer was transferred to a clean tube and evaporated to dryness with nitrogen gas. Samples were reconstituted in 250 µl of mobile phase and 100 µl was analyzed.

| Chromatographic conditions and apparatus
The system consisted of a 2695 separations module, a 2487 UV absorbance detector, and a computer equipped with Empower software (Waters). The compounds were separated on a Symmetry RP 18 (4.6 × 150 mm, 5 µm) column with a Symmetry Shield RP 18 (3.8 mm × 5 mm × 5µm) guard column. The mobile phase was a mixture of 0.1% formic acid in water and acetonitrile (50:50 v/v).
Absorbance was measured at 254 nm with a flow rate of 1.1 ml/min and the column temperature was maintained at 23°C.

| Calibration
Calibration plasma samples were obtained from six different cats not treated with ponazuril, to verify the possibility of any interfering matrix compounds near the retention time of ponazuril, and were prepared exactly as study plasma samples. The standard curve was composed of 0.1, 0.25, 0.5, 1, 2.5, 5, 10 and 25 μg/ml concentrations, which were chosen based on expected results of study samples. Standard curves for plasma analysis were prepared by fortifying untreated, pooled plasma with ponazuril to produce a linear concentration range of 0.1 -25 µm/ml.

| RE SULTS
The average recovery of ponazuril for the quality control (QC) samples (0.3, 7.5, and 15 µg/ml) and the LLOQ was 101% ± 0.5%. This was obtained for the liquid-liquid extraction method from feline plasma using a reversed phase high performance liquid chromatography method. The intra-assay variability for the QC samples ranged from 3.7% to 9% while the accuracy was 100%. The interassay variability ranged from 4.9% to 7.6% and the accuracy ranged from 96% to 104%. The lower limit of quantification (LLOQ) was 0.1 µg/ml. The accuracy and precision for the method was consid-

Sarcocystis neurona infection in horses. Ponazuril is an effective
anti-coccidial medication in chickens, calves, and buffalo. (Epe et al., 2005;Ghanem et al., 2008;Laczay et al., 1995) Previous pharmacokinetic studies have determined absorption of singledose oral administration of ponazuril in cattle, goats, horses, llamas, and swine. (Dirikolu et al., 2009;Furr & Kennedy, 2000;Love et al., 2015;Prado et al., 2011;Zou et al., 2014) Pharmacokinetic parameters vary dramatically by species. Both Dirikolu, et al. and Prado, et al. proposed that the variability of half-life among species may reflect a difference in the rate of oral absorption rather than elimination rate. (Dirikolu et al., 2009;Prado et al., 2011) The rate of absorption can be affected by binding to food in the gastrointestinal tract. The earliest detection of plasma ponazuril above the LLOQ occurred at 30 min in 50% of the subjects, with detection in all cats at 1 hr. The t max was 14.67 ± 7.45 hr, indicating absorption rate was much faster in cats than llamas, goats, swine, and cattle; (Dirikolu et al., 2009;Love et al., 2015;Prado et al., 2011;Zou et al., 2014) however, the determination of t max can be dependent on sampling schedule and the clinical significance of this difference is unknown. The C max ranged from 5.4 to 10.4 µg/ml with a mean of 7.49 ± 2.06 µg/ml, which is higher than cattle or swine but lower than llamas, horses, and goats. (Dirikolu et al., 2009;Furr & Kennedy, 2000;Love et al., 2015;Prado et al., 2011;Zou et al., 2014) It should be noted for the C max comparisons that these other studies used lower dosages than the cats in this study, with llamas receiving 20 mg/kg, goats and horses receiving 10 mg/kg, and cattle and pigs receiving 5 mg/kg. The mean t½ of ponazuril in cats was similar to that of goats, llamas, and swine, whereas it was greater than two-fold longer than in cattle or horses. (Dirikolu et al., 2009; TA B L E 1 Pharmacokinetic parameters (mean ± SD) in cats following oral administration of 50 mg/kg ponazuril (n = 6) MRT 0-∞ (h) 208 ± 67 Note: Elimination half-life (t½), elimination rate constant (λz), maximum plasma concentration (C max ), time to maximum plasma concentration (t max ) , area under the plasma concentration-time curve from time 0 to last point (AUC 0-last ), area under the plasma concentration-time curve from time 0 to infinity (AUC 0-∞ ), percent of the AUC 0-∞ extrapolated to infinity (AUC extrap ), mean residence time (MRT 0-∞ ). Furr & Kennedy, 2000;Love et al., 2015;Prado et al., 2011;Zou et al., 2014).
Limitations of this study include the inability to study bioavailability and the sampling timeframe. An injectable formulation of ponazuril is not available; therefore, the oral bioavailability of ponazuril in cats cannot be determined from our study; however, future studies could consider preparing ponazuril in a suitable vehicle for intravenous administration. Sample collection was stopped 14 days after ponazuril administration, at which point ponazuril was still measurable. When the last measured concentrations are above the assay lower limit of quantification, parameters such as halflife and mean residence time can be artifactually elevated, (Smith et al., 2018) as the ability to detect lower concentrations for a lon-

CO N FLI C T O F I NTE R E S T
The authors disclose no conflict of interest.

AUTH O R CO NTR I B UTI O N
CDB contributed to sample collection, data analysis, and manuscript writing. SC led analytical method development, sample and data analysis, and contributed to study design and manuscript preparation. JSS was involved in sample collection, data analysis, and manuscript preparation. JES contributed to overall study execution and manuscript preparation. JCW contributed to overall study execution and manuscript preparation. BKD was involved in study design, sample collection, data analysis, and manuscript preparation. All authors have read and approved the final manuscript.

F I G U R E 1
Mean plasma concentration-time curve for ponazuril following oral administration of a single dose (50 mg/kg) to 6 healthy cats. Values reported are mean ± SD