Pharmacokinetics of tildipirosin in beagle dogs

Tildipirosin (20,23-di-piperidinyl-mycaminosyl-tylonolide, TD, Cas No 328898-40-4) is a semisynthetic derivative of the naturally occurring 16-membered macrolide tylosin. It is exclusively used in veterinary practice. Tildipirosin has been approved for parenteral treatment of respiratory disease in cattle and swine (EMA, 2010). This macrolide antibiotic is rapidly absorbed and extensively distributed to tissues where they achieve multifold higher concentrations relative to those observed in pigs and cattle plasma samples (Anadon & Reeve-johnson, 1999; Norcia et al., 2004).

Due to the extensive use of TD, several studies on the pharmacokinetics, pharmacodynamics, particularly, distribution, and metabolism have been conducted in various species, including pigs (Rose et al., 2013; Torres et al., 2016) and cattle (Menge et al., 2012). In pigs, it is postulated that the metabolism of tildipirosin proceeds by reduction and sulfate conjugation with subsequent hydration (or ring opening), by demethylation, by dihydroxylation, and by S-cysteine and S-glutathione conjugation (EMA, 2015). In cattle, it is postulated that metabolism of tildipirosin proceeds by cleavage of the mycaminose sugar moiety, by reduction and sulfate conjugation with subsequent hydration, by demethylation, by mono- or dihydroxylation with subsequent dehydration, and by S-cysteine and S-glutathione conjugation (EMA, 2015). Tildipirosin (Zuprevo^®) is labeled for use in swine and cattle (FDA, 2012). While it is not approved for use in dogs, there are numerous anecdotal reports of clinical effectiveness of TD in treating canine respiratory diseases. EMA reported pharmacokinetic characteristics of TD via oral route in beagle dogs (EMA, 2010). However, no information is available on the pharmacokinetic characteristics of TD via i.v and i.m. in dogs which could be accessed. The aim of this study was to fully characterize pharmacokinetic profiles of TD in dogs following intravenous (i.v.) and intramuscular (i.m.) administration, respectively.

Tildipirosin (Zuprevo^® 40 mg/ml; MSD Animal Health) was used in this study. The study protocols were approved by the Animal Use and Care Administrative Advisory Committee of the China Agricultural University. Both male and female beagle dogs, aged 1.5–2.5 years and weighing 9–12 kg, were used (three males and three females per group). In study 1, groups A, B, and C were administered i.m. at 2 mg (n = 6), 4 mg (n = 6), and 6 mg (n = 6) per kg, respectively. In study 2, the dogs were administered i.v. at 2 mg/kg (n = 6). Prior to study, all animals were acclimatized for 1 week with commercial standard feed and free access to fresh water. Blood samples of 2 ml were collected from the cephalic vein directly into heparinized tubes prior to drug administration and 5, 15, 30 min, 1, 2, 4, 8, 10 hr, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 14 days postadministration. The samples were centrifuged at 2,280 g for 10 min, and the plasma samples were stored at −20°C prior to further analysis.

The extraction procedures were according to published documents with mild modification (Rose et al., 2013): Protein precipitation of 200 μl of plasma was conducted by extraction with 600 μl acetonitrile and then vortex-mixed for 2 min. The mixture was centrifuged at 32,630 g for 10 min at 4°C. The transferred supernatants were evaporated to dryness under nitrogen flow at 50°C. Afterward, the residues were finally dissolved in 300 μl acetonitrile/water (v/v 1: 9, 0.1% formic acid). Subsequently, the mixture was vortexed for 5 min and centrifuged at 32,630 g for 15 min at 4°C. The supernatants were collected and filtrated through a 0.22-μm microbore cellulose membrane, and analyzed through Ultra high-pressure liquid chromatography mass/mass spectrometer (UPLC-MS/MS).

The plasma samples and TD standards were detected using an Acquity UPLC–MS/MS system (Waters Co., USA) with BEH C₁₈ column (2.1 × 100 mm, 1.7 μm). About 0.1% formic acid (solvent A) and acetonitrile (solvent B) were used as mobile phase with a flow rate of 0.35 ml/min according to the following gradient elution program: 0–0.5 min, 98% solvent A; 0.5–1.5 min, 98%–20% solvent A; 1.5–2 min, 20%–2% solvent A; 2.0–2.8 min, 2%–2% solvent A; 2.8–2.81 min, 2%–98% solvent A; and 2.81–3.3 min, 98% solvent A. Typical mass conditions were as follows: source temperature, 120°C, capillary voltage, 2.8 kV, and ESI⁺. The parent ion was m/z 735.1, while the quantified and qualified ions were m/z 98 and m/z 174.2, acquired at the same cone voltage, 65 V, with the collision energy of 35 eV, respectively. This method has been thoroughly validated with the limit of quantification of 5 ng/ml. The calibration curves were in good linearity cover the range of 5–1,000 ng/ml with the coefficient of correlation higher than 0.999746. The interday and intraday coefficients of variation at three different concentrations (20, 200, 800 ng/ml) were all below 7.43%, meanwhile the mean recoveries ranged from 91.90 to 108.01%, meeting the requirements of criteria of Guidance for Industry Bioanalytical Method Validation (FDA, 2013).

To determine the degree of exposure following administration of a drug (such as AUC), and pharmacokinetic parameters, such as clearance, elimination half-life, T_max, and C_max, noncompartmental analysis is generally the preferred method to use, because it requires fewer assumptions than model-based approaches (Gabrielsson & Weiner, 2012). Plasma pharmacokinetic (PK) parameters were calculated through noncompartmental analysis model 200 (intravenous or extravascular dosing, linear/log trapezoidal method, 1/y weighting) in WinNonlin™ software (version 6.4; Pharsight Corporation, Mountain View, CA, USA). The main parameters after i.m. injection such as C_max, AUC, and T_max were observed data. Intramuscular bioavailability (F%) was gained from the ratio of the areas under the plasma concentration curve.

There were no visible adverse effects following i.v and i.m. injection of TD at different doses of the drug. However, at much higher dose of TD in dogs (180 mg kg⁻¹day⁻¹ in a 4-week toxicity study) gastrointestinal disturbances have been reported (EMA, 2010) and at 300 mg kg⁻¹day⁻¹ in a maximum tolerated dose study, CNS disturbances have been reported. The NOEL was 10 mg kg⁻¹day⁻¹. The mean PK parameters following i.m. administration of TD at 2, 4, and 6 mg/kg and i.v. administration of 2 mg/kg, and the absolute bioavailability (F%) are summarized in Table 1. In agreement with the EMA report (EMA, 2010), TD plasma concentrations did not differ significantly between genders. The mean plasma concentrations vs. time curves after each administration are plotted on a semilogarithmic in Figure 1 and Figure 2.

To our best knowledge, this is the first report on the PK properties of TD via i.v and i.m. administration in dogs. At all three dosage levels, plasma concentrations above the lower limit of quantification (LLOQ) of 5 ng/ml were determined up to the collection time point 14 days after i.m. administration. Similar to C_max, the area under the plasma concentration vs. time curve increased in a dose-dependent manner. The areas under the concentration–time curve in dogs are different from those of pigs and cattle, which could be due to interspecies differences. The dose linearity was observed within the dose range of 2–6 mg/kg. The mean time to reach C_max after the i.m. injection of TD suggested that it is rapidly absorbed after i.m. administration. The mean value of T_1/2λz for i.m. administration of TD at doses of 2, 4, and 6 mg/kg is equivalent to 3, 3.7, and 4 days. The long persistence of TD in the body is demonstrated further by a pronounced T_1/2λz for which consistency was demonstrated for matrix and administration route. The mean residence time (MRT) was validated for this. Almost without exception, TD, like most macrolides, has been classified as time-dependent killing drugs and therefore is best described by the PK/PD parameter time above MIC (T > MIC) for newer macrolides appears to be the best correlate with therapeutic effect. For example, a previous study reported that MIC₉₀ of TD for Actinobacillus pleuropneumoniae in pigs was 2,000 ng/ml (Rose et al., 2013). The concentration of tildipirosin was approximately 100 times higher in porcine lung than plasma during days 1–14 after i.m. administration (Rose et al., 2013). Although in the current study, the C_max of TD (approximately 1,000 ng/ml) in canine plasma after 4 mg/kg i.m. was not higher than the MIC₉₀ for that bacterium (Rose et al., 2013), it can be assumed that TD concentration in canine lungs was probably higher than that MIC₉₀ for a few days. Further investigation is necessary to get detailed information of PK/PD relationship of TD to confirm this assumption in dogs.

These PK characteristics of TD after i.m. injection suggested that TD is rapidly distributed into tissues due to the high lipid solubility and then released into the plasma. In Figure 1, there is artifactual fluctuation in TD concentrations. In addition, high individual variability was observed and the results were very different from those in EMA 2010. This could be attributed to individual physiologic variability, differences in fat deposition, and variations in gastrointestinal, hepatic, and renal functions. Meanwhile, the use of a single drug administration site could have led to variable drug absorption rates among individual animals. The variability in drug absorption rate is likely to appear when there is a limited surface area of absorption which leads to drug pooling. Furthermore, in virtue of that TD is an extended release formulation (Rose et al., 2013), T_1/2λz following i.m. injection could be impacted by “flip-flop” kinetics where the slower and extended absorption process may complicate the estimation of the terminal elimination rates. Another potential artifactual cause of the fluctuation could be entero-hepatic recirculation. Two absorption peaks may be observed if the drug enters entero-hepatic recirculation. Further work is needed to study this phenomenon.

In conclusion, tildipirosin administered by single i.m. administration to dogs showed long half-life, high bioavailability, and a strong dose–response relationship with rapid absorption. The main parameters could provide a foundation for the use of TD in dog. Further investigation is necessary to get deeper information of PK/PD relationship of tildipirosin.