J. vet. Pharmacol. Therap. 38, 471--474. doi: 10.1111/jvp.12222.

Pharmacokinetics of tulathromycin after subcutaneous injection in North American bison (Bison bison) K. BACHTOLD*

Bachtold, K., Alcorn, J., Matus, J., Boison, J., Woodbury, M. Pharmacokinetics of tulathromycin after subcutaneous injection in North American bison (Bison bison). J. vet. Pharmacol. Therap. 38, 471–474.


*College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada; †Centre for Veterinary Drug Residues (CVDR), Canadian Food Inspection Agency (CFIA), Saskatoon, SK, Canada; ‡ Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada

Tulathromycin is approved for the treatment of respiratory disease in cattle and swine. It is intended for long-acting, single-dose injection therapy (Draxxin), making it particularly desirable for use in bison due to the difficulty in handling and ease of creating stress in these animals. The pharmacokinetic properties of tulathromycin in bison were investigated. Ten wood bison received a single 2.5 mg/kg subcutaneous injection of Draxxin. Serum concentrations were measured by liquid chromatography–mass spectrometry (LC-MS) detection. Tulathromycin demonstrated early maximal serum concentrations, extensive distribution, and slow elimination characteristics. The mean maximum serum concentration (Cmax) was 195 ng/mL at 1.04 h (tmax) postinjection. The mean area under the serum concentration–time curve, extrapolated to infinity (AUC0–inf), was 9341 ngh/mL. The mean apparent volume of distribution (Vd/F) and clearance (Cls/F) was 111 L/kg and 0.4 L/h/kg, respectively, and the mean half-life (t1/2) was 214 h (8.9 days). Compared to values for cattle, Cmax and AUC0–inf were lower in bison, while the Vd/F was larger and the t1/2 longer. Tissue distribution and clinical efficacy studies in bison are needed to confirm the purported extensive distribution of tulathromycin into lung tissue and to determine whether a 2.5 mg/kg subcutaneous dosage is adequate for bison. (Paper received 20 October 2014; accepted for publication 18 February 2015) Murray Woodbury, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK, S7N 5B4, Canada. E-mail: [email protected]

The commercial bison industry continues to prosper in today’s economy (NBA, 2013). With inevitable intensification of production practices infectious disease, including respiratory infections (e.g., Mycoplasma bovis), has become an important cause of death in bison (Janardhan et al., 2010; Dyer et al., 2008). In North America, there are no antibiotic drugs approved for use in bison (Woodbury, 2012). Significant species differences in pharmacokinetics known to exist with many drug classes (Toutain et al., 2010), including the antimicrobials, bring into question the practice of treating bison with antimicrobials approved for use in cattle at the recommended cattle dose. An emergent need exists to determine species-specific drug therapy regimens to mitigate the morbidity and mortality associated with infectious diseases. Tulathromycin is a macrolide antibiotic approved in Canada and USA for the treatment of bovine and swine bacterial respiratory disease (APVMA, 2007; Evans, 2005; Pfizer, Inc, 2005; EMEA, 2004). It is widely used bovine respiratory disease asso© 2015 John Wiley & Sons Ltd

ciated with Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis and for the reduction of morbidity associated with respiratory disease in feedlot calves caused by the above named organisms during the first 14 days in the feedlot when administered at the time of arrival. (Zoetis Canada, 2014; APVMA, 2007; Pfizer, Inc, 2005). In cattle and swine, tulathromycin undergoes rapid absorption (Cmax < 1 h) and extensive distribution to tissues. Elimination half-lives typically range between 3 and 5 days. Metabolism is minimal, and tulathromycin is eliminated primarily as unchanged parent drug by biliary excretion (APVMA, 2007; Evans, 2005; Nowakowski et al., 2004; Benchaoui et al., 2004; G aler et al., 2004). Tulathromycin is formulated as a long-acting, single-dose injection therapy (Draxxin) (APVMA, 2007; Evans, 2005). Its chemical structure consists of three polar amine groups, which allows for greater tissue penetration and persistent activity in the lung (APVMA, 2007; Evans, 2005; Kilgore et al., 2005; 471

472 K. Bachtold et al.

G aler et al., 2004). Single-dose therapies are desirable in the bison industry where the difficulty associated with animal handling is a critical barrier to effective therapy. Despite its routine off-label use in bison, a review of the published literature failed to find any reference to the study of tulathromycin in this species. This study describes the single subcutaneous dose pharmacokinetics of tulathromycin in bison with a comparison to published cattle parameters to predict the adequacy of the recommended cattle dose for use in bison. Ten wood bison (Bison bison athabascae, female, 4–7 years) were housed at the Specialized Livestock Research Facility, University of Saskatchewan, and all bison had no known history of macrolide exposure. This study was approved by the University of Saskatchewan’s Animal Research Ethics Board and adhered to the Canadian Council on Animal Care guidelines for humane animal use. Bison were given a single 2.5 mg/kg bodyweight subcutaneous injection of commercially available 100 mg/mL Draxxin (Zoetis Canada, Kirkland, QC, Canada) in the side of the neck (therapeutic dose approved for cattle). Blood samples (10 mL) were collected by jugular venipuncture just prior to dose administration and at 1, 2, 4, 6, 12 h, and 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 21, 23, and 25 days postinjection. Serum was transferred to microcentrifuge tubes and stored at 80 °C until analysis. Serum tulathromycin concentrations were analyzed by a validated LC-MS method. Briefly, serum samples (0.5 mL) were subject to protein precipitation with acetonitrile at pH 6.8, and tulathromycin extracted from the supernatant using solid phase extraction (Bond Elut–CBA, Agilent Technologies, Santa Clara, CA, USA). Eluates were evaporated to dryness, reconstituted in 200 lL methanol and 300 lL 1% formic acid, and filtered. Five micro litre was injected onto a Poroshell 120 ECC18 column (2.1 9 50 mm, 2.7 lm, Agilent Technologies). The mobile phase, 5 mM ammonium formate with 0.1% formic acid (A) and 100% methanol (B), was delivered at 0.35 mL/min under gradient conditions: 80% A for 1.0 min, 25% A until 6.5 min, 5% A until 9.0 min, and a return to 80% A at 9.2 min. A Waters Alliance 2695 high-performance liquid chromatography was coupled to a Waters Micromass ZQ mass spectrometer, and electrospray ionization was used in positive ionization mode. The double charge ion [M + 2H]+2 at m/z 403.9 was monitored in selected ion monitoring mode and used for quantification. The calibration curve was prepared by spiking blank bison serum with working solutions (10 lg/mL and 100 ng/mL in acetonitrile) initially prepared from a tulathromycin stock solution (1000 lg/mL in acetonitrile) and processed identically as the study samples. The calibration curve was linear from 0.8 to 100 ng/mL (r2 > 0.99). The limit of detection was statistically calculated as 0.3 ng/mL. Recovery ranged from 76 to 80% and matrix enhancement was also noted. Intra- and interassay accuracy and precision ranged from 2 to 14%. Quality control samples at three different concentrations performed in duplicate were assessed as acceptance criteria for individual analytical runs. Pharmacokinetic parameters were estimated from concentration vs. time data for each individual bison

using a noncompartmental approach (GraphPad Prism software, 5.0, GraphPad Software, Inc., La Jolla, CA, USA). PK parameter estimates were then reported as mean  SD. Extensive interindividual variation in tulathromycin serum concentration vs. time profiles following a subcutaneous injection was observed in bison (Fig. 1). No bison demonstrated any adverse effects from the commercial formulation of tulathromycin. Table 1 reports the mean and standard deviation of pharmacokinetic parameter estimates in bison. Maximal serum concentrations (Cmax) ranged from 30 to 506 ng/mL (mean = 195 ng/mL), which was achieved in the majority of animals within 1–2 h (median of 1.04 h) after dosing (tmax). However, in two bison, Cmax was achieved 12 h postdose. The AUC0–inf values were between 3582 and 19339 ngh/mL (mean = 9341 ngh/mL). The apparent volume of distribution

Fig. 1. Natural logarithmic mean tulathromycin serum concentrations (+standard deviation) over time following a 2.5 mg/kg subcutaneous injection of Draxxin to 10 female bison.

Table 1. Mean and standard deviation pharmacokinetic parameter estimates from bison compared to estimates reported for cattle in the literature. All animals received a 2.5 mg/kg bw subcutaneous injection of tulathromycin (Draxxin)

Bison Mean  SD (n = 10)

Nowakowski et al., 2004 Cattle Mean  SD (n = 6)

0.0033  0.0006 9341  5087

0.0077  0.0012 18700  1800


500  400 1.8  3.0

PK parameter k (per hour) AUC0–inf (ngh/mL) Cmax (ng/mL) tmax (h) Cl/F (L/h/kg) Vd/F (L/kg) Half-life (h) AUMC0–inf (lgh2/mL) MRT (h)

195 4.2* 0.4 111 214 1985

157 4.6 0.2 66 43 1132

11 90

Galer et al., 2004 Cattle Mean  SD (n = 6)

300  400 0.7  3.0

110  40

208  40

*Median = 1.04 h. © 2015 John Wiley & Sons Ltd

Pharmacokinetics of tulathromycin in bison 473

(Vd/F) ranged between 34 and 175 L/kg and apparent systemic clearance (ClS/F) between 0.1–0.7 L/h/kg (mean = 0.4 L/h/kg). Half-lives ranged between 173 and 312 h (7 and 13 days) (mean = 214 h (8.9 days)) and mean residence time was 208 h. To the best of our knowledge, we are the first to report tulathromycin pharmacokinetics in bison following a dosage regimen recommended for cattle. Tulathromycin reached maximal serum concentrations within 1–2 h after subcutaneous injection, which is consistent with values reported in cattle (G aler et al., 2004; Nowakowski et al., 2004). The mean Cmax (195 ng/mL) was lower in bison compared to cattle (300 ng/mL (G aler et al., 2004) and 500 ng/mL (Nowakowski et al., 2004)). Total body exposure to tulathromycin was lower in bison (AUC0–inf = 9341 ngh/mL) compared to cattle (AUC0–inf = 18700 ngh/mL) (Nowakowski et al., 2004). The combination of lower Cmax and total body exposure suggests bioavailability of tulathromycin following subcutaneous injection is lower in bison relative to cattle. This might, in part, explain the larger apparent volume of distribution in bison (Vd/F = 111 L/kg) relative to cattle (Vd/F = 11 L/kg) (Nowakowski et al., 2004). However, the half-life of tulathromycin was approximately two times longer in bison (214 h) compared to cattle (90 h (G aler et al., 2004) and 110 h (Nowakowski et al., 2004)). As half-life is a hybrid function of volume of distribution and systemic clearance, the longer halflife suggests a larger volume of distribution in this species or possibly slower elimination kinetics. Interestingly, tulathromycin pharmacokinetic characteristics reported in other species such as swine (Huang et al., 2012; Benchaoui et al., 2004), goats (Young et al., 2010), and foals (Scheuch et al., 2007) tend to exhibit similar values to cattle. More recent studies with the newer macrolides indicate that total drug exposure to the pathogen is more predictive of therapeutic efficacy than other indices (Evans, 2005; Kilgore et al., 2005; Benchaoui et al., 2004; Nowakowski et al., 2004; Zhanel et al., 2001; Nightingale, 1997). The newer macrolides achieve prolonged half-lives in the target tissue, allowing for persistent pathogen exposure to the drug (Kilgore et al., 2005; Nowakowski et al., 2004). In cattle, tulathromycin extensively distributes into tissue with greater lung AUC values than plasma. The lung half-life of tulathromycin in cattle is 184 h (8 days), significantly longer than the plasma half-life at 90 h, and maximal lung concentrations considerably exceed the MIC90 values for bacterial pathogens in cattle (Nowakowski et al., 2004; Evans, 2005). Significant tissue distribution and residence time of tulathromycin in lung may explain the clinical efficacy of this drug in cattle. In the present study, the plasma AUC0–inf of tulathromycin was twofold lower in bison relative to values reported in cattle likely due to lower bioavailability following subcutaneous injection. Such data might question the clinical efficacy of a single 2.5 mg/kg subcutaneous dose in bison. Yet, the twofold greater half-life suggests a larger volume of distribution in bison. Such data warrant an evaluation of tulathromycin concentrations and half-life at the site of infection (lungs) to © 2015 John Wiley & Sons Ltd

understand the potential clinical efficacy of tulathromycin in bison (Benchaoui et al., 2004; Nowakowski et al., 2004; Nightingale, 1997). Further research is needed to confirm whether a dosage regimen recommended for cattle will produce efficacious therapeutic outcomes for infectious respiratory disease in bison and to determine an appropriate meat withdrawal period following use of tulathromycin in bison.

ACKNOWLEDGMENTS This study was supported by funding from the Alberta Livestock and Meat Agency (ALMA) and the Saskatchewan AgriFood Innovation Fund. Tulathromycin analytical standard was a kind gift from Pfizer Inc. (Groton, CT). Special thanks to the CVDR/CFIA in Saskatoon, SK, Canada for their help with method development and use of their laboratory space and MS facilities. Thank you to Dr. Elsie-Dawn Parsons for organizing and carrying out sample collection from animals used in the study.

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Pharmacokinetics of tulathromycin after subcutaneous injection in North American bison (Bison bison).

Tulathromycin is approved for the treatment of respiratory disease in cattle and swine. It is intended for long-acting, single-dose injection therapy ...
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