Introduction

Tulathromycin A (Figure 1) is a semi-synthetic macrolide antibiotic approved in the European Union and the United States for the treatment of respiratory disease in bovine (BRD, caused by Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis) and swine (SRD,caused by Actinobacillus pleuropneumoniae, P. multocida, Mycoplasma hyopneumoniae, Haemophilus parasuis, and Bordetella bronchiseptica).[1]The agent penetrates gram-negative bacteria well, and it exhibits mixed bacteriostatic and bactericidal activity. Tulathromycin A is formulated as a ready-to-use, sterile aqueous solution, and the packaged concentration of 100 mg tulathromycin A/ml allows low-volume dosing. This agent is characterized by rapid absorption from the injection site, extensive distribution to tissue, and slow elimination, thereby providing high, prolonged drug concentration in the lungs. Studies show that a single dose of tulathromycin A is effective in treating cattle and swine with respiratory disease and in preventing high-risk cattle from developing respiratory disease.[2]

Tulathromycin A(MW: 806.1) is an effective protein synthesis inhibitor that targets the 50s ribosomal subunit of bacteria and stimulate the dissociation of peptidyl-tRNA from the ribosome during the translocation process. This inhibition prevents the bacteria from being able to make vital proteins and stops them growing and multiplying. Following administration at the recommended dosage (2.5 mg/kg b.w., single subcutaneous injection in cattle, and intramuscular in pigs), the drug shows a rapid absorption, a high bioavailability, significant tissue distribution,and a long plasma elimination half-life.[1]

Pharmacokinetics overview

Some of the pharmacokinetic features of tulathromycin A are a consequence of the chemical characteristics of the drug molecule, which has three nitrogen/amine functional groups. Due to its unique chemical structure, tulathromycin A is the first member of a novel subclass of macrolides known as triamilides. Each amine group can be positively charged at the appropriate pH. Depending on the basic amino group in the molecule, the acid dissociation constant (pKa) ranges from 8.6 to 9.6. In addition, tulathromycin A is approximately 50 times more soluble in hydrophilic than in hydrophobic media.

The pharmacokinetics of tulathromycin A has been evaluated in multiple species, including cattle, pigs, goats, foals, and mice.The pharmacokinetic parameters available in the literature for multiple species are summarized in reference 1. In cattle and swine, tulathromycin A is characterized by a rapid rate of absorption and large systemic availability after subcutaneous (s.c.) and intramuscular (i.m.) administration. In contrast,oral administration of tulathromycin A, using a compounded formulation, had a lower systemic availability (~ 50%). In plasma, tulathromycin A has a long terminal half-life,ranging across species from ~2 to ~5 days except in mice in which the terminal half-life is <20 h. The longest terminal half-life in plasma is seen in foals (~140 h).Most of the tulathromycin A is not biotransformed; therefore, it is mainly eliminated unchanged by biliary and renal excretion. In pigs, most of the dose (two-thirds) is eliminated in the feces, while in cattle, only half is eliminated in the manure. Also, in plasma, the drug is not considerably bound to proteins (unbound fractions range from 0.53 to 0.68). In cattle, linear pharmacokinetics is observed with s.c.doses ranging from 1.27 mg/kg body weight (BW) to 5.0 mg/kg BW . No pharmacokinetic differences have been observed in castrated male versus female calves.[3]

In general, tulathromycin A is absorbed rapidly and extensively after parenteral administration. In plasma, tulathromycin A shows a long terminal half-life in most species, but a relatively shorter terminal half-life than in lung homogenate samples. In all the species studied, tulathromycin A shows notable accumulation and long persistence in lung tissue. This is reflected in the large lung homogenate/plasma ratio and the long mean residence time value. There exists much criticism regarding the value of information generated using lung homogenate samples.[3]

Detection method for tulathromycin A

Over the past years, several liquid chromatography mass spectrometry methods (LC-MS/MS) have been developed to determine tulathromycin A in different biological matrices, including plasma, serum, synovial fluid, milk, and other tissues. However,to date, no LC-MS/MS approaches have been validated in seminal plasma and urine. This could be useful to obtain information on its concentrations when used to treat genital tract infections, allowing to develop an appropriate and effective antibiotic therapy in animals.This work proposes the validation of a single LC-MS/MS approach,previously developed in pig serum,12 for tulathromycin  A quantification in bull plasma, seminal plasma, and urine, to be applied in the context of a pharmacokinetic study in bulls.

The approach is based on a quick protein precipitation with acetonitrile, filtration, and sample dilution before injection, allowing to rapidly process large batches of samples. Analytes separation was obtained using a BEH C18 (50×2.1mm, 1.7μm) column,maintained at 40℃ with a chromatographic run of 5 min. The method was fully validated over concentration ranges suitable for field levels of tulathromycin  A found in each matrix (0.01–1 μg/ml for plasma, 0.05–5 μg/ml for seminal plasma, and 0.1–10 μg/ml for urine), showing good linearity during each day of testing (R2 always>0.99). Accuracy and precision were within ±15% at all QC concentrations in all the three matrices. Furthermore, the use of tulathromycine-d7 as internal standard mitigated the potential impacts of matrix effect. The validated technique was successfully applied to samples collected during a pharmacokinetic study in bulls,allowing to monitor tulathromycin A concentrations over time in the three matrices. To our knowledge, this is the first validated approach for LC-MS/MS quantification of tulathromycin  A in seminal plasma and urine.[1]

References

[1]Barbarossa A, Bardhi A, Gazzotti T, Mari G, Pagliuca G. A single LC-MS/MS validated method for tulathromycin quantification in plasma, seminal plasma, and urine to be applied in a pharmacokinetic study in bull. Drug Test Anal. 2022;14(8):1525-1531. doi:10.1002/dta.3270 

[2]Evans NA. Tulathromycin: an overview of a new triamilide antibiotic for livestock respiratory disease. Vet Ther. 2005;6(2):83-95.

[3]Villarino N, Brown SA, Martín-Jiménez T. Understanding the pharmacokinetics of tulathromycin: a pulmonary perspective. J Vet Pharmacol Ther. 2014;37(3):211-221. doi:10.1111/jvp.12080