Subcutaneous pharmacokinetic interaction of tulathromycin With flunixin meglumine in goats

ARTICLE INFO The pharmacokinetic aspects of tulathromycin(2.5 mg/kg) administered alone and in combination with flunixin meglumine (2.2 mg/kg) after a single subcutaneous (SC) administration, werestudied in clinically healthy goats. The animals were divided into two groups: the 1 st group was given tulathromycin alone and the 2 nd group was given tulathromycin concurrently with flunixin meglumine. Serum concentrations of tulathromycin were determined using microbiological assay method. Tulathromycin was rapidly absorbed with a half-life of absorption (t(0.5)ab) of 0.54 h and the peak plasma concentration (Cmax) was 3.7ug/ml was attained after 0.98 h (Tmax). Flunixin significantly altered the pharmacokinetics of tulathromycin by increasing its absorption and delay its elimination from body where t0.5(ab)were 0.54 and0.34 h and the elimination half-lives (t0.5(el)) were 1.35 and 1.8 h, for alone and combination groups, respectively. Significant decreases (39.8%) in the area under the curve (AUC) and (22.6%) in the elimination rate constant (Kel) from the central compartment were found following coadministration with flunixin compared with administration of tulathromycin alone. It was concluded that the combination of tulathromycin and flunixin negatively altered the kinetics of tulathromycin. Article history:


Introduction
Macrolide antibiotics are antibacterial agents used as veterinary drugs in foodproducing animals with either a curative or prophylactic aim (Codony et al., 2002). It active against Gram-positive bacteria, they target the bacterial ribosome and inhibit bacterial protein biosynthesis (Leal et al., 2001). Triamilides are semisynthetic derivatives of the natural product, erythromycin, and are characterized bythe presence of three polar aminegroups (tribasic) that differentiate themstructurally from other macrolides (Letavic et al., 2002). Tulathromycin is the first member of a new macrolide class, the triamilides, developed exclusively forveterinary use (Evans, 2005).Newer macrolides, such as tulathromycin, have been designed with modified configurations to enhance invitro and in vivo antibacterial properties along with increasing bioavailability, lung tissue penetration, and extended tissue half-lives (Benchaoui, et «/.,2004;Retsemea& Fu, 2001). Tulathromycin demonstrates better tissue penetration and longerhalf-lives than older macrolides due to its lipophilic properties (Benchaoui eta/., 2004;Evans, 2005). This activity can provide unique therapeutic advantage in treating bacterial respiratory infections in livestock species. Brunton et al. (2008)recorded that in addition to impacting enhanced tissue and cellular penetration characteristic of all macrolides, this novel structure (tulathromycin) conveys desirable antibacterial properties particularly against Gram negative respiratory bacteria. Tulathromycin is more efficacious injectable macrolide antibiotic used for the treatment of pneumonia of ruminants compared with other antibiotics in recent years (Venner eta/., 2007;Nutsch et a/., 2005;Godinho et a/., 2005;Skogerboe eta/., 2005 andRobb et a/., 2007). Tulathromycin injectable solution is effective as a means of mass treatment to prevent bovine respiratory disease (BRD) and reduce the number of retreats and chronics in stocker calves (Richeson, 2008 andNutsch, 2005). Tulathromycin is used for treatmentand prevention of BRD associated with Mannheimia haemolytica, Pasteurella multocida, Histophilus somni and Mycoplasma bovis. Also, It is used for treatment of infectious bovine keratoconjunctivitis (IBK) associated with Moraxella bovi (CVMP, 2002). Flunixinis non steroidal antiinflammatory drug (NSAID) inhibiting cycloxygenase enzymes in the arachidonic acid cascade, thus block the formation of cycloxygenase derived eicosanoid inflammatory mediators (Landoni et al., 1995;Cheng et al., 1998). Flunixin is widely used in veterinary medicine, to treat the musculoskeletal conditions, endotoxic shock, acute mastitis, endotoxemia, and calf pneumonia (Anderson et al., 1991;Welsh & Nolan, 1995;Odensvik& Magnusson, 1996;Rantala et al., 2002). Due to its anti-inflammatory, analgesic, and antipyretic effects (Mckellar et al., 1989;Beretta et al., 2005).Consequently, the present study describes some pharmacokinetics aspects of tulathromycin after single subcutaneous administration in goats. Also, to assess the effect of co-administration of flunixin on pharmacokinetic behavior of tulathromycin. Material and Methods Drugs: Tulathromycin 100 mg ml-1 was supplied as an injectable solution (Draxxin®) by animal health division Pfizer Company, Cairo, Egypt. Flunixin meglumine (Flunidyne) is a product of ArabcoMed, Egypt. Animals: Ten apparently healthy, male and female Egyptian goats (3-9 months old and mean body weightof (12-23 kg) were used. Animals were obtained from a local market at Beni-Suef governorate kept under good hygienic condition and fed barseem free access to water. Methods: Experimental design: the animals were randomly divided into two group's five goats each. Animals of first group administered a single dose of 2.5 mg kg-1tulathromycin subcutaneously (Clothier et al., 2011, Young et al., 2011Grismer et al., 2014), while the 2nd was injected2.5 mg kg-1tulathromycin with 2.2 mg kg-1flunixin subcutaneously (Konigssonet al., 2003). Blood samples were collected via vein puncture from jugular vein before and 0. 083, 0.167, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48 and 72 hours post-administration. Blood samples were left to clot then centrifuged at 3000 revolution per minute for 15 minutes to obtain clear serum that was kept frozen at -20 °C until assayed.

Drug bioassay Samples
were assayed by microbiological assay according to the method of Arret et al. (1971) using Bacillus subtiles (ATCC 6633) as a test organism.
Standard tulathromycin concentrations of 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5, 5, 10 and 20 ug ml-1were prepared in antibiotic-free goat serum and phosphate buffer saline (pH 8). The minimal detectable limit for the assay method was 0.078ugml-1. Semilogarithmic plots of the inhibition zone diameter versus standard tulathromycin concentrations in serum and phosphate buffer were linear with typical correlation coefficient of 0.992 (for the standard curve). The difference of inhibition zone diameter between the solutions of the drug in serum and buffer was used to calculate the in-vitro protein binding tendency of tulathromycin according to Craig and Suh (1991) by the following equation: Protein binding % = Zone of inhibition inbuffer-Zone of inhibition in serum x 100 Zone of inhibition in buffer Pharmacokinetic analysis: A computerized curve strippingprogram (R Strip; Micromath Scientific Software, Salt Lake City, UT, USA) was used toanalyze the concentration-time curves for each individual animal using the statistical moment theory (Gibaldi and Perrier, 1982). Following SC administration, The Cmax (maximum serum concentration) and tmax (time of maximum serum concentration) were taken directly from the curve. The terminal elimination half-life (t0.5(ei)) and absorption half-life (t0.5(ab)) were calculated as ln2/Kel or ln2/Kab, respectively, where Kel and Kab are the elimination and absorption rate constants, respectively. The area under serum concentration-time curve (AUC) and area under the first moment curve (AUMC) were calculated by the method of trapezoids and extrapolation to infinitywas performed. Results were expressed asmean and standard error (S.E). Standard errors werecalculated from the mean data according to Snedecorand Cochran (1976).

Figure (1):
Semi-logarithmic graph depicting the time-concentration of tulathromycin in serum of goats after a single subcutaneous injection of 2.5 mg kg -1 b.wt alone (■) and with flunixin (A).

Results:
Disposition of tulathromycin in serum after subcutaneous injection was best fitted by the 2compartment open pharmacokinetic model ( Figure  1).The pharmacokinetic parameters of tulathromycin following a single subcutaneous administration of 2.5 mg kg -1 b.wt alone and with flunixin are recorded in table (1). The results of the present study revealed that tulathromycin was rapidly absorbed following a single subcutaneous injection alone and with flunixin with to.5(ab) of 0.54 and0.34 h and maximum serum concentrations (Cmax) of3.7 and 2.59ug ml -1 were achieved at (tmax) of0.98 and 0.95 h., respectively. The elimination halflives (t0.5(el)) were1.35 and 1.8 h. for tulathromycin alone and with flunixin, respectively. The in-vitro serum protein-binding tendency was calculated to be 18.72%.   (1995). Effect of flunixinmeglumine on the thresholds to mechanical stimulation in healthy and lame sheep. Research in Veterinary Science, 58, 61-66 Whittem, T., Firth, E.C., Hodge, H., and Turner, K., (1996). Pharmacokinetic interactions between repeated dose phenylbutazone and gentamicin in the horse.