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Pharmacokinetics of gamithromycin after intravenous and subcutaneous administration in broiler chickens
Pharmacokinetics of gamithromycin after intravenous and subcutaneous administration in broiler chickens A. Watteyn,1 E. Plessers, H. Wyns, S. De Baere, P. De Backer, and S. Croubels Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium plasma concentration-time curve (AUC0→∞), and α and β half-life of elimination (t1/2el α and t1/2el β) were 3,998 h∙ng/mL, 0.90 h, and 14.12 h, respectively. Similar values were obtained after a SC bolus injection, i.e., 4,095 h∙ng/mL, 0.34 h, and 11.63 h, for AUC0→∞, t1/2el α, and t1/2el β, respectively. The mean maximum plasma concentration (889.46 ng/mL) appeared at 0.13 h. Gamithromycin showed a large volume of distribution after IV as well as SC administration, 27.08 and 20.89 L/kg, respectively, and a total body clearance of 1.61 and 1.77 L/h∙kg, respectively. The absolute bioavailability was 102.4%, showing that there is a complete absorption of gamithromycin after a SC bolus injection of 6 mg/kg of BW.
Key words: gamithromycin, pharmacokinetic, chicken, absolute bioavailability, subcutaneous 2013 Poultry Science 92:1516–1522 http://dx.doi.org/10.3382/ps.2012-02932
INTRODUCTION The group of macrolide antibiotics contains structurally similar compounds, which are classified as macrocyclic lactones with 12 to 20 carbon atoms in the lactone ring. On this lactone ring, several combinations of deoxy sugars can be attached by glycosidic linkages. The parent molecule of the macrolides is erythromycin and originates from the organism Streptomyces erythreus. There are numerous natural or synthetic erythromycinderived compounds (Papich and Riviere, 2009). The spectrum of activity of the macrolides is mainly against gram-positive microorganisms and also against many intracellular bacteria. The place of action is at the 23S ribosomal RNA in the 50S subunit of ribosomes, where they bind to different bases of the peptidyl transferase center and prevent protein elongation during the translocation process (Cobos-Trigueros et al., 2009). This action results in an inhibition of the growth of the bacteria. In general, macrolides act bacteriostatic, but at ©2013 Poultry Science Association Inc. Received November 21, 2012. Accepted February 18, 2013. 1 Corresponding author: [email protected]
higher doses they can also be bactericidal. It is known that the 14- and 15-membered macrolides have a timedependent action (Tamaoki, 2004). Figure 1 depicts the chemical structure of gamithromycin in comparison with the parent molecule erythromycin. Gamithromycin is a 15-membered semi-synthetic macrolide, with a uniquely positioned alkylated nitrogen at the 7a carbon of the lactone ring, which is typical for azalides. Information on the pharmacokinetics (PK) of gamithromycin is limited. Because the drug is only registered for cattle, PK studies in other species are rare. In Europe, gamithromycin is only available as an injectable formulation for subcutaneous (SC) use, known as Zactran (Merial Ltd., North Brunswick, NJ; EMA, 2008). Huang et al. (2010) described the PK of gamithromycin in cattle. Because of an extended elimination half-life (t1/2el = ± 50 h at the recommended dose of 6 mg/kg of BW SC), gamithromycin is considered to be a longacting antibiotic. It has a large volume of distribution in cattle (Vd = 24.9 L/kg), with accumulation in lung tissue. Therefore, gamithromycin is a therapeutic and preventive antibiotic in the treatment of bovine respiratory disease, associated with Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. The low
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ABSTRACT Gamithromycin is a new macrolide antibiotic that is only registered for use in cattle to treat respiratory disorders such as bovine respiratory disease. The aim of this study was to determine the pharmacokinetics of gamithromycin in broiler chickens. Gamithromycin (6 mg/kg of BW) was injected intravenously (IV) or subcutaneously (SC) to six 4-wk-old chickens in a parallel study design, and blood was collected at different time points postadministration. Quantification of gamithromycin in plasma was performed using an in-house validated liquid chromatography-tandem mass spectrometry method and the pharmacokinetics analyzed according to a 2-compartmental model. Following IV administration, the mean area under the
PHARMACOKINETICS OF GAMITHROMYCIN IN BROILERS
The maximum plasma concentration (Cmax) is achieved quickly [time to maximum plasma concentration (tmax) = 1 h at a dose of 6 mg/kg SC]. The absolute bioavailability of a subcutaneous injection is 110%. Berghaus et al. (2012) also evaluated PK and pharmacodynamics of the drug in foals because gamithromycin might be useful in the treatment of Rhodococcus equi and Streptococcus zooepidemicus infections in horses. These authors obtained similar PK variables and parameters as Huang et al. (2010) described in cattle. Furthermore, the gamithromycin MIC90 of these bacteria establish its high activity (0.125 and 1.0 µg/ mL for S. zooepidemicus and macrolide-susceptible R. equi, respectively). Pharmacokinetic variables of gamithromycin in pigs are described by H. Wyns, E. Meyer, E. Plessers, A. Watteyn, S. De Baere, P. De Backer, and S. Croubels (Department of Pharmacology, Toxicology, and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Belgium, personal communication). A higher clearance (Cl) is observed (1.69 L/h∙kg) compared with cattle (0.712 L/h∙kg), resulting in a shorter t1/2el (16.03 h). Gamithromycin could be effective in the treatment of respiratory diseases in poultry, infections such as Ornithobacterium rhinotracheale or Mycoplasma gallisepticum. It is acknowledged that the well-known macrolides, such as erythromycin, azithromycin, and clarithromycin, have immunomodulatory capacities (Kanoh and Rubin, 2010). Kovaleva et al. (2012) suggested that macrolides can temper the inflammatory response at different levels (cytokines, inflammatory cells, and structural cells), independent of their antibacterial activity. They considered several in vitro and in vivo studies with different macrolide antibiotics and pathogens. Consequently, gamithromycin might have similar immunomodulatory properties on inflammation in avian species. Particularly for broilers, a model of lipopolysaccharide (LPS)induced inflammation was developed at our laboratory to investigate drugs affecting the acute phase response (Baert et al., 2005; De Boever et al., 2009, 2010). To assess gamithromycin in a model of inflammation, its PK properties in chickens must first be determined. As an example, the time of LPS-administration is preferably done at tmax of the drug. However, there are no PK studies other than the ones previously mentioned in cattle and foals. Therefore, the aim of this study was to determine the PK properties of gamithromycin in broilers after intravenous (IV) and SC administration.
MATERIALS AND METHODS minimum inhibitory concentrations (MIC) confirm the high activity of gamithromycin against these pathogens. The MIC90 for M. haemolytica, P. multocida, and H. somni are 0.5, 1, and 1 µg/mL, respectively (EMA, 2008).
Birds Twelve 4-wk-old female broiler chickens (Ross, local commercial poultry farm) were housed according to the requirements of the European Union (Council of Europe, 2007). They were kept together in a floor pen
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Figure 1. The chemical structure of erythromycin (C37H67NO13, 733.93 g/mol; part A) and gamithromycin (C40H76N2O12, 777.04 g/ mol; part B). Erythromycin consists of a 14-membered lactone ring with 2 sugars, clandinose and desosamine. Gamithromycin is composed of a 15-membered lactone ring with the same 2 sugars as erythromycin and with a unique alkylated nitrogen at the 7a carbon of the lactone ring.
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with wood shavings. The light cycle was the same as in the commercial farm (20L:4D). The birds acclimatized to water and feed ad libitum for 4 d. The mean BW ± SD of the chickens was 1.369 ± 0.082 kg. Because of the high concentration of lipids in chicken plasma after feeding (Ferlazzo et al., 2011), the plasma was more viscous, resulting in higher interference in the analytical method. Therefore, feed was withdrawn from 12 h before until 6 h after gamithromycin administration. After the experiment, the birds were humanely euthanized with T61 (Intervet, Ukkel, Belgium). The animal experiment was approved by the Ethical Committee of the Faculty of Veterinary Medicine, Ghent University (EC 2012/031).
The veterinary drug Zactran (Merial Ltd., North Brunswick, NJ) contains 150 mg/mL (15.0% wt/vol) gamithromycin as the active substance in a buffered solution. This commercially available formulation was diluted with aqua ad injectabilia up to a concentration of 25 mg/mL (2.5% wt/vol). The analytical standards of gamithromycin and d5-gamithromycin were kindly donated by Merial Ltd. (North Brunswick, NJ) and stored at 2 to 8°C. Stock solutions of 1 mg/mL of gamithromycin and d5gamithromycin were prepared in MeOH and stored at ≤−15°C for at least 1 mo. Working solutions of 0.025, 0.050, 0.10, 0.25, 0.50, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, and 100 µg/mL of gamithromycin were prepared in HPLC water. For the internal standard (IS, d5-gamithromycin), a working solution of 1.0 µg/mL was prepared in HPLC water. The working solutions were stored at 2 to 8°C for at least 1 mo. The solvents that were used for the preparation of the HPLC mobile phase (water and acetonitrile, ACN) were of ultra-pure liquid chromatography grade and were obtained from Biosolve (Valkenswaard, the Netherlands). All other solvents and reagents were of HPLC grade (water, ACN, and MeOH) or analytical grade (formic acid, ammonium acetate, glacial acetic acid, and ammonium hydroxide) and were purchased from VWR (Leuven, Belgium). The OSTRO protein precipitation and phospholipid removal 96-well plates (25 mg) were obtained from Waters (Zellik, Belgium).
Experimental Protocol The animal experiment was performed as a single dose parallel study with 12 female chickens. Six animals received an IV bolus of 6 mg of gamithromycin/kg of BW in the wing vein. The other 6 were injected with the same dose, SC at the neck. Both administrations were carried out with a 25-Ga needle (0.5 × 16 mm, BD, Drogheda, Ireland). Blood (1 mL) was collected by venipuncture from the leg vein into heparinized tubes
Gamithromycin Analysis Quantification of gamithromycin in chicken plasma samples was performed using a validated HPLC method with tandem mass spectrometric detection (LC-MS/ MS). An aliquot (12.5 µL) of the IS working solution was added to 100 µL of plasma sample. After vortex mixing, the plasma sample was transferred to an OSTRO 96-well plate, followed by the addition of 300 µL of 1% formic acid in ACN to each well. Protein precipitation was enhanced by aspirating the samples 3 times using a multichannel pipette. The sample was passed through the well plate by the application of a vacuum (2,000 Pa) for 10 min. The samples were diluted by the addition of 500 µL of ultra-pure liquid chromatography water, followed by gentle vortexing of the 96-well plate. A 2.5-µL aliquot was injected onto the tandem MS detection instrument. The LC system consisted of a quaternary, lowpressure mixing pump with vacuum degassing, type Surveyor MSpump Plus, and an autosampler with temperature-controlled tray and column oven, type Autosampler Plus, from ThermoFisher Scientific (Breda, the Netherlands). Chromatographic separation was achieved on a Hypersil Gold (50 mm × 2.1 mm i.d., dp: 1.9 µm) column in combination with a precolumn of the same type (10 mm × 2.1 mm i.d., dp: 3 µm), both from ThermoFisher Scientific. The temperatures of the column oven and autosampler tray were set at 50 and 5°C, respectively. The LC column effluent was interfaced to a TSQ Quantum Ultra triple quadrupole mass spectrometer, equipped with a heated electrospray ionization probe operating in the positive ionization mode (all from ThermoFisher Scientific). The MS acquisition was performed in the selected reaction monitoring mode. For gamithromycin, the 2 most intense product ions were monitored (i.e., m/z 777.45 > 619.35 for quantification and m/z 777.45 > 157.80 for identification, respectively). For the IS, only one selected reaction monitoring transition was monitored (m/z 782.45 > 624.35). The method was validated and the following parameters were successfully evaluated: linearity: 2.5 to 10,000 ng/mL, within-run and between-run accuracy and precision (concentration level 25, 250, and 2,500 ng/mL, each n = 6), limit of quantification (LOQ, 2.5 ng/mL, n = 6) and detection (0.01 ng/mL, n = 6), specificity, and carry-over. All values below the LOQ were not included in the plasma concentration-time curves and the PK analysis.
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Veterinary Drug, Chemicals, and Solutions
(Vacutest Kima, Novolab, Geraardsbergen, Belgium) at different time points before (time 0) and postadministration (p.a.; 0.08, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, and 12 h, once daily from second d until the tenth and once on d 12 and 14). Samples were centrifuged at 1,500 × g at 4°C for 10 min, the plasma was then collected and stored at ≤−15°C until analysis.
PHARMACOKINETICS OF GAMITHROMYCIN IN BROILERS
PK and Statistical Analysis
F (%) =
AUC0→∞ SC × 100. AUC0→∞ IV
The data are expressed as mean ± SD and were statistically analyzed by means of single-factor ANOVA, using SPSS Statistics 19 (IBM SPSS software, New York, NY). A value of P < 0.05 was considered significant.
RESULTS No adverse effects were noticed after administration of the drug. The mean plasma concentration-time curves of gamithromycin after IV and SC injection are plotted in Figure 2. Figure 2A represents the mean plasma concentration over a period of 72 h p.a., whereas Figure 2B gives a detail of the first 12 h p.a. The plasma concentration was below the LOQ at 48 and 72 h after IV and SC administration, respectively. The PK parameters of gamithromycin calculated in a 2-compartmental model are summarized in Table 1. Following an IV bolus administration, the mean AUC0→∞ was 3,998 ± 1,060 h∙ng/mL, and the t1/2el α and t1/2el β were 0.90 and 14.12 h, respectively. Similar values were calculated for the SC administration: 4,094 ± 1,645 h∙ng/mL, 0.34 h, and 11.63 h for the AUC0→∞, t1/2el α, and t1/2el β, respectively. There were significant differences only between the kel α and the t1/2el α of the IV and SC bolus injections. The Cmax (889.46 ± 382.16 ng/mL) appeared at 0.13 ± 0.04 h. Gamithromycin showed a large Vd after IV and SC administration, 27.08 ± 7.41 L/kg and 20.89 ± 7.48 L/kg, respectively, and a Cl of 1.61 ± 0.52 and 1.77 ± 0.98 L/h∙kg, respectively. An absolute bioavailability of 102.4 ± 51.0% could be calculated after a SC injection of 6 mg/kg of BW.
DISCUSSION Gamithromycin is a recently developed second-generation macrolide registered to be employed in cattle only. Therefore, only a few PK and pharmacodynamic
studies have been performed using this macrolide. No research has been done in poultry. Although drinking water medication is the most commonly used route of drug administration in intensively reared poultry, the use of this antibiotic in the drinking water was infeasible because gamithromycin is a water-insoluble molecule. No water-soluble formulations of this macrolide are currently available. Therefore, we performed the PK study with the only commercialized formulation of gamithromycin for SC use. The plasma concentration-time curves of gamithromycin in broiler chickens showed a 2-compartmental course with a fast distribution from plasma to tissue. Gamithromycin is fully absorbed after SC administration in cattle and therefore showed a high bioavailability (F = 110%, Huang et al., 2010). We found similar results in broiler chickens. The absorption of gamithromycin after SC administration in chickens was very fast, with a t1/2abs of 0.021 h. The drug reached the maximum plasma concentration substantially faster (at 0.13 h) in comparison with cattle and foals, where a Cmax of 1 h was observed (Huang et al., 2010; Berghaus et al., 2012). Pigs seemed to have an intermediate tmax (0.63 h; H. Wyns, E. Meyer, E. Plessers, A. Watteyn, S. De Baere, P. De Backer, and S. Croubels, Department of Pharmacology, Toxicology, and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Belgium, personal communication). The volume of distribution at steady state was similar in chickens, cattle and pigs (29.16, 24.9, and 31.03 L/kg, respectively). This is an extremely high value, which confirms the 2-compartmental distribution model and probably results in high tissue concentrations. Moreover, macrolides have excellent penetration into lung tissue (Zuckerman et al., 2011). In chickens, the clearance was higher than in cattle (1.61 vs. 0.71 L/ h∙kg, respectively), which resulted in a shorter half-life of elimination after IV as well as SC administration (14.12 and 11.63 h, respectively, in broiler chickens and 44.9 and 50.8 h, respectively, in cattle). Remarkably, pigs appeared to have similar values for clearance (1.69 L/h∙kg) and half-life of elimination (16.03 h) compared with chickens (H. Wyns, E. Meyer, E. Plessers, A. Watteyn, S. De Baere, P. De Backer, and S. Croubels, Department of Pharmacology, Toxicology, and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Belgium, personal communication). Foals seemed to have an intermediate half-life of elimination after intramuscular (IM) administration (39.1 h). High clearance was also seen in PK studies of other drugs in birds in comparison with mammals, which is consistent with higher metabolic rates in birds (Baert and De Backer, 2003; Toutain and Bousquet-Melou, 2004; Neirinckx et al., 2011; Singh et al., 2011; Watteyn et al., 2013). Only a few studies described the PK of commonly used and registered macrolide antibiotics for chickens (Kowalski et al., 2002; Goudah et al., 2004; Abu-Basha et al., 2007). The first generation macrolide erythromycin eliminates quickly after IV, IM, SC, and oral
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The PK properties were analyzed using the software program WINNONLIN, Version 6.2.0 (Pharsight, Sunnyvale, CA). A 2-compartmental model was used to determine the area under the plasma concentrationtime curve (AUC0→∞), calculated by the linear trapezoidal rule with extrapolation to infinity; absorption, α, and β elimination rate constant (kabs, kel α, and kel β, respectively); half-life of absorption (t1/2abs), t1/2el α and t1/2el β; volume of distribution at steady state (Vss); volume of distribution of the first and second compartment (V1 and V2); Cl; Cmax; and tmax. The absolute bioavailability (F) was calculated from the following equation:
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(PO) bolus administration of 30 mg/kg of BW with a t1/2el of 5.3, 3.9, 2.6, and 4.1 h, respectively (Goudah et al., 2004). Tylosin has even a faster t1/2el, namely 0.52 and 2.07 h after IV and PO administration of 10 mg/kg of BW, respectively (Kowalski et al., 2002). The bioavailability of erythromycin after IM and PO was very high (92.5 and 109.3%, respectively), whereas it was only 68.8% after SC administration. The oral bioavailability of tylosin was much lower, only between 30.7 and 34.0%. Despite this incomplete absorption, it should guarantee the occurrence of a therapeutic level in plasma and tissue, according to some investigators (Kowalski et al., 2002). On the contrary, tilmicosin, also a semi-synthetic macrolide, has a very long t1/2el (45.0–47.4 h), whereas the clearance is comparable with that of gamithromycin (1.18–1.28 L/h; Abu-Basha et al., 2007). This is due to the smaller volume of distri-
bution (about 1 L/kg). In spite of a long residence in the body, tilmicosin is safe to apply in broiler chickens (EMA, 1998). It was reported that gamithromycin has the ability to reduce the morbidity of bacterial bovine respiratory disease in cattle (Baggott et al., 2011); therefore, it would also be of value to treat some bacterial respiratory infections in poultry. Recently, a study of Gerchman et al. (2011) demonstrated a decreased susceptibility of M. gallisepticum to tylosin and tilmicosin in broiler. Moreover, azalides and its derivatives, such as gamithromycin, might reduce the increased resistance development against the second generation macrolides. Furthermore, azalides accumulate rapidly in tissues and reach high concentrations in the respiratory tract, also have a prolonged postantibiotic effect, build up high concentrations in macrophages and circulating phagocytes, and have a
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Figure 2. Part A: Plasma concentration-time profiles (mean ± SD) of gamithromycin in broiler chickens after intravenous (IV; n = 6) and subcutaneous (SC; n = 6) bolus administration of 6 mg/kg of BW. Part B illustrates the plasma concentration-time profile during the first 12 h postadministration (p.a.).
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Table 1. The pharmacokinetic properties of gamithromycin in broiler chickens after intravenous (IV; n = 6) and subcutaneous (SC; n = 6) bolus administration of 6 mg/kg of BW (mean ± SD) Unit
AUC0→∞ kabs kel α kel β t1/2 abs t1/2 el α t1/2 el β Vss V1 V2 Cl tmax C0 Cmax F
h∙ng/mL /h /h /h h h h L/kg L/kg L/kg L/h∙kg h ng/mL ng/mL %
IV
SC
3,998 ± 1,060 — 7.69 ± 2.35 0.049 ± 0.010 — 0.90A2 14.12A 29.16 ± 7.20 2.09 ± 1.26 27.08 ± 7.41 1.61 ± 0.52 — 3,626.96 ± 1,804.99 — —
4,094 ± 1,645 32.70 ± 13.41 2.02 ± 0.67* 0.060 ± 0.035 0.021A 0.34A* 11.63A — 6.67 ± 2.82* 20.89 ± 7.48 1.77 ± 0.98 0.13 ± 0.04 — 889.46 ± 382.16 102.4 ± 51.0
1AUC, area under the plasma concentration-time curve; k , absorption rate constant; k abs el α, α elimination rate constant; kel β, β elimination rate constant; t1/2abs, half-life of absorption; t1/2el α, half-life of elimination of α-phase; t1/2el β, half-life of elimination of β-phase; Vss, volume of distribution at steady state; V1, volume of distribution of compartment 1; V2, volume of distribution of compartment 2; Cl, clearance; tmax, time to maximum plasma concentration; Cmax, maximum plasma concentration; F, absolute bioavailability. 2A: harmonic mean. *Significant difference (P < 0.05).
long half-life of elimination (Retsema and Fu, 2001; Baggott et al., 2011). In conclusion, gamithromycin has a complete and rapid absorption after SC administration in broiler chickens. It shows a large volume of distribution, which suggests that high tissue concentrations can be reached. The slow elimination of gamithromycin, on the other hand, could be a problem to commercialize the drug for meat birds. The PK properties presented in this study will be used for future pharmacodynamic studies of inflammatory processes in broiler chickens because it is supposed that gamithromycin could have antiinflammatory and immunomodulatory properties. The main aim of this study was to investigate the presence of these interesting properties of the antibiotic. Currently, oral application of gamithromycin in the broiler industry is impossible because of the lack of a water-soluble formulation. The future development of such a formulation of gamithromycin could be under consideration. Subsequently, PK/pharmacodynamic studies for oral administration could be performed.
ACKNOWLEDGMENTS The authors thank G. Antonissen, M. Devreese, J. Goossens, A. Osselaere, and A. Van den Bussche (Department of Pharmacology, Toxicology, and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Belgium) for their aid in the animal experiment.
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Gerchman, I., S. Levisohn, I. Mikula, L. Manso-Silvan, and I. Lysnyansky. 2011. Characterization of in vivo-acquired resistance to macrolides of Mycoplasma gallisepticum strains isolated from poultry. Vet. Res. 42:90. Goudah, A., K. Abo El Sooud, and A. M. Abd El-Aty. 2004. Pharmacokinetics and tissue residue profiles of erythromycin in broiler chickens after different routes of administration. Dtsch. Tierarztl. Wochenschr. 111:162–165. Huang, R. A., L. T. Letendre, N. Banav, J. Fisher, and B. Somerville. 2010. Pharmacokinetics of gamithromycin in cattle with comparison of plasma and lung tissue concentrations and plasma antibacterial activity. J. Vet. Pharmacol. Ther. 33:227–237. Kanoh, S., and B. K. Rubin. 2010. Mechanisms of action and clinical application of macrolides as immunomodulatory medications. Clin. Microbiol. Rev. 23:590–615. Kovaleva, A., H. H. F. Remmelts, G. T. Rijkers, A. I. M. Hoepelman, D. H. Biesma, and J. J. Oosterheert. 2012. Immunomodulatory effects of macrolides during community-acquired pneumonia: A literature review. J. Antimicrob. Chemother. 67:530–540. Kowalski, C., Z. Rolinski, R. Zan, and W. Wawron. 2002. Pharmacokinetics of tylosin in broiler chickens. Pol. J. Vet. Sci. 5:127–130. Neirinckx, E., S. Croubels, S. De Boever, J. P. Remon, T. Bosmans, S. Daminet, P. De Backer, and C. Vervaet. 2011. Species comparison of enantioselective oral bioavailability and pharmacokinetics of ketoprofen. Res. Vet. Sci. 91:415–421.
Key words: gamithromycin, pharmacokinetic, chicken, absolute bioavailability, subcutaneous 2013 Poultry Science 92:1516–1522 http://dx.doi.org/10.3382/ps.2012-02932
INTRODUCTION The group of macrolide antibiotics contains structurally similar compounds, which are classified as macrocyclic lactones with 12 to 20 carbon atoms in the lactone ring. On this lactone ring, several combinations of deoxy sugars can be attached by glycosidic linkages. The parent molecule of the macrolides is erythromycin and originates from the organism Streptomyces erythreus. There are numerous natural or synthetic erythromycinderived compounds (Papich and Riviere, 2009). The spectrum of activity of the macrolides is mainly against gram-positive microorganisms and also against many intracellular bacteria. The place of action is at the 23S ribosomal RNA in the 50S subunit of ribosomes, where they bind to different bases of the peptidyl transferase center and prevent protein elongation during the translocation process (Cobos-Trigueros et al., 2009). This action results in an inhibition of the growth of the bacteria. In general, macrolides act bacteriostatic, but at ©2013 Poultry Science Association Inc. Received November 21, 2012. Accepted February 18, 2013. 1 Corresponding author: [email protected]
higher doses they can also be bactericidal. It is known that the 14- and 15-membered macrolides have a timedependent action (Tamaoki, 2004). Figure 1 depicts the chemical structure of gamithromycin in comparison with the parent molecule erythromycin. Gamithromycin is a 15-membered semi-synthetic macrolide, with a uniquely positioned alkylated nitrogen at the 7a carbon of the lactone ring, which is typical for azalides. Information on the pharmacokinetics (PK) of gamithromycin is limited. Because the drug is only registered for cattle, PK studies in other species are rare. In Europe, gamithromycin is only available as an injectable formulation for subcutaneous (SC) use, known as Zactran (Merial Ltd., North Brunswick, NJ; EMA, 2008). Huang et al. (2010) described the PK of gamithromycin in cattle. Because of an extended elimination half-life (t1/2el = ± 50 h at the recommended dose of 6 mg/kg of BW SC), gamithromycin is considered to be a longacting antibiotic. It has a large volume of distribution in cattle (Vd = 24.9 L/kg), with accumulation in lung tissue. Therefore, gamithromycin is a therapeutic and preventive antibiotic in the treatment of bovine respiratory disease, associated with Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. The low
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ABSTRACT Gamithromycin is a new macrolide antibiotic that is only registered for use in cattle to treat respiratory disorders such as bovine respiratory disease. The aim of this study was to determine the pharmacokinetics of gamithromycin in broiler chickens. Gamithromycin (6 mg/kg of BW) was injected intravenously (IV) or subcutaneously (SC) to six 4-wk-old chickens in a parallel study design, and blood was collected at different time points postadministration. Quantification of gamithromycin in plasma was performed using an in-house validated liquid chromatography-tandem mass spectrometry method and the pharmacokinetics analyzed according to a 2-compartmental model. Following IV administration, the mean area under the
PHARMACOKINETICS OF GAMITHROMYCIN IN BROILERS
The maximum plasma concentration (Cmax) is achieved quickly [time to maximum plasma concentration (tmax) = 1 h at a dose of 6 mg/kg SC]. The absolute bioavailability of a subcutaneous injection is 110%. Berghaus et al. (2012) also evaluated PK and pharmacodynamics of the drug in foals because gamithromycin might be useful in the treatment of Rhodococcus equi and Streptococcus zooepidemicus infections in horses. These authors obtained similar PK variables and parameters as Huang et al. (2010) described in cattle. Furthermore, the gamithromycin MIC90 of these bacteria establish its high activity (0.125 and 1.0 µg/ mL for S. zooepidemicus and macrolide-susceptible R. equi, respectively). Pharmacokinetic variables of gamithromycin in pigs are described by H. Wyns, E. Meyer, E. Plessers, A. Watteyn, S. De Baere, P. De Backer, and S. Croubels (Department of Pharmacology, Toxicology, and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Belgium, personal communication). A higher clearance (Cl) is observed (1.69 L/h∙kg) compared with cattle (0.712 L/h∙kg), resulting in a shorter t1/2el (16.03 h). Gamithromycin could be effective in the treatment of respiratory diseases in poultry, infections such as Ornithobacterium rhinotracheale or Mycoplasma gallisepticum. It is acknowledged that the well-known macrolides, such as erythromycin, azithromycin, and clarithromycin, have immunomodulatory capacities (Kanoh and Rubin, 2010). Kovaleva et al. (2012) suggested that macrolides can temper the inflammatory response at different levels (cytokines, inflammatory cells, and structural cells), independent of their antibacterial activity. They considered several in vitro and in vivo studies with different macrolide antibiotics and pathogens. Consequently, gamithromycin might have similar immunomodulatory properties on inflammation in avian species. Particularly for broilers, a model of lipopolysaccharide (LPS)induced inflammation was developed at our laboratory to investigate drugs affecting the acute phase response (Baert et al., 2005; De Boever et al., 2009, 2010). To assess gamithromycin in a model of inflammation, its PK properties in chickens must first be determined. As an example, the time of LPS-administration is preferably done at tmax of the drug. However, there are no PK studies other than the ones previously mentioned in cattle and foals. Therefore, the aim of this study was to determine the PK properties of gamithromycin in broilers after intravenous (IV) and SC administration.
MATERIALS AND METHODS minimum inhibitory concentrations (MIC) confirm the high activity of gamithromycin against these pathogens. The MIC90 for M. haemolytica, P. multocida, and H. somni are 0.5, 1, and 1 µg/mL, respectively (EMA, 2008).
Birds Twelve 4-wk-old female broiler chickens (Ross, local commercial poultry farm) were housed according to the requirements of the European Union (Council of Europe, 2007). They were kept together in a floor pen
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Figure 1. The chemical structure of erythromycin (C37H67NO13, 733.93 g/mol; part A) and gamithromycin (C40H76N2O12, 777.04 g/ mol; part B). Erythromycin consists of a 14-membered lactone ring with 2 sugars, clandinose and desosamine. Gamithromycin is composed of a 15-membered lactone ring with the same 2 sugars as erythromycin and with a unique alkylated nitrogen at the 7a carbon of the lactone ring.
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with wood shavings. The light cycle was the same as in the commercial farm (20L:4D). The birds acclimatized to water and feed ad libitum for 4 d. The mean BW ± SD of the chickens was 1.369 ± 0.082 kg. Because of the high concentration of lipids in chicken plasma after feeding (Ferlazzo et al., 2011), the plasma was more viscous, resulting in higher interference in the analytical method. Therefore, feed was withdrawn from 12 h before until 6 h after gamithromycin administration. After the experiment, the birds were humanely euthanized with T61 (Intervet, Ukkel, Belgium). The animal experiment was approved by the Ethical Committee of the Faculty of Veterinary Medicine, Ghent University (EC 2012/031).
The veterinary drug Zactran (Merial Ltd., North Brunswick, NJ) contains 150 mg/mL (15.0% wt/vol) gamithromycin as the active substance in a buffered solution. This commercially available formulation was diluted with aqua ad injectabilia up to a concentration of 25 mg/mL (2.5% wt/vol). The analytical standards of gamithromycin and d5-gamithromycin were kindly donated by Merial Ltd. (North Brunswick, NJ) and stored at 2 to 8°C. Stock solutions of 1 mg/mL of gamithromycin and d5gamithromycin were prepared in MeOH and stored at ≤−15°C for at least 1 mo. Working solutions of 0.025, 0.050, 0.10, 0.25, 0.50, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, and 100 µg/mL of gamithromycin were prepared in HPLC water. For the internal standard (IS, d5-gamithromycin), a working solution of 1.0 µg/mL was prepared in HPLC water. The working solutions were stored at 2 to 8°C for at least 1 mo. The solvents that were used for the preparation of the HPLC mobile phase (water and acetonitrile, ACN) were of ultra-pure liquid chromatography grade and were obtained from Biosolve (Valkenswaard, the Netherlands). All other solvents and reagents were of HPLC grade (water, ACN, and MeOH) or analytical grade (formic acid, ammonium acetate, glacial acetic acid, and ammonium hydroxide) and were purchased from VWR (Leuven, Belgium). The OSTRO protein precipitation and phospholipid removal 96-well plates (25 mg) were obtained from Waters (Zellik, Belgium).
Experimental Protocol The animal experiment was performed as a single dose parallel study with 12 female chickens. Six animals received an IV bolus of 6 mg of gamithromycin/kg of BW in the wing vein. The other 6 were injected with the same dose, SC at the neck. Both administrations were carried out with a 25-Ga needle (0.5 × 16 mm, BD, Drogheda, Ireland). Blood (1 mL) was collected by venipuncture from the leg vein into heparinized tubes
Gamithromycin Analysis Quantification of gamithromycin in chicken plasma samples was performed using a validated HPLC method with tandem mass spectrometric detection (LC-MS/ MS). An aliquot (12.5 µL) of the IS working solution was added to 100 µL of plasma sample. After vortex mixing, the plasma sample was transferred to an OSTRO 96-well plate, followed by the addition of 300 µL of 1% formic acid in ACN to each well. Protein precipitation was enhanced by aspirating the samples 3 times using a multichannel pipette. The sample was passed through the well plate by the application of a vacuum (2,000 Pa) for 10 min. The samples were diluted by the addition of 500 µL of ultra-pure liquid chromatography water, followed by gentle vortexing of the 96-well plate. A 2.5-µL aliquot was injected onto the tandem MS detection instrument. The LC system consisted of a quaternary, lowpressure mixing pump with vacuum degassing, type Surveyor MSpump Plus, and an autosampler with temperature-controlled tray and column oven, type Autosampler Plus, from ThermoFisher Scientific (Breda, the Netherlands). Chromatographic separation was achieved on a Hypersil Gold (50 mm × 2.1 mm i.d., dp: 1.9 µm) column in combination with a precolumn of the same type (10 mm × 2.1 mm i.d., dp: 3 µm), both from ThermoFisher Scientific. The temperatures of the column oven and autosampler tray were set at 50 and 5°C, respectively. The LC column effluent was interfaced to a TSQ Quantum Ultra triple quadrupole mass spectrometer, equipped with a heated electrospray ionization probe operating in the positive ionization mode (all from ThermoFisher Scientific). The MS acquisition was performed in the selected reaction monitoring mode. For gamithromycin, the 2 most intense product ions were monitored (i.e., m/z 777.45 > 619.35 for quantification and m/z 777.45 > 157.80 for identification, respectively). For the IS, only one selected reaction monitoring transition was monitored (m/z 782.45 > 624.35). The method was validated and the following parameters were successfully evaluated: linearity: 2.5 to 10,000 ng/mL, within-run and between-run accuracy and precision (concentration level 25, 250, and 2,500 ng/mL, each n = 6), limit of quantification (LOQ, 2.5 ng/mL, n = 6) and detection (0.01 ng/mL, n = 6), specificity, and carry-over. All values below the LOQ were not included in the plasma concentration-time curves and the PK analysis.
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Veterinary Drug, Chemicals, and Solutions
(Vacutest Kima, Novolab, Geraardsbergen, Belgium) at different time points before (time 0) and postadministration (p.a.; 0.08, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, and 12 h, once daily from second d until the tenth and once on d 12 and 14). Samples were centrifuged at 1,500 × g at 4°C for 10 min, the plasma was then collected and stored at ≤−15°C until analysis.
PHARMACOKINETICS OF GAMITHROMYCIN IN BROILERS
PK and Statistical Analysis
F (%) =
AUC0→∞ SC × 100. AUC0→∞ IV
The data are expressed as mean ± SD and were statistically analyzed by means of single-factor ANOVA, using SPSS Statistics 19 (IBM SPSS software, New York, NY). A value of P < 0.05 was considered significant.
RESULTS No adverse effects were noticed after administration of the drug. The mean plasma concentration-time curves of gamithromycin after IV and SC injection are plotted in Figure 2. Figure 2A represents the mean plasma concentration over a period of 72 h p.a., whereas Figure 2B gives a detail of the first 12 h p.a. The plasma concentration was below the LOQ at 48 and 72 h after IV and SC administration, respectively. The PK parameters of gamithromycin calculated in a 2-compartmental model are summarized in Table 1. Following an IV bolus administration, the mean AUC0→∞ was 3,998 ± 1,060 h∙ng/mL, and the t1/2el α and t1/2el β were 0.90 and 14.12 h, respectively. Similar values were calculated for the SC administration: 4,094 ± 1,645 h∙ng/mL, 0.34 h, and 11.63 h for the AUC0→∞, t1/2el α, and t1/2el β, respectively. There were significant differences only between the kel α and the t1/2el α of the IV and SC bolus injections. The Cmax (889.46 ± 382.16 ng/mL) appeared at 0.13 ± 0.04 h. Gamithromycin showed a large Vd after IV and SC administration, 27.08 ± 7.41 L/kg and 20.89 ± 7.48 L/kg, respectively, and a Cl of 1.61 ± 0.52 and 1.77 ± 0.98 L/h∙kg, respectively. An absolute bioavailability of 102.4 ± 51.0% could be calculated after a SC injection of 6 mg/kg of BW.
DISCUSSION Gamithromycin is a recently developed second-generation macrolide registered to be employed in cattle only. Therefore, only a few PK and pharmacodynamic
studies have been performed using this macrolide. No research has been done in poultry. Although drinking water medication is the most commonly used route of drug administration in intensively reared poultry, the use of this antibiotic in the drinking water was infeasible because gamithromycin is a water-insoluble molecule. No water-soluble formulations of this macrolide are currently available. Therefore, we performed the PK study with the only commercialized formulation of gamithromycin for SC use. The plasma concentration-time curves of gamithromycin in broiler chickens showed a 2-compartmental course with a fast distribution from plasma to tissue. Gamithromycin is fully absorbed after SC administration in cattle and therefore showed a high bioavailability (F = 110%, Huang et al., 2010). We found similar results in broiler chickens. The absorption of gamithromycin after SC administration in chickens was very fast, with a t1/2abs of 0.021 h. The drug reached the maximum plasma concentration substantially faster (at 0.13 h) in comparison with cattle and foals, where a Cmax of 1 h was observed (Huang et al., 2010; Berghaus et al., 2012). Pigs seemed to have an intermediate tmax (0.63 h; H. Wyns, E. Meyer, E. Plessers, A. Watteyn, S. De Baere, P. De Backer, and S. Croubels, Department of Pharmacology, Toxicology, and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Belgium, personal communication). The volume of distribution at steady state was similar in chickens, cattle and pigs (29.16, 24.9, and 31.03 L/kg, respectively). This is an extremely high value, which confirms the 2-compartmental distribution model and probably results in high tissue concentrations. Moreover, macrolides have excellent penetration into lung tissue (Zuckerman et al., 2011). In chickens, the clearance was higher than in cattle (1.61 vs. 0.71 L/ h∙kg, respectively), which resulted in a shorter half-life of elimination after IV as well as SC administration (14.12 and 11.63 h, respectively, in broiler chickens and 44.9 and 50.8 h, respectively, in cattle). Remarkably, pigs appeared to have similar values for clearance (1.69 L/h∙kg) and half-life of elimination (16.03 h) compared with chickens (H. Wyns, E. Meyer, E. Plessers, A. Watteyn, S. De Baere, P. De Backer, and S. Croubels, Department of Pharmacology, Toxicology, and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Belgium, personal communication). Foals seemed to have an intermediate half-life of elimination after intramuscular (IM) administration (39.1 h). High clearance was also seen in PK studies of other drugs in birds in comparison with mammals, which is consistent with higher metabolic rates in birds (Baert and De Backer, 2003; Toutain and Bousquet-Melou, 2004; Neirinckx et al., 2011; Singh et al., 2011; Watteyn et al., 2013). Only a few studies described the PK of commonly used and registered macrolide antibiotics for chickens (Kowalski et al., 2002; Goudah et al., 2004; Abu-Basha et al., 2007). The first generation macrolide erythromycin eliminates quickly after IV, IM, SC, and oral
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The PK properties were analyzed using the software program WINNONLIN, Version 6.2.0 (Pharsight, Sunnyvale, CA). A 2-compartmental model was used to determine the area under the plasma concentrationtime curve (AUC0→∞), calculated by the linear trapezoidal rule with extrapolation to infinity; absorption, α, and β elimination rate constant (kabs, kel α, and kel β, respectively); half-life of absorption (t1/2abs), t1/2el α and t1/2el β; volume of distribution at steady state (Vss); volume of distribution of the first and second compartment (V1 and V2); Cl; Cmax; and tmax. The absolute bioavailability (F) was calculated from the following equation:
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(PO) bolus administration of 30 mg/kg of BW with a t1/2el of 5.3, 3.9, 2.6, and 4.1 h, respectively (Goudah et al., 2004). Tylosin has even a faster t1/2el, namely 0.52 and 2.07 h after IV and PO administration of 10 mg/kg of BW, respectively (Kowalski et al., 2002). The bioavailability of erythromycin after IM and PO was very high (92.5 and 109.3%, respectively), whereas it was only 68.8% after SC administration. The oral bioavailability of tylosin was much lower, only between 30.7 and 34.0%. Despite this incomplete absorption, it should guarantee the occurrence of a therapeutic level in plasma and tissue, according to some investigators (Kowalski et al., 2002). On the contrary, tilmicosin, also a semi-synthetic macrolide, has a very long t1/2el (45.0–47.4 h), whereas the clearance is comparable with that of gamithromycin (1.18–1.28 L/h; Abu-Basha et al., 2007). This is due to the smaller volume of distri-
bution (about 1 L/kg). In spite of a long residence in the body, tilmicosin is safe to apply in broiler chickens (EMA, 1998). It was reported that gamithromycin has the ability to reduce the morbidity of bacterial bovine respiratory disease in cattle (Baggott et al., 2011); therefore, it would also be of value to treat some bacterial respiratory infections in poultry. Recently, a study of Gerchman et al. (2011) demonstrated a decreased susceptibility of M. gallisepticum to tylosin and tilmicosin in broiler. Moreover, azalides and its derivatives, such as gamithromycin, might reduce the increased resistance development against the second generation macrolides. Furthermore, azalides accumulate rapidly in tissues and reach high concentrations in the respiratory tract, also have a prolonged postantibiotic effect, build up high concentrations in macrophages and circulating phagocytes, and have a
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Figure 2. Part A: Plasma concentration-time profiles (mean ± SD) of gamithromycin in broiler chickens after intravenous (IV; n = 6) and subcutaneous (SC; n = 6) bolus administration of 6 mg/kg of BW. Part B illustrates the plasma concentration-time profile during the first 12 h postadministration (p.a.).
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Table 1. The pharmacokinetic properties of gamithromycin in broiler chickens after intravenous (IV; n = 6) and subcutaneous (SC; n = 6) bolus administration of 6 mg/kg of BW (mean ± SD) Unit
AUC0→∞ kabs kel α kel β t1/2 abs t1/2 el α t1/2 el β Vss V1 V2 Cl tmax C0 Cmax F
h∙ng/mL /h /h /h h h h L/kg L/kg L/kg L/h∙kg h ng/mL ng/mL %
IV
SC
3,998 ± 1,060 — 7.69 ± 2.35 0.049 ± 0.010 — 0.90A2 14.12A 29.16 ± 7.20 2.09 ± 1.26 27.08 ± 7.41 1.61 ± 0.52 — 3,626.96 ± 1,804.99 — —
4,094 ± 1,645 32.70 ± 13.41 2.02 ± 0.67* 0.060 ± 0.035 0.021A 0.34A* 11.63A — 6.67 ± 2.82* 20.89 ± 7.48 1.77 ± 0.98 0.13 ± 0.04 — 889.46 ± 382.16 102.4 ± 51.0
1AUC, area under the plasma concentration-time curve; k , absorption rate constant; k abs el α, α elimination rate constant; kel β, β elimination rate constant; t1/2abs, half-life of absorption; t1/2el α, half-life of elimination of α-phase; t1/2el β, half-life of elimination of β-phase; Vss, volume of distribution at steady state; V1, volume of distribution of compartment 1; V2, volume of distribution of compartment 2; Cl, clearance; tmax, time to maximum plasma concentration; Cmax, maximum plasma concentration; F, absolute bioavailability. 2A: harmonic mean. *Significant difference (P < 0.05).
long half-life of elimination (Retsema and Fu, 2001; Baggott et al., 2011). In conclusion, gamithromycin has a complete and rapid absorption after SC administration in broiler chickens. It shows a large volume of distribution, which suggests that high tissue concentrations can be reached. The slow elimination of gamithromycin, on the other hand, could be a problem to commercialize the drug for meat birds. The PK properties presented in this study will be used for future pharmacodynamic studies of inflammatory processes in broiler chickens because it is supposed that gamithromycin could have antiinflammatory and immunomodulatory properties. The main aim of this study was to investigate the presence of these interesting properties of the antibiotic. Currently, oral application of gamithromycin in the broiler industry is impossible because of the lack of a water-soluble formulation. The future development of such a formulation of gamithromycin could be under consideration. Subsequently, PK/pharmacodynamic studies for oral administration could be performed.
ACKNOWLEDGMENTS The authors thank G. Antonissen, M. Devreese, J. Goossens, A. Osselaere, and A. Van den Bussche (Department of Pharmacology, Toxicology, and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Belgium) for their aid in the animal experiment.
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Gerchman, I., S. Levisohn, I. Mikula, L. Manso-Silvan, and I. Lysnyansky. 2011. Characterization of in vivo-acquired resistance to macrolides of Mycoplasma gallisepticum strains isolated from poultry. Vet. Res. 42:90. Goudah, A., K. Abo El Sooud, and A. M. Abd El-Aty. 2004. Pharmacokinetics and tissue residue profiles of erythromycin in broiler chickens after different routes of administration. Dtsch. Tierarztl. Wochenschr. 111:162–165. Huang, R. A., L. T. Letendre, N. Banav, J. Fisher, and B. Somerville. 2010. Pharmacokinetics of gamithromycin in cattle with comparison of plasma and lung tissue concentrations and plasma antibacterial activity. J. Vet. Pharmacol. Ther. 33:227–237. Kanoh, S., and B. K. Rubin. 2010. Mechanisms of action and clinical application of macrolides as immunomodulatory medications. Clin. Microbiol. Rev. 23:590–615. Kovaleva, A., H. H. F. Remmelts, G. T. Rijkers, A. I. M. Hoepelman, D. H. Biesma, and J. J. Oosterheert. 2012. Immunomodulatory effects of macrolides during community-acquired pneumonia: A literature review. J. Antimicrob. Chemother. 67:530–540. Kowalski, C., Z. Rolinski, R. Zan, and W. Wawron. 2002. Pharmacokinetics of tylosin in broiler chickens. Pol. J. Vet. Sci. 5:127–130. Neirinckx, E., S. Croubels, S. De Boever, J. P. Remon, T. Bosmans, S. Daminet, P. De Backer, and C. Vervaet. 2011. Species comparison of enantioselective oral bioavailability and pharmacokinetics of ketoprofen. Res. Vet. Sci. 91:415–421.