Research in Veterinary Science 96 (2014) 160–163
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Pharmacokinetics of gamithromycin after intravenous and subcutaneous administration in pigs H. Wyns ⇑, E. Meyer, E. Plessers, A. Watteyn, S. De Baere, P. De Backer, S. Croubels Department of Pharmacology, Toxicology and Biochemistry, Ghent University, Faculty of Veterinary Medicine, Salisburylaan 133, 9820 Merelbeke, Belgium
a r t i c l e
i n f o
Article history: Received 24 July 2013 Accepted 24 November 2013
Keywords: Gamithromycin Pharmacokinetics Pig Absolute bioavailability Subcutaneous
a b s t r a c t The aim of this study was to investigate the pharmacokinetic properties of gamithromycin in pigs after an intravenous (i.v.) or subcutaneous (s.c.) bolus injection of 6 mg/kg body weight. The plasma concentrations of gamithromycin were determined using a validated high-performance liquid chromatography– tandem mass spectrometry method, and the pharmacokinetics were noncompartmentally analysed. Following i.v. administration, the mean area under the plasma concentration–time curve extrapolated to infinity (AUCinf) and the mean elimination half-life (t1/2kz) were 3.67 ± 0.75 lg.h/mL and 16.03 h, respectively. The volume of distribution at steady state (Vss) and the plasma clearance were 31.03 ± 6.68 L/kg and 1.69 ± 0.33 L/h.kg, respectively. The mean residence time (MRTinf) was 18.84 ± 4.94 h. Gamithromycin administered subcutaneously to pigs demonstrated a rapid and complete absorption, with a mean maximal plasma concentration (Cmax) of 0.41 ± 0.090 lg/ml at 0.63 ± 0.21 h and a high absolute bioavailability of 118%. None of the reported pharmacokinetic variables significantly differed between both administration routes. Ó 2013 Elsevier Ltd. All rights reserved.
Macrolide antibiotics are classified as macrocyclic lactone rings containing 12–20 carbon atoms with diverse combinations of deoxy sugars attached to this ring by glycosidic linkages. The antibacterial action of macrolides comprises the inhibition of protein synthesis by binding to the 50S ribosomal subunit of prokaryotic organisms and consequently inhibiting the translocation (Papich and Riviere, 2009). In addition to the anti-infectious properties, these drugs have been reported to influence a variety of inflammatory processes, such as the release of cytokines and mediators, the migration of neutrophils and the oxidative burst in phagocytes. A significant decrease of the production of the pro-inflammatory cytokines TNF-a, IL-1b and IL-6 was reported after in vitro lipopolysaccharide (LPS) stimulation and treatment with tilmicosin and tylosin. Moreover, macrolides are also characterised by an extensive accumulation into leukocytes and lung tissues, achieving much higher tissue concentrations compared to those observed in plasma (Nightingale, 1997; Ianaro et al., 2000; Cao et al., 2006; Tauber and Nau, 2008; EMA, 2008a; Buret, 2010). Via introduction of a nitrogen atom into the macrolactone ring, a novel class of macrolide antibiotics, the so-called azalides, was generated. Azythromycin was the first azalide which has been ⇑ Corresponding author. Tel.: +32 9 264 73 50; fax: +32 9 264 74 97. E-mail address: [email protected] (H. Wyns). 0034-5288/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2013.11.012
associated with remarkable pharmacokinetics, such as a high tissue distribution, metabolic stability and a high tolerability compared to other macrolide antibiotics (Mutak, 2007). In veterinary medicine, gamithromycin is a 15-membered semisynthetic macrolide antibiotic of the azalide subclass which has been recently developed for the treatment and prevention of bovine respiratory disease (BRD) associated with Mannheimia haemolytica, Pasteurella multocida and Histophilus somni (Huang et al., 2010). Bacteria, such as Actinobacillus pleuropneumoniae, P. multocida, Haemophilus parasuis and Mycoplasma hyopneumoniae are major pathogens involved in swine respiratory disease (SRD). In growing pigs, respiratory infections are responsible for severe economical losses and reduced animal welfare. Other second generation macrolide antibiotics, such as tulathromycin (DraxxinÒ) and tildipirosin (ZuprevoÒ), have been approved for treatment of SRD in pigs (EMA, 2008a, 2011; Rose et al., 2013). The aim of this study was to determine the PK properties of gamithromycin in pigs, whereafter the characteristics of this antibiotic can be used in future research to investigate the immunomodulatory properties in an in vitro and in vivo porcine LPS inflammation model (Wyns et al., 2013). Twelve clinically healthy male pigs (Landrace) with a mean body weight (BW) of 24.81 ± 1.65 kg were randomly divided in two groups. The study was conducted according to a single dose
H. Wyns et al. / Research in Veterinary Science 96 (2014) 160–163
parallel design. The pigs received a bolus injection of 6 mg/kg BW gamithromycin (ZactranÒ, Merial), either intravenously (i.v.) in the ear vein (n = 6) or subcutaneously (s.c.) in the flank region (n = 6). Blood was collected by venepuncture of the jugular vein into EDTA-coated tubes (Vacutest Kima) before administration (time 0 h); at 0.25, 0.50, 0.75, 1, 2, 4, 6, 8, 10, 12 h post administration (p.a.) and once daily from day 2 to day 14 p.a. Blood samples were centrifuged and plasma was stored at 615 °C until analysis. The animal experiment was approved by the Ethical Committee of the Faculty of Veterinary Medicine of Ghent University (EC2011/159). After a solid phase extraction, using HybridSPEÒ-Phospholipid cartridges, the quantification of gamithromycin in porcine plasma was performed using a validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) method, which was previously described in detail by Watteyn et al. (2013). The PK properties were noncompartmentally determined by means of WinNonlinÒ, version 6.2.0 software program (Pharsight Corporation). Values below the limit of quantification of 5 ng/mL were not included in the analysis. The mean area under the plasma concentration–time curve was calculated using the linear trapezoidal method from time 0 to the last time point with a quantifiable concentration (AUClast) and the AUC extrapolated to infinity (AUCinf). The absolute bioavailability (F) was calculated from the following equation:
Fð%Þ ¼
AUC0!1s:c: 100 AUC0!1i:v:
The data are presented as mean ± standard deviation (SD), except for F, and were statistically analysed by means of single-factor analysis of variance (ANOVA), using SPSS Version 20.0 software for Windows. The level of significance was a = 0.05. No adverse effects following i.v. or s.c. administration were observed during the course of the study. Semi-logaritmic plots of the mean plasma concentration–time curves following i.v. and s.c. administration are presented in Fig. 1. Quantifiable concentrations of gamithromycin in plasma were present for 48 and 72 h after i.v. and s.c. administration, respectively. The PK properties of gamithromycin (mean ± SD) are summarized in Table 1. Following an i.v. bolus injection of 6 mg/kg BW gamithromycin, the AUCinf was 3.67 ± 0.75 lg.h/mL. The elimination half-life (t1/2kz) was 16.03 h. The volume of distribution at steady state (Vss) and clearance (Cl) were 31.03 L/kg and 1.69 ± 0.33 L/h.kg, respectively.
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The mean residence time extrapolated to infinity (MRTinf) was 18.84 ± 4.94 h. The volume of distribution at steady state (Vss) was 31.03 ± 6.68 L/kg. Following a s.c. bolus injection of 6 mg/kg BW gamithromycin, the AUCinf was 4.31 ± 1.14 lg.h/mL. A maximal plasma concentration (Cmax) of 0.41 ± 0.090 lg/mL was reached at 0.63 ± 0.21 h (tmax). The t1/2kz and MRTinf were 18.76 and 24.41 ± 9.17 h, respectively. The absolute bioavailability of gamithromycin was 117.6%. None of the reported pharmacokinetic properties significantly differed between both administration routes. In the current study in pigs, a higher clearance of gamithromycin was observed compared to cattle (1.69 vs 0.71 L/h.kg), resulting in a shorter t1/2kz (16.03 vs 44.9 h in pigs and cattle, respectively) after i.v. administration of gamithromycin. The Vss, on the other hand, was quite comparable between both species (31.03 and 24.90 L/kg, respectively) (Huang et al., 2010). Notwithstanding a generally more pronounced metabolic rate in birds, similar values for clearance and t1/2kz (1.61 L/h.kg and 14.12 h, respectively) were recently observed in broiler chickens (Watteyn et al., 2013). In addition, the Vss was high in both species (31.03 and 29.16 L/kg in pigs and broiler chickens, respectively). Macrolide antibiotics are lipophilic molecules and are subsequently well absorbed and extensively distributed in body fluids and tissues (Zuckerman et al., 2011). Conversely, binding to plasma proteins can considerably restrict this extravascular distribution (Huang et al., 2010). In pigs, the mean plasma protein binding of gamithromycin was only 23% (EMA, 2008b). In comparison with other macrolide antibiotics, gamithromycin showed a relatively short t1/2kz. The clearance of gamithromycin was considerably higher than that of tulathromycin (0.58 L/h.kg, Benchaoui et al., 2004 and 0.18 L/h.kg, Wang et al., 2011). In this respect, tulathromycin, approved for treatment of bacterial SRD, showed a t1/2kz of 67.5–76.5, 75.6 and 78.7 h after an i.v., i.m. and oral bolus of 2.5 mg/kg BW, respectively (Benchaoui et al., 2004; Wang et al., 2011). Likewise, tildipirosin, a semi-synthetic tylosin analogue also approved for SRD, revealed a very slow elimination with a t1/2kz of 97 h following a single i.m. injection of 6 mg/kg BW (Rose et al., 2013). On the other hand, tylosin, a first generation macrolide antibiotic mainly used to treat pneumonia and dysentery in pigs, has a t1/2kz of 4.52 and 24.5 h after i.v. and i.m. administration of 10 mg/kg BW, respectively. The total body clearance of tylosin is comparable to that of gamithromycin (1.88 L/h.kg, Prats et al., 2002). While the short t1/2kz of tylosin after i.v. administration
Fig. 1. Mean (± SD) plasma concentration–time profiles of gamithromycin after i.v. (n = 6) and s.c. (n = 6) bolus administration of 6 mg/kg BW in pigs.
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Table 1 Pharmacokinetic properties of gamithromycin in pigs after i.v. (n = 6) and s.c. (n = 6) bolus administration of 6 mg/kg BW (mean ± SD). Parameter
Unit
i.v.
s.c.
AUClast AUCinf kz t1/2kz MRTinf Vss Cl tmax Cmax F
lg.h/mL lg.h/mL
3.24 ± 0.56 3.67 ± 0.75 0.043 ± 0.011 16.03a 18.84 ± 4.94 31.03 ± 6.68 1.69 ± 0.33 – – –
3.48 ± 1.90 4.31 ± 1.14 0.037 ± 0.009 18.76a 24.41 ± 9.17 – – 0.63 ± 0.21 0.41 ± 0.090 117.6
/h h h L/kg L/h.kg h lg/mL %
AUClast, area under the plasma concentration–time curve from time 0 to the last time point with a quantifiable concentration; AUCinf, AUC extrapolated to infinity; kz, elimination rate constant; t1/2kz, half-life of elimination; MRTinf, mean residence time extrapolated to infinity; Vss, volume of distribution at steady state; Cl, clearance; tmax, time to maximum plasma concentration; Cmax, maximum plasma concentration; F, absolute bioavailability. a Harmonic mean.
can be attributed to the smaller Vss (9.7 L/kg), the extended t1/2kz after i.m. administration is a reflection of a continuous, but slow release from the injection site (Prats et al., 2002). Tilmicosin is a macrolide antibiotic synthesized from tylosin which has been recommended for the treatment and prevention of bacterial pneumonia in pigs as a feed formulation. It has a t1/2kz of 25.3 and 20.7 h after oral bolus administration of 20 and 40 mg/kg, respectively (Shen et al., 2005). Gamithromycin shows a very high absolute bioavailability of 118% after s.c. administration in pigs. Similarly, tylosin has a bioavailability of around 95% after i.m. injection (Prats et al., 2002). The bioavailability of tulathromycin after i.m. administration was >87%, whereas the oral bioavailability was only 51% (Benchaoui et al., 2004; Wang et al., 2011). For gamithromycin, minimum inhibitory concentration (MIC) values of 0.25 and 2 lg/mL were established in pigs for M. hyopneumoniae (J-strain) and A. pleuropneumoniae (ATCC 27090 reference strain), respectively (unpublished data). Of each pathogen, only one isolate was included. Therefore, these values can only be regarded as a first indication of the susceptibility. For tulathromycin, a MIC90 of 0.06 and 16 lg/mL was described for M. hyopneumoniae and A. pleuropneumoniae, respectively, in pigs (Benchaoui et al., 2004; Godinho, 2008). For tildipirosin, on the other hand, a MIC90 value of 8 lg/mL was reported for A. pleuropneumoniae (EMA, 2011), yet recent research of Rose et al. (2013) indicated that a MIC90 value of 2 lg/mL would be more plausible. In general, time above the MIC (t > MIC) is the PK/PD index considered to be best correlated with clinical efficacy of conventional macrolides. However, for newer advanced generation macrolide antibiotics such as azithromycin, the plasma AUC/MIC ratio would also be an appropriate index (Nightingale, 1997; Van Bambeke and Tulkens, 2001). Taking the MIC of 0.25 lg/mL for M. hyopneumoniae into account, it can be suggested that plasma levels are indeed higher for a certain period of time. In contrast, but in accordance with other macrolide antibiotics, plasma concentrations of gamithromycin never exceed the MIC value of 2 lg/mL of A. pleuropneumoniae in this study. Nevertheless, it is generally accepted that plasma pharmacokinetics and plasma concentrations of macrolides are poor predictors of the antimicrobial activity and clinical efficacy of these drugs. Tissue concentrations at the site of infection would rather be more significant to comprehend the pharmacodynamic properties for this class of antibiotics (Nightingale, 1997). In this respect, it can be stated that for tulathromycin, concentrations were 24.9–181 times higher in the lung than those measured in plasma of pigs (Benchaoui et al., 2004). In addition, it was observed that bovine
lung concentrations of gamithromycin were 247–410 times higher than in plasma over the time course from 1 to 15 days after treatment (Huang et al., 2010). In accordance with other macrolide antibiotics, the Vss of gamithromycin is also very high and consequently much higher concentrations can be expected in the pig’s lung compared to those in plasma. In conclusion, gamithromycin administered subcutaneously to pigs demonstrated a fast and complete absorption. The high Vss indicates a distinct tissue penetration. Although no lung concentrations are available, we still would like to suggest that, in analogy to other second generation macrolides, gamithromycin might be advantageous for the treatment of bacterial respiratory diseases in pigs. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgements The authors would like to thank Antonissen G., Devreese M., Goossens J., Lambrecht J., Vandenbroucke V., Van den Bussche A. for the technical assistance during the animal experiment. References Benchaoui, H.A., Nowakowski, M., Sherington, J., Rowan, T.G., Sunderland, S.J., 2004. Pharmacokinetics and lung tissue concentrations of tulathromycin in swine. Journal of Veterinary Pharmacology and Therapeutics 27, 203–210. Buret, A.G., 2010. Immuno-modulation and anti-inflammatory benefits of antibiotics: the example of tilmicosin. Canadian Journal of Veterinary Research 74, 1–10. Cao, X.Y., Dong, M., Shen, J.Z., Wu, B.B., Wu, C.M., Du, X.D., Wang, Z., Qi, Y.T., Li, B.Y., 2006. Tilmicosin and tylosin have anti-inflammatory properties via modulation of COX-2 and iNOS gene expression and production of cytokines in LPS-induced macrophages and monocytes. International Journal of Antimicrobial Agents 27, 431–438. EMA, 2008a. The European Agency for the Evaluation of Medicinal Products. Draxxin: EPAR – Product Information. Available from: http:// www.ema.europa.eu/docs/en_GB/document_library/EPAR__Product_Information/veterinary/000077/WC500063309.pdf. (Date latest accessed: 08 July 2013). EMA, 2008b. The European Agency for the Evaluation of Medicinal Products. Zactran: EPAR – Scientific Discussion. http://www.ema.europa.eu/docs/en_GB/ document_library/EPAR_-_Scientific_Discussion/veterinary/000129/ WC500068716.pdf. (Date latest accessed: 08 July 2013). EMA, 2011. The European Agency for the Evaluation of Medicinal Products. Zuprevo: EPAR – Product Information. http://www.ema.europa.eu/docs/ en_GB/document_library/EPAR_-_Product_Information/veterinary/002009/ WC500106578.pdf. (Date latest accessed: 08 July 2013). Godinho, K.S., 2008. Susceptibility testing of tulathromycin: interpretative breakpoints and susceptibility of field isolates. Veterinary Microbiology 129, 426–432. Huang, R.A., Letendre, L.T., Banav, N., Fischer, J., Somerville, B., 2010. Pharmacokinetics of gamithromycin in cattle with comparison of plasma and lung tissue concentrations and plasma antibacterial activity. Journal of Veterinary Pharmacology and Therapeutics 33, 227–237. Ianaro, A., Ialenti, A., Maffia, P., Sautebin, L., Rombolà, L., Carnuccio, R., Iuvone, T., D’Acquisto, F., Di Rosa, M., 2000. Anti-inflammatory activity of macrolide antibiotics. Journal of Pharmacology and Experimental Therapeutics 292, 156– 163. Mutak, S., 2007. Azalides from azithromycin to new azalide derivatives. Journal of Antibiotics 60, 85–122. Nightingale, C.H., 1997. Pharmacokinetics and pharmacodynamics of newer macrolides. The Pediatric Infectious Disease Journal 16, 438–443. Papich, M.G., Riviere, J.E., 2009. Chloramphenicol and derivatives, macrolides, lincosamides, and miscellaneous antimicrobials. In: Riviere, J.E., Papich, M.G. (Eds.), Veterinary Pharmacology and Therapeutics, nineth ed., Ames, Iowa, USA, pp. 945–982. Prats, C., El Korchi, G., Francesch, R., Arboix, M., Pérez, B., 2002. Disposition kinetics of tylosin administered intravenously and intramuscularly to pigs. Research in Veterinary Science 73, 141–144. Rose, M., Menge, M., Bohland, C., Zschiesche, E., Wilhelm, C., Kilp, S., Metz, W., Allan, M., Röpke, R., Nürnberger, M., 2013. Pharmacokinetics of tildipirosin in porcine
H. Wyns et al. / Research in Veterinary Science 96 (2014) 160–163 plasma, lung tissue, and bronchial fluid and effects of test conditions on in vitro activity against reference strains and field isolates of Actinobacillus pleuropneumoniae. Journal of Veterinary Pharmacology and Therapeutics 36, 140–153. Shen, J., Li, C., Jiang, H., Zhang, S., Guo, P., Ding, S., Li, X., 2005. Pharmacokinetics of tilmicosin after oral administration in swine. American Journal of Veterinary Research 66, 1071–1074. Tauber, S.C., Nau, R., 2008. Immunomodulatory properties of antibiotics. Current Molecular Pharmacology 1, 68–79. Van Bambeke, F., Tulkens, P.M., 2001. Macrolides: pharmacokinetics and pharmacodynamics. International Journal of Antimicrobial Agents 18, S17–S23. Wang, X., Tao, Y.F., Huang, L.L., Chen, D.M., Yin, S.Z., Ihsan, A., Zhou, W., Su, S.J., Liu, Z.L., Pan, Y.H., Yuan, Z.H., 2011. Pharmacokinetics of tulathromycin and its
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metabolite in swine administered with an intravenous bolus injection and a single gavage. Journal of Veterinary Pharmacology and Therapeutics 35, 282– 289. Watteyn, A., Plessers, E., Wyns, H., De Baere, S., De Backer, P., Croubels, S., 2013. Pharmacokinetics of gamithromycin after intravenous and subcutaneous administration in broiler chickens. Poultry Science 92, 1516–1522. Wyns, H., Croubels, S., Demeyere, K., Watteyn, A., De Backer, P., Meyer, E., 2013. Development of a cytometric bead array screening tool for the simultaneous detection of pro-inflammatory cytokines in porcine plasma. Veterinary Immunology and Immunopathology 151, 28–36. Zuckerman, J.M., Qamar, F., Bono, B.R., 2011. Review of macrolides (azithromycin, clarithromycin), ketolids (telithromycin) and glycylcyclines (tigecycline). Medical Clinics of North America 95, 761–791, viii.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Pharmacokinetics of gamithromycin after intravenous and subcutaneous administration in pigs H. Wyns ⇑, E. Meyer, E. Plessers, A. Watteyn, S. De Baere, P. De Backer, S. Croubels Department of Pharmacology, Toxicology and Biochemistry, Ghent University, Faculty of Veterinary Medicine, Salisburylaan 133, 9820 Merelbeke, Belgium
a r t i c l e
i n f o
Article history: Received 24 July 2013 Accepted 24 November 2013
Keywords: Gamithromycin Pharmacokinetics Pig Absolute bioavailability Subcutaneous
a b s t r a c t The aim of this study was to investigate the pharmacokinetic properties of gamithromycin in pigs after an intravenous (i.v.) or subcutaneous (s.c.) bolus injection of 6 mg/kg body weight. The plasma concentrations of gamithromycin were determined using a validated high-performance liquid chromatography– tandem mass spectrometry method, and the pharmacokinetics were noncompartmentally analysed. Following i.v. administration, the mean area under the plasma concentration–time curve extrapolated to infinity (AUCinf) and the mean elimination half-life (t1/2kz) were 3.67 ± 0.75 lg.h/mL and 16.03 h, respectively. The volume of distribution at steady state (Vss) and the plasma clearance were 31.03 ± 6.68 L/kg and 1.69 ± 0.33 L/h.kg, respectively. The mean residence time (MRTinf) was 18.84 ± 4.94 h. Gamithromycin administered subcutaneously to pigs demonstrated a rapid and complete absorption, with a mean maximal plasma concentration (Cmax) of 0.41 ± 0.090 lg/ml at 0.63 ± 0.21 h and a high absolute bioavailability of 118%. None of the reported pharmacokinetic variables significantly differed between both administration routes. Ó 2013 Elsevier Ltd. All rights reserved.
Macrolide antibiotics are classified as macrocyclic lactone rings containing 12–20 carbon atoms with diverse combinations of deoxy sugars attached to this ring by glycosidic linkages. The antibacterial action of macrolides comprises the inhibition of protein synthesis by binding to the 50S ribosomal subunit of prokaryotic organisms and consequently inhibiting the translocation (Papich and Riviere, 2009). In addition to the anti-infectious properties, these drugs have been reported to influence a variety of inflammatory processes, such as the release of cytokines and mediators, the migration of neutrophils and the oxidative burst in phagocytes. A significant decrease of the production of the pro-inflammatory cytokines TNF-a, IL-1b and IL-6 was reported after in vitro lipopolysaccharide (LPS) stimulation and treatment with tilmicosin and tylosin. Moreover, macrolides are also characterised by an extensive accumulation into leukocytes and lung tissues, achieving much higher tissue concentrations compared to those observed in plasma (Nightingale, 1997; Ianaro et al., 2000; Cao et al., 2006; Tauber and Nau, 2008; EMA, 2008a; Buret, 2010). Via introduction of a nitrogen atom into the macrolactone ring, a novel class of macrolide antibiotics, the so-called azalides, was generated. Azythromycin was the first azalide which has been ⇑ Corresponding author. Tel.: +32 9 264 73 50; fax: +32 9 264 74 97. E-mail address: [email protected] (H. Wyns). 0034-5288/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2013.11.012
associated with remarkable pharmacokinetics, such as a high tissue distribution, metabolic stability and a high tolerability compared to other macrolide antibiotics (Mutak, 2007). In veterinary medicine, gamithromycin is a 15-membered semisynthetic macrolide antibiotic of the azalide subclass which has been recently developed for the treatment and prevention of bovine respiratory disease (BRD) associated with Mannheimia haemolytica, Pasteurella multocida and Histophilus somni (Huang et al., 2010). Bacteria, such as Actinobacillus pleuropneumoniae, P. multocida, Haemophilus parasuis and Mycoplasma hyopneumoniae are major pathogens involved in swine respiratory disease (SRD). In growing pigs, respiratory infections are responsible for severe economical losses and reduced animal welfare. Other second generation macrolide antibiotics, such as tulathromycin (DraxxinÒ) and tildipirosin (ZuprevoÒ), have been approved for treatment of SRD in pigs (EMA, 2008a, 2011; Rose et al., 2013). The aim of this study was to determine the PK properties of gamithromycin in pigs, whereafter the characteristics of this antibiotic can be used in future research to investigate the immunomodulatory properties in an in vitro and in vivo porcine LPS inflammation model (Wyns et al., 2013). Twelve clinically healthy male pigs (Landrace) with a mean body weight (BW) of 24.81 ± 1.65 kg were randomly divided in two groups. The study was conducted according to a single dose
H. Wyns et al. / Research in Veterinary Science 96 (2014) 160–163
parallel design. The pigs received a bolus injection of 6 mg/kg BW gamithromycin (ZactranÒ, Merial), either intravenously (i.v.) in the ear vein (n = 6) or subcutaneously (s.c.) in the flank region (n = 6). Blood was collected by venepuncture of the jugular vein into EDTA-coated tubes (Vacutest Kima) before administration (time 0 h); at 0.25, 0.50, 0.75, 1, 2, 4, 6, 8, 10, 12 h post administration (p.a.) and once daily from day 2 to day 14 p.a. Blood samples were centrifuged and plasma was stored at 615 °C until analysis. The animal experiment was approved by the Ethical Committee of the Faculty of Veterinary Medicine of Ghent University (EC2011/159). After a solid phase extraction, using HybridSPEÒ-Phospholipid cartridges, the quantification of gamithromycin in porcine plasma was performed using a validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) method, which was previously described in detail by Watteyn et al. (2013). The PK properties were noncompartmentally determined by means of WinNonlinÒ, version 6.2.0 software program (Pharsight Corporation). Values below the limit of quantification of 5 ng/mL were not included in the analysis. The mean area under the plasma concentration–time curve was calculated using the linear trapezoidal method from time 0 to the last time point with a quantifiable concentration (AUClast) and the AUC extrapolated to infinity (AUCinf). The absolute bioavailability (F) was calculated from the following equation:
Fð%Þ ¼
AUC0!1s:c: 100 AUC0!1i:v:
The data are presented as mean ± standard deviation (SD), except for F, and were statistically analysed by means of single-factor analysis of variance (ANOVA), using SPSS Version 20.0 software for Windows. The level of significance was a = 0.05. No adverse effects following i.v. or s.c. administration were observed during the course of the study. Semi-logaritmic plots of the mean plasma concentration–time curves following i.v. and s.c. administration are presented in Fig. 1. Quantifiable concentrations of gamithromycin in plasma were present for 48 and 72 h after i.v. and s.c. administration, respectively. The PK properties of gamithromycin (mean ± SD) are summarized in Table 1. Following an i.v. bolus injection of 6 mg/kg BW gamithromycin, the AUCinf was 3.67 ± 0.75 lg.h/mL. The elimination half-life (t1/2kz) was 16.03 h. The volume of distribution at steady state (Vss) and clearance (Cl) were 31.03 L/kg and 1.69 ± 0.33 L/h.kg, respectively.
161
The mean residence time extrapolated to infinity (MRTinf) was 18.84 ± 4.94 h. The volume of distribution at steady state (Vss) was 31.03 ± 6.68 L/kg. Following a s.c. bolus injection of 6 mg/kg BW gamithromycin, the AUCinf was 4.31 ± 1.14 lg.h/mL. A maximal plasma concentration (Cmax) of 0.41 ± 0.090 lg/mL was reached at 0.63 ± 0.21 h (tmax). The t1/2kz and MRTinf were 18.76 and 24.41 ± 9.17 h, respectively. The absolute bioavailability of gamithromycin was 117.6%. None of the reported pharmacokinetic properties significantly differed between both administration routes. In the current study in pigs, a higher clearance of gamithromycin was observed compared to cattle (1.69 vs 0.71 L/h.kg), resulting in a shorter t1/2kz (16.03 vs 44.9 h in pigs and cattle, respectively) after i.v. administration of gamithromycin. The Vss, on the other hand, was quite comparable between both species (31.03 and 24.90 L/kg, respectively) (Huang et al., 2010). Notwithstanding a generally more pronounced metabolic rate in birds, similar values for clearance and t1/2kz (1.61 L/h.kg and 14.12 h, respectively) were recently observed in broiler chickens (Watteyn et al., 2013). In addition, the Vss was high in both species (31.03 and 29.16 L/kg in pigs and broiler chickens, respectively). Macrolide antibiotics are lipophilic molecules and are subsequently well absorbed and extensively distributed in body fluids and tissues (Zuckerman et al., 2011). Conversely, binding to plasma proteins can considerably restrict this extravascular distribution (Huang et al., 2010). In pigs, the mean plasma protein binding of gamithromycin was only 23% (EMA, 2008b). In comparison with other macrolide antibiotics, gamithromycin showed a relatively short t1/2kz. The clearance of gamithromycin was considerably higher than that of tulathromycin (0.58 L/h.kg, Benchaoui et al., 2004 and 0.18 L/h.kg, Wang et al., 2011). In this respect, tulathromycin, approved for treatment of bacterial SRD, showed a t1/2kz of 67.5–76.5, 75.6 and 78.7 h after an i.v., i.m. and oral bolus of 2.5 mg/kg BW, respectively (Benchaoui et al., 2004; Wang et al., 2011). Likewise, tildipirosin, a semi-synthetic tylosin analogue also approved for SRD, revealed a very slow elimination with a t1/2kz of 97 h following a single i.m. injection of 6 mg/kg BW (Rose et al., 2013). On the other hand, tylosin, a first generation macrolide antibiotic mainly used to treat pneumonia and dysentery in pigs, has a t1/2kz of 4.52 and 24.5 h after i.v. and i.m. administration of 10 mg/kg BW, respectively. The total body clearance of tylosin is comparable to that of gamithromycin (1.88 L/h.kg, Prats et al., 2002). While the short t1/2kz of tylosin after i.v. administration
Fig. 1. Mean (± SD) plasma concentration–time profiles of gamithromycin after i.v. (n = 6) and s.c. (n = 6) bolus administration of 6 mg/kg BW in pigs.
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H. Wyns et al. / Research in Veterinary Science 96 (2014) 160–163
Table 1 Pharmacokinetic properties of gamithromycin in pigs after i.v. (n = 6) and s.c. (n = 6) bolus administration of 6 mg/kg BW (mean ± SD). Parameter
Unit
i.v.
s.c.
AUClast AUCinf kz t1/2kz MRTinf Vss Cl tmax Cmax F
lg.h/mL lg.h/mL
3.24 ± 0.56 3.67 ± 0.75 0.043 ± 0.011 16.03a 18.84 ± 4.94 31.03 ± 6.68 1.69 ± 0.33 – – –
3.48 ± 1.90 4.31 ± 1.14 0.037 ± 0.009 18.76a 24.41 ± 9.17 – – 0.63 ± 0.21 0.41 ± 0.090 117.6
/h h h L/kg L/h.kg h lg/mL %
AUClast, area under the plasma concentration–time curve from time 0 to the last time point with a quantifiable concentration; AUCinf, AUC extrapolated to infinity; kz, elimination rate constant; t1/2kz, half-life of elimination; MRTinf, mean residence time extrapolated to infinity; Vss, volume of distribution at steady state; Cl, clearance; tmax, time to maximum plasma concentration; Cmax, maximum plasma concentration; F, absolute bioavailability. a Harmonic mean.
can be attributed to the smaller Vss (9.7 L/kg), the extended t1/2kz after i.m. administration is a reflection of a continuous, but slow release from the injection site (Prats et al., 2002). Tilmicosin is a macrolide antibiotic synthesized from tylosin which has been recommended for the treatment and prevention of bacterial pneumonia in pigs as a feed formulation. It has a t1/2kz of 25.3 and 20.7 h after oral bolus administration of 20 and 40 mg/kg, respectively (Shen et al., 2005). Gamithromycin shows a very high absolute bioavailability of 118% after s.c. administration in pigs. Similarly, tylosin has a bioavailability of around 95% after i.m. injection (Prats et al., 2002). The bioavailability of tulathromycin after i.m. administration was >87%, whereas the oral bioavailability was only 51% (Benchaoui et al., 2004; Wang et al., 2011). For gamithromycin, minimum inhibitory concentration (MIC) values of 0.25 and 2 lg/mL were established in pigs for M. hyopneumoniae (J-strain) and A. pleuropneumoniae (ATCC 27090 reference strain), respectively (unpublished data). Of each pathogen, only one isolate was included. Therefore, these values can only be regarded as a first indication of the susceptibility. For tulathromycin, a MIC90 of 0.06 and 16 lg/mL was described for M. hyopneumoniae and A. pleuropneumoniae, respectively, in pigs (Benchaoui et al., 2004; Godinho, 2008). For tildipirosin, on the other hand, a MIC90 value of 8 lg/mL was reported for A. pleuropneumoniae (EMA, 2011), yet recent research of Rose et al. (2013) indicated that a MIC90 value of 2 lg/mL would be more plausible. In general, time above the MIC (t > MIC) is the PK/PD index considered to be best correlated with clinical efficacy of conventional macrolides. However, for newer advanced generation macrolide antibiotics such as azithromycin, the plasma AUC/MIC ratio would also be an appropriate index (Nightingale, 1997; Van Bambeke and Tulkens, 2001). Taking the MIC of 0.25 lg/mL for M. hyopneumoniae into account, it can be suggested that plasma levels are indeed higher for a certain period of time. In contrast, but in accordance with other macrolide antibiotics, plasma concentrations of gamithromycin never exceed the MIC value of 2 lg/mL of A. pleuropneumoniae in this study. Nevertheless, it is generally accepted that plasma pharmacokinetics and plasma concentrations of macrolides are poor predictors of the antimicrobial activity and clinical efficacy of these drugs. Tissue concentrations at the site of infection would rather be more significant to comprehend the pharmacodynamic properties for this class of antibiotics (Nightingale, 1997). In this respect, it can be stated that for tulathromycin, concentrations were 24.9–181 times higher in the lung than those measured in plasma of pigs (Benchaoui et al., 2004). In addition, it was observed that bovine
lung concentrations of gamithromycin were 247–410 times higher than in plasma over the time course from 1 to 15 days after treatment (Huang et al., 2010). In accordance with other macrolide antibiotics, the Vss of gamithromycin is also very high and consequently much higher concentrations can be expected in the pig’s lung compared to those in plasma. In conclusion, gamithromycin administered subcutaneously to pigs demonstrated a fast and complete absorption. The high Vss indicates a distinct tissue penetration. Although no lung concentrations are available, we still would like to suggest that, in analogy to other second generation macrolides, gamithromycin might be advantageous for the treatment of bacterial respiratory diseases in pigs. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgements The authors would like to thank Antonissen G., Devreese M., Goossens J., Lambrecht J., Vandenbroucke V., Van den Bussche A. for the technical assistance during the animal experiment. References Benchaoui, H.A., Nowakowski, M., Sherington, J., Rowan, T.G., Sunderland, S.J., 2004. Pharmacokinetics and lung tissue concentrations of tulathromycin in swine. Journal of Veterinary Pharmacology and Therapeutics 27, 203–210. Buret, A.G., 2010. Immuno-modulation and anti-inflammatory benefits of antibiotics: the example of tilmicosin. Canadian Journal of Veterinary Research 74, 1–10. Cao, X.Y., Dong, M., Shen, J.Z., Wu, B.B., Wu, C.M., Du, X.D., Wang, Z., Qi, Y.T., Li, B.Y., 2006. Tilmicosin and tylosin have anti-inflammatory properties via modulation of COX-2 and iNOS gene expression and production of cytokines in LPS-induced macrophages and monocytes. International Journal of Antimicrobial Agents 27, 431–438. EMA, 2008a. The European Agency for the Evaluation of Medicinal Products. Draxxin: EPAR – Product Information. Available from: http:// www.ema.europa.eu/docs/en_GB/document_library/EPAR__Product_Information/veterinary/000077/WC500063309.pdf. (Date latest accessed: 08 July 2013). EMA, 2008b. The European Agency for the Evaluation of Medicinal Products. Zactran: EPAR – Scientific Discussion. http://www.ema.europa.eu/docs/en_GB/ document_library/EPAR_-_Scientific_Discussion/veterinary/000129/ WC500068716.pdf. (Date latest accessed: 08 July 2013). EMA, 2011. The European Agency for the Evaluation of Medicinal Products. Zuprevo: EPAR – Product Information. http://www.ema.europa.eu/docs/ en_GB/document_library/EPAR_-_Product_Information/veterinary/002009/ WC500106578.pdf. (Date latest accessed: 08 July 2013). Godinho, K.S., 2008. Susceptibility testing of tulathromycin: interpretative breakpoints and susceptibility of field isolates. Veterinary Microbiology 129, 426–432. Huang, R.A., Letendre, L.T., Banav, N., Fischer, J., Somerville, B., 2010. Pharmacokinetics of gamithromycin in cattle with comparison of plasma and lung tissue concentrations and plasma antibacterial activity. Journal of Veterinary Pharmacology and Therapeutics 33, 227–237. Ianaro, A., Ialenti, A., Maffia, P., Sautebin, L., Rombolà, L., Carnuccio, R., Iuvone, T., D’Acquisto, F., Di Rosa, M., 2000. Anti-inflammatory activity of macrolide antibiotics. Journal of Pharmacology and Experimental Therapeutics 292, 156– 163. Mutak, S., 2007. Azalides from azithromycin to new azalide derivatives. Journal of Antibiotics 60, 85–122. Nightingale, C.H., 1997. Pharmacokinetics and pharmacodynamics of newer macrolides. The Pediatric Infectious Disease Journal 16, 438–443. Papich, M.G., Riviere, J.E., 2009. Chloramphenicol and derivatives, macrolides, lincosamides, and miscellaneous antimicrobials. In: Riviere, J.E., Papich, M.G. (Eds.), Veterinary Pharmacology and Therapeutics, nineth ed., Ames, Iowa, USA, pp. 945–982. Prats, C., El Korchi, G., Francesch, R., Arboix, M., Pérez, B., 2002. Disposition kinetics of tylosin administered intravenously and intramuscularly to pigs. Research in Veterinary Science 73, 141–144. Rose, M., Menge, M., Bohland, C., Zschiesche, E., Wilhelm, C., Kilp, S., Metz, W., Allan, M., Röpke, R., Nürnberger, M., 2013. Pharmacokinetics of tildipirosin in porcine
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