- Email: [email protected]
Pharmacokinetics after intravenous, intramuscular and subcutaneous administration of difloxacin in sheep
Research in Veterinary Science 83 (2007) 234–238 www.elsevier.com/locate/rvsc
Pharmacokinetics after intravenous, intramuscular and subcutaneous administration of difloxacin in sheep P. Marı´n *, E. Ferna´ndez-Varo´n, E. Escudero, C.M. Ca´rceles Department of Pharmacology, Faculty of Veterinary Medicine, University of Murcia, Campus de Espinardo, 30071 Murcia, Spain Accepted 15 January 2007
Abstract The disposition kinetics of difloxacin, a fluoroquinolone antibiotic, after intravenous (IV), intramuscular (IM) and subcutaneous (SC) administration were determined in sheep at a single dose of 5 mg/kg. The concentration–time data were analysed by compartmental (after IV dose) and non-compartmental pharmacokinetics method (after IV, IM and SC administration). Plasma concentrations of difloxacin were determined by high performance liquid chromatography with fluorescence detection. Steady-state volume of distribution (Vss) and clearance (Cl) of difloxacin after IV administration were 1.68 ± 0.21 L/kg and 0.21 ± 0.03 L/h kg, respectively. Following IM and SC administration difloxacin achieved maximum plasma concentration of 1.89 ± 0.55 and 1.39 ± 0.14 mg/L at 2.42 ± 1.28 and 5.33 ± 1.03 h, respectively. The absolute bioavailabilities after IM and SC routes were 99.92 ± 26.50 and 82.35 ± 25.65%, respectively. Based on these kinetic parameters, difloxacin is likely to be effective in sheep. 2007 Elsevier Ltd. All rights reserved. Keywords: Difloxacin; Pharmacokinetics; Sheep; Bioavailability
1. Introduction Fluoroquinolones are a group of antimicrobials that have become widely used in veterinary medicine. These compounds are regarded as almost the ‘‘ideal’’ antimicrobial agents because of their broad spectrum of activity, clinically advantageous pharmacokinetic properties and low toxicity, which represents a considerable advance over other classes of antimicrobial agents. Because the fluoroquinolones are relatively new antimicrobial agents, extensive research is needed to develop and implement appropriate specific dosing regimens that can maximize their clinical efficacy for use in animals and reduce the selection for resistant pathogens, particularly, as these drugs are used for the treatment of multi-drug resistant infection in humans (Walker, 2000).
*
Corresponding author. Tel.: +34 968 398404; fax: +34 968 364147. E-mail address: [email protected] (P. Marı´n).
0034-5288/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2007.01.005
Difloxacin is a fluoroquinolone carboxylic acid antimicrobial agent with high in vitro activity against a wide range of gram-positive and gram-negative aerobes and anaerobes (Granneman et al., 1986). Like other fluoroquinolones, difloxacin is a concentration-dependent bactericidal agent, it acts by inhibiting bacterial DNA topoisomerases II and IV (Drlica and Zhao, 1997; Wolfson and Hooper, 1989). It has been shown to be effective in the treatment of experimentally induced pneumonic pasteurellosis in calves (Olchowy et al., 2000) and experimental Staphylococcus aureus endocarditis in rabbits (Boscia et al., 1988). The pharmacokinetic properties of difloxacin have been evaluated in several species as non-lactating goats (Atef et al., 2002), rabbits (Abd El-Aty et al., 2005) and horses (Ferna´ndez-Varo´n et al., 2005a), but not yet in sheep. Thus, the purpose of the study reported here was to determine the pharmacokinetics of difloxacin after intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration to sheep, and to investigate the bioavailability after the extravascular routes.
P. Marı´n et al. / Research in Veterinary Science 83 (2007) 234–238
2. Materials and methods 2.1. Animals Six clinically healthy Seguren˜a female sheep weighing between 46.2 and 58.6 kg and aged from 5 to 7 years were obtained from the farm of the University of Murcia. The animals were housed and fed an antibiotic-free diet for at least 30 days preceding the study. For each treatment period of the cross-over, they were observed daily for general health, and clinical observations were made prior to injection and at 2, 10 and 24 h post-injection. Alfalfa hay and water was provided ad libitum together with a drug-free concentrate. The study was approved by the Bioethics Committee of the University of Murcia.
235
of plasma blank samples spiked with different amounts of drug and treated as any samples, with the peak areas of the same standards prepared in phosphate buffer. Each point was established from an average of five determinations. The mean percentage recoveries of difloxacin from plasma was 95.68 ± 4.29%. The assay precision (R.S.D.) was assessed by expressing the standard deviation of repeated measurements as a percentage of the mean value. Intra-day precision was estimated from six replicates of three standard samples used for calibration curves (R.S.D. < 6.5%). Inter-day precision was estimated from the analysis of standard samples on three separate days (R.S.D. < 9.2%). The limit of quantification (LOQ) of difloxacin in plasma was chosen as the concentrations of the lowest concentration level on the calibration curves for which the RDS was <15% (LOQ: 5 lg/L).
2.2. Experimental design 2.5. Pharmacokinetic analysis A cross-over design (2 · 2 · 2) was used in three phases. Each animal received single IV bolus, IM and SC injections of difloxacin 5% (Dicural, Fort Dodge Veterinaria, S.A.) at a dose of 5 mg/kg with at least 15-day washout period. For the IV administration, the solution was injected into the left jugular vein and blood samples (4 mL) were collected from the contralateral jugular vein into heparinized tubes. Subcutaneous injections were administered under the skin of the back at a single location in the thoraco-lumbar region lateral of the mid-line and IM injections into the semimembranous muscle. Blood samples were collected at 0 (pre-treatment), 0.083, 0.167, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72 and 96 h post-dosing, and each sample was collected by individual venipuncture. Samples were centrifuged at 1500g for 15 min and the plasma taken and stored at –45 C until assayed.
The concentration–time data obtained after IV treatment in each individual animal were initially fitted to one-, two- and three-exponential equations by the retroprojection method (Gibaldi and Perrier, 1982). The PKCALC computer program (Shumaker, 1986) was used to obtain the best estimates of the parameters of these equations. The final curve fitting was carried out using non-linear regression with the MULTI computer program and the Gauss–Newton damping algorithm (Yamaoka et al., 1981). Akaike’s Information Criterion (Yamaoka et al., 1978) was used to determine the number of compartments used in the pharmacokinetic analysis and the most appropriate weighting for the data. The data points were weighted with the inverse of the squared fitted value. The general polyexponential equation is:
2.3. Analytical method
C¼
n X
C i ekit
i¼1
Plasma concentrations of difloxacin were measured using a HPLC method previously reported (Ferna´ndezVaro´n et al., 2006a). Difloxacin pure substance (Fort-Dodge, Madrid) was used for quality controls. Ciprofloxacin (Vita Pharma, Madrid) was used as internal standard. 2.4. Method validation Quality controls were prepared from a pool of sheep plasma spiked with nine concentrations of difloxacin between 5 and 2000 lg/L. Plasma aliquots were stored at 45 C until assayed. Aliquots of quality controls were extracted as above and 60 lL was injected into the chromatographic system. Standard curves were obtained by unweighted linear regression of difloxacin peak areas versus known concentrations. Each point was established from an average of five determinations. Correlation coefficients (r) were >0.99% for calibration curves. The percentage recovery was determined by comparing the peak areas
where C is the plasma concentration of drug; t is the time after drug administration; Ci and ki are the intercept and slope, respectively, of the different disposition phases and e is the base of natural logarithm. Pharmacokinetic parameters were obtained from the individual fitted equations (Gibaldi and Perrier, 1982). The distribution and elimination half-lives were calculated as t12k1 = ln 2/k1 and t12kz = ln 2/kz, respectively. Non-compartmental pharmacokinetics parameters were estimated using WinNonlin Professional (version 5.1, Pharsight Corporation, CA, USA). WinNonlin model 200 was used for extravascular administrations and model 201 for intravenous administration. Non-compartmental parameters calculated were: area under the plasma concentration–time curve (AUC) using the linear trapezoidal rule with extrapolation to time infinity, mean residence time (MRT), plasma clearance (Cl), and apparent volume of distribution at steady state (Vss). Mean absorption times were calculated as MAT = MRTSC,IM – MRTIV.
236
P. Marı´n et al. / Research in Veterinary Science 83 (2007) 234–238
Bioavailability (F) was calculated by the method of corresponding areas: ½F ð%Þ ¼ AUCSC;IM 100=AUCIV 2.6. Statistical analysis Descriptive statistical parameters as mean, standard deviation and coefficient of variation were calculated. Harmonic means were calculated for the half-lives of elimination. The Wilcoxon Rank Sum test and Student’s t test were used to test parameters for significant differences between IV, IM and SC administration (Powers, 1990). The SPSS (Version 11.0) statistical software was used. 3. Results Clinical examination of all the sheep before and after each administration did not reveal any abnormalities. No local or systemic adverse reactions occurred after IV, IM and SC injection of difloxacin. Difloxacin plasma concentrations versus time data after IV administration could best be described by a two-compartment open model. The mean (±S.D.) plasma concentrations of difloxacin at the times of sample collection after IV, IM and SC administration are plotted in Fig. 1. Mean (±S.D.) pharmacokinetic parameters are presented in Table 1. Difloxacin was detected in plasma up to 72 h after IV and SC administration and 96 h after IM dosing. 4. Discussion The results demonstrated that difloxacin is quickly and widely distributed after IV administration with a half-life (t12k1) of 1.53 h and a Vss of 1.68 ± 0.21 L/kg, which suggest good penetration through biological membranes. This Vss is in agreement with values found in other species such as horses (Vss = 1.02 L/kg, Ferna´ndez-Varo´n et al., 2006a), rabbits (Vss = 1.51 L/kg, Abd El-Aty et al., 2005) and goats (Vss = 1.11 L/kg, Atef et al., 2002). Elimination half-lives (t12kz = 11.43 h IV, t12kz = 13.89 h IM and t12kz = 12.02 h SC) in this study were longer than those described for difloxacin in horses (t12kz = 2.66 h IV and t12kz = 5.72 h IM, Ferna´ndez-Varo´n et al., 2006a), pigs and chicken (t12kz = 7.92 and 4.10 h IV, respectively, Inui et al., 1998) and those reported for other fluoroquinolones in sheep (McKellar et al., 1998; Soback et al., 1994) and goats (Ferna´ndez-Varo´n et al., 2006b). Consequently the systemic clearance of difloxacin in our study (Cl = 0.21 ± 0.03 L/h kg) was slower than those described in the afore-mentioned manuscripts. Differences in the kinetic parameters are relatively common, and frequently related to interspecies variation, age, breed, health status of the animals and/or the assay method used (Haddad et al., 1985). Following IM and SC administration, difloxacin showed bioavailabilities of 99.92 ± 26.50 and 82.35 ± 25.65%,
Fig. 1. Experimental plasma concentrations (mean ± S.D.) of difloxacin at a single dose of 5 mg/kg bodyweight (n = 6). (a) Semilogarithmic plot after intravenous administration. (b) Semilogarithmic plot after intramuscular administration. (c) Semilogarithmic plot after subcutaneous administration.
respectively. These results indicated that difloxacin was well absorbed following both routes of administration, these data are similar to those reported for danofloxacin in sheep (McKellar et al., 1998), and for difloxacin in horses (Ferna´ndez-Varo´n et al., 2006a) and rabbits (Abd El-Aty et al., 2005).
P. Marı´n et al. / Research in Veterinary Science 83 (2007) 234–238 Table 1 Pharmacokinetic parameters (mean ± S.D.) of difloxacin in sheep after intravenous, intramuscular and subcutaneous administration at a single dose of 5 mg/kg bodyweight (n = 6) Parameters
Units
Intravenous
Intramuscular
Subcutaneous
C1 Cz k1 kz t12k1c t12kzc Vss AUC MRT Cl MAT Cmax Tmax F (%)
mg/L mg/L h1 h1 H H L/kg mg h/L h L/h kg h mg/L h –
6.83 ± 2.30 0.58 ± 0.14 0.45 ± 0.12 0.06 ± 0.01 1.53 ± 0.37 11.43 ± 0.88 1.68 ± 0.21 24.23 ± 3.18 8.07 ± 0.83 0.21 ± 0.03 – – – –
– – – 0.05 ± 0.01 – 13.89 ± 2.42 – 23.62 ± 3.81 15.83 ± 4.27a – 7.76 ± 4.64 1.89 ± 0.55 2.42 ± 1.28 99.92 ± 26.50
– – – 0.06 ± 0.01 – 12.02 ± 2.83 – 19.40 ± 3.93b 12.62 ± 1.29a – 4.55 ± 1.59 1.39 ± 0.14 5.33 ± 1.03b 82.35 ± 25.65b
C1, intercept of the ordinate by the fastest disposition slope minus the intercept of the next fastest disposition slope; Cz, intercept of the slowest disposition slope with the ordinate; t12k1, the disposition half-life associated with the initial slope (k1) of a semilogarithmic concentration–time curve; t12kz, the elimination half-life associated with the terminal slope (kz) of a semilogarithmic concentration–time curve; Vss, the apparent volume of distribution at steady state; Cl, the total body clearance of drug from the plasma; AUC, the area under the plasma concentration–time curve from zero to infinity; MRT, mean residence time; F, the fraction of the administered dose systemically available (bioavailability); Tmax, the time to reach peak or maximum plasma concentration following intramuscular and subcutaneous administration; MAT, mean absorption time; Cmax, the peak or maximum plasma concentration following intramuscular and subcutaneous administration. a Significantly different from IV (P < 0.05). b Significantly different from IM (P < 0.05). c Harmonic mean.
After SC administration of difloxacin, peak plasma concentration (Cmax) 1.39 ± 0.14 mg/L was attained at 5.33 ± 1.03 h post administration. The drug was slowly absorbed, showing some influence of the absorption phase in the disposition of the drug, because MAT was shorter than MRTiv (Toutain and Bousquet-Me´lou, 2004). For this reason, it is necessary to do further study with design of multiple dose regimens for calculation accumulation factor. However, after IM dose of difloxacin the absorption was faster compared to SC dose, the Cmax and tmax values (1.89 ± 0.55 lg/L and 2.42 ± 1.28 h, respectively) were close to the corresponding data reported in horses (Ferna´ndez-Varo´n et al., 2006a). Maximum plasma concentrations (Cmax) were significantly different (p < 0.05) after IM and SC administrations. Since fluoroquinolones are drugs acting in a concentration-dependent manner, it is considered that the ratios Cmax/MIC90 and AUC0–24/MIC90 are the best parameters for predicting their antimicrobial effect (Lode et al., 1998). Previous investigations have shown that for fluoroquinolones Cmax/MIC90 > 3 produced 99% reduction in bacterial count and Cmax/MIC90 of 8 or greater prevented the emergence of resistant organisms (Craig, 1998). Furthermore AUC24/MIC90 > 100 h should be achieved to
237
give maximum clinical and bacteriology efficacy (Turnidge, 1999). Minimal inhibitory concentration data of difloxacin against ovine bacterial isolates have not been reported up to now, so further studies are necessary to predict clinical efficacy of difloxacin in sheep. Fluoroquinolones have low MIC values against many Gram-negative bacteria (Walker, 2000). Abd El-Aty et al. (2005) have reported MIC of difloxacin against E. coli of 0.065 lg/ml, and similar results have been published by Stamm et al. (1986) (MICs = 0.03–0.2 lg/ml). So difloxacin could be effective by the IM and SC route at 5 mg/kg against bacterial isolates with MIC 6 0.19 and MIC 6 0.17 lg/ml, respectively, taking into account values of PK/PD parameters of Cmax/MIC90 P 8 and AUC24/ MIC90 P 100 h. Moreover the efficacy of the drug against respiratory infections of lambs (Mavrogianni and Fthenakis, 2005), calves (Paulsen et al., 1992; Olchowy et al., 2000), dogs (van den Hoven et al., 2000) and poultry (Kempf et al., 1998) has been documented. It is conclude, that in view of general adverse reactions were not observed in any sheep, and the favourable pharmacokinetic properties such as long half-life and high bioavailability, difloxacin administered at 5 mg/kg after IM and SC dose could be effective in sheep. However, further studies are needed to establish a multiple dosage regimen and clinical efficacy. Acknowledgements Thanks are due to Fort-Dodge for supplying difloxacin pure substance and to J. Carrizosa for his assistance with the experiments. References Abd El-Aty, A.M., Goudah, A., Ismail, M., Shimoda, M., 2005. Disposition kinetics of difloxacin in rabbit after intravenous and intramuscular injection of dicural. Veterinary Research Communications 29, 297–304. Atef, M., El-Banna, H.A., Abd El-Aty, A.M., Goudah, A., 2002. Pharmacokinetics of difloxacin in goats. Deutsche Tiera¨rztliche Wochenschrift 109, 320–323. Boscia, J.A., Kobasa, W.D., Kaye, D., 1988. Comparison of difloxacin, enoxacin and cefazolin for the treatment of experimental Staphylococcus aureus endocarditis. Antimicrobial Agents and Chemotherapy 32, 262–264. Craig, W.A., 1998. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clinical Infectious Diseases 26, 1–10. Drlica, K., Zhao, X., 1997. DNA gyrase, topoisomerase IV, and the 4quinolones. Microbiology and Molecular Biology Reviews 61, 377– 392. Ferna´ndez-Varo´n, E., Ca´rceles, C.M., Marı´n, P., Martos, N., Escudero, E., Ayala, I., 2006a. Pharmacokinetics of difloxacin after intravenous, intramuscular and intragastric administration to horses. American Journal of Veterinary Research 67, 1076–1081. Ferna´ndez-Varo´n, E., Villamayor, L., Escudero, E., Espuny, A., Ca´rceles, C.M., 2006b. Pharmacokinetics and milk penetration of moxifloxacin after intravenous and subcutaneous administration to lactating goats. The Veterinary Journal 172, 302–307.
238
P. Marı´n et al. / Research in Veterinary Science 83 (2007) 234–238
Gibaldi, M., Perrier, P., 1982. Pharmacokinetics, second ed. Marcel Dekker, New York. Granneman, G.R., Snyder, K.M., Shu, V.S., 1986. Difloxacin metabolism and pharmacokinetics in humans after single oral doses. Antimicrobial Agents and Chemotherapy 30, 689–693. Haddad, N.S., Pedersoli, W.M., Ravis, W.R., Fazeli, M.H., Carson, R.L., 1985. Combined pharmacokinetics of gentamicin in pony mares after a single intravenous and intramuscular administration. American Journal of Veterinary Research 46, 2004–2007. van den Hoven, R., Wagenaar, J.A., Walker, R.D., 2000. In vitro activity of difloxacin against canine bacterial isolates. Journal of Veterinary Diagnostic Investigation 12, 218–223. Inui, T., Taira, T., Matsushita, T., Endo, T., 1998. Pharmacokinetic properties and oral bioavailabities of difloxacin in pig and chicken. Xenobiotica 28 (9), 887–893. Kempf, I., van den Hoven, R., Gesbert, F., Guittet, M., 1998. Efficacy of difloxacin in growing broiler chicken for the control of infection due to pathogenic Mycoplasma gallisepticum. Journal of Veterinary Medicine B 45, 305–310. Lode, H., Borner, K., Koeppe, P., 1998. Pharmacodynamics of fluoroquinolones. Clinical Infectious Disease 27, 33–39. Mavrogianni, V.S., Fthenakis, G.C., 2005. Efficacy of difloxacin against respiratory infections of lambs. Journal of Veterinary Pharmacology and Therapeutics 28, 325–328. McKellar, Q., Gibson, I., McCormack, R., 1998. Pharmacokinetics and tissue disposition of danofloxacin in sheep. Biopharmaceutics and Drug Disposition 19, 123–129. Olchowy, T.W.J., TerHune, T.N., Herrick, R.L., 2000. Efficacy of difloxacin in calves experimentally infected with Mannheimia haemolytica. American Journal of Veterinary Research 61, 710–713. Paulsen, D.B., Corstvet, R.E., Mcclure, J.R., Enright, F.M., McBride, J.W., McDonough, K.C., 1992. Use of an indwelling bronchial
catheter model of bovine pneumonic pasteurellosis for evaluation of therapeutic efficacy of various compounds. American Journal of Veterinary Research 53, 679–683. Power, J., 1990. Statistical analysis of pharmacokinetics data. Journal of Veterinary Pharmacology and Therapeutics 13, 113–120. Shumaker, R.C., 1986. PKCALC: a basic interactive computer program for statistical and pharmacokinetic analysis of data. Drug Metabolism Reviews 17, 331–348. Soback, S., Gips, M., Bialer, M., Bor, A., 1994. Effect of lactation on single dose pharmacokinetics of norfloxacin in ewes. Antimicrobial Agents and Chemotherapy 38, 2336–2339. Stamm, J.M., Hanson, C.W., Chu, D.T., Bailer, R., Vojtko, C., Fernandes, P.B., 1986. In vitro evaluation of A-56619 (difloxacin) and A-56620: new aryl-fluoroquinolones. Antimicrobial Agents and Chemotherapy 29, 193–200. Toutain, P.L., Bousquet-Me´lou, A., 2004. Bioavailability and its assessment. Journal of Veterinary Pharmacology and Therapeutics 27, 455– 466. Turnidge, J., 1999. Pharmacokinetics and pharmacodynamics of fluoroquinolones. Drugs 58, 29–36. Walker, R.D., 2000. Fluoroquinolones. In: Prescott, J.F., Baggot, J.D., Walker, R.D. (Eds.), Antimicrobial Therapy in Veterinary Medicine, third ed. Iowa State University Press, Ames, USA, pp. 315–338. Wolfson, J.S., Hooper, D.C., 1989. Fluoroquinolone antimicrobial agents. Clinical Microbiology Reviews 2, 378–424. Yamaoka, K., Tamigawara, Y., Uno, J., 1981. A pharmacokinetic analysis program (MULTI) for microcomputers. Journal of Pharmacobio-Dynamics 4, 879–885. Yamaoka, K., Nakagawa, T., Uno, T., 1978. Application of Akaike’s Information Criterion (AIC) in the evaluation of linear pharmacokinetic equations. Journal Pharmacokinetics Biopharmaceutics 6, 165– 175.
Pharmacokinetics after intravenous, intramuscular and subcutaneous administration of difloxacin in sheep P. Marı´n *, E. Ferna´ndez-Varo´n, E. Escudero, C.M. Ca´rceles Department of Pharmacology, Faculty of Veterinary Medicine, University of Murcia, Campus de Espinardo, 30071 Murcia, Spain Accepted 15 January 2007
Abstract The disposition kinetics of difloxacin, a fluoroquinolone antibiotic, after intravenous (IV), intramuscular (IM) and subcutaneous (SC) administration were determined in sheep at a single dose of 5 mg/kg. The concentration–time data were analysed by compartmental (after IV dose) and non-compartmental pharmacokinetics method (after IV, IM and SC administration). Plasma concentrations of difloxacin were determined by high performance liquid chromatography with fluorescence detection. Steady-state volume of distribution (Vss) and clearance (Cl) of difloxacin after IV administration were 1.68 ± 0.21 L/kg and 0.21 ± 0.03 L/h kg, respectively. Following IM and SC administration difloxacin achieved maximum plasma concentration of 1.89 ± 0.55 and 1.39 ± 0.14 mg/L at 2.42 ± 1.28 and 5.33 ± 1.03 h, respectively. The absolute bioavailabilities after IM and SC routes were 99.92 ± 26.50 and 82.35 ± 25.65%, respectively. Based on these kinetic parameters, difloxacin is likely to be effective in sheep. 2007 Elsevier Ltd. All rights reserved. Keywords: Difloxacin; Pharmacokinetics; Sheep; Bioavailability
1. Introduction Fluoroquinolones are a group of antimicrobials that have become widely used in veterinary medicine. These compounds are regarded as almost the ‘‘ideal’’ antimicrobial agents because of their broad spectrum of activity, clinically advantageous pharmacokinetic properties and low toxicity, which represents a considerable advance over other classes of antimicrobial agents. Because the fluoroquinolones are relatively new antimicrobial agents, extensive research is needed to develop and implement appropriate specific dosing regimens that can maximize their clinical efficacy for use in animals and reduce the selection for resistant pathogens, particularly, as these drugs are used for the treatment of multi-drug resistant infection in humans (Walker, 2000).
*
Corresponding author. Tel.: +34 968 398404; fax: +34 968 364147. E-mail address: [email protected] (P. Marı´n).
0034-5288/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2007.01.005
Difloxacin is a fluoroquinolone carboxylic acid antimicrobial agent with high in vitro activity against a wide range of gram-positive and gram-negative aerobes and anaerobes (Granneman et al., 1986). Like other fluoroquinolones, difloxacin is a concentration-dependent bactericidal agent, it acts by inhibiting bacterial DNA topoisomerases II and IV (Drlica and Zhao, 1997; Wolfson and Hooper, 1989). It has been shown to be effective in the treatment of experimentally induced pneumonic pasteurellosis in calves (Olchowy et al., 2000) and experimental Staphylococcus aureus endocarditis in rabbits (Boscia et al., 1988). The pharmacokinetic properties of difloxacin have been evaluated in several species as non-lactating goats (Atef et al., 2002), rabbits (Abd El-Aty et al., 2005) and horses (Ferna´ndez-Varo´n et al., 2005a), but not yet in sheep. Thus, the purpose of the study reported here was to determine the pharmacokinetics of difloxacin after intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration to sheep, and to investigate the bioavailability after the extravascular routes.
P. Marı´n et al. / Research in Veterinary Science 83 (2007) 234–238
2. Materials and methods 2.1. Animals Six clinically healthy Seguren˜a female sheep weighing between 46.2 and 58.6 kg and aged from 5 to 7 years were obtained from the farm of the University of Murcia. The animals were housed and fed an antibiotic-free diet for at least 30 days preceding the study. For each treatment period of the cross-over, they were observed daily for general health, and clinical observations were made prior to injection and at 2, 10 and 24 h post-injection. Alfalfa hay and water was provided ad libitum together with a drug-free concentrate. The study was approved by the Bioethics Committee of the University of Murcia.
235
of plasma blank samples spiked with different amounts of drug and treated as any samples, with the peak areas of the same standards prepared in phosphate buffer. Each point was established from an average of five determinations. The mean percentage recoveries of difloxacin from plasma was 95.68 ± 4.29%. The assay precision (R.S.D.) was assessed by expressing the standard deviation of repeated measurements as a percentage of the mean value. Intra-day precision was estimated from six replicates of three standard samples used for calibration curves (R.S.D. < 6.5%). Inter-day precision was estimated from the analysis of standard samples on three separate days (R.S.D. < 9.2%). The limit of quantification (LOQ) of difloxacin in plasma was chosen as the concentrations of the lowest concentration level on the calibration curves for which the RDS was <15% (LOQ: 5 lg/L).
2.2. Experimental design 2.5. Pharmacokinetic analysis A cross-over design (2 · 2 · 2) was used in three phases. Each animal received single IV bolus, IM and SC injections of difloxacin 5% (Dicural, Fort Dodge Veterinaria, S.A.) at a dose of 5 mg/kg with at least 15-day washout period. For the IV administration, the solution was injected into the left jugular vein and blood samples (4 mL) were collected from the contralateral jugular vein into heparinized tubes. Subcutaneous injections were administered under the skin of the back at a single location in the thoraco-lumbar region lateral of the mid-line and IM injections into the semimembranous muscle. Blood samples were collected at 0 (pre-treatment), 0.083, 0.167, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72 and 96 h post-dosing, and each sample was collected by individual venipuncture. Samples were centrifuged at 1500g for 15 min and the plasma taken and stored at –45 C until assayed.
The concentration–time data obtained after IV treatment in each individual animal were initially fitted to one-, two- and three-exponential equations by the retroprojection method (Gibaldi and Perrier, 1982). The PKCALC computer program (Shumaker, 1986) was used to obtain the best estimates of the parameters of these equations. The final curve fitting was carried out using non-linear regression with the MULTI computer program and the Gauss–Newton damping algorithm (Yamaoka et al., 1981). Akaike’s Information Criterion (Yamaoka et al., 1978) was used to determine the number of compartments used in the pharmacokinetic analysis and the most appropriate weighting for the data. The data points were weighted with the inverse of the squared fitted value. The general polyexponential equation is:
2.3. Analytical method
C¼
n X
C i ekit
i¼1
Plasma concentrations of difloxacin were measured using a HPLC method previously reported (Ferna´ndezVaro´n et al., 2006a). Difloxacin pure substance (Fort-Dodge, Madrid) was used for quality controls. Ciprofloxacin (Vita Pharma, Madrid) was used as internal standard. 2.4. Method validation Quality controls were prepared from a pool of sheep plasma spiked with nine concentrations of difloxacin between 5 and 2000 lg/L. Plasma aliquots were stored at 45 C until assayed. Aliquots of quality controls were extracted as above and 60 lL was injected into the chromatographic system. Standard curves were obtained by unweighted linear regression of difloxacin peak areas versus known concentrations. Each point was established from an average of five determinations. Correlation coefficients (r) were >0.99% for calibration curves. The percentage recovery was determined by comparing the peak areas
where C is the plasma concentration of drug; t is the time after drug administration; Ci and ki are the intercept and slope, respectively, of the different disposition phases and e is the base of natural logarithm. Pharmacokinetic parameters were obtained from the individual fitted equations (Gibaldi and Perrier, 1982). The distribution and elimination half-lives were calculated as t12k1 = ln 2/k1 and t12kz = ln 2/kz, respectively. Non-compartmental pharmacokinetics parameters were estimated using WinNonlin Professional (version 5.1, Pharsight Corporation, CA, USA). WinNonlin model 200 was used for extravascular administrations and model 201 for intravenous administration. Non-compartmental parameters calculated were: area under the plasma concentration–time curve (AUC) using the linear trapezoidal rule with extrapolation to time infinity, mean residence time (MRT), plasma clearance (Cl), and apparent volume of distribution at steady state (Vss). Mean absorption times were calculated as MAT = MRTSC,IM – MRTIV.
236
P. Marı´n et al. / Research in Veterinary Science 83 (2007) 234–238
Bioavailability (F) was calculated by the method of corresponding areas: ½F ð%Þ ¼ AUCSC;IM 100=AUCIV 2.6. Statistical analysis Descriptive statistical parameters as mean, standard deviation and coefficient of variation were calculated. Harmonic means were calculated for the half-lives of elimination. The Wilcoxon Rank Sum test and Student’s t test were used to test parameters for significant differences between IV, IM and SC administration (Powers, 1990). The SPSS (Version 11.0) statistical software was used. 3. Results Clinical examination of all the sheep before and after each administration did not reveal any abnormalities. No local or systemic adverse reactions occurred after IV, IM and SC injection of difloxacin. Difloxacin plasma concentrations versus time data after IV administration could best be described by a two-compartment open model. The mean (±S.D.) plasma concentrations of difloxacin at the times of sample collection after IV, IM and SC administration are plotted in Fig. 1. Mean (±S.D.) pharmacokinetic parameters are presented in Table 1. Difloxacin was detected in plasma up to 72 h after IV and SC administration and 96 h after IM dosing. 4. Discussion The results demonstrated that difloxacin is quickly and widely distributed after IV administration with a half-life (t12k1) of 1.53 h and a Vss of 1.68 ± 0.21 L/kg, which suggest good penetration through biological membranes. This Vss is in agreement with values found in other species such as horses (Vss = 1.02 L/kg, Ferna´ndez-Varo´n et al., 2006a), rabbits (Vss = 1.51 L/kg, Abd El-Aty et al., 2005) and goats (Vss = 1.11 L/kg, Atef et al., 2002). Elimination half-lives (t12kz = 11.43 h IV, t12kz = 13.89 h IM and t12kz = 12.02 h SC) in this study were longer than those described for difloxacin in horses (t12kz = 2.66 h IV and t12kz = 5.72 h IM, Ferna´ndez-Varo´n et al., 2006a), pigs and chicken (t12kz = 7.92 and 4.10 h IV, respectively, Inui et al., 1998) and those reported for other fluoroquinolones in sheep (McKellar et al., 1998; Soback et al., 1994) and goats (Ferna´ndez-Varo´n et al., 2006b). Consequently the systemic clearance of difloxacin in our study (Cl = 0.21 ± 0.03 L/h kg) was slower than those described in the afore-mentioned manuscripts. Differences in the kinetic parameters are relatively common, and frequently related to interspecies variation, age, breed, health status of the animals and/or the assay method used (Haddad et al., 1985). Following IM and SC administration, difloxacin showed bioavailabilities of 99.92 ± 26.50 and 82.35 ± 25.65%,
Fig. 1. Experimental plasma concentrations (mean ± S.D.) of difloxacin at a single dose of 5 mg/kg bodyweight (n = 6). (a) Semilogarithmic plot after intravenous administration. (b) Semilogarithmic plot after intramuscular administration. (c) Semilogarithmic plot after subcutaneous administration.
respectively. These results indicated that difloxacin was well absorbed following both routes of administration, these data are similar to those reported for danofloxacin in sheep (McKellar et al., 1998), and for difloxacin in horses (Ferna´ndez-Varo´n et al., 2006a) and rabbits (Abd El-Aty et al., 2005).
P. Marı´n et al. / Research in Veterinary Science 83 (2007) 234–238 Table 1 Pharmacokinetic parameters (mean ± S.D.) of difloxacin in sheep after intravenous, intramuscular and subcutaneous administration at a single dose of 5 mg/kg bodyweight (n = 6) Parameters
Units
Intravenous
Intramuscular
Subcutaneous
C1 Cz k1 kz t12k1c t12kzc Vss AUC MRT Cl MAT Cmax Tmax F (%)
mg/L mg/L h1 h1 H H L/kg mg h/L h L/h kg h mg/L h –
6.83 ± 2.30 0.58 ± 0.14 0.45 ± 0.12 0.06 ± 0.01 1.53 ± 0.37 11.43 ± 0.88 1.68 ± 0.21 24.23 ± 3.18 8.07 ± 0.83 0.21 ± 0.03 – – – –
– – – 0.05 ± 0.01 – 13.89 ± 2.42 – 23.62 ± 3.81 15.83 ± 4.27a – 7.76 ± 4.64 1.89 ± 0.55 2.42 ± 1.28 99.92 ± 26.50
– – – 0.06 ± 0.01 – 12.02 ± 2.83 – 19.40 ± 3.93b 12.62 ± 1.29a – 4.55 ± 1.59 1.39 ± 0.14 5.33 ± 1.03b 82.35 ± 25.65b
C1, intercept of the ordinate by the fastest disposition slope minus the intercept of the next fastest disposition slope; Cz, intercept of the slowest disposition slope with the ordinate; t12k1, the disposition half-life associated with the initial slope (k1) of a semilogarithmic concentration–time curve; t12kz, the elimination half-life associated with the terminal slope (kz) of a semilogarithmic concentration–time curve; Vss, the apparent volume of distribution at steady state; Cl, the total body clearance of drug from the plasma; AUC, the area under the plasma concentration–time curve from zero to infinity; MRT, mean residence time; F, the fraction of the administered dose systemically available (bioavailability); Tmax, the time to reach peak or maximum plasma concentration following intramuscular and subcutaneous administration; MAT, mean absorption time; Cmax, the peak or maximum plasma concentration following intramuscular and subcutaneous administration. a Significantly different from IV (P < 0.05). b Significantly different from IM (P < 0.05). c Harmonic mean.
After SC administration of difloxacin, peak plasma concentration (Cmax) 1.39 ± 0.14 mg/L was attained at 5.33 ± 1.03 h post administration. The drug was slowly absorbed, showing some influence of the absorption phase in the disposition of the drug, because MAT was shorter than MRTiv (Toutain and Bousquet-Me´lou, 2004). For this reason, it is necessary to do further study with design of multiple dose regimens for calculation accumulation factor. However, after IM dose of difloxacin the absorption was faster compared to SC dose, the Cmax and tmax values (1.89 ± 0.55 lg/L and 2.42 ± 1.28 h, respectively) were close to the corresponding data reported in horses (Ferna´ndez-Varo´n et al., 2006a). Maximum plasma concentrations (Cmax) were significantly different (p < 0.05) after IM and SC administrations. Since fluoroquinolones are drugs acting in a concentration-dependent manner, it is considered that the ratios Cmax/MIC90 and AUC0–24/MIC90 are the best parameters for predicting their antimicrobial effect (Lode et al., 1998). Previous investigations have shown that for fluoroquinolones Cmax/MIC90 > 3 produced 99% reduction in bacterial count and Cmax/MIC90 of 8 or greater prevented the emergence of resistant organisms (Craig, 1998). Furthermore AUC24/MIC90 > 100 h should be achieved to
237
give maximum clinical and bacteriology efficacy (Turnidge, 1999). Minimal inhibitory concentration data of difloxacin against ovine bacterial isolates have not been reported up to now, so further studies are necessary to predict clinical efficacy of difloxacin in sheep. Fluoroquinolones have low MIC values against many Gram-negative bacteria (Walker, 2000). Abd El-Aty et al. (2005) have reported MIC of difloxacin against E. coli of 0.065 lg/ml, and similar results have been published by Stamm et al. (1986) (MICs = 0.03–0.2 lg/ml). So difloxacin could be effective by the IM and SC route at 5 mg/kg against bacterial isolates with MIC 6 0.19 and MIC 6 0.17 lg/ml, respectively, taking into account values of PK/PD parameters of Cmax/MIC90 P 8 and AUC24/ MIC90 P 100 h. Moreover the efficacy of the drug against respiratory infections of lambs (Mavrogianni and Fthenakis, 2005), calves (Paulsen et al., 1992; Olchowy et al., 2000), dogs (van den Hoven et al., 2000) and poultry (Kempf et al., 1998) has been documented. It is conclude, that in view of general adverse reactions were not observed in any sheep, and the favourable pharmacokinetic properties such as long half-life and high bioavailability, difloxacin administered at 5 mg/kg after IM and SC dose could be effective in sheep. However, further studies are needed to establish a multiple dosage regimen and clinical efficacy. Acknowledgements Thanks are due to Fort-Dodge for supplying difloxacin pure substance and to J. Carrizosa for his assistance with the experiments. References Abd El-Aty, A.M., Goudah, A., Ismail, M., Shimoda, M., 2005. Disposition kinetics of difloxacin in rabbit after intravenous and intramuscular injection of dicural. Veterinary Research Communications 29, 297–304. Atef, M., El-Banna, H.A., Abd El-Aty, A.M., Goudah, A., 2002. Pharmacokinetics of difloxacin in goats. Deutsche Tiera¨rztliche Wochenschrift 109, 320–323. Boscia, J.A., Kobasa, W.D., Kaye, D., 1988. Comparison of difloxacin, enoxacin and cefazolin for the treatment of experimental Staphylococcus aureus endocarditis. Antimicrobial Agents and Chemotherapy 32, 262–264. Craig, W.A., 1998. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clinical Infectious Diseases 26, 1–10. Drlica, K., Zhao, X., 1997. DNA gyrase, topoisomerase IV, and the 4quinolones. Microbiology and Molecular Biology Reviews 61, 377– 392. Ferna´ndez-Varo´n, E., Ca´rceles, C.M., Marı´n, P., Martos, N., Escudero, E., Ayala, I., 2006a. Pharmacokinetics of difloxacin after intravenous, intramuscular and intragastric administration to horses. American Journal of Veterinary Research 67, 1076–1081. Ferna´ndez-Varo´n, E., Villamayor, L., Escudero, E., Espuny, A., Ca´rceles, C.M., 2006b. Pharmacokinetics and milk penetration of moxifloxacin after intravenous and subcutaneous administration to lactating goats. The Veterinary Journal 172, 302–307.
238
P. Marı´n et al. / Research in Veterinary Science 83 (2007) 234–238
Gibaldi, M., Perrier, P., 1982. Pharmacokinetics, second ed. Marcel Dekker, New York. Granneman, G.R., Snyder, K.M., Shu, V.S., 1986. Difloxacin metabolism and pharmacokinetics in humans after single oral doses. Antimicrobial Agents and Chemotherapy 30, 689–693. Haddad, N.S., Pedersoli, W.M., Ravis, W.R., Fazeli, M.H., Carson, R.L., 1985. Combined pharmacokinetics of gentamicin in pony mares after a single intravenous and intramuscular administration. American Journal of Veterinary Research 46, 2004–2007. van den Hoven, R., Wagenaar, J.A., Walker, R.D., 2000. In vitro activity of difloxacin against canine bacterial isolates. Journal of Veterinary Diagnostic Investigation 12, 218–223. Inui, T., Taira, T., Matsushita, T., Endo, T., 1998. Pharmacokinetic properties and oral bioavailabities of difloxacin in pig and chicken. Xenobiotica 28 (9), 887–893. Kempf, I., van den Hoven, R., Gesbert, F., Guittet, M., 1998. Efficacy of difloxacin in growing broiler chicken for the control of infection due to pathogenic Mycoplasma gallisepticum. Journal of Veterinary Medicine B 45, 305–310. Lode, H., Borner, K., Koeppe, P., 1998. Pharmacodynamics of fluoroquinolones. Clinical Infectious Disease 27, 33–39. Mavrogianni, V.S., Fthenakis, G.C., 2005. Efficacy of difloxacin against respiratory infections of lambs. Journal of Veterinary Pharmacology and Therapeutics 28, 325–328. McKellar, Q., Gibson, I., McCormack, R., 1998. Pharmacokinetics and tissue disposition of danofloxacin in sheep. Biopharmaceutics and Drug Disposition 19, 123–129. Olchowy, T.W.J., TerHune, T.N., Herrick, R.L., 2000. Efficacy of difloxacin in calves experimentally infected with Mannheimia haemolytica. American Journal of Veterinary Research 61, 710–713. Paulsen, D.B., Corstvet, R.E., Mcclure, J.R., Enright, F.M., McBride, J.W., McDonough, K.C., 1992. Use of an indwelling bronchial
catheter model of bovine pneumonic pasteurellosis for evaluation of therapeutic efficacy of various compounds. American Journal of Veterinary Research 53, 679–683. Power, J., 1990. Statistical analysis of pharmacokinetics data. Journal of Veterinary Pharmacology and Therapeutics 13, 113–120. Shumaker, R.C., 1986. PKCALC: a basic interactive computer program for statistical and pharmacokinetic analysis of data. Drug Metabolism Reviews 17, 331–348. Soback, S., Gips, M., Bialer, M., Bor, A., 1994. Effect of lactation on single dose pharmacokinetics of norfloxacin in ewes. Antimicrobial Agents and Chemotherapy 38, 2336–2339. Stamm, J.M., Hanson, C.W., Chu, D.T., Bailer, R., Vojtko, C., Fernandes, P.B., 1986. In vitro evaluation of A-56619 (difloxacin) and A-56620: new aryl-fluoroquinolones. Antimicrobial Agents and Chemotherapy 29, 193–200. Toutain, P.L., Bousquet-Me´lou, A., 2004. Bioavailability and its assessment. Journal of Veterinary Pharmacology and Therapeutics 27, 455– 466. Turnidge, J., 1999. Pharmacokinetics and pharmacodynamics of fluoroquinolones. Drugs 58, 29–36. Walker, R.D., 2000. Fluoroquinolones. In: Prescott, J.F., Baggot, J.D., Walker, R.D. (Eds.), Antimicrobial Therapy in Veterinary Medicine, third ed. Iowa State University Press, Ames, USA, pp. 315–338. Wolfson, J.S., Hooper, D.C., 1989. Fluoroquinolone antimicrobial agents. Clinical Microbiology Reviews 2, 378–424. Yamaoka, K., Tamigawara, Y., Uno, J., 1981. A pharmacokinetic analysis program (MULTI) for microcomputers. Journal of Pharmacobio-Dynamics 4, 879–885. Yamaoka, K., Nakagawa, T., Uno, T., 1978. Application of Akaike’s Information Criterion (AIC) in the evaluation of linear pharmacokinetic equations. Journal Pharmacokinetics Biopharmaceutics 6, 165– 175.