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Disposition kinetics and pharmacokinetics–pharmacodynamic integration of difloxacin against Staphylococcus aureus isolates from rabbits
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Research in Veterinary Science 84 (2008) 90–94 www.elsevier.com/locate/rvsc
Disposition kinetics and pharmacokinetics–pharmacodynamic integration of difloxacin against Staphylococcus aureus isolates from rabbits E. Ferna´ndez-varo´n a, C.M. Ca´rceles a, P. Marı´n a, D. Vancraeynest b, A. Montes c, J. Sotillo c, J.D. Garcı´a-Martı´nez c,* a Department of Pharmacology, Faculty of Veterinary Medicine, University of Murcia, Murcia, Spain Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, Belgium Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University of Murcia, Campus de Espinardo, 30071 Murcia, Spain b
c
Accepted 11 April 2007
Abstract The pharmacokinetics of difloxacin were studied following intravenous (IV), subcutaneous (SC) and oral administration of 5 mg/kg to healthy white New Zealand rabbits (n = 6). Difloxacin concentrations were determined by HPLC assay with fluorescence detection. Minimal inhibitory concentrations (MICs) assay of difloxacin against different strains of S. aureus from different european countries was performed in order to compute the main pharmacodynamic surrogate markers. The plasma difloxacin clearance (Cl) for the IV route was (mean ± SD) 0.41 ± 0.05 L/h kg. The steady-state volume of distribution (Vss) was 1.95 ± 0.17 L/kg. The terminal half-life ðt1=2kz Þ was (mean ± SD) 4.19 ± 0.34 h, 7.53 ± 1.32 h and 8.00 ± 0.45 h after IV, IM and oral, respectively. From this data, it seems that a 5 mg/kg dose difloxacin would be effective by SC and oral routes in rabbits against bacterial isolates with MIC 6 0.1 lg/mL. 2007 Elsevier Ltd. All rights reserved. Keywords: Difloxacin; Pharmacokinetics; Pharmacodynamics; S. aureus; MIC; Rabbits
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), including most species and strains of Klebsiella spp., Staphylococcus spp., Escherichia coli, Enterobacter, Campylobacter, Shigella, Proteus, Pasteurella spp., Mycoplasma spp., Rickettsia spp., and Chlamydia (Stamm et al., 1986; Abd El-Aty et al., 2005). Similar to other fluoroquinolones difloxacin has a large volume of distribution, low plasma protein binding and relatively low minimal inhibitory concentration (MICs) against target microorganisms (Spreng et al., 1995; Brown, 1996). In recent years, a large number of pharmacokinetics
*
Corresponding author. Tel.: +34 968 398328; fax: +34 968 364147. E-mail address: [email protected] (J.D. Garcı´a-Martı´nez).
0034-5288/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2007.04.002
studies with fluorquinolones have been conducted in several species (Aliabadi and Lees, 2001; Ferna´ndez-Varo´n et al., 2005, 2006a, 2007; Martinez et al., 2006). Consideration of rabbits has changed from production animals to companion animal which makes us reconsider strategies in the treatment of infectious diseases in this specie. Difloxacin pharmacokinetics has been reported in dogs (Frazier et al., 2000; Heinen, 2002), goats (Atef et al., 2002), mares (Adams et al., 2005), chickens and pigs (Inui et al., 1998) and rabbits (Abd El-Aty et al., 2005). The present study was designed to determine the complete plasma pharmacokinetics of difloxacin following intravenous, subcutaneous and oral administration to rabbits and its activity against different strains of S. aureus. Six New Zealand white rabbits of both sexes weighing between 3.2 and 4.5 kg were obtained from the Laboratory Animal Farm of the University of Murcia. They did not receive any drug treatment
E. Ferna´ndez-varo´n et al. / Research in Veterinary Science 84 (2008) 90–94
before the study. The study was approved by the bioethics committee of the University of Murcia. Thirty rabbit S. aureus strains were tested. Sixteen of these strains were highly virulent strains, isolated from commercial rabbitries with chronic problems of staphylococcosis (Hermans et al., 1999). The other strains were low virulence strains. The strains were isolated in Belgium (11), France (12), Greece (1) and Spain (6). The MICs were determined using the NCCLS (National Committee for Clinical Laboratory Standards) agar dilution method (NCCLS, 2002). A cross-over design was used in three phases (2 · 2 · 2), with two washout periods of 15 days. Dicural 50 mg/mL (Fort Dodge, Madrid, Spain) were administered by intravenous and subcutaneous route at single doses of 5 mg/kg bodyweight. For oral administration a solution was prepared from Dicural Tablets by the Pharmacy Service of the Clinic Veterinary Hospital of the University of Murcia and administered by nasogastric tube. Blood samples (0.5 mL) were obtained by inserting a 24gauge catheter into the marginal ear vein, and allowing the blood to drip into a 2 mL heparinized syringe. Blood was then placed in a tube. Blood samples were collected at 0, 5 (IV), 10, 15, 30, 45 min and 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 32 and 72 h following drug administration and centrifuged at 1500g for 15 min within 30 min after collection. Plasma was immediately removed and stored at 45 C until assayed. Plasma concentrations of difloxacin were measured using a modified HPLC method (Siefert et al., 1999). Quality controls were prepared from a pool of blank rabbit plasma spiked with seven concentrations of difloxacin between 5 and 2000 lg/L. Correlation coefficients (r) were >0.98 for calibration curves. The average (±SE) recovery obtained was 92.4 ± 0.68%. Intraday precision (RSD) was <7%. Inter-day precision was <9%. The limit of quantification (LOQ) of difloxacin in plasma was 5 lg/L. Compartimental analysis was performed using a retroprojection method (Gibaldi and Perrier, 1982) and the PKCALC computer program (Shumaker, 1986). The final curve fitting was carried out using non-linear regression with the MULTIFIT computer program (Yamaoka et al., 1981). Akaike’s Information Criterion (AIC) (Yamaoka et al., 1978) was used to determine the number of compartments. Pharmacokinetic parameters were obtained from the individual fitted equations (Gibaldi and Perrier, 1982). The absorption, distribution and elimination halflives were calculated as t1=2a ¼ ln 2=K a ; t1=2k1 ¼ ln 2=k1 and t1=2kz ¼ ln 2=kz , respectively. Area under the concentration–time curve (AUC) was calculated using the linear trapezoidal rule with extrapolation to infinity. Mean Residence Time was calculated as MRT = AUMC/AUC. Mean absorption times were calculated as MAT = MRTSC, ORAL – MRTIV. The systemic clearance as Cl = Dose/AUC. The apparent volume of distribution (area method) and apparent volume of distribution at steady state were calculated as
91
Vz = Dose/(AUC Æ kz) and Vss = (Dose Æ AUMC)/AUC2, respectively. Bioavailability (F) was calculated by the method of corresponding areas. Descriptive statistical parameters as mean (harmonic mean for half-lives), standard deviation and coefficient of variation were calculated. The Wilcoxon Rank Sum test was used to test parameters for significant differences between IV with respect to SC and oral administration (Powers, 1990) (P < 0.05). The parameters most commonly correlated with clinical outcome of antimicrobials include the ratio of maximum concentration to minimum inhibitory concentration (Cmax/MIC), the ratio of the 24-h area under the curve at steady-state to MIC (AUC24/MIC) and the duration of time that serum levels exceed the MIC (T > MIC). For a concentration-dependent drug such as difloxacin, clinical response usually correlates with AUC24/MIC and Cmax/ MIC (Drusano et al., 1993; Lode et al., 1998; Toutain et al., 2002; Toutain and Lees, 2004). Mean (±SD) plasma concentrations of difloxacin following IV, SC and oral administration are given in Fig. 1 and mean (±SD) values for pharmacokinetic parameters are given in Table 1. No adverse effects were observed in any of the rabbits following IV, SC and oral administration of difloxacin at 5 mg/ kg. The difloxacin plasma concentration versus time data after IV administration could best be described by a two compartment open model. The disposition of SC and orally administered difloxacin in rabbits was best described by a one-compartment model. Significant differences were found between IV and SC for MRT, t1=2kz and AUC, between IV and oral for t1=2kz , AUC and MRT, and between SC and oral for MRT, MAT and Cmax (P < 0.05). The MICs values and PK/PD ratios are shown in Table 2. Difloxacin has a wide distribution in rabbits with a Vss of 1.95 ± 0.17 L/kg suggesting penetration through biological membranes and into tissues. This Vss obtained is large
Fig. 1. Experimental plasma concentrations (mean ± SD) of difloxacin at a single dose of 5 mg/kg bodyweight (n = 6). Semilogarithmic plot after IV (n), SC (}) and oral (h) administration.
E. Ferna´ndez-varo´n et al. / Research in Veterinary Science 84 (2008) 90–94
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Table 1 Pharmacokinetic parameters (mean ± SD) of difloxacin in rabbits after intravenous, subcutaneous and oral administration at a dose of 5 mg/kg bodyweight (n = 6) Parameters
Units
Intravenous
Subcutaneous
Oral
C1 Cz k1 kz t1=2k1 t1=2kz Vz Vss AUC AUMC MRT Cl Ka t1/2Ka MAT Cmax Tmax F
lg/L lg/L h1 h1 h h L/kg L/kg mg h/L mg h2/L h L/h kg h1 h h lg/L h %
3772.33 ± 423 1571.76 ± 254 1.31 ± 0.11 0.17 ± 0.01 0.53 ± 0.04 4.19 ± 0.34 2.47 ± 0.34 1.95 ± 0.17 12.73 ± 1.42 61.73 ± 12.51 4.82 ± 0.51 0.41 ± 0.05 – – – – – –
– – – 0.09 ± 0.01 – 7.53 ± 1.32a – – 10.79 ± 1.64a 56.33 ± 13.3 5.18 ± 0.50a – 4.11 ± 0.47 0.17 ± 0.02 0.37 ± 0.06 879.5 ± 20.9 0.96 ± 0.09 84.65 ± 6.47
– – – 0.09 ± 0.01 – 8.00 ± 0.45a – – 9.64 ± 0.38a 49.36 ± 6.22 5.11 ± 0.51a,b – 3.82 ± 0.28 0.18 ± 0.01 0.30 ± 0.02b 756.30 ± 20.9b 1.02 ± 0.06 76.36 ± 7.36
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. t1=2k1 : the disposition halflife associated with the initial slope (k1) of a semilogarithmic concentration–time curve. t1=2kz : the elimination half-life associated with the terminal slope (kz) of a semilogarithmic concentration–time curve. Vz: the apparent volume of distribution calculated by the area method. 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. AUMC: area under the moment curve. 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 subcutaneous and oral administration. Ka: absorption rate constant (first-order). t1/2a: absorption half-life. MAT: mean absorption time. Cmax: the peak or maximum plasma concentration following subcutaneous and oral administration. a Significantly different from IV (P < 0.05). b Significantly different from SC (P < 0.05). Table 2 Minimal inhibitory concentrations on rabbit Staphylococcus aureus strains (n = 30) and surrogate markers of efficacy from pharmacokinetics parameters Species
Origin
S. aureus S. aureus ATCC 29213 E. coli ATCC 25922
Rabbit Control
Number of strains with difloxacin MIC (lg/ml) of 60.03
Control
0.06
0.12
0.25
0.5
1
2
13 x
12
1
2
4
8
16
32
64
128
>128
2
x
PK/PD parameters
Subcutaneous
Oral
MIC = 0.12 mg/L AUC24/MIC Cmax/MIC
89.91 7.32
80.33 6.30
MIC = 0.25 mg/L AUC24/MIC Cmax/MIC
43.16 3.5
38.56 3.02
MIC = 0.5 mg/L AUC24/MIC Cmax/MIC
21.58 1.75
19.28 1.51
and in agreement with the Vss (1.5 L/kg) reported by Abd El-Aty et al. (2005) for difloxacin and by Ferna´ndez-Varo´n et al. (2005a) for moxifloxacin (1.95 L/kg) both in rabbits, but twofold higher than that for enrofloxacin (Vss = 0.93 L/kg; Broome et al., 1991). The t1=2kz (4.19 ± 0.34 h) after IV dosing was longer than that for difloxacin in rabbits (3.25 h; Abd El-Aty et al., 2005). However, some differences in the previous study,
such as the use of a microbiological assay, time of sample collection (until 48 h) and t1=2kz calculated by non-compartimental model can be responsible for this shorter t1=2kz prediction. The t1=2kz values estimated following SC and oral administration were 7.53 ± 1.32 h and 8.00 ± 0.45 h respectively. These are significantly longer than that observed following IV injection indicating an influence of the absorption process.
E. Ferna´ndez-varo´n et al. / Research in Veterinary Science 84 (2008) 90–94
Difloxacin was well absorbed following SC and oral administration with an absolute bioavailability (F) of 84.65 ± 6.47% and 76.36 ± 7.36%, respectively. Animal models using different quinolones showed that a AUC24/MIC ratio of about 100 h or Cmax/MIC ratio of 10 should be achieved to give maximum clinical and bacterial efficacy (Turnidge, 1999). MIC values of difloxacin against rabbit isolates of S. aureus from different European countries have been tested in the present work (Table 2). AUC24/MIC and Cmax/MIC ratios were calculated, and from these ratios, difloxacin appears to present optimum ratios for bacteria with MIC of 0.12 mg/L mainly for the SC route. However, from this data and according to MIC90 of 0.5 mg/L obtained, difloxacin would not be appropriate for treating S. aureus infections in rabbits. Moreover, it is necessary to interpret the clinical implications of these benchmark ratios with caution since the therapeutic effect of antimicrobial agents reflect a complex array of variables including microbial susceptibility, host pharmacokinetics, the pathophysiology of the disease condition, the site of the infection, pathogen virulence factors, host immune status, if the infection is acute or chronic, and drug anti-inflammatory activities. Therefore, it would be helpful to have the results from studies that explore the target AUC24/MIC and Cmax/MIC ratios needed to maximize the likelihood of achieving the desired therapeutic outcome when difloxacin is administered to rabbits. In conclusion, the systemic difloxacin exposure achieved in rabbits following either SC or oral administration is consistent with the predicted blood levels needed for a positive therapeutic outcome for S. aureus strains with a MIC of 0.12 mg/L but not for the MIC90 of 0.5 mg/L obtained. Therefore, it would be necessary to assay higher doses of difloxacin to reach optimal ratios of the surrogate markers and to establish repeated dosage regimens and clinical efficacy. Acknowledgements The authors thank Fort-Dodge for supplying difloxacin pure substance and to Arlette Van de Kerckhove for her skilled technical assistance. References Abd El-Aty, A.M., Goudah, A., Ismail, M., Mounir, S.M., Shimoda, M., 2005. Disposition kinetics of difloxacin in rabbit after intravenous and intramuscular injection of dicural. Veterinary Research Communications 28, 297–304. Adams, A.R., Haines, G.R., Brown, M.P., Gronwall, R., Merritt, R., 2005. Pharmacokinetics of difloxacin and its concentration in body fluids and endometrial tissues of mares after repeated intragastric administration. Canadian Journal of Veterinary Research 69 (3), 229– 235. Aliabadi, F.S., Lees, P., 2001. Pharmacokinetics and pharmacodynamics of danofloxacin in serum and tissue fluids of goats following
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intravenous and intramuscular administration. American Journal of Veterinary Research 62 (12), 1979–1989. Atef, M., El-Banna, H.A., Abd el-Aty, A.M., Goudah, A., 2002. Pharmacokinetics of difloxacin in goats. Deutsche Tierarztliche Wochenschrif 109 (7), 320–323. Broome, R.L., Brooks, D.L., Babish, J.G., Copeland, D.D., Conzelman, G.M., 1991. Pharmacokinetic properties of enrofloxacin in rabbits. American Journal of Veterinary Research 52 (11), 1835–1841. Brown, S.A., 1996. Fluoroquinolones in animal health. Journal of Veterinary Pharmacology and Therapeutics 19, 1–14. Drusano, G.L., Johnson, D.E., Rosen, M., Standiford, H., 1993. Pharmacodynamics of a fluoroquinolone antimicrobial agent in a neutropenic rat model of Pseudomonas sepsis. Antimicrobial Agents and Chemotherapy 37, 483–490. Ferna´ndez-Varo´n, E., Bovaira, M.J., Espuny, A., Escudero, E., Vancraeynest, D., Ca´rceles, C.M., 2005. Pharmacokinetic–pharmacodynamic integration of moxifloxacin in rabbits after intravenous, intramuscular and oral administration. Journal of Veterinary Pharmacology and Therapeutics 27, 57–60. Ferna´ndez-Varo´n, E., Villamayor, L., Escudero, E., Espuny, A., Ca´rceles, C.M., 2006a. Pharmacokinetic and milk penetration of moxifloxacin after intravenous and subcutaneous administration to lactating goats. The Veterinary Journal 172 (2), 302–307. Ferna´ndez-Varo´n, E., Marin, P., Escudero, E., Vancraeynest, D., Ca´rceles, C.M., 2007. Pharmacokinetic–pharmacodynamic integration of danofloxacin after intravenous, intramuscular and subcutaneous administration to rabbits. Journal of Veterinary Pharmacology and Therapeutics 30 (1), 18–24. Frazier, D.L., Thompson, L., Trettien, A., Evans, E.I., 2000. Comparison of fluoroquinolone pharmacokinetic parameters after treatment with marbofloxacin, enrofloxacin, and difloxacin in dogs. Journal of Veterinary Pharmacology and Therapeutics 23, 293–302. Gibaldi, M., Perrier, P., 1982. Pharmacokinetics, 2nd ed. Marcel Dekker, New York, pp. 445–449. 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. Heinen, E., 2002. Comparative serum pharmacokinetics of the fluoroquinolones enrofloxacin, difloxacin, marbofloxacin, and orbifloxacin in dogs after single oral administration. Journal of Veterinary Pharmacology and Therapeutics 25, 1–5. Hermans, K., De Herdt, P., Devriese, L.A., Hendrickx, W., Godard, C., Haesebrouck, F., 1999. Colonisation of rabbits with Staphylococcus aureus in flocks with and without chronic staphylococcosis. Veterinary Microbiology 67, 37–46. Inui, T., Taira, T., Matsushita, T., Endo, T., 1998. Pharmacokinetic properties and oral bioavailabities of difloxacin in pig and chicken. Xenobiotica 9, 887–893. Lode, H., Borner, K., Koeppe, P., 1998. Pharmacodynamics of fluoroquinolones. Clinical Infectious Diseases 27, 33–39. Martinez, M., McDermott, P., Walker, R., 2006. Pharmacology of the fluoroquinolones: a perspective for the use in domestic animals. The Veterinary Journal 172 (1), 10–28. NCCLS., 2002. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard. NCCLS document, M31-A2 (ISBN: 1-56238-461-9). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087, USA, 2002. Powers, J., 1990. Statistical analysis of pharmacokinetic data. Journal of Veterinary Pharmacology and Therapeutics 13, 113–120. Shumaker, RC., 1986. PKCALC: a basic interactive computer program for statistical and pharmacokinetic analysis of data. Drug Metabolism Reviews 17, 331–348. Siefert, H.M., Domdey-Bette, A., Henninger, F., Hucke, F., Kohlsdorfer, C., Steinke, W., Stass, H.H., 1999. Pharmacokinetics of the 8methoxyquinolone, moxifloxacin: a comparison in humans and other mammalian species. Journal of Antimicrobial Chemotherapy 43 (Suppl. B), 69–76.
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Research in Veterinary Science 84 (2008) 90–94 www.elsevier.com/locate/rvsc
Disposition kinetics and pharmacokinetics–pharmacodynamic integration of difloxacin against Staphylococcus aureus isolates from rabbits E. Ferna´ndez-varo´n a, C.M. Ca´rceles a, P. Marı´n a, D. Vancraeynest b, A. Montes c, J. Sotillo c, J.D. Garcı´a-Martı´nez c,* a Department of Pharmacology, Faculty of Veterinary Medicine, University of Murcia, Murcia, Spain Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, Belgium Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University of Murcia, Campus de Espinardo, 30071 Murcia, Spain b
c
Accepted 11 April 2007
Abstract The pharmacokinetics of difloxacin were studied following intravenous (IV), subcutaneous (SC) and oral administration of 5 mg/kg to healthy white New Zealand rabbits (n = 6). Difloxacin concentrations were determined by HPLC assay with fluorescence detection. Minimal inhibitory concentrations (MICs) assay of difloxacin against different strains of S. aureus from different european countries was performed in order to compute the main pharmacodynamic surrogate markers. The plasma difloxacin clearance (Cl) for the IV route was (mean ± SD) 0.41 ± 0.05 L/h kg. The steady-state volume of distribution (Vss) was 1.95 ± 0.17 L/kg. The terminal half-life ðt1=2kz Þ was (mean ± SD) 4.19 ± 0.34 h, 7.53 ± 1.32 h and 8.00 ± 0.45 h after IV, IM and oral, respectively. From this data, it seems that a 5 mg/kg dose difloxacin would be effective by SC and oral routes in rabbits against bacterial isolates with MIC 6 0.1 lg/mL. 2007 Elsevier Ltd. All rights reserved. Keywords: Difloxacin; Pharmacokinetics; Pharmacodynamics; S. aureus; MIC; Rabbits
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), including most species and strains of Klebsiella spp., Staphylococcus spp., Escherichia coli, Enterobacter, Campylobacter, Shigella, Proteus, Pasteurella spp., Mycoplasma spp., Rickettsia spp., and Chlamydia (Stamm et al., 1986; Abd El-Aty et al., 2005). Similar to other fluoroquinolones difloxacin has a large volume of distribution, low plasma protein binding and relatively low minimal inhibitory concentration (MICs) against target microorganisms (Spreng et al., 1995; Brown, 1996). In recent years, a large number of pharmacokinetics
*
Corresponding author. Tel.: +34 968 398328; fax: +34 968 364147. E-mail address: [email protected] (J.D. Garcı´a-Martı´nez).
0034-5288/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2007.04.002
studies with fluorquinolones have been conducted in several species (Aliabadi and Lees, 2001; Ferna´ndez-Varo´n et al., 2005, 2006a, 2007; Martinez et al., 2006). Consideration of rabbits has changed from production animals to companion animal which makes us reconsider strategies in the treatment of infectious diseases in this specie. Difloxacin pharmacokinetics has been reported in dogs (Frazier et al., 2000; Heinen, 2002), goats (Atef et al., 2002), mares (Adams et al., 2005), chickens and pigs (Inui et al., 1998) and rabbits (Abd El-Aty et al., 2005). The present study was designed to determine the complete plasma pharmacokinetics of difloxacin following intravenous, subcutaneous and oral administration to rabbits and its activity against different strains of S. aureus. Six New Zealand white rabbits of both sexes weighing between 3.2 and 4.5 kg were obtained from the Laboratory Animal Farm of the University of Murcia. They did not receive any drug treatment
E. Ferna´ndez-varo´n et al. / Research in Veterinary Science 84 (2008) 90–94
before the study. The study was approved by the bioethics committee of the University of Murcia. Thirty rabbit S. aureus strains were tested. Sixteen of these strains were highly virulent strains, isolated from commercial rabbitries with chronic problems of staphylococcosis (Hermans et al., 1999). The other strains were low virulence strains. The strains were isolated in Belgium (11), France (12), Greece (1) and Spain (6). The MICs were determined using the NCCLS (National Committee for Clinical Laboratory Standards) agar dilution method (NCCLS, 2002). A cross-over design was used in three phases (2 · 2 · 2), with two washout periods of 15 days. Dicural 50 mg/mL (Fort Dodge, Madrid, Spain) were administered by intravenous and subcutaneous route at single doses of 5 mg/kg bodyweight. For oral administration a solution was prepared from Dicural Tablets by the Pharmacy Service of the Clinic Veterinary Hospital of the University of Murcia and administered by nasogastric tube. Blood samples (0.5 mL) were obtained by inserting a 24gauge catheter into the marginal ear vein, and allowing the blood to drip into a 2 mL heparinized syringe. Blood was then placed in a tube. Blood samples were collected at 0, 5 (IV), 10, 15, 30, 45 min and 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 32 and 72 h following drug administration and centrifuged at 1500g for 15 min within 30 min after collection. Plasma was immediately removed and stored at 45 C until assayed. Plasma concentrations of difloxacin were measured using a modified HPLC method (Siefert et al., 1999). Quality controls were prepared from a pool of blank rabbit plasma spiked with seven concentrations of difloxacin between 5 and 2000 lg/L. Correlation coefficients (r) were >0.98 for calibration curves. The average (±SE) recovery obtained was 92.4 ± 0.68%. Intraday precision (RSD) was <7%. Inter-day precision was <9%. The limit of quantification (LOQ) of difloxacin in plasma was 5 lg/L. Compartimental analysis was performed using a retroprojection method (Gibaldi and Perrier, 1982) and the PKCALC computer program (Shumaker, 1986). The final curve fitting was carried out using non-linear regression with the MULTIFIT computer program (Yamaoka et al., 1981). Akaike’s Information Criterion (AIC) (Yamaoka et al., 1978) was used to determine the number of compartments. Pharmacokinetic parameters were obtained from the individual fitted equations (Gibaldi and Perrier, 1982). The absorption, distribution and elimination halflives were calculated as t1=2a ¼ ln 2=K a ; t1=2k1 ¼ ln 2=k1 and t1=2kz ¼ ln 2=kz , respectively. Area under the concentration–time curve (AUC) was calculated using the linear trapezoidal rule with extrapolation to infinity. Mean Residence Time was calculated as MRT = AUMC/AUC. Mean absorption times were calculated as MAT = MRTSC, ORAL – MRTIV. The systemic clearance as Cl = Dose/AUC. The apparent volume of distribution (area method) and apparent volume of distribution at steady state were calculated as
91
Vz = Dose/(AUC Æ kz) and Vss = (Dose Æ AUMC)/AUC2, respectively. Bioavailability (F) was calculated by the method of corresponding areas. Descriptive statistical parameters as mean (harmonic mean for half-lives), standard deviation and coefficient of variation were calculated. The Wilcoxon Rank Sum test was used to test parameters for significant differences between IV with respect to SC and oral administration (Powers, 1990) (P < 0.05). The parameters most commonly correlated with clinical outcome of antimicrobials include the ratio of maximum concentration to minimum inhibitory concentration (Cmax/MIC), the ratio of the 24-h area under the curve at steady-state to MIC (AUC24/MIC) and the duration of time that serum levels exceed the MIC (T > MIC). For a concentration-dependent drug such as difloxacin, clinical response usually correlates with AUC24/MIC and Cmax/ MIC (Drusano et al., 1993; Lode et al., 1998; Toutain et al., 2002; Toutain and Lees, 2004). Mean (±SD) plasma concentrations of difloxacin following IV, SC and oral administration are given in Fig. 1 and mean (±SD) values for pharmacokinetic parameters are given in Table 1. No adverse effects were observed in any of the rabbits following IV, SC and oral administration of difloxacin at 5 mg/ kg. The difloxacin plasma concentration versus time data after IV administration could best be described by a two compartment open model. The disposition of SC and orally administered difloxacin in rabbits was best described by a one-compartment model. Significant differences were found between IV and SC for MRT, t1=2kz and AUC, between IV and oral for t1=2kz , AUC and MRT, and between SC and oral for MRT, MAT and Cmax (P < 0.05). The MICs values and PK/PD ratios are shown in Table 2. Difloxacin has a wide distribution in rabbits with a Vss of 1.95 ± 0.17 L/kg suggesting penetration through biological membranes and into tissues. This Vss obtained is large
Fig. 1. Experimental plasma concentrations (mean ± SD) of difloxacin at a single dose of 5 mg/kg bodyweight (n = 6). Semilogarithmic plot after IV (n), SC (}) and oral (h) administration.
E. Ferna´ndez-varo´n et al. / Research in Veterinary Science 84 (2008) 90–94
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Table 1 Pharmacokinetic parameters (mean ± SD) of difloxacin in rabbits after intravenous, subcutaneous and oral administration at a dose of 5 mg/kg bodyweight (n = 6) Parameters
Units
Intravenous
Subcutaneous
Oral
C1 Cz k1 kz t1=2k1 t1=2kz Vz Vss AUC AUMC MRT Cl Ka t1/2Ka MAT Cmax Tmax F
lg/L lg/L h1 h1 h h L/kg L/kg mg h/L mg h2/L h L/h kg h1 h h lg/L h %
3772.33 ± 423 1571.76 ± 254 1.31 ± 0.11 0.17 ± 0.01 0.53 ± 0.04 4.19 ± 0.34 2.47 ± 0.34 1.95 ± 0.17 12.73 ± 1.42 61.73 ± 12.51 4.82 ± 0.51 0.41 ± 0.05 – – – – – –
– – – 0.09 ± 0.01 – 7.53 ± 1.32a – – 10.79 ± 1.64a 56.33 ± 13.3 5.18 ± 0.50a – 4.11 ± 0.47 0.17 ± 0.02 0.37 ± 0.06 879.5 ± 20.9 0.96 ± 0.09 84.65 ± 6.47
– – – 0.09 ± 0.01 – 8.00 ± 0.45a – – 9.64 ± 0.38a 49.36 ± 6.22 5.11 ± 0.51a,b – 3.82 ± 0.28 0.18 ± 0.01 0.30 ± 0.02b 756.30 ± 20.9b 1.02 ± 0.06 76.36 ± 7.36
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. t1=2k1 : the disposition halflife associated with the initial slope (k1) of a semilogarithmic concentration–time curve. t1=2kz : the elimination half-life associated with the terminal slope (kz) of a semilogarithmic concentration–time curve. Vz: the apparent volume of distribution calculated by the area method. 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. AUMC: area under the moment curve. 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 subcutaneous and oral administration. Ka: absorption rate constant (first-order). t1/2a: absorption half-life. MAT: mean absorption time. Cmax: the peak or maximum plasma concentration following subcutaneous and oral administration. a Significantly different from IV (P < 0.05). b Significantly different from SC (P < 0.05). Table 2 Minimal inhibitory concentrations on rabbit Staphylococcus aureus strains (n = 30) and surrogate markers of efficacy from pharmacokinetics parameters Species
Origin
S. aureus S. aureus ATCC 29213 E. coli ATCC 25922
Rabbit Control
Number of strains with difloxacin MIC (lg/ml) of 60.03
Control
0.06
0.12
0.25
0.5
1
2
13 x
12
1
2
4
8
16
32
64
128
>128
2
x
PK/PD parameters
Subcutaneous
Oral
MIC = 0.12 mg/L AUC24/MIC Cmax/MIC
89.91 7.32
80.33 6.30
MIC = 0.25 mg/L AUC24/MIC Cmax/MIC
43.16 3.5
38.56 3.02
MIC = 0.5 mg/L AUC24/MIC Cmax/MIC
21.58 1.75
19.28 1.51
and in agreement with the Vss (1.5 L/kg) reported by Abd El-Aty et al. (2005) for difloxacin and by Ferna´ndez-Varo´n et al. (2005a) for moxifloxacin (1.95 L/kg) both in rabbits, but twofold higher than that for enrofloxacin (Vss = 0.93 L/kg; Broome et al., 1991). The t1=2kz (4.19 ± 0.34 h) after IV dosing was longer than that for difloxacin in rabbits (3.25 h; Abd El-Aty et al., 2005). However, some differences in the previous study,
such as the use of a microbiological assay, time of sample collection (until 48 h) and t1=2kz calculated by non-compartimental model can be responsible for this shorter t1=2kz prediction. The t1=2kz values estimated following SC and oral administration were 7.53 ± 1.32 h and 8.00 ± 0.45 h respectively. These are significantly longer than that observed following IV injection indicating an influence of the absorption process.
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Difloxacin was well absorbed following SC and oral administration with an absolute bioavailability (F) of 84.65 ± 6.47% and 76.36 ± 7.36%, respectively. Animal models using different quinolones showed that a AUC24/MIC ratio of about 100 h or Cmax/MIC ratio of 10 should be achieved to give maximum clinical and bacterial efficacy (Turnidge, 1999). MIC values of difloxacin against rabbit isolates of S. aureus from different European countries have been tested in the present work (Table 2). AUC24/MIC and Cmax/MIC ratios were calculated, and from these ratios, difloxacin appears to present optimum ratios for bacteria with MIC of 0.12 mg/L mainly for the SC route. However, from this data and according to MIC90 of 0.5 mg/L obtained, difloxacin would not be appropriate for treating S. aureus infections in rabbits. Moreover, it is necessary to interpret the clinical implications of these benchmark ratios with caution since the therapeutic effect of antimicrobial agents reflect a complex array of variables including microbial susceptibility, host pharmacokinetics, the pathophysiology of the disease condition, the site of the infection, pathogen virulence factors, host immune status, if the infection is acute or chronic, and drug anti-inflammatory activities. Therefore, it would be helpful to have the results from studies that explore the target AUC24/MIC and Cmax/MIC ratios needed to maximize the likelihood of achieving the desired therapeutic outcome when difloxacin is administered to rabbits. In conclusion, the systemic difloxacin exposure achieved in rabbits following either SC or oral administration is consistent with the predicted blood levels needed for a positive therapeutic outcome for S. aureus strains with a MIC of 0.12 mg/L but not for the MIC90 of 0.5 mg/L obtained. Therefore, it would be necessary to assay higher doses of difloxacin to reach optimal ratios of the surrogate markers and to establish repeated dosage regimens and clinical efficacy. Acknowledgements The authors thank Fort-Dodge for supplying difloxacin pure substance and to Arlette Van de Kerckhove for her skilled technical assistance. References Abd El-Aty, A.M., Goudah, A., Ismail, M., Mounir, S.M., Shimoda, M., 2005. Disposition kinetics of difloxacin in rabbit after intravenous and intramuscular injection of dicural. Veterinary Research Communications 28, 297–304. Adams, A.R., Haines, G.R., Brown, M.P., Gronwall, R., Merritt, R., 2005. Pharmacokinetics of difloxacin and its concentration in body fluids and endometrial tissues of mares after repeated intragastric administration. Canadian Journal of Veterinary Research 69 (3), 229– 235. Aliabadi, F.S., Lees, P., 2001. Pharmacokinetics and pharmacodynamics of danofloxacin in serum and tissue fluids of goats following
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