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Pharmacokinetics of florfenicol after intravenous and intramuscular administration in New Zealand White rabbits
Research in Veterinary Science 87 (2009) 102–105
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Pharmacokinetics of florfenicol after intravenous and intramuscular administration in New Zealand White rabbits F. Koc a,*, M. Ozturk b, Y. Kadioglu b, E. Dogan c, L.E. Yanmaz c, Z. Okumus c a
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Atatürk University, Erzurum 25400, Turkey Department of Analytical Chemistry, Faculty of Pharmacy, Atatürk University, Erzurum, Turkey c Department of Surgery, Faculty of Veterinary Medicine, Atatürk University, Erzurum, Turkey b
a r t i c l e
i n f o
Article history: Accepted 22 October 2008
Keywords: Pharmacokinetic Florfenicol Rabbit Plasma
a b s t r a c t The pharmacokinetic disposition and bioavailability of florfenicol (FF) were determined after single intravenous (i.v.) and intramuscular (i.m.) administrations of 25 mg/kg b.w. to ten healthy New Zealand White rabbits. Plasma FF concentrations were determined by high-performance liquid chromatography (HPLC). The plasma pharmacokinetic values for FF were best described by a one-compartment open model. The elimination half-life (t1/2b) was different (p < 0.05) however, the area under curve (AUC) was similar (p > 0.05) after i.v. and i.m. administrations. FF was rapidly eliminated (t1/2b 1.49 ± 0.23 h), slowly absorbed and high (F, 88.75 ± 0.22%) after i.m. injection. In addition, FF was widely distributed to the body tissues (Vss 0.98 ± 0.05 L/kg) after i.v. injection. In this study the time that plasma concentration exceeded the concentration of 2 lg/mL was approximately 6 h. For bacteria with MIC of 2 lg/mL, frequent administration at this dose would be needed to maintain the concentration above the MIC. However, it is possible that rabbit pathogens may have MIC values less than 2 lg/mL which would allow for less frequent administration. Further studies are necessary to identify the range of MIC values for rabbit pathogens and to identify the most appropriate PK-PD parameter needed to predict an effective dose. Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
1. Introduction FF is a synthetic broad-spectrum antibiotic in veterinary treatment of infectious diseases. It is a novel antibiotic that belongs to the chloramphenicol (CP) family, but it is used only in animals (Papich and Riviere, 2001). The compounds act by inhibiting bacterial protein synthesis by binding to 50 S and 70 S subunits in the ribosomes (Cannon et al., 1990). Moreover, FF prevents bacterial enzymatic acetylation; consequently, this product has more antibacterial activity than CP and TP (Cannon et al., 1990). Because it is both activity against CP-resistant pathogens (Neu and Fu, 1980; Syriopoulou et al., 1981) and low adverse effects (Paape et al., 1990) it has been widely used in veterinary clinics to treat bacterial diseases (Booker et al., 1997; Jim et al., 1999; Angelos et al., 2000). The pharmacokinetic disposition of FF has been extensively investigated in calves, horses, poultry, pigs, fish and sheep (Varma et al., 1986; Adams et al., 1987; Mestorino et al., 1993; Martinsen et al., 1993; El-Banna, 1998; Soback et al., 1995; Lobell et al., 1994; Afifi et al., 1997; Li et al., 2002; Jianzhong et al., 2004) but in rabbits
* Corresponding author. Tel.: +90 442 631 4193x1007; fax: +90 442 631 4188. E-mail address: [email protected] (F. Koc).
has scarcely been documented (El-Aty et al., 2004; Park et al., 2007). In this study, both used analytical method and dose were different from previous reported two studies. The aim of this study was to determine FF (25 mg/kg b.w.) kinetic disposition in plasma and bioavailability after i.m. and i.v. administration in healthy New Zealand White rabbits.
2. Materials and methods 2.1. Reagents and instruments All solvents were HPLC grade and obtained from J.T. Baker (Deventer, Netherlands). FF (99.6% assay purity) and CP (99.7% assay purity) analytical standards and injectable formulation (Nuflor, 300 mg/mL, was commercial preparation of FF) were provided by the Schering-Plough (Segre, France). HPLC was supplied from a Thermoseparations Spectra Series. The HPLC system consisted of a P 4000 gradient pump coupled with a Spectra System UV 6000 LP photodiode array detection system, Thermoseparations AS 3000 autosampler and software Chromoquest 30. The column was reverse phase nucleosil C18 (4.6 250 mm, 5 lm particle size) and it was supplied from Phenomenex (CA, USA).
0034-5288/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2008.10.010
F. Koc et al. / Research in Veterinary Science 87 (2009) 102–105
2.2. Animals and experimental design Ten healthy rabbits (New Zealand White) approximately 6–12 months old were used in this study. Of ten rabbits, five animals were female (2.50 ± 0.41 kg), five male (2.78 ± 0.35 kg). Before use, they were housed for two weeks in Experimental Research Centrum of Ataturk University. These animals were fed pelleted feed (antibacterial-free) and water ad libitum. Animal experiments were performed in an ethically proper way by following guidelines as set by the Ethical Committee of Ataturk University (Report No.: 03/2007). The rabbits were divided into two groups (n = 5, each group). Before i.v. and i.m. administrations, the restrain devices were placed in the marginal vein each animal. The blood (2 mL) samples were collected from the restrain devices for control (at 0 min). Following, in group One, florfenicol was administrated single dose i.m. injection of 25 mg/kg b.w. into semimembraneous muscle. Blood samples were taken from restrain devices of each rabbit and collected in tubes containing heparin as anticoagulant at 5, 10, 15, 30, 45, 60, 90 min, 2, 4, 6, 8, 12, 18 and 24 h after drug administration. In group Two, single dose (i.v.) FF (25 mg/kg b.w.) was injected into right the marginal vein. Blood samples were taken from the restrain devices of each rabbit (opposite ear) and collected in tubes with anticoagulant (heparin) at the same times. Samples were centrifuged within 1 h after collection and plasma samples were stored frozen (20 °C) until analysis. All the samples were analysed within one week after the experiments. 2.3. Analytical method The chromatographic method was performed as described by Kowalski et al. (2005). CP was used as an internal standard in the analytical method. The mobile phase consisted of a mixture of acetonitrile–water at a ratio of 25:75 (v/v) and adjusted to pH of 2.7 with 85% orthophosphoric acid. The injection volume was 20 lL, monitoring wavelength was at 224 nm, and oven temperature was at 2425 °C and the flow rate was 1.5 mL/min. Calibration curve was prepared in the range of 0.125.0 lg/mL (n = 6). CP (internal standard) was added to spike samples 0.5 mL at 10.0 lg/mL concentration. 2.4. Sample preparation The extraction procedure was also achieved as described by Kowalski et al. (2005). Briefly, the frozen plasma samples (0.5 mL) were thawed at a temperature of 25 °C (room temperature) and added 0.2 mL 1.0 M sodium hydroxide and 3 mL of ethyl acetate. The mixture was mechanically shacked and centrifuged for 15 min at 1650g. The organic layer was evaporated at 50 °C under nitrogen and supernatant was dissolved in 0.5 mL of mobile phase. Following again it was centrifuged at 11,200g during 10 min and 20 lL volume was injected into HPLC system. 2.5. Validation The following parameters were determined for validation of method: linearity, precision (RSD), accuracy, limit of detection (LOD), limit of quantitation (LOQ), recovery, reproducibility. For linearity, calibration curve was calculated automatically using software (0.125.0 lg/mL). The recoveries were calculated as the percentage using extraction process after spiking from 0.1 to 25.0 lg/ mL with eight different levels of florfenicol (n = 6). The inter-and intra-day reproducibility was determined at 1, 5 and 10 lg/mL concentrations. LOD’s were calculated on the basis to be 3 of
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signal-to-noise ratio spiking at low concentrations the plasma samples. LOQ was calculated to be 10 of signal-to-noise ratio. 2.6. Pharmacokinetic and statistical analysis Florfenicol plasma concentration vs. time plot of each animal was analysed using the computer program WinNonlin version 4.1 (Pharsight, Mountain View, CA, USA). The proper model was chosen by minimum Akaike’s information criterion estimation (Yamaoka et al., 1978). Pharmacokinetic parameters for each animal were analysed using by one-compartmental open model. The absorption half-life (t1/2abs), the elimination half-life (t1/2b = 0.693/b), the total body clearance (Cltot) and the apparent steadystate volume of distribution (Vss) and bioavailability (F% = AUCi.m/ AUCi.v.) were calculated. Areas under the plasma concentration– time curves for both i.v. (AUCi.v.0?1) and i.m. (AUCi.m.0?1) studies were calculated by the method of trapezoids. Peak plasma concentrations (Cmax) of drug and times to reach peak concentration (tmax) for the i.m. study were determined from the individual plasma concentration–time curves. All results are presented as mean ± SD. Harmonic means were calculated for t½b and MRT. Mann–Whitney U test was used to test for significant difference in t½b. Comparison of AUCs was made using the Independent Samples t-test. Statistical significance was assigned at p < 0.05. 3. Results The HPLC analysis for the determination of FF was high degree of reproducibility. Calibration curves had an r2 value of >0.99. Intra-assay variations were determined by measuring six replicates (n = 6) of three standard samples used for calibration curves. The intra-assay variation coefficient was 6.29 ± 2.58. Inter-assay precision was determined by assaying the three standard samples on three separate days. The inter-assay variation coefficient was 6.90 ± 3.53. The average of inter- and intra-day precision (RSD) was <7. The accuracy ranged from 95% to 108% and mean recovery was 85 ± 6.33%. LOD and LOQ were found to be 0.03 and 0.1 lg/mL, respectively. After i.v. and i.m. administration to rabbits at a single dose (25 mg/kg b.w.), plasma concentration of FF time curves were shown (Fig. 1). The pharmacokinetic parameters for FF were shown in Table 1. After i.v. administration, its t1/2b, MRT, AUC, Vss, and Cltot, of FF were 1.21 ± 0.09 h, 1.75 ± 0.12 h, 44.59 ± 1.38 lg h/mL, 0.98 ± 0.05 L/kg, and 0.56 ± 0.02 L/kg/h, respectively. Following i.m. injection, its t1/2b, t1/2abs, AUC, tmax, Cmax and F were 1.49 ± 0.23 h, 0.83 ± 0.24 h, 39.10 ± 10.12 lg h/mL, 1.56 ± 0.13 h, 8.65 ± 2.19 lg/mL, and 88.75 ± 0.22%, respectively. 4. Discussion In this study, all rabbits were clinically healthy and there were not observed any side effects after administration of FF by a single dose i.m. and i.v. The elimination half-life (t1/2b) was different (p < 0.05) however, the area under curve (AUC) was similar (p > 0.05), after i.v. and i.m. administrations. Following i.v. injection, t1/2b of FF in rabbits was 1.21 ± 0.09 h in the present study. This value was similar to 1.54 ± 0.20 h (El-Aty et al., 2004) and 0.90 ± 0.20 h (Park et al., 2007) in rabbits. However, this finding was shorter than in calves (3.18 ± 1.01 h), cattle (159 min), turkeys (2.34 ± 0.44 h), broiler chickens (173.25 min), turtles (from 2 to 7.8 h), (Decraene et al., 1997; Lobell et al., 1994; Switała et al., 2007; Afifi et al., 1997; Stamper et al., 2003).
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F. Koc et al. / Research in Veterinary Science 87 (2009) 102–105
Fig. 1. Semilogarithmic plot of plasma concentrations–time curves of florfenicol after single dose i.v. and i.m. administrations at a dose of 25 mg/kg b.w. to New Zealand White rabbits (n = 5).
Table 1 Pharmacokinetic parameters determined after i.v. and i.m. administration of florfenicol (25 mg/kg) (n = 5). Variables
i.v. Mean ± SD (n = 5)
i.m. Mean ± SD (n = 5)
P<
t1/2abs (h) t1/2ba (h) MRTa (h) AUC (lg h/mL) Vss (L/kg) Cltot (L/kg/h) tmax (h) Cmax (lg/mL) F (%)
NA 1.21 ± 0.09 1.74 ± 0.12 44.59 ± 1.38 0.98 ± 0.05 0.56 ± 0.02 NA NA NA
0.83 ± 0.24 1.49 ± 0.23 NA 39.10 ± 10.12 NA NA 1.56 ± 0.13 8.65 ± 2.19 88.75 ± 0.22
– 0.028 – 0.26 – – – – –
t1/2abs: Absorption half-life; t1/2b: the elimination half-life; MRT: mean resistance time; AUC: area under the concentrations time curves; Vss: mean volume of distribution at steady-state; Cltot: total body clearance; tmax: time to maximum concentration; Cmax: maximum concentration; F%: bioavailability; NA: not applicable. a Harmonic mean.
The short half-life of its elimination indicates that the drug was rapidly eliminated from the bodies of rabbits. Cltot of FF was 0.56 ± 0.02 L/kg/h in this study. The result was somewhat different from reported (0.34 ± 0.11 L/kg/h) by El-Aty et al. (2004) in rabbits. However, this value was in agreement with reported (0.63 ± 0.06 L/kg/h) by Park et al. (2007) in the same animal species. The result of this study was higher than 0.23 and 0.16 L/kg/h in lactating cows (Mestorino et al., 1993; Soback et al., 1995), 0.22 L/kg/h in cattle (Lobell et al., 1994), 0.15 L/kg/h in non-lactating dairy cows (Bretzlaff et al., 1987), 0.171, 0.172 L/ kg/h, 0.22 ± 0.05 L/kg/h in calves (Varma et al., 1986; Adams et al., 1987; Decraene et al., 1997), 0.25 ± 0.02 L/kg/h, in sheep (Jianzhong et al., 2004), 0.33 ± 0.04, 0.30 ± 0.03 and 0.27 ± 0.03 L/ kg/h, in camels, sheep and goats, respectively (Ali et al., 2003). Park et al. (2008) reported that Cltot of FF in the animal species, smaller body weight, was higher than bigger body weight animals. The result of this study was in agreement with previous report (Park et al., 2008). As known that, Vss is an important indication of the diffusion of the drug in tissues (Galinsky and Svensson, 1995). In the present study, Vss was large (0.98 ± 0.05 L/kg). This result was in agreement with that of 0.94 ± 0.19 L/kg reported in rabbits (Park et al. 2007). FF is a lipophilic drug and the Vss may be related to physiochemical characteristics of drug (Ali et al., 2003). However, this finding was inconsistent with the result reported (0.57 ± 0.85 L/kg) by El-Aty et al. (2004) in rabbits. In this study, after i.m. injection, Cmax of the drug reached 8.65 ± 2.19 lg/mL at 1.56 ± 0.13 h (tmax) and it was determined un-
til 12 h (Fig. 1) and F% value was 88 ± 0.22%. It was reported that Cmax was high (21.65 lg/mL), tmax was rapid (0.5 h) and F% was high (88.25%) in rabbits (El-Aty et al., 2004). In this study, Cmax and tmax were inconsistent with the reported by El-Aty et al. (2004). These differences in parameters may be related to difference in analytical methods used in the two studies (Lane et al., 2004; Park et al., 2007). It is known that the different methods may occur different results from the same species (Atef et al., 2001). El-Aty et al. (2004) reported that used method was microbiological assay and measured only the unbound and microbiological active compound in their study. In this study used method was HPLC, which measures the total concentration of FF (Atef et al., 2001). In addition, these values in this study were different from previous reports in different animal species including, broiler chickens, lactating cows, turtles, Egyptian goats, Atlantic salmon (Mestorino et al., 1993; Soback et al., 1995; Stamper et al., 2003; Atef et al., 2001; Martinsen et al., 1993). Consequently, in our study, the absorption of drug was slow and higher. These differences in absorption from the i.m. injection site may be due to differences in regional blood flow from different muscle tissues of animal species. After i.v. administration, MRT of the drug was 1.75 ± 0.12. Park et al. (2007) reported that 1.50 ± 0.34 h after i.v. injection in rabbits. It was determined that following i.v. and i.m. administration MRT was 1.69 ± 0.67 and 4.34 ± 0.38 h, respectively (El-Aty et al., 2004). After i.v. administration, MRT of florfenicol in the present study was similar to two studies. Until now, not only the minimum inhibitory concentrations (MICs) of FF for bacteria isolates from rabbits but also studies on efficacy of it in terms of pharmacokinetic/pharmacodynamic (PK/PD) approaches have not yet been reported. However, previous studies in other animal species showed that the susceptibility the MIC values of FF ranging from 0.25–2 lg/mL for many various bacterial pathogens in cows, pigs and fish (Bretzlaff et al., 1987; Ueda et al., 1995; Ho et al., 2000). In this study the time that plasma concentration exceeded the concentration of 2 lg/mL was approximately 6 h (Fig. 1). The value of 2 lg/ mL is used as the breakpoint for susceptible pathogens as defined by the CLSI for swine and cattle respiratory pathogens. For bacteria with MIC of 2 lg/mL, frequent administration at this dose would be needed to maintain the concentration above the MIC. However, it is possible that rabbit pathogens may have MIC values less than 2 lg/mL which would allow for less frequent administration. Further studies are necessary to identify the range of MIC values for rabbit pathogens and to identify the most appropriate PK-PD parameter needed to predict an effective dose.
F. Koc et al. / Research in Veterinary Science 87 (2009) 102–105
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electrophoresis and high-performance liquid chromatography for the analysis of florfenicol. J. Pharm. Biomed. Anal. 39, 983–989. Lane, V.M., Villarroel, A., Wetzlich, S.E, Clifford, A., Taylor, I., Craigmill, A.L., 2004. Intravenous and subcutaneous pharmacokinetics of florfenicol in sheep. J. Vet. Pharmacol. Ther. 27, 191–196. Li, J.Z., Fung, K.F., Chen, Z.L., Zhang, J., 2002. Tissue pharmacokinetics of florfenicol in pigs experimentally infected with Actinobacillus pleuropneumoniae. Eur. J. Drug Metab. Pharmacokinet. 27, 265–271. Lobell, R.D., Varma, K.J., Johnson, J.C., Sams, R.A., Gerken, D.F., Ashcraft, S.M., 1994. Pharmacokinetics of florfenicol following intravenous and intramuscular doses to cattle. J. Vet. Pharmacol. Ther. 17, 253–258. Martinsen, B., Horsbeg, T.E., Varma, K.J., Sams, R., 1993. Single dose pharmacokinetic study of florfenicol in Atlantic salmon (Salmo salar) in seawater at 11 °C. Aquaculture 112, 1–11. Mestorino, N., Landoni, M.F., Alt, M., Errecalde, J.O., 1993. The pharmacokinetics of thiamphenicol in lactating cows. Vet. Res. Commun. 17, 295–303. Neu, H.C., Fu, K.P., 1980. In vitro activity of chloramphenicol and thiamphenicol analogs. Antimicrob. Agents Chemother. 18, 311–316. Paape, M.J., Miller, R.H., Ziv, G., 1990. Effects of florfenicol, chloramphenicol and thiamphenicol on phagocytosis, chemiluminescence, and morphology of bovine polymorphonuclear neutrophil leukocytes. J. Dairy Sci. 73, 1734– 1744. Papich, M.G., Riviere, J.E., 2001. Chloramphenicol and derivatives, macrolides, lincosamides and miscellaneous antimicrobials. In: Adams, H.R. (Ed.), Veterinary Pharmacology and Therapeutics, Iowa State University Press, Ames IA, pp. 868–897 (Chapter 44). Park, B.K., Lim, J.H., Kim, M.S., Hwang, Y.H., Yun, H.I., 2007. Pharmacokinetics of florfenicol and its major metabolite, florfenicol amine, in rabbits. J. Vet. Pharmacol. Ther. 30, 32–36. Park, B.K., Lim, J.H., Kim, M.S., Hwang, Y.H., Yun, H.I., 2008. Pharmacokinetics of florfenicol and its metabolite, florfenicol amine, in dogs. Res. Vet. Sci. 84, 85–89. Soback, S., Paape, M.J., Filep, R., Varma, K.J., 1995. Florfenicol pharmacokinetics in lactating cows after intravenous, intramuscular and intramammary administration. J. Vet. Pharmacol. Ther. 18, 413–417. Stamper, M.A., Papich, M.G., Lewbart, G.A., May, S.B., Plummer, D.D., Stoskopf, M.K., 2003. Pharmacokinetics of florfenicol in loggerhead sea turtles (Caretta caretta) after single intravenous and intramuscular injections. J. Zoo Wildlife Med. 34, 3–8. Syriopoulou, V.P., Harding, A.L., Goldmann, D.A., Smith, A.L., 1981. In vitro antibacterial activity of fluorinated analogs of chloramphenicol and thiamphenicol. Antimicrob. Agent Chemother. 19, 294–297. Switała, M., Hrynyk, R., Smutkiewicz, A., Jaworski, K., Pawlowski, P., Okoniewski, P., Grabowski, T., Debowy, J., 2007. Pharmacokinetics of florfenicol, thiamphenicol, and chloramphenicol in turkeys. J. Vet. Pharmacol. Ther. 30, 145–150. Ueda, Y., Ohtsuki, S., Narukawa, N., 1995. Efficacy of florfenicol on experimental Actinobacillus pleuropneumonia in pigs. J. Vet. Med. Sci. 57, 261–265. Varma, K.J., Adams, P.E., Powers, T.E., Powers, J.D., Lamendola, J.F., 1986. Pharmacokinetics of florfenicol in veal calves. J. Vet. Pharmacol. Ther. 9, 412– 425. Yamaoka, K., Nakagawa, T., Uno, T., 1978. Application of Akaike’s information criterion (AIC) in the evaluation of linear pharmacokinetic equations. J. Pharm. Biopharm. 6, 165–175.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Pharmacokinetics of florfenicol after intravenous and intramuscular administration in New Zealand White rabbits F. Koc a,*, M. Ozturk b, Y. Kadioglu b, E. Dogan c, L.E. Yanmaz c, Z. Okumus c a
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Atatürk University, Erzurum 25400, Turkey Department of Analytical Chemistry, Faculty of Pharmacy, Atatürk University, Erzurum, Turkey c Department of Surgery, Faculty of Veterinary Medicine, Atatürk University, Erzurum, Turkey b
a r t i c l e
i n f o
Article history: Accepted 22 October 2008
Keywords: Pharmacokinetic Florfenicol Rabbit Plasma
a b s t r a c t The pharmacokinetic disposition and bioavailability of florfenicol (FF) were determined after single intravenous (i.v.) and intramuscular (i.m.) administrations of 25 mg/kg b.w. to ten healthy New Zealand White rabbits. Plasma FF concentrations were determined by high-performance liquid chromatography (HPLC). The plasma pharmacokinetic values for FF were best described by a one-compartment open model. The elimination half-life (t1/2b) was different (p < 0.05) however, the area under curve (AUC) was similar (p > 0.05) after i.v. and i.m. administrations. FF was rapidly eliminated (t1/2b 1.49 ± 0.23 h), slowly absorbed and high (F, 88.75 ± 0.22%) after i.m. injection. In addition, FF was widely distributed to the body tissues (Vss 0.98 ± 0.05 L/kg) after i.v. injection. In this study the time that plasma concentration exceeded the concentration of 2 lg/mL was approximately 6 h. For bacteria with MIC of 2 lg/mL, frequent administration at this dose would be needed to maintain the concentration above the MIC. However, it is possible that rabbit pathogens may have MIC values less than 2 lg/mL which would allow for less frequent administration. Further studies are necessary to identify the range of MIC values for rabbit pathogens and to identify the most appropriate PK-PD parameter needed to predict an effective dose. Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
1. Introduction FF is a synthetic broad-spectrum antibiotic in veterinary treatment of infectious diseases. It is a novel antibiotic that belongs to the chloramphenicol (CP) family, but it is used only in animals (Papich and Riviere, 2001). The compounds act by inhibiting bacterial protein synthesis by binding to 50 S and 70 S subunits in the ribosomes (Cannon et al., 1990). Moreover, FF prevents bacterial enzymatic acetylation; consequently, this product has more antibacterial activity than CP and TP (Cannon et al., 1990). Because it is both activity against CP-resistant pathogens (Neu and Fu, 1980; Syriopoulou et al., 1981) and low adverse effects (Paape et al., 1990) it has been widely used in veterinary clinics to treat bacterial diseases (Booker et al., 1997; Jim et al., 1999; Angelos et al., 2000). The pharmacokinetic disposition of FF has been extensively investigated in calves, horses, poultry, pigs, fish and sheep (Varma et al., 1986; Adams et al., 1987; Mestorino et al., 1993; Martinsen et al., 1993; El-Banna, 1998; Soback et al., 1995; Lobell et al., 1994; Afifi et al., 1997; Li et al., 2002; Jianzhong et al., 2004) but in rabbits
* Corresponding author. Tel.: +90 442 631 4193x1007; fax: +90 442 631 4188. E-mail address: [email protected] (F. Koc).
has scarcely been documented (El-Aty et al., 2004; Park et al., 2007). In this study, both used analytical method and dose were different from previous reported two studies. The aim of this study was to determine FF (25 mg/kg b.w.) kinetic disposition in plasma and bioavailability after i.m. and i.v. administration in healthy New Zealand White rabbits.
2. Materials and methods 2.1. Reagents and instruments All solvents were HPLC grade and obtained from J.T. Baker (Deventer, Netherlands). FF (99.6% assay purity) and CP (99.7% assay purity) analytical standards and injectable formulation (Nuflor, 300 mg/mL, was commercial preparation of FF) were provided by the Schering-Plough (Segre, France). HPLC was supplied from a Thermoseparations Spectra Series. The HPLC system consisted of a P 4000 gradient pump coupled with a Spectra System UV 6000 LP photodiode array detection system, Thermoseparations AS 3000 autosampler and software Chromoquest 30. The column was reverse phase nucleosil C18 (4.6 250 mm, 5 lm particle size) and it was supplied from Phenomenex (CA, USA).
0034-5288/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2008.10.010
F. Koc et al. / Research in Veterinary Science 87 (2009) 102–105
2.2. Animals and experimental design Ten healthy rabbits (New Zealand White) approximately 6–12 months old were used in this study. Of ten rabbits, five animals were female (2.50 ± 0.41 kg), five male (2.78 ± 0.35 kg). Before use, they were housed for two weeks in Experimental Research Centrum of Ataturk University. These animals were fed pelleted feed (antibacterial-free) and water ad libitum. Animal experiments were performed in an ethically proper way by following guidelines as set by the Ethical Committee of Ataturk University (Report No.: 03/2007). The rabbits were divided into two groups (n = 5, each group). Before i.v. and i.m. administrations, the restrain devices were placed in the marginal vein each animal. The blood (2 mL) samples were collected from the restrain devices for control (at 0 min). Following, in group One, florfenicol was administrated single dose i.m. injection of 25 mg/kg b.w. into semimembraneous muscle. Blood samples were taken from restrain devices of each rabbit and collected in tubes containing heparin as anticoagulant at 5, 10, 15, 30, 45, 60, 90 min, 2, 4, 6, 8, 12, 18 and 24 h after drug administration. In group Two, single dose (i.v.) FF (25 mg/kg b.w.) was injected into right the marginal vein. Blood samples were taken from the restrain devices of each rabbit (opposite ear) and collected in tubes with anticoagulant (heparin) at the same times. Samples were centrifuged within 1 h after collection and plasma samples were stored frozen (20 °C) until analysis. All the samples were analysed within one week after the experiments. 2.3. Analytical method The chromatographic method was performed as described by Kowalski et al. (2005). CP was used as an internal standard in the analytical method. The mobile phase consisted of a mixture of acetonitrile–water at a ratio of 25:75 (v/v) and adjusted to pH of 2.7 with 85% orthophosphoric acid. The injection volume was 20 lL, monitoring wavelength was at 224 nm, and oven temperature was at 2425 °C and the flow rate was 1.5 mL/min. Calibration curve was prepared in the range of 0.125.0 lg/mL (n = 6). CP (internal standard) was added to spike samples 0.5 mL at 10.0 lg/mL concentration. 2.4. Sample preparation The extraction procedure was also achieved as described by Kowalski et al. (2005). Briefly, the frozen plasma samples (0.5 mL) were thawed at a temperature of 25 °C (room temperature) and added 0.2 mL 1.0 M sodium hydroxide and 3 mL of ethyl acetate. The mixture was mechanically shacked and centrifuged for 15 min at 1650g. The organic layer was evaporated at 50 °C under nitrogen and supernatant was dissolved in 0.5 mL of mobile phase. Following again it was centrifuged at 11,200g during 10 min and 20 lL volume was injected into HPLC system. 2.5. Validation The following parameters were determined for validation of method: linearity, precision (RSD), accuracy, limit of detection (LOD), limit of quantitation (LOQ), recovery, reproducibility. For linearity, calibration curve was calculated automatically using software (0.125.0 lg/mL). The recoveries were calculated as the percentage using extraction process after spiking from 0.1 to 25.0 lg/ mL with eight different levels of florfenicol (n = 6). The inter-and intra-day reproducibility was determined at 1, 5 and 10 lg/mL concentrations. LOD’s were calculated on the basis to be 3 of
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signal-to-noise ratio spiking at low concentrations the plasma samples. LOQ was calculated to be 10 of signal-to-noise ratio. 2.6. Pharmacokinetic and statistical analysis Florfenicol plasma concentration vs. time plot of each animal was analysed using the computer program WinNonlin version 4.1 (Pharsight, Mountain View, CA, USA). The proper model was chosen by minimum Akaike’s information criterion estimation (Yamaoka et al., 1978). Pharmacokinetic parameters for each animal were analysed using by one-compartmental open model. The absorption half-life (t1/2abs), the elimination half-life (t1/2b = 0.693/b), the total body clearance (Cltot) and the apparent steadystate volume of distribution (Vss) and bioavailability (F% = AUCi.m/ AUCi.v.) were calculated. Areas under the plasma concentration– time curves for both i.v. (AUCi.v.0?1) and i.m. (AUCi.m.0?1) studies were calculated by the method of trapezoids. Peak plasma concentrations (Cmax) of drug and times to reach peak concentration (tmax) for the i.m. study were determined from the individual plasma concentration–time curves. All results are presented as mean ± SD. Harmonic means were calculated for t½b and MRT. Mann–Whitney U test was used to test for significant difference in t½b. Comparison of AUCs was made using the Independent Samples t-test. Statistical significance was assigned at p < 0.05. 3. Results The HPLC analysis for the determination of FF was high degree of reproducibility. Calibration curves had an r2 value of >0.99. Intra-assay variations were determined by measuring six replicates (n = 6) of three standard samples used for calibration curves. The intra-assay variation coefficient was 6.29 ± 2.58. Inter-assay precision was determined by assaying the three standard samples on three separate days. The inter-assay variation coefficient was 6.90 ± 3.53. The average of inter- and intra-day precision (RSD) was <7. The accuracy ranged from 95% to 108% and mean recovery was 85 ± 6.33%. LOD and LOQ were found to be 0.03 and 0.1 lg/mL, respectively. After i.v. and i.m. administration to rabbits at a single dose (25 mg/kg b.w.), plasma concentration of FF time curves were shown (Fig. 1). The pharmacokinetic parameters for FF were shown in Table 1. After i.v. administration, its t1/2b, MRT, AUC, Vss, and Cltot, of FF were 1.21 ± 0.09 h, 1.75 ± 0.12 h, 44.59 ± 1.38 lg h/mL, 0.98 ± 0.05 L/kg, and 0.56 ± 0.02 L/kg/h, respectively. Following i.m. injection, its t1/2b, t1/2abs, AUC, tmax, Cmax and F were 1.49 ± 0.23 h, 0.83 ± 0.24 h, 39.10 ± 10.12 lg h/mL, 1.56 ± 0.13 h, 8.65 ± 2.19 lg/mL, and 88.75 ± 0.22%, respectively. 4. Discussion In this study, all rabbits were clinically healthy and there were not observed any side effects after administration of FF by a single dose i.m. and i.v. The elimination half-life (t1/2b) was different (p < 0.05) however, the area under curve (AUC) was similar (p > 0.05), after i.v. and i.m. administrations. Following i.v. injection, t1/2b of FF in rabbits was 1.21 ± 0.09 h in the present study. This value was similar to 1.54 ± 0.20 h (El-Aty et al., 2004) and 0.90 ± 0.20 h (Park et al., 2007) in rabbits. However, this finding was shorter than in calves (3.18 ± 1.01 h), cattle (159 min), turkeys (2.34 ± 0.44 h), broiler chickens (173.25 min), turtles (from 2 to 7.8 h), (Decraene et al., 1997; Lobell et al., 1994; Switała et al., 2007; Afifi et al., 1997; Stamper et al., 2003).
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F. Koc et al. / Research in Veterinary Science 87 (2009) 102–105
Fig. 1. Semilogarithmic plot of plasma concentrations–time curves of florfenicol after single dose i.v. and i.m. administrations at a dose of 25 mg/kg b.w. to New Zealand White rabbits (n = 5).
Table 1 Pharmacokinetic parameters determined after i.v. and i.m. administration of florfenicol (25 mg/kg) (n = 5). Variables
i.v. Mean ± SD (n = 5)
i.m. Mean ± SD (n = 5)
P<
t1/2abs (h) t1/2ba (h) MRTa (h) AUC (lg h/mL) Vss (L/kg) Cltot (L/kg/h) tmax (h) Cmax (lg/mL) F (%)
NA 1.21 ± 0.09 1.74 ± 0.12 44.59 ± 1.38 0.98 ± 0.05 0.56 ± 0.02 NA NA NA
0.83 ± 0.24 1.49 ± 0.23 NA 39.10 ± 10.12 NA NA 1.56 ± 0.13 8.65 ± 2.19 88.75 ± 0.22
– 0.028 – 0.26 – – – – –
t1/2abs: Absorption half-life; t1/2b: the elimination half-life; MRT: mean resistance time; AUC: area under the concentrations time curves; Vss: mean volume of distribution at steady-state; Cltot: total body clearance; tmax: time to maximum concentration; Cmax: maximum concentration; F%: bioavailability; NA: not applicable. a Harmonic mean.
The short half-life of its elimination indicates that the drug was rapidly eliminated from the bodies of rabbits. Cltot of FF was 0.56 ± 0.02 L/kg/h in this study. The result was somewhat different from reported (0.34 ± 0.11 L/kg/h) by El-Aty et al. (2004) in rabbits. However, this value was in agreement with reported (0.63 ± 0.06 L/kg/h) by Park et al. (2007) in the same animal species. The result of this study was higher than 0.23 and 0.16 L/kg/h in lactating cows (Mestorino et al., 1993; Soback et al., 1995), 0.22 L/kg/h in cattle (Lobell et al., 1994), 0.15 L/kg/h in non-lactating dairy cows (Bretzlaff et al., 1987), 0.171, 0.172 L/ kg/h, 0.22 ± 0.05 L/kg/h in calves (Varma et al., 1986; Adams et al., 1987; Decraene et al., 1997), 0.25 ± 0.02 L/kg/h, in sheep (Jianzhong et al., 2004), 0.33 ± 0.04, 0.30 ± 0.03 and 0.27 ± 0.03 L/ kg/h, in camels, sheep and goats, respectively (Ali et al., 2003). Park et al. (2008) reported that Cltot of FF in the animal species, smaller body weight, was higher than bigger body weight animals. The result of this study was in agreement with previous report (Park et al., 2008). As known that, Vss is an important indication of the diffusion of the drug in tissues (Galinsky and Svensson, 1995). In the present study, Vss was large (0.98 ± 0.05 L/kg). This result was in agreement with that of 0.94 ± 0.19 L/kg reported in rabbits (Park et al. 2007). FF is a lipophilic drug and the Vss may be related to physiochemical characteristics of drug (Ali et al., 2003). However, this finding was inconsistent with the result reported (0.57 ± 0.85 L/kg) by El-Aty et al. (2004) in rabbits. In this study, after i.m. injection, Cmax of the drug reached 8.65 ± 2.19 lg/mL at 1.56 ± 0.13 h (tmax) and it was determined un-
til 12 h (Fig. 1) and F% value was 88 ± 0.22%. It was reported that Cmax was high (21.65 lg/mL), tmax was rapid (0.5 h) and F% was high (88.25%) in rabbits (El-Aty et al., 2004). In this study, Cmax and tmax were inconsistent with the reported by El-Aty et al. (2004). These differences in parameters may be related to difference in analytical methods used in the two studies (Lane et al., 2004; Park et al., 2007). It is known that the different methods may occur different results from the same species (Atef et al., 2001). El-Aty et al. (2004) reported that used method was microbiological assay and measured only the unbound and microbiological active compound in their study. In this study used method was HPLC, which measures the total concentration of FF (Atef et al., 2001). In addition, these values in this study were different from previous reports in different animal species including, broiler chickens, lactating cows, turtles, Egyptian goats, Atlantic salmon (Mestorino et al., 1993; Soback et al., 1995; Stamper et al., 2003; Atef et al., 2001; Martinsen et al., 1993). Consequently, in our study, the absorption of drug was slow and higher. These differences in absorption from the i.m. injection site may be due to differences in regional blood flow from different muscle tissues of animal species. After i.v. administration, MRT of the drug was 1.75 ± 0.12. Park et al. (2007) reported that 1.50 ± 0.34 h after i.v. injection in rabbits. It was determined that following i.v. and i.m. administration MRT was 1.69 ± 0.67 and 4.34 ± 0.38 h, respectively (El-Aty et al., 2004). After i.v. administration, MRT of florfenicol in the present study was similar to two studies. Until now, not only the minimum inhibitory concentrations (MICs) of FF for bacteria isolates from rabbits but also studies on efficacy of it in terms of pharmacokinetic/pharmacodynamic (PK/PD) approaches have not yet been reported. However, previous studies in other animal species showed that the susceptibility the MIC values of FF ranging from 0.25–2 lg/mL for many various bacterial pathogens in cows, pigs and fish (Bretzlaff et al., 1987; Ueda et al., 1995; Ho et al., 2000). In this study the time that plasma concentration exceeded the concentration of 2 lg/mL was approximately 6 h (Fig. 1). The value of 2 lg/ mL is used as the breakpoint for susceptible pathogens as defined by the CLSI for swine and cattle respiratory pathogens. For bacteria with MIC of 2 lg/mL, frequent administration at this dose would be needed to maintain the concentration above the MIC. However, it is possible that rabbit pathogens may have MIC values less than 2 lg/mL which would allow for less frequent administration. Further studies are necessary to identify the range of MIC values for rabbit pathogens and to identify the most appropriate PK-PD parameter needed to predict an effective dose.
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