Pharmacokinetics of danofloxacin following intravenous and intramuscular administration in donkeys

Abstract

Danofloxacin (DNF) is a synthetic antibacterial agent of the fluoroquinolone group, developed specifically for use in veterinary medicine. The drug possesses good in vitro activity against a variety of pathogens, including gram-positive and gram-negative bacteria, mycoplasmas, and intracellular pathogens, such as Brucella and Chlamdia species; but it has poor activity against anaerobes (Wolfson & Hooper, 1985; Neu, 1987; McKellar et al., 1998; Aliabadi et al., 2003a). Danofloxacin shares with other fluoroquinolones, such as enrofloxacin and ciprofloxacin, a wide spectrum of activity, a large volume of distribution and activity at low concentrations (Van Cutsem et al., 1990; Spreng et al., 1995; Brown et al., 1996). Pharmacokinetic properties of different fluoroquinolones have been extensively studied on several animal species (Knoll et al., 1999; Atef et al., 2001; Fernandez-Varon et al., 2006). In addition, some fluoroquinolones, such as DNF, moxifloxacin, marbofloxacin and enrofloxacin, are being used widely in horses (Bertone et al., 2000; Carretero et al., 2002; Gardner et al., 2004). Pharmacokinetic disposition of DNF has been evaluated in many animal species including cattle, goats, sheep, camels, rabbit, chickens, turkeys, pig and horses (Knoll et al., 1999; McKellar et al., 1999; Lindecrona et al., 2000; Atef et al., 2001; Aliabadi et al., 2003a,b; Shojaee Aliabadi & Lees, 2003; Haritova et al., 2006; Fernandez-Varon et al., 2006, 2007). There is a paucity of data available on the pharmacokinetics of drugs used in donkeys including antibiotics, as donkeys are often neglected species in domestic animals. Different classes of drugs used in horses and ruminants are commonly extrapolated to donkeys without optimization of dosing regimens and determination of pharmacokinetic and pharmacodynamic properties. Because of the lack of registered drugs for donkeys, antimicrobials licensed for horses or ruminants are used for treatment of bacterial infections in this species with same dose rates. In the literature, data are available only on the pharmacokinetics of gentamicin, sulfamethoxazole with trimethoprim and marbofloxacin in donkeys (Welfare et al., 1996; Peck et al., 2002; González et al., 2007). It has been reported that donkeys have a greater capacity to metabolize certain drugs compared with horses; thus higher dosage or shorter intervals are required for maintaining effective concentrations (Welfare et al., 1996; Matthews et al., 1997Coakley et al., 1999; Peck et al., 2002). Therefore, the aim of the present study was to determine the pharmacokinetic properties of DNF in donkeys following intravenous (i.v.) and intramuscular (i.m.) administration at single dose of 1.25 mg/kg bodyweight. Six native breed donkeys (Equus asinus) which ranged in age from 2 to 5 years and weighted 90–125 kg were used in this study. The animals were kept indoors and had clover hay and water available ad libitum throughout the course of the study. This study was approved by Animal Ethic Committee of University of Adnan Menderes. The animals were allocated into two groups of three such that the mean weight of animals in each group was similar and the donkeys were identified by unique freeze brand or natural markings. Danofloxacin was administered according to a two-phase crossover design protocol. In phase I, group I received i.v. the commercially available injectable solution of DNF (Advocin®, 2.5% w/v, Pfizer, Turkey) at a dose of 1.25 mg/kg bodyweight and group II received i.m. the same formulation at the same dose rate into gluteal muscle. A 2-week washout period was allowed between the two phases. Heparinized blood samples (5 mL) were collected 1 h prior to drug administration and 5, 15, 30, 45 min and 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 32, 36 and 48 h post-treatment. Blood samples were centrifuged at 5000 g for 20 min and plasma was transferred to plastic tubes. All the plasma samples were stored at −20 °C until the determination of drug concentration. In addition, serum samples were also collected to determine creatin kinase (CK) activity after i.m. administration. The samples were stored at 4 °C for 2 h until CK activity (expressed in U/L), which was measured preinjection and after i.m. injection using a commercial available kit (CK EE547; Linear Chemicals®, Barcelona, Spain). The parent compound of DNF in plasma was analysed using validated high-performance liquid chromatography (HPLC) following a liquid–liquid phase extraction procedure with a minor modification of the methods described by (Garcia et al., 2000). The mobile phase was a mixture of acetonitrile–water to which triflouroacetic acid was added (0.1% v/v) and delivered (Agilent 1100 Series QuatPump; Waldbronn, Germany) isocratically at a flow rate of 1 mL/min. A Nemesis nucleosil C18 column (4 μ, 150 × 4.6 mm) (Phenomenex, Cheshire, UK) with nucleosil C18 guard column (Phenomenex) was used for the analysis. The column was maintained at 40 °C throughout the analysis in a column oven. The eluate was continuously monitored using fluorescence detection for DNF (Agilent 1100 Series; Waldbronn, Germany) (λex 280 nm and λem 440 nm). The analytical method used for DNF in donkey plasma was validated prior to the start of the studies. Recoveries of the two molecules under study were measured by comparison of the peak areas from spiked plasma samples with the areas resulting from direct injections of standards. The inter-assay precision of the extraction and chromatography procedures was evaluated by processing replicate aliquots of drug-free donkey plasma samples containing known amounts of the drugs on different days. Calibration graph for DNF was prepared (linear range 0.01–10 μg/mL). The slope of the lines between peak areas and drug concentration was determined by least squares linear regression and showed correlation coefficients between 0.997 and 0.999. The limit of quantification (LOQ) was 0.01 μg/mL for plasma analysis. This value being the lowest concentrations detected with a coefficient of variations (CV) lower than 20%. The assay validation of DNF indicated a percentage of recovery of 93.71% (n = 36), intra-assay variation (CV) of 7.49 (n = 16), inter-assay CV of 6.86 (n = 20). The plasma concentration vs. time curves determined after each treatment (i.v. and i.m.) in individual animals were semi-logarithmically fitted with winnonlin software program (Version 4.1; Pharsight Corporation, Mountain View, CA, USA). Pharmacokinetic parameters for each animal were analysed using noncompartmental model analysis for both i.v. and i.m administrations (Gibaldi & Perrier, 1982). The pharmacokinetic parameters are reported as mean ± SD. Mean pharmacokinetic parameters after i.v. and i.m. administration were statistically compared using Student’s t-test. Mean parameters were considered significantly different at P < 0.05. No adverse response was observed for any of the treatments during the study. Semi-logarithmic plot of mean plasma concentration vs. time curves for DNF following i.v. and i.m. dose of 1.25 mg/kg bodyweight are shown in Fig. 1. The pharmacokinetic data associated with each route of drug administrations are given in Table 1. Danofloxacin had a large volume of distribution (Vdss) of 5.95 ± 1.00 L/kg, an elimination half-life (t1/2λz) of 7.25 ± 0.91 h and a clearance (Cl) of 1.00 ± 0.17 L·h/kg after i.v. administration. Following i.m. administration it was well adsorbed with an absolute bioavailability (F) of 100.54 ± 8.91% and a mean absorption time (MAT) of 3.12 ± 1.54 h, and achieved quickly a maximum plasma concentration (Cmax) of 0.15 ± 0.05 μg/mL at 0.88 ± 0.41 h. The mean residence time (MRT) observed after i.m. administration (9.10 h) is significantly longer compared with the value observed after i.v. route (5.98 h). Mean (±SD) plasma concentration profiles of danofloxacin following intravenous and intramuscular administration (1.25 mg/kg) to donkeys (n = 6). Serum CK activity at first 8 h after i.m. administration increased nine-fold compared with preinjection levels (Fig. 2). Danofloxacin was absorbed rapidly from the i.m. injection site and reached the peak of plasma concentration at 0.88 h in donkeys. Similar results were reported for cattle (Giles et al., 1991a), sheep (McKellar et al., 1998) and goats (Atef et al., 2001). Moreover significantly longer t1/2λz and MRT were observed following i.m. route compared with after i.v. administration in the present study. Mean (±SD) serum creatine kinase activity (U/L) after intramuscular administration of danofloxacin (1.25 mg/kg) to donkeys (n = 6). It is widely known that horses exhibit poor tolerability to irritant drugs when injected i.m. Kaartinen et al. (1997) reported that tissue reactions (e.g. swelling and tenderness) to enrofloxacin, and serum CK activity increased 10-fold. In another studies, CK activity was increased three-fold (12 h) for danofloxacin (Fernandez-Varon et al., 2006) after i.m. administration in horses. In this study, CK activity increased up to nine-fold (8 h) compared with preinjection levels. The systemic bioavailability (100.54%) of DNF after i.m. was almost complete in donkeys and higher than horses with 88% bioavailability (Fernandez-Varon et al., 2006). This reflects that tissue irritation does not appear to affect bioavailability of DNF in donkeys after i.m. administration. The plasma pharmacokinetic parameters determined following i.v. and i.m. administration of DNF to donkeys are not similar to those reported for horses at same dosage (Fernandez-Varon et al., 2006). Danafloxacin was more rapidly cleared in donkeys (1.00 L/h/kg) compared with horses (Cl: 0.34 L/h/kg) and following i.m. administration DNF reached a lower Cmax in donkeys (0.15 μg/mL) than in horses (0.35 μg/mL). Moreover, DNF displayed almost three times larger volume of distribution in donkeys (5.95 L/kg) compared with horses (2.00 L/kg) at same dosage. Although, the area under the concentration time curves (AUCs) of DNF after i.v. and i.m. administration were similar in donkeys (1.30 and 1.28 μg·h/mL, respectively), these values were almost three times smaller than those observed in horses after i.v. (3.80 μg·h/mL) and i.m. (3.32 μg·h/mL) administration (Fernandez-Varon et al., 2006). It has been suggested that serum concentrations related to the minimal inhibitory concentration (MIC) values are the best indicator of response to infections (Nix et al., 1991). PK/PD relationships between AUC from zero to 24 h and MIC, Cmax and MIC and time during which plasma concentrations exceed the MIC have been particularly useful in optimizing efficacy (McKellar et al., 2004). Although MIC values of DNF against equine pathogens are unknown, the serum concentrations of DNF exceeded MIC of 0.06 μg/mL against respiratory pathogens in cattle (Giles et al., 1991b; McKellar et al., 1998, 1999). Danofloxacin has a concentration dependent activity (McKellar et al., 2004); therefore, it is desirable to achieve a high Cmax after a single dose. Considering the plasma concentration in horses, higher loading doses of DNF may require in donkeys to provide effective plasma concentrations.