Pharmacokinetics of a single intramuscular injection of cefquinome in buffalo calves

Cefquinome is one of the fourth-generation cephalosporins developed exclusively for use in animals and has been used in the treatment of respiratory tract diseases, acute mastitis, and foot rot in cattle, calf septicemia, respiratory diseases in pigs, metritis,mastitis, and agalactia syndrome in sows, foal septicemia, and respiratory tract diseases in horses (CVMP, 1995, 1999, 2003). Pharmacokinetics of cefquinome following intramuscular (IM) administration have been reported in lactating cows (Ehinger, Schmidt, & Kietzmann, 2006), goats (Dumka, Dinakaran, Bibhuti, & Satyavan, 2013), sheep (Tohamy, 2011; Uney, Altan, & Elmas, 2011), and camels (Al-Taher, 2010), and the pharmacokinetics following intravenous (IV) administration have been reported in buffalo calves (Dinakaran, Dumka, Ranjan, Balaje, & Sidhu, 2013), horses (Dumka et al., 2013) and goats (Dumka et al., 2013). However, there are no published report on the disposition and bioavailability of cefquinome following IM administration in buffalo calves. Keeping the above facts in view and considering the common route of drug administration in field conditions, this study was conducted to investigate the disposition of cefquinome following single IM administration.

Six male buffalo calves (6–8 months old) weighing 120.50 ± 5.67 kg were used in this study. The animals were maintained on green fodder, and concentrate mixture and water were provided ad libitum. All animals were healthy, and none of them were treated with antibiotics for 1 month prior to the trial. The experimental protocol followed the ethical guidelines on the proper care and use of animals and was approved by the Institutional Animal Ethics Committee of Guru Angad Dev Veterinary and Animal Sciences University (Protocol number VMC/2010/IAEC/1976-92). Cefquinome sulfate (Cobactan® 2.5%, Intervet, Pune, India) was administered as single IM dose of 2 mg/kg in the lower third region of the neck. Blood samples (4–6 ml) were collected in heparinized test tubes by venepuncture from the jugular vein at 0, 2.5, 5, 10, 15, 30, and 45 min, and 1, 1.5, 2, 4, 6, 8, 12, and 24 hr after IM administration. Plasma was separated from the collected blood samples by centrifugation at 1,300 g for 15 min at 4°C and stored at −20°C until analysis.

Plasma cefquinome concentrations were quantified using a modified HPLC/UV method previously described by Uney et al., 2011. Briefly, 200 μl of plasma was added to 2-ml microcentrifuge tube, and subsequently, 400 μl of methanol was added and vortexed for 10 s. After centrifugation at 4000 g for 10 min, 300 μl of clear supernatant was pipetted into a fresh vial, 150 μl of water was added and mixed for 10 s, and the mixed clear supernatant (300 μl) was pipetted into an autosampler vial.

The HPLC system (PerkinElmer, series 200, Waltham, MA, USA) was equipped with a single pump, a degasser and an autosampler injector. The reverse-phase chromatography was performed with an analytical C₁₈ column (Sun fire, Particle size 5 μ, 4.6 × 150 mm, Waters, USA). The optimized method used binary gradient mobile phase with water containing 0.1% trifluoroacetic acid (Sigma-Aldrich, Bengaluru, India) as mobile phase A and acetonitrile (Sigma-Aldrich) as mobile phase B. The gradient elution was programmed with the ratio of mobile phases A:B as 90:10 for 7 min followed by 50:50 for 3 min and 90:10 for 5 min. The detection was performed using a UV/VIS detector set at 268 nm. The volume of injection was 50 μl, and the flow rate was 0.9 ml/min. The TotalChrom software (version 6.1) was used for instrument control and data analysis. The retention time of cefquinome was about 4.3 min, and calibration curve for cefquinome was linear at concentrations of 0.05–50 μg/ml with correlation coefficients (r) above .999. The limit of detection (LOD) and quantification (LOQ) were determined by signal-to-noise ratio evaluations of samples spiked from 0.01 to 0.1 μg/ml. The LOD and LOQ of cefquinome were determined to be 0.02 μg/ml and 0.04 μg/ml, respectively. The intraday and interday assay precision levels were lower than 5 and 6%, respectively, and accuracy levels ranged from 0.66 to 1.98% and from 0.1 to 2.1%, respectively. The extraction recoveries of cefquinome from plasma were 89.00 ± 5.05%, 91.90 ± 4.13%, and 93.60 ± 3.15% for the low- (0.5 μg/ml), medium- (10 μg/ml), and high (50 μg/ml)-quality control samples, respectively.

The appropriate pharmacokinetic model was determined by visual examination of individual concentration–time curves, and PK parameters were calculated using standard pharmacokinetic formulae (Gibaldi & Perrier, 1982). The peak concentration in plasma (C_max) and the time to C_max (T_max) were determined from individual plasma concentration–time curves. The pharmacokinetic variables of cefquinome are expressed as mean ± SE. Intramuscular bioavailability (%F) was calculated using the equation:

% F = ([AUC_IM/D_IM]/[AUC_IV/D_IV]) × 100.

The area under the plasma cefquinome concentration curve following IV injection of 2 mg/kg has been reported by the present author (Dinakaran et al., 2013) in a study conducted along with this study by crossover design model. Hence, the AUC_IV for calculation of bioavailability was used from the reported article.

The time for which the plasma drug concentrations remain above or equal to minimal inhibitory concentration (MIC) value is calculated using the formula:

% T > MIC = ln (D/[V_d(area) × MIC]) × (t_1/2β/ln(2)) × (100/τ)

where T > MIC is the time interval (in percent) during which the plasma concentration is above or equal to the MIC values, ln is natural logarithm, D is the proposed dose, V_d(area) is the volume of distribution, t_1/2β is the terminal elimination half-life, and τ is the dose interval (Turnidge, 1998).

No clinical signs of adverse effects or intolerance were observed to cefquinome after IM injection. The mean plasma concentration–time curve for cefquinome in buffalo calves following single IM administration is depicted in Figure 1. Following IM injection, a peak plasma concentration (C_max) of 6.93 ± 0.58 μg/ml was achieved at T_max of 0.5 hr. Mean pharmacokinetic parameters are presented in Table 1. Table 2 shows the calculated %T > MIC for cefquinome based on the estimated pharmacokinetic parameters obtained following IM injection in buffalo calves for 12- and 24-hr dosing interval.

The plasma concentration–time profile following single IM dose of cefquinome in buffalo calves was best described by a mono-compartment open model which is similar to that `described in goats (Dumka et al., 2013) and camels (Al-Taher, 2010). However, a two-compartment model was shown to provide the best fit for IM cefquinome plasma concentration–time data in sheep (Uney et al., 2011). Cefquinome was rapidly absorbed after IM administration, with absorption half-life of 0.16 ± 0.05 hr. Rapid absorption of cefquinome has also been reported in cattle (CVMP, 1995) and sheep (Uney et al., 2011) after IM administration. The absolute bioavailability (F) of cefquinome following IM administration was 86.30 ± 3.95%, which indicates good absorption of the drug from the site of injection. Similar high bioavailability of cefquinome has also been confirmed in different animal species such as horses (89.2%–103.70%), piglets (83.7%–107%), sheep (89.31%), and goats (>100%) after IM administration (Allan & Thomas, 2003; Batzias, 2009; Li et al., 2008; Uney et al., 2011). Rapid absorption and high value of bioavailability revealed that IM administration is likely to be as effective as IV injection in the treatment of mild-to-moderate bacterial infections in buffalo species.

The elimination half-life of cefquinome in this study was 3.73 ± 0.10 hr, which was similar to the t_1/2β following IV administration in buffalo calves (3.56 ± 0.05 hr) (Dinakaran et al., 2013), cattle (1.5–3 hr) (CVMP, 1995), and the t_1/2β in 1-year-old sheep (3.91 hr) (Uney et al., 2011), following IM administration.

Cefquinome is a beta-lactam antimicrobial and acts as a time-dependent bactericidal drug (Thomas, Thomas, & Wilhelm, 2006). It is generally recommended that T > MIC should be at least 50% of the dosage interval to ensure an optimal bactericidal effect (Toutain & Lees, 2004). Considering the reported MIC_90s (0.06–0.39 μg/ml) for Escherichia coli, Pasteruella multocida, and Streptococcus agalactiae (Chin, Gu, Fang, & Neu, 1992; Deshpande, Pfaller, & Jones, 2000; Limbert et al., 1991; Murphy, Erwin, & Jones, 1994; Orden, Ruiz, Garcia, Cid, & Fuente, 1999; Sheldon, Bushnell, Montgomery, & Rycroft, 2004; Thomas et al., 2006; Wallmann et al., 2006), the T > MIC has been calculated for MICs of 0.06, 0.125, and 0.39 μg/ml. The experimental data presented here show that cefquinome at a dose of 2 mg/kg body weight at 24 hr interval is sufficient to maintain T > MIC above 58% following IM injection, respectively, for bacteria with MIC values ≤0.39 μg/ml. This dosage regimen meets pharmacokinetic–pharmacodynamic criteria predicting a successful therapy for susceptible bacteria with MIC ≤ 0.39 μg/ml, although this prediction is not based on the PD parameters from the pathogens of buffaloes. The calculated dosage regimen is hence suggested for potential clinical testing of cefquinome in buffalo calves against susceptible microorganisms.

Rapid absorption and high bioavailability of cefquinome indicate that IM injection is as effective as IV injection in the treatment of mild-to-moderate bacterial infections in buffalo species. The suggested dosage regimen has to be validated in the diseased model. The PK parameters like mean elimination half-life etc. need to be verified in a larger population of buffalo calves, and variations in dosage regimens may be required.

CONFLICT OF INTERESTS

The authors declare that they have no conflict of interests.