Animals
Six clinically healthy Murciano-Granadina female lactating goats weighing between 40.5 and 56 kg and aged from 2.5 to 3.5 years from the Caprine Farm of the University of Murcia were used. The animals were housed and fed an antibiotic-free diet for at least 30 days preceding the study. For each treatment period of the cross-over study, they were observed daily for general health, and clinical observations were made prior to injection and at 2, 10 and 24 h post-injection. Alfalfa hay and water was provided ad libitum together with a drug-free concentrate. The study was approved by the Bioethics Committee of the University of Murcia.
Experimental design
A cross-over design (2 × 2 × 2) was used in three phases. Each animal received either a single IV or SC injection of ceftiofur sodium (Excenell® 4 g, Pfizer, Madrid, Spain) at a dose of 2.2 mg/kg or a SC-LA administration of 6.6 mg/kg with at least a 15-day washout period.
For the IV administration, the solution was injected into the left jugular vein and blood samples (4 mL) were collected from the contralateral jugular vein. SC and SC-LA injections were administered under the skin of the back at a single location in the thoraco-lumbar region lateral of the mid-line. Blood samples were collected at 0 (pre-treatment), 0.083, 0.167, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 32, 48, 72, 96 and 120 h post-dosing. Samples were centrifuged at 1500 g for 15 min and the serum taken and stored at −90 °C until assayed.
Milk samples for analysis were collected from each goat after complete evacuation of the udder by manual stripping of each gland immediately before dosing on the day of treatment administration (time 0) and at 1, 2, 4, 6, 8, 10, 12, 24, 32, 48, 72, 96 and 120 h after administration. After shaking the milk to homogenize, a 4–5 mL sample was collected and stored at −90 °C until assayed.
Gel preparation
Gel was prepared on a weight basis using the cold method [13]. Concentrations of P407 and ceftiofur reported here are expressed as weight percentage (% wt/wt). An amount of P407 sufficient to yield 25 % and Carboxymethylcellulose sodium to yield 2 % gel was slowly added at 4 °C and ceftiofur sodium sufficient to yield a 20 % concentration was dissolved in the cold solution.
Analytical method
Serum and milk samples were analyzed for concentrations of ceftiofur, desfuroylceftiofur, and related metabolites by reduction and derivatization to desfuroylceftiofuracetamide (DCA) [14]. Ceftiofur and desfuroylceftiofur-related metabolites were extracted from serum and milk following a reduction step through the addition of 1,4-dithioerythritol solution (20 mg/mL in 0.1 M ammonium acetate, pH = 8.9). An internal standard consisting of a cefotaxime solution (200 μg/mL) was incorporated in this step. Following a 30-min incubation step at 50 °C to fully reduce the thioester bond in ceftiofur and desfuroylceftiofur-related metabolites, the resulting desfuroylceftiofur was captured on a C18 solid-phase extraction columns (Oasis HLB SPE cartridges, Waters, Barcelona, Spain) and further derivatized with iodoacetamide to create desfuroylacetamide (DCA). DCA was removed from the column with 30:70 acetonitrile: 0.01 M ammonium acetate with 0.1 % trifluoroacetic acid providing a final injection equal to isocratic HPLC conditions (15 % acetonitrile: 85 % 0.01 M ammonium acetate (0.1 % trifluoroacetic acid)). The HPLC separation was performed using a reverse-phase Kinetex TM PFP C18 column (250 × 4.6 mm; 5 μm) with an injection volume of 100 μL. Ultraviolet detector was set at 240 nm.
Method validation
Quality controls were prepared from a pool of blank goat serum or milk spiked with seven concentrations of ceftiofur between 0.10 and 10 μg/mL. Serum and milk aliquots were stored at −90 ° C until assay. Aliquots of quality controls were extracted as above and 100 μL was injected into the chromatographic system. Standard curves were obtained by unweighted linear regression of ceftiofur and cefotaxime peak areas versus known concentrations. Each point was established from an average of five determinations. Correlations coefficients (r) were >0.99 for calibration curves. The percentage recovery was determined by comparing the peak areas of serum and milk blank samples spiked with different amounts of drug and treated as any samples, with the peak areas of the same standards prepared in phosphate buffer. Each point was established from an average of five determinations. The mean percentage recoveries of ceftiofur from serum and milk were 84.79 and 88.16 %, respectively. The assay precision (R.S.D.) was assessed by expressing the standard deviation of repeated measurements as a percentage of the mean value. Serum intra-day precision was estimated from six replicates of three standard samples used for calibration curves (R.S.D. < 5.83 %). Milk intra-day precision was R.S.D. < 10.2 %. Inter-day precision was estimated from the analysis of standard samples (serum or milk) on three separate days. Serum inter-day obtained a R.S.D. < 4.91 %. Milk inter-day assay obtained a R.S.D. < 6.54 %. The limit of quantification (LOQ) and the limit of detection (LOD) for was 0.1 μg/mL for serum and milk.
Bacterial strains, MIC, MBC and MPC determination
Five field strains of M. haemolytica isolated from goats affected by pneumonia in Spain were used. The strains were stored at −80 °C in a nutrient broth enriched with 15 % glycerol until assayed. The MIC of ceftiofur was determined in goat serum and Mueller-Hinton broth (MHB, Fluka analytical, Madrid, Spain) using the microdilution method recommended by CLSI [15]. The assay was performed in U-bottomed, 96-well, custom-designed microtiter plates. After overnight incubation at 37 °C on tryptone soya blood agar plates, bacterial suspensions equal to a 0.5 McFarland standard were further diluted and 10 μL were added to the plates to achieve a final inoculum of 5 × 105 CFU/mL. The plates contained antimicrobial dilutions ranging from 0.03 to 128 μg/mL in 90 μL/well of goat serum or MHB, the final volume was 100 μL. Assays were incubated at 37 °C and observed after 24 h. The MIC was taken as the lowest drug concentration that inhibited visible growth. MBC was established by plate count as the concentration of antibacterial to reduce a 3log10 (99.9 % killing) the initial inoculum, in accordance with CLSI guidelines [15].
The MPC was measured by agar dilution using a method previously described [16]. Briefly, the content of overnight cultures of each strain of M. haemolytica (5 plate per isolate) in TSBA was transferred to 100 mL of MHB and incubated overnight at 37 °C with shaking at 200 rpm. The next day, bacterial suspensions were estimated to have concentrations close to 3 · 108 CFU/mL by turbidity measurements. Then, cultures were concentrated by centrifugation at 5000 g for 30 min at 5 °C and re-suspended in 3 mL of fresh MHB. Aliquots of 200 μL containing 1010 CFU were inoculated in TSBA plates previously prepared with ceftiofur over a range from 0.03 to 128 μg/mL. Plates were incubated for 48 h at 37 °C and screened visually each 24 h for growth. The MPC was recorder as the lowest concentration that prevented the growth of colonies.
For MIC, MBC and MPC assays, a non-inoculated plate was included as a negative control, and an inoculated plate without drug as a positive control. Also, S. aureus ATCC 29213 and E. coli ATCC 25922 strains were used as controls. All determinations were performed in duplicate, and the geometric mean was calculated.
In vitro antimicrobial growth (time-kill) curves
Time kill curves were obtained in a second trial after MIC, MBC and MPC determination. In vitro and ex vivo activity of ceftiofur against M. haemolytica was obtained using a method previously described [17]. Eight to 10 colonies from overnight cultures in TSBA of each strain were used to inoculate 9 mL of MHB and incubated overnight at 37 °C with shaking at 200 rpm.
For in vitro assays, 480 μL of MHB or goat serum were spiked with 10 μL of concentrated solutions of ceftiofur. The final samples contained antimicrobial concentrations at 0 (control), 0.25, 0.5, 1, 2, 4, 8 and 16 multiples of MIC previously obtained in each fluid. A total of 10 μL of stationary-phase bacterial culture was added to give a final concentration of approximately 5 · 106 CFU/ml. Aliquots of 25 μL were sampled from each culture at 0, 1, 2, 4, 8 and 24 h. Counts were determined by serial dilution in saline and spread on TSBA agar plates [18]. For ex vivo testing, serum samples from goat which had received ceftiofur IV, SC or SC-LA at 2.2 or 6.6 mg/kg respectively, were collected at 0, 1, 2, 4, 12, 24, 32, 48, and 72 h. Concentrations of ceftiofur in these samples were previously determined with the HPLC method described in the above section. Then, a total of 10 μL of stationary-phase bacterial culture was added, and aliquots of 25 μL were sampled from each culture at 0, 1, 2, 4, 8 and 24 h using the same methodology described for in vitro time kill curves. Counts were determined by serial dilution in saline and spread on TSBA agar plates. The lower limit of detection was 40 CFU/mL for both in vivo and ex vivo assays. Time kill curves were performed at 37 °C with shaking at 200 r.p.m.
Pharmacokinetic analysis
The serum ceftiofur time-concentration data were analysed by non-compartment methods using WinNonLin 5.2 (Pharsight Corp, Mountain View, Calif). The area under the concentration-time Curve (AUC) was calculated using the linear trapezoidal rule with extrapolation to time infinity. Mean Residence Time was calculated as MRT = AUMC/AUC. Mean absorption times were calculated as MAT = MRTSC, SC-LA – MRTIV and Ka was calculated non-compartment methods as Ka = 1/MAT. The systemic clearance was estimated as Cl = Dose/AUC. The apparent volume of distribution at steady state were calculated as Vss = (Dose · AUMC)/AUC2. The apparent volume of distribution (area method) was calculated as Vz = Dose/ (AUC · λz). Biovailability (F) was calculated by the method of corresponding areas:
$$ \left[\mathrm{F}\left(\%\right) = \left({{\mathrm{AUC}}_{\mathrm{SC}}}_{,\ \mathrm{S}\mathrm{C}\hbox{-} \mathrm{L}\mathrm{A}}\cdotp\;{\mathrm{Dose}}_{\mathrm{IV}}\right)\times 100\ /\ \left({\mathrm{AUC}}_{\mathrm{IV}}\cdotp\;{\mathrm{Dose}}_{\mathrm{SC},\ \mathrm{S}\mathrm{C}\hbox{-} \mathrm{L}\mathrm{A}}\right)\right] $$
Statistical analysis
Descriptive statistical parameters as mean, standard deviation and coefficient of variation were calculated. Harmonic means were calculated for the half-lives of elimination. A Kruskal-Wallis analysis of variance, followed by a Dunn multiple comparison test if applicable was used to test parameters for significant differences between IV, SC and SC-LA administration. Dose-dependent (AUC and Cmax) parameters were corrected with dose to test for statistical differences. The level of significance was P ≤ 0.05. The statistical software used was SPSS (IBM SPSS Statistics, Version 19.0. Armonk, NY: IBM Corp).