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    Figure 1—

    Mean ± SD plasma concentration of CFAE following SC administration of a single dose of CCFA (40 mg/kg) to each of 6 female New Zealand White rabbits. A blood sample was collected from each rabbit immediately before (0 minutes), at 5 and 30 minutes after, and at 1, 1.5, 2, 3, 4, 8, 12, 24, 48, 72, 95, 120, 144, and 168 hours after drug injection.

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  • 32. Hope KL, Tell LA, Byrne BA, et al. Pharmacokinetics of a single intramuscular injection of ceftiofur crystalline-free acid in American black ducks (Anas rubripes). Am J Vet Res 2012; 73: 620627.

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  • 33. Valitutto MT, Newton AL, Raphael BL, et al. Pharmacokinetics of a long-acting formulation of ceftiofur (ceftiofur crystalline free acid) administered intramuscularly in the ringneck dove (Streptopelia risoria), in Proceedings. Annu Conf Am Assoc Zoo Vet 2010; 223224.

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Pharmacokinetics and safety of ceftiofur crystalline free acid in New Zealand White rabbits (Oryctolagus cuniculus)

Sara GardhouseWilliam T. Pritchard Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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David Sanchez-Migallon GuzmanDepartment of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Sherry CoxDepartment of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

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Philip H. KassDepartment of Population Health and Reproduction, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Tracy L. DrazenovichDepartment of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Barbara A. ByrneDepartment of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Michelle G. HawkinsDepartment of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Abstract

OBJECTIVE To determine the pharmacokinetics and adverse effects following SC administration of ceftiofur crystalline free acid (CCFA) in New Zealand White rabbits.

ANIMALS 6 adult sexually intact female New Zealand White rabbits.

PROCEDURES Each rabbit was administered 40 mg of CCFA/kg SC. A blood sample was obtained immediately before (0 minutes), at 5 and 30 minutes after, and at 1, 1.5, 2, 3, 4, 8, 12, 24, 48, 72, 95, 120, 144, and 168 hours after administration, and plasma concentrations of ceftiofur free acid equivalents (CFAE) were measured. Pharmacokinetic parameters were calculated. For each rabbit, body weight, food consumption, fecal output, and injection site were monitored. Minimum inhibitory concentrations of ceftiofur for 293 bacterial isolates from rabbit clinical samples were determined.

RESULTS Mean ± SD peak plasma concentration of CFAE and time to maximum plasma concentration were 33.13 ± 10.15 μg/mL and 1.75 ± 0.42 hours, respectively. The mean terminal half-life of CFAE was 42.6 ± 5.2 hours. Plasma CFAE concentration was > 4 μg/mL for approximately 24 hours and > 1 μg/mL for at least 72 hours after CCFA administration. An apparently nonpainful subcutaneous nodule developed at the injection site in 3 of 6 rabbits.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that CCFA (40 mg/kg) could be administered SC every 24 to 72 hours to New Zealand White rabbits to treat infections with ceftiofur-susceptible bacteria. Single-dose administration of CCFA resulted in minimal adverse effects. Additional studies are needed to evaluate the effects of repeated CCFA administration in New Zealand White rabbits.

Abstract

OBJECTIVE To determine the pharmacokinetics and adverse effects following SC administration of ceftiofur crystalline free acid (CCFA) in New Zealand White rabbits.

ANIMALS 6 adult sexually intact female New Zealand White rabbits.

PROCEDURES Each rabbit was administered 40 mg of CCFA/kg SC. A blood sample was obtained immediately before (0 minutes), at 5 and 30 minutes after, and at 1, 1.5, 2, 3, 4, 8, 12, 24, 48, 72, 95, 120, 144, and 168 hours after administration, and plasma concentrations of ceftiofur free acid equivalents (CFAE) were measured. Pharmacokinetic parameters were calculated. For each rabbit, body weight, food consumption, fecal output, and injection site were monitored. Minimum inhibitory concentrations of ceftiofur for 293 bacterial isolates from rabbit clinical samples were determined.

RESULTS Mean ± SD peak plasma concentration of CFAE and time to maximum plasma concentration were 33.13 ± 10.15 μg/mL and 1.75 ± 0.42 hours, respectively. The mean terminal half-life of CFAE was 42.6 ± 5.2 hours. Plasma CFAE concentration was > 4 μg/mL for approximately 24 hours and > 1 μg/mL for at least 72 hours after CCFA administration. An apparently nonpainful subcutaneous nodule developed at the injection site in 3 of 6 rabbits.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that CCFA (40 mg/kg) could be administered SC every 24 to 72 hours to New Zealand White rabbits to treat infections with ceftiofur-susceptible bacteria. Single-dose administration of CCFA resulted in minimal adverse effects. Additional studies are needed to evaluate the effects of repeated CCFA administration in New Zealand White rabbits.

Rabbits are commonly kept as pets and often require veterinary care. According to the 2012 AVMA US Pet Ownership and Demographics source book, there are 3.21 million companion rabbits in US homes, and rabbits are the most common of all the small mammal exotic companion animals.1 Rabbits are frequently brought to veterinarians with signs of illness or injury, which commonly require treatment with antimicrobial drugs.2

Safe antimicrobial choices are limited in rabbits owing to the susceptibility of the normal flora in the rabbit gastrointestinal tract to some common orally administered antimicrobials and to a lack of objective information regarding the use of many antimicrobials in these species.2 Rabbits are at risk for gram-positive and gram-negative bacterial infections, and there is a need for more treatment options with antimicrobials that provide a broad spectrum of activity and can be administered less frequently to minimize stress and discomfort. One of the most common medical problems in companion rabbits is dental disease, and common sequelae to dental disease in rabbits are periapical tooth infections, bacterial osteomyelitis, and facial abscess formation.3 Such abscesses result in considerable morbidity and death and are a common reason for antimicrobial treatment. Oral administration of antimicrobials (eg, β-lactams, macrolides, and clindamycin) commonly given for treatment of abscesses in other mammals will typically result in fatal dysbiosis and enterotoxemia in rabbits.3 Antimicrobials such as the fluoroquinolones and trimethoprim-sulfamethoxazole are safe to administer orally to rabbits but are ineffective against the anaerobic bacteria commonly identified in these abscesses.3 To our knowledge, the data currently available on doses and dosing frequency of antimicrobials from pharmacokinetic and safety studies in rabbits are scant, and there are only a small number of safe antimicrobial options.4,5 All of the antimicrobials known to be effective in rabbits require administration once to multiple times a day to be effective.3 A commonly used injectable antimicrobial for the treatment of abscesses in rabbits is penicillin G procaine, for which an 8-hour IM dosing frequency is recommended on the basis of pharmacokinetic and MIC data6; however, anecdotally, dosing every 24 hours is commonly undertaken to minimize patient handling.6,7 Although likely to cause fatal dysbiosis in rabbits if administered orally, an injectable formulation of this medication has been used successfully without adverse effects.6 In addition to the aforementioned challenges, physical restraint is required to administer antimicrobials in oral or injectable formulations to rabbits, which can result in stress and discomfort.8

In rabbits, the intestinal flora are predominantly anaerobic, gram-negative bacteria of the genus Bacteroides.9 Other facultative anaerobic bacteria that are isolated from the intestinal tract of rabbits include gram-positive genera (eg, Bacillus, Enterococcus, and Staphylococcus) and gram-negative genera (eg, Enterobacter and Escherichia).9 Many of the antimicrobials commonly administered orally in other species are effective against the bacteria found in the rabbit gastrointestinal tract. Destruction of the normal flora through antimicrobial administration results in proliferation of pathogenic flora, a change in the intestinal pH, and progression to enterotoxemia caused by an overgrowth of Clostridium spiroforme.2 Assessments of the effects of antimicrobials on the gastrointestinal system in rabbits range in complexity from observation of clinical signs of diarrhea and anorexia to analysis of histologic alterations or bacterial population changes in the gastrointestinal tract.10 Effects on the intestinal flora can potentially be observed with parenteral administration of some antimicrobials, which should be considered by veterinarians before they undertake any clinical treatment of rabbits.

Ceftiofur is a third-generation cephalosporin antimicrobial that has activity in domestic animals against a variety of gram-positive and gram-negative bacteria, including Escherichia coli and species of Proteus, Klebsiella, Salmonella, Shigella, and Enterobacter.11,12 However, it is important to note that although ceftiofur is considered a third-generation cephalosporin on the basis of its structure, it does not have activity against Pseudomonas aeruginosa as do other third-generation cephalosporins.13,14 Similar to other cephalosporins, ceftiofur is a time-dependent antimicrobial that inhibits bacterial cell wall synthesis and is usually bactericidal.11 Several different formulations of ceftiofur exist, but there are only anecdotal reports on the use of short-acting ceftiofur formulations in rabbits.15 A newer, injectable formulation of ceftiofur (CCFAa) is a United States FDA-approved, sustained-release cephalosporin antimicrobial developed as a single-dose treatment of bacterial infections in cattle and swine and is labeled for the treatment (administered every 4 days) of Streptococcus equi subsp zooepidemicus infection in horses.11 Its bactericidal nature and broad-spectrum and long-acting properties make it desirable as a potential antimicrobial treatment for rabbits. Owing to considerable variation in pharmacokinetics among mammalian species, CCFA should be evaluated in each target species.16–18 To our knowledge, there are no reports of the use of this antimicrobial in rabbits.

The purpose of the study reported here was to determine the pharmacokinetics and adverse effects of CCFA following SC administration in New Zealand White rabbits. The intent was to determine the safety of drug administration and provide a basis for dosage recommendations. We hypothesized that CCFA administered SC (in the interscapular region) once at a dose of 40 mg/kg would maintain plasma drug concentrations that exceeded the MIC for susceptible rabbit bacterial isolates for at least 24 hours and that minimal to no adverse effects would be detected in the treated rabbits.

Materials and Methods

Animals

Six adult (age range, 6 months to 2.5 years) sexually intact female New Zealand White rabbits were used in 2 experiments. The mean ± SD weight of the rabbits was 4.56 ± 0.62 kg. All rabbits were transferred from a previous teaching protocol in which they had been administered sedatives; therefore, a period of 1 month was allowed to elapse prior to the start of the study. Two of the rabbits were initially used in a pilot dosing experiment; a 1-month washout period was provided between the pilot experiment and the primary experiment period. Housing, diet, and husbandry were standardized for each of the 6 rabbits. Each rabbit was fed 89 g of commercial rabbit pelletsb and 2 timothy hay cubes daily,c and water was provided ad libitum via a bottle. Rabbits were housed individually during the study. One week prior to the start of the pilot study, a physical examination and assessments of PCV, total protein concentration, and WBC count (with a differential leukocyte count) were performed to verify that each rabbit was healthy. The study was approved by the University of California-Davis Institutional Care and Use Committee.

Dosage selection, drug administration, and blood sample collection

In the pilot experiment, 2 rabbits were administered 20 mg of CCFAa/kg SC into the interscapular space. For each rabbit, a 4 × 4-cm patch of fur was shaved at the region of the injection site. A black permanent marker was used to circle the injection site. A blood sample (1 mL) was collected from a lateral saphenous or cephalic vein immediately before (0 minutes), at 5 and 30 minutes after, and at 1, 1.5, 2, 3, 4, 8, 12, 24, 48, 72, 95, 120, 144, and 168 hours after injection. A 95-hour sample (instead of a 96-hour sample) was collected because of scheduling conflicts. Collected blood samples were stored on ice until centrifugation for 10 minutes at 3,000 × g. Plasma was separated and stored in cryotubes at −80°C until analysis. On the basis of results of the pilot experiment and allometric scaling calculations,19 a dose of 40 mg of CCFA/kg was chosen for the primary experiment. In the primary experiment, drug administration and blood sample collection were performed in the same manner as described for the pilot experiment. The day of drug administration in the primary experiment was designated as day 0.

Plasma sample analysis

Analysis of CFAE concentration in plasma samples was conducted with reversed-phase high-performance liquid chromatography. The system consisted of a 2695 separations module and a 2487 UV detector.d Separation was attained on a C18 columne (internal diameter, 4.6 mm; length, 250 mm; and particle size, 5 μm) with a 5-μm guard column.f The mobile phase was a mixture of 0.1% trifluroracetic acid in water (A) and 0.1% trifluroracetic acid in acetonitrile (B). The mixture was pumped at a starting gradient of 90% A and 10% B, was adjusted to 75% A and 25% B over a period of 25 minutes, and was reset to initial conditions over a period of 3 minutes. The drug was quantified by means of UV detection at 265 nm at a flow rate of 1.0 mL/min. The column was at ambient temperature (approx 22°C).

Ceftiofur was extracted from each plasma sample with a slightly modified derivitization method that converted ceftiofur and all desfuroylceftiofur metabolites to desfuroylceftiofur acetamide.20 Briefly, each previously frozen plasma sample was thawed and vortexed, and 100 μL of plasma was transferred to a clean test tube to which 15 μL of an internal standard (cefotaxime [100 μg/mL]) was added. Seven milliliters of 0.4% dithioerythritol in borate buffer was added, and then the tube was placed in a water bath (50°C) for 15 minutes. The tube was removed and allowed to cool to room temperature, after which 1.5 mL of iodoacetamide buffer was added. The solution was passed through an extraction column.g The column was eluted with a 5% glacial acetic acid in methanol solution, which was evaporated to dryness under a steady stream of nitrogen gas. Each sample was reconstituted in 200 μL of mobile phase, and 50 μL was injected into the high-performance liquid chromatography system.

Standard curves for plasma analysis were prepared by spiking untreated plasma samples with ceftiofur, which produced a linear concentration range of 0.1 to 100 μg/mL. Spiked standards were treated exactly as were plasma samples. Mean percentage calculated recovery was 99% for ceftiofur. For ceftiofur, intra-assay variability ranged from 0.7% to 4.5% and interassay variability ranged from 3.6% to 8.8%. The lower limit of quantification was 0.1 μg/mL.

Pharmacokinetic analysis

Pharmacokinetic analysis of time-versus-plasma concentration data was performed with commercial software.h Noncompartmental pharmacokinetic variables (terminal-phase half-life [t1/2,λz], Cmax, Tmax, and AUC0–∞) were determined for each rabbit.

Determination of MICs of ceftiofur and target CFAE plasma concentration

Minimum inhibitory concentrations of ceftiofur for bacteria (293 isolates) that had been isolated from various rabbit clinical samples were determined. The clinical samples had been submitted to the William R. Pritchard Veterinary Medical Teaching Hospital Microbiology Laboratory at the University of California-Davis during the period from 2000 through 2014. Bacteria were isolated on 5% sheep blood agar, MacConkey agar, or both and incubated at 35°C in a 5% CO2 atmosphere. Identification of bacterial isolates was performed by use of conventional microbiological methods with spot tests, tubed media, and bacterial identification strips.i

The broth microdilution method was used to perform antimicrobial susceptibility testing in accordance with the standards of the Clinical Laboratory Standards Institute.21,f Briefly, 2 mL of brain-heart infusion broth was inoculated and incubated at 35°C without CO2 for 4 to 5 hours. The broth culture was then added dropwise to saline (0.9% NaCl) solution to achieve a McFarland standard of 0.5 as determined by use of a nephelometer. Ten microliters of each suspension was diluted in 11 mL of cation-adjusted Mueller-Hinton broth that contained N-Tris (hydroxymethyl) methyl-2-aminoethane sulfonic acid; for determination of antimicrobial susceptibility testing, microwell platesc were inoculated with 50 μL of diluted bacterial suspension/well. Plates were incubated at 35°C without CO2 overnight (approx 16 hours). The MIC of ceftiofur for each bacterial isolate was determined, as was the MIC required to inhibit growth of 50% of the bacterial isolates and the MIC90.

The period during which plasma CFAE concentration was 1 to 4 times the MIC of ceftiofur for each bacterial isolate was used to determine a range for frequency of administration at the CCFA dosage evaluated; a multiple of 1 to 4 was chosen in an attempt to account for inclusion of results for any inactive ceftiofur metabolites.

Safety of drug administration

Each rabbit's weight, food consumption (ie, food intake measured by weight), number of fecal pellets produced in each 24-hour period, and fecal pellet diameters (based on the mean of a random subset of 10 pellets produced each day) were measured every 24 hours. The effects on these variables were evaluated once daily during 3 periods of handling, and changes over time during each of these periods were evaluated. The 3 periods included a pretreatment period (days −4 to −1, when the rabbits were handled once daily for weighing), a treatment period (days 0 to 7, when the rabbits were handled during weighing and blood sample collection), and a posttreatment period (days 8 to 14, when the rabbits were handled once daily for weighing). Rabbits were monitored daily for adverse effects. Each rabbit's injection site was also monitored by visual examination and palpation to detect any changes during these handling periods.

Statistical analysis

The dependent variables of interest in the study included daily weight (kg), number of fecal pellets produced and mean fecal pellet diameter (cm) in 24 hours, and weight of food pellets consumed per day (g). A mixed-effects random intercept ANOVA was used to examine the potential effects of the pretreatment, treatment, and posttreatment periods on each dependent variable, while statistically controlling for the number of handling events in each period. Mixed-effects random intercept linear regression analysis was used to examine the potential effects of time (day) on each dependent variable, while statistically controlling for the number of handling events; this analysis was restricted to days 0 through 14. Post hoc contrasts of treatment effects were adjusted with a Bonferroni approach to preserve a nominal significance level of α ≤ 0.05. The assumption of linearity between time and the dependent variables was assessed, and quadratic values of time were included when appropriate. All statistical analyses were performed with statistical software.k

Results

During the pilot and primary experiments, all rabbits appeared to remain healthy (no diarrhea, lethargy, or anorexia) with minimal adverse effects of drug administration. The mean ± SD plasma concentration-time profile of CFAE following SC administration of CCFA at a dose of 40 mg/kg in the 6 rabbits was evaluated (Figure 1). Mean ± SD values of various pharmacokinetic parameters were calculated (Table 1). Ceftiofur absorption in the study rabbits was rapid, with a mean ± SD Cmax of 33.13 ± 10.15 μg/mL; Tmax was 1.75 ± 0.42 hours after SC administration of CCFA.

Figure 1—
Figure 1—

Mean ± SD plasma concentration of CFAE following SC administration of a single dose of CCFA (40 mg/kg) to each of 6 female New Zealand White rabbits. A blood sample was collected from each rabbit immediately before (0 minutes), at 5 and 30 minutes after, and at 1, 1.5, 2, 3, 4, 8, 12, 24, 48, 72, 95, 120, 144, and 168 hours after drug injection.

Citation: American Journal of Veterinary Research 78, 7; 10.2460/ajvr.78.7.796

Table 1—

Mean ± SD noncompartmental CFAE pharmacokinetic parameters derived after SC administration of a single dose (40 mg/kg) of CFFA to each of 6 adult female New Zealand white rabbits.

Pharmacokinetic parameterMean ± SD
t1/2 (h)*42.6 ± 5.2
λz (1/h)0.02 ± 0.002
Tmax (h)1.75 ± 0.42
Cmax (μg/mL)33.13 ± 10.15
AUC0–∞ (h•μg/mL)497 ± 138
MRT0–∞ (h)43.1 ± 6.11

A blood sample was collected from each rabbit immediately before (0 minutes), at 5 and 30 minutes after, and at 1, 1.5, 2, 3, 4, 8, 12, 24, 48, 72, 95, 120, 144, and 168 hours after drug injection.

Harmonic mean.

λz = Elimination rate constant. MRT0–∞ = Mean residence time from time 0 to infinity. t1/2 = Terminal half-life.

The ceftiofur MIC testing revealed that 195 of 293 (67%) aerobic bacterial isolates from rabbit clinical samples had a ceftiofur MIC ≤ 1.0 μg/mL. Thirteen additional aerobic isolates had a ceftiofur MIC between the cutpoints ≤ 1.0 μg/mL and ≤ 4.0 μg/mL, resulting in a total of 71% of isolates with a ceftiofur MIC ≤ 4.0 μg/mL The MIC data for microorganisms for which > 10 isolates were cultured (ie, Bordetella bronchiseptica, E coli, non-Enterobacteriaceae spp, nonfermentor group 3, Pasteurella multocida, other Pasteurella spp, Pseudomonas aeruginosa, Staphylococcus aureus, coagulase-negative Staphylococcus group, and Streptococcus spp) were summarized (Table 2).

Table 2—

Ranges of MIC of ceftiofur for bacteria isolated from clinical samples obtained from rabbits.

MicroorganismNo. of isolatesMIC range (μg/mL)Isolates for which ceftiofur MIC was ≤ 1 μg/mL (%)Isolates for which ceftiofur MIC was ≤ 4 μg/mL (%)
Bordetella bronchiseptica17≤ 0.5 to 45.85.8
Escherichia coli15≤ 0.25 to 886.786.7
Non–Enterobacteriaceae spp14≤ 0.25 to > 485.785.7
Nonfermentor group 310≤ 0.5 to 490.090.0
Pasteurella multocida37≤ 0.25 to 0.5100.0100.0
Other Pasteurella spp17≤ 0.25 to > 494.194.1
Pseudomonas aeruginosa52≤ 0.5 to > 45.89.6
Staphylococcus aureus17≤ 0.5 to 482.488.2
Coagulase-negative Staphylococcus group35≤ 0.25 to > 474.388.6
Streptococcus spp11≤ 0.25 to 1100.0100.0

The clinical samples had been submitted to the William R. Pritchard Veterinary Medical Teaching Hospital Microbiology Laboratory at the University of California-Davis during the period of 2000 to 2014. A broth microdilution method was used to perform antimicrobial susceptibility testing in accordance with the standards of the Clinical Laboratory Standards Institute. Minimum inhibitory concentrations of ceftiofur for 293 bacterial isolates from rabbit clinical samples were determined; data are reported for microorganisms for which the number of isolates was ≥ 10.

Bacterial isolates with a ceftiofur MIC90 ≤ 1.0 μg/mL included Actinobacillus spp, Arcanobacterium pyogenes, Citrobacter spp, Corynebacterium spp, Gemella haemolysans, Micrococcus spp, Moraxella spp, Myroides spp, nonfermentor group 3, Pantoea spp, P multocida, other Pasteurella spp, Proteus mirabilis, Providencia rettgeri, Serratia spp, and Streptococcus spp. Among the bacterial isolates, only Enterobacter spp had a ceftiofur MIC90 ≤ 4.0 μg/mL; the MIC required to inhibit growth of 50% of those bacterial isolates was ≤ 1.0 μg/mL. The target MIC of ceftiofur was therefore determined as ≤ 1.0 μg/mL

In the present study, the only adverse effect detected following CCFA administration was reaction (bruising and subcutaneous nodule formation) at the injection site in 3 of the 6 rabbits. In 1 rabbit, the reaction resolved without complication; in the 2 other affected rabbits, the reaction was still present at the time of study completion (14 days after CCFA administration). None of the rabbits had obvious signs of pain or discomfort associated with the injection site reaction.

During the posttreatment period (days 8 to 14, when the rabbits were handled once daily for weighing), there was a significant (P = 0.001) increase in mean weight of the rabbits, compared with that during the treatment period (days 0 to 7, when the rabbits were handled during weighing and blood sample collection). During the posttreatment period, there were significantly (P < 0.001) more fecal pellets produced each day, compared with findings for the pretreatment period (days −4 to −1, when the rabbits were handled once daily for weighing) and treatment period. There were no significant differences in mean fecal pellet diameter each day among the rabbits during any treatment period or across treatment periods. In the posttreatment period, rabbits consumed significantly (P < 0.001) more food pellets per day than they did in the treatment period.

Discussion

In the present study, a pharmacokinetic dose profile of a sustained-release formulation of the third-generation cephalosporin CCFA in female New Zealand White rabbits was established. This antimicrobial was elected for evaluation because of its gram-positive and gram-negative coverage and its perceived benefit of reduced frequency of administration.11 In the study rabbits, the pharmacokinetic profile of CCFA following a single SC dose of 40 mg/kg was examined, and the results were evaluated in light of measured ceftiofur MICs for various bacterial isolates from rabbit clinical samples.

In the present study, CCFA absorption following SC administration was rapid, with a mean Tmax of approximately 2 hours after administration. When the pharmacokinetic results of CCFA in the study rabbits were compared with data from other species, many of the pharmacokinetic parameters differed. Although the terminal half-life of CCFA is similar for all mammals evaluated,17,18,22–26 the Tmax was much shorter in the rabbits of the present study, compared with findings for other evaluated species except neonatal foals. This may reflect faster absorption in rabbits and, potentially, in foals.17,18,22–26 The Cmax of CCFA was much higher in rabbits than in all other evaluated species; the Cmax in rabbits was most similar to but still higher than that in neonatal foals, which may be attributable to the much higher dose administered to the rabbits in the present study17,18,22–26 The AUC0–∞ was much larger in rabbits than in other evaluated species,17,18,22–26 indicating that the total amount of drug absorbed by rabbits was higher and the plasma drug concentration was detectable for a longer period, both of which may be a result of the higher dose administered given that AUC0–∞ is affected by dose.27

There is a single reportl on the use of ceftiofur sodium administered via the IM and IV routes in rabbits. In rabbits infected with susceptible pathogens that had a ceftiofur MIC ≤ 1 μg/mL, ceftiofur sodium administered IV or IM at a dose of 2.2 mg/kg was found to be effective in maintaining plasma drug concentrations at ≥ 1 μg/mL for up to 12 hours.l Compared with findings following IM administration of ceftiofur sodium, the elimination half-life of CCFA administered IV was much longer (42.6 ± 5.2 hours) than that for ceftiofur sodium administered IM (2.84 ± 0.68 hours) in that study.l Area under the curve for CCFA was significantly larger (497 ± 138 h•μg/mL) than that for ceftiofur sodium administered IM (20.11 ± 2.49 h•μg/mL).l The Cmax was 33.13 ± 10.15 μg/mL for CCFA, compared with 994 ± 1.35 μg/mL for ceftiofur sodium.l The Tmax was 1.75 ± 0.42 hours for CCFA, compared with 0.25 hours for ceftiofur sodium.l These data indicate potential benefits of the sustained-release formulation in rabbits to facilitate decreased frequency of injections.

Metabolism of ceftiofur is rapid, compared with that of its active metabolite desfuroylceftiofur.22 However, desfuroylceftiofur is not stable in vitro; thus, ceftiofur MICs are based solely on the parent drug, even though both desfuroylceftiofur and desfuroylceftiofur conjugates exert antimicrobial activity in vivo.22 The effectiveness of a single dose of CCFA depends on the MIC of ceftiofur for the organism being targeted.22 The antimicrobial action of CCFA is time dependent; therefore, the efficacy and dosing frequency of CCFA are determined by the interval during which desfuroylceftiofur concentration in the affected tissue exceeds the ceftiofur MIC for a specific organism.22 Additionally, protein binding, tissue diffusion, differences in bacterial populations (including intrinsic resistance factors), and local inflammatory changes will affect the clinical efficacy of the drug administered.28,29 Free tissue concentrations at the site of the infection are frequently lower than serum concentrations, a contributing factor to the clinical efficacy of CCFA treatment that is important to consider in future studies.25,28 The degree of protein binding of CCFA in rabbits is unknown; however, in many other species, protein binding is high, meaning that free drug concentrations are often markedly reduced, compared with the total serum drug concentrations in those species.22,25 Tissue concentration of CCFA was not evaluated in the rabbits of the present study, but would be an important variable to investigate in future studies.

In veterinary medicine, drug dosages for many species are commonly extrapolated from data for another species.22 However, differences in body mass, metabolism, circulation time, density of capillaries per unit measure of a specific tissue, respiratory gas exchange surface, rate of drug clearance, and other factors make extrapolation of dosages a challenge.30 When species-specific pharmacokinetic data are not available, allometric scaling may be used to determine therapeutic doses.19 However, absorption and elimination of long-acting medications vary considerably among species, and the dosages should be determined from species-specific data.22 The results of the present study in rabbits have highlighted the concern regarding pharmacokinetic data extrapolation, given that the dose of CCFA used was 6 times that of most mammalian doses; the variability of doses among species is also evident from avian data, wherein doses of CCFA range from 10 to 50 mg/kg in ducks, red-tailed hawks, and doves.16–18,23–26,31–33

In the present study, the only adverse effect detected following CCFA administration was local reaction at the injection site in 3 of the 6 rabbits. In 2 of those 3 rabbits, the reaction was still present at the completion of the study (14 days after CCFA administration). However, the injection site reactions did not appear to be associated with pain or with any clinical effects. The injection site reactions could have been attributable to the large volume of the oil-based carrier of the CCFA that was injected, rather than to actual abscess formation.16,22 The areas of reaction were not aspirated to obtain samples for microbial culture in the present study. Overall, safety data for this study should be interpreted cautiously, given the single drug administration, small number of treated animals, and absence of a control group. Additionally, more extensive safety testing with multiple dosages and histologic examination of tissues at the site of injection is warranted. In clinical settings, most rabbits that would be treated with this antimicrobial would receive > 1 dose, and concerns for more marked injection site reactions might arise. Many drugs form a repository site when administered SC, and reactions can be caused by the drug or the vehicle used for delivery of the drug. It is important to note that there were no obvious systemic effects (eg, diarrhea, lethargy, or anorexia) in the rabbits of the present study as a result of administration of CCFA. It could be speculated that the increased food consumption and fecal pellet production that occurred once the treatment period was complete were effects of decreases in stress (ie, cessation of blood sample collection and acclimatization to handling when being weighed). Further studies of treatment of rabbits with CCFA and the inclusion of control groups are needed to determine whether this is the case.

Limitations of the study reported here included a small sample size. The smallest possible number of rabbits was used to complete the pharmacokinetic analysis, but a larger number of rabbits (with equal numbers of males and females) would have been advantageous to evaluate interindividual variability. However, the SDs of the data acquired were small, which suggested that inclusion of additional rabbits may not have changed the data significantly. Previous studies34–36 have revealed differences in pharmacokinetics for other therapeutic agents between male and female rabbits. The absence of a control group made it a challenge to interpret the safety data in the present study. In addition, without examination of biopsy specimens of the injection site tissue reactions, it is difficult to know whether the reactions were attributable to abscess formation or a local inflammatory response to the oil-based carrier. Additional research in rabbits should be conducted to evaluate the pharmacokinetics of CCFA after administration of multiple doses, given that bioaccumulation could result in a lower frequency of administration.

The MIC values generated in the present study were based on in vitro MICs of ceftiofur; therefore, it is very difficult to provide accurate frequency of administration recommendations without knowing how much of the CFAE is active. A range of frequency of administration was provided, which will depend on the bacteria being targeted as well as the MIC of that isolate. The high end of the target MIC was 4 μg/mL, which was selected in an effort to account for inadvertent inclusion of results for any inactive ceftiofur metabolites present, given that no information exists on inactive metabolites in lagomorphs. Absorption of CCFA following SC administration in rabbits was rapid; target concentrations were reached by 30 minutes following injection and maintained for 24 hours for bacteria with a ceftiofur MIC ≤ 4 μg/mL or for 72 hours for bacteria with a ceftiofur MIC ≤ 1 μg/mL.

Ideally, clinical use of CCFA should be based on results of bacterial culture and antimicrobial susceptibility testing, with the awareness that administration of CCFA in rabbits is an extralabel use of the drug and that no cephalosporins should be used in rabbits that are intended for human consumption. Given the findings of the study reported here, a CCFA dosage of 40 mg/kg, SC, administered every 24 to 72 hours should provide effective plasma concentrations to inhibit susceptible bacteria, but it may result in the formation of nonpainful subcutaneous nodules in rabbits.

Acknowledgments

Supported by the Center for Companion Animal Health, University of California-Davis.

ABBREVIATIONS

AUC

Area under the curve

AUC0-∞

Area under the plasma concentration-time curve from time 0 to infinity

CCFA

Ceftiofur crystalline free acid

CFAE

Ceftiofur free acid equivalents

Cmax

Maximum plasma concentration

MIC

Minimum inhibitory concentration

MIC90

Minimum inhibitory concentration required to inhibit growth of 90% of bacterial isolates

Tmax

Time to maximum plasma concentration

Footnotes

a.

Excede, swine injectable, 100 mg/mL, Zoetis, Madison, NJ.

b.

Laboratory Rabbit Diet HF #5236, LabDiet, St Louis, Mo.

c.

LabDiet Timothy Hay Cubes, LabDiet, St Louis, Mo.

d.

2695 separations module and 2487 UV detector, Waters, Milford, Mass.

e.

Symmetry C18 column, Waters Corp, Milford, Mass.

f.

Symmetry C18 guard column, Waters Corp, Milford Mass.

g.

prewet Oasis HLB, Waters Corp, Mildford Mass.

h.

Phoenix 6.4, Certara, LP, Princeton, NJ.

i.

API BioMerieux, Durham, NC.

j.

Sensititre, ThermoFisher, Waltham, Mass.

k.

Stata, version 13.1/IC, StatCorp LP, College Station, Tex.

l.

Uney K, Altan F, Yazar E, et al. Pharmacokinetics of ceftiofur after single intravenous and intramuscular injections in rabbits, in Proceedings (abstr). World Cong Infect Dis 2015;110.

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Contributor Notes

Address correspondence to Dr. Guzman (guzman@ucdavis.edu).