Respiratory tract disease causes substantial morbidity and death on commercial sheep-raising operations and was responsible for 5.1% of nonpredator-related deaths of adult sheep in 2014.1,2 Mannhemia haemolytica and Pasteurella multocida, the bacterial pathogens most frequently associated with lower respiratory tract disease in sheep, are also associated with pneumonia in cattle.3,4 Thus, antimicrobials approved by the US FDA for the treatment of BRD in cattle may also be effective for the treatment of pneumonia in sheep. There are at least 16 antimicrobial compounds approved by the FDA for use in cattle for the treatment, prevention, or control of BRD, available in over 100 commercially available products in various concentrations and formulations and labeled for various routes of administration.5 In contrast, there are only 5 antimicrobials (ceftiofur sodium, tilmicosin, penicillin G procaine, oxytetracycline hydrochloride, and neomycin) approved by the FDA for the treatment, prevention, or control of respiratory tract disease in sheep.6–9
Florfenicol is a fluorinated derivative of thiamphenicol. It is a bacteriostatic antimicrobial with a broad spectrum of activity against gram-negative and gram-positive bacteria including M haemolytica, P multocida, and some isolates of Escherichia coli, Staphylococcus aureus, and Trueperella pyogenes.10–12 The Clinical Laboratories Standards Institute has established only a few breakpoints for florfenicol in livestock. In cattle, the florfenicol breakpoint is ≤ 2 μg/mL for susceptible isolates of M haemolytica, P multocida, and Histophilus somni.13 In swine, the florfenicol breakpoint is ≤ 4 μg/mL for susceptible isolates of Salmonella enterica serotype Cholerasuis and ≤ 2 μg/L for susceptible isolates of Streptococcus suis, Actinobacillus pleuropneumoniae, and Bordetella bronchiseptica.13 Florfenicol has good penetration of CSF in calves,14 tear fluid in sheep,15 and synovial fluid in cattle.16 It is approved by the FDA for parenteral administration to beef and nonlactating dairy cattle for the treatment and control of BRD associated with M haemolytica, P multocida, and H somni, as well as for treatment of bovine interdigital phlegmon (foot rot) associated with Fusobacterium necrophorum and Bacteroides melaninogenicus.17 Florfenicol is also used for the treatment of nonrespiratory tract diseases in sheep including Campylobacter jejuni–induced abortion18 and foot rot19 and has been suggested for use as treatment for infectious keratoconjunctivitis in sheep.15
Currently, there are 2 formulations of florfenicol commercially available for parenteral administration to livestock in the United States. One formulation contains florfenicol (300 mg/mL) in the excipient N-methyl-2-pyrrolidone (formulation A) and is labeled for the treatment and prevention of BRD associated with M haemolytica, P multocida, and H somni and treatment of foot rot associated with F necrophorum and B melaninogenicus in cattle at a dosage of 20 mg/kg, IM, twice with a 48-interval between doses or 40 mg/kg, SC, once.17 The other formulation contains florfenicol (300 mg/mL) in the excipient 2-pyrrolidone and triacetin (formulation B) and is labeled for the treatment of BRD associated with M haemolytica, P multocida, H somni, and Mycoplasma bovis in beef and nonlactating dairy cattle at a dosage of 40 mg/kg, SC, once.20
The pharmacokinetics of formulation A has been determined following I V, IM, and SC administration to sheep,21–25 goats,26,27 and camelids.21,28,29 Limited pharmacokinetic research has been performed for formulation B, although the pharmacokinetics of formulations A and B has been compared following administration to feedlot calves for the treatment of BRD.30 To our knowledge, the pharmacokinetics of formulations A and B following administration to sheep has not been compared. The objective of the study reported here was to compare the pharmacokinetics of those 2 formulations following administration of a single dose of 20 mg/kg, IM, and 40 mg/kg, SC, to healthy adult sheep.
Materials and Methods
Animals
The study was approved by the University of California-Davis Institutional Animal Care and Use Committee (protocol No. 15471) and was conducted at the University of California-Davis Hopland Research and Extension Center. Sixteen adult mixed-breed sheep (8 ewes and 8 wethers) with ages ranging from 12 to 24 months and a median weight of 37.5 kg (range, 32 to 44 kg) were used for the study. Prior to study initiation, all sheep were determined to be healthy on the basis of results of a physical examination. The sheep were housed in a single pen that included a shelter and outdoor access, were fed alfalfa hay, and had ad libitum access to water.
Study design
The study had a 2-way crossover design. Initially, sheep were divided into 2 groups on the basis of sex; then, the sheep within each group were stratified on the basis of weight. The study population was then resorted into 4 subgroups, each of which contained 2 ewes and 2 wethers and had a similar weight distribution. Each subgroup was randomly assigned by pulling numbers from a hat to receive florfenicol formulation A (florfenicol [300 mg/mL] with the excipient N-methyl-2-pyrrolidone) or B (florfenicol [300 mg/mL] with the excipient 2-pyrrolidone and triacetin) by the IM or SC route such that 1 group received formulation Aa (florfenicol dose, 20 mg/kg) by the IM route, 1 group received formulation Bb (florfenicol dose, 20 mg/kg) by the IM route, 1 group received formulation A (florfenicol dose, 40 mg/kg) by the SC route, and 1 group received formulation B (florfenicol dose, 40 mg/kg) by the SC route. Following a 2-week washout period, each group was administered the opposite formulation at the same dose and by the same route as the initial formulation administered. Both formulations were injected into the cervical musculature or region during IM and SC administration, respectively. For each sheep, the volume of the assigned formulation required was calculated and preloaded into a syringe. To ensure dosing accuracy, syringes used for florfenicol administration were weighed prior to filling, after they were filled with the calculated volume, and after the drug was injected.
Blood sample collection
Florfenicol administration was designated as day 1. For each sheep on day 0 prior to administration of the first formulation, a catheterc was aseptically placed in a jugular vein for blood sample collection. The catheter was flushed with 6 mL of heparinized saline solution (0.9% NaCl solutiond with 1% sodium heparine) immediately after placement and every 6 hours thereafter until blood sample collection was initiated and after each blood sample collection. It was removed immediately after collection of the last blood sample. The process was repeated for administration of the second formulation, except the catheter was placed in the opposite jugular vein.
For each sheep, a blood sample (10 mL) was obtained and placed into a blood collection tubef containing lithium heparin as an anticoagulant immediately before (0 hours) and at 0.25, 0.50, 0.75, 1.0, 1.5, 2, 4, 6, 8, 12, 18, and 24 hours after florfenicol administration. Prior to each sample collection, 10 mL of blood was withdrawn from the catheter and discarded to ensure that the sample collected for analysis was not diluted with any residual heparinized saline solution from the preceding catheter flush. Blood samples were centrifuged at 2,000 X g at 37°C for 15 minutes. Plasma was harvested from each sample and stored at approximately −18°C for up to 36 hours until transport to the analytical laboratory, after which they were stored at −80°C until analysis.
Determination of plasma florfenicol concentration
The florfenicol concentration in each plasma sample was determined as described.22 Briefly, plasma samples were deproteinated with acetone and then extracted with ethyl acetate. The ethyl acetate was dried, and the residue was dissolved in a mobile phase that consisted of 35% acetonitrile in water for analysis. Sample analysis was performed with high-performance liquid chromatographyg and UV detection at 224 nm on a dual-wavelength absorbance detector. A 5-μm, 4.6 × 250-mm column with a 5-μm, 4.6 × 12.5-mm precolumnh was used. The mean recovery of florfenicol was 96.2%, the limit of quantification was 0.05 μg/mL, and the limit of detection was 0.02 μg/mL. The pharmacokinetic assay was linear within florfenicol concentrations of 0.05 to 5 μg/mL, and the intraday and inter-day coefficients of variation were 7.7% and 13.4%, respectively.
Pharmacokinetic analysis
Individual plasma florfenicol concentration-time data were tabulated for all sheep, and the data were plotted by use of a commercially available program.i A semilogarithmic scale was used to visually evaluate concentration-time data for the 2 formulations and routes of administration. The Cmax(obs), tmax(obs), and Thigh(obs) were determined directly from the concentration-time data. The area under the plasma concentration-time curve from time 0 to infinity area under the plasma concentration-time curve from time 0 to the last quantifiable measurement, λz, and t1/2β of florfenicol were estimated by use of noncompartmental methods performed by a commercially available software program.j
Statistical analysis
The data distribution for each pharmacokinetic parameter was evaluated for normality by means of the Shapiro-Wilk test and use of a commercial software program.i Results indicated that none of the pharmacokinetic parameters were normally distributed. The data underwent a logarithmic transformation, were rechecked for normality, and still had nonparametric distributions. Therefore, pharmacokinetic parameters were compared between formulations A and B by use of a Mann-Whitney test performed with a statistical software program,k and results were reported as the median (range). For all analyses, values of P < 0.05 were considered significant.
MIC data
The MICs of florfenicol for ovine bacterial isolates identified between January 1, 2011, and December 31, 2016, at 4 veterinary diagnostic laboratories (University of California-Davis Veterinary Medical Teaching Hospital Clinical Microbiology Laboratory, South Dakota Animal Disease Research and Diagnostic Laboratory, University of Missouri Veterinary Medical Diagnostic Laboratory, and Iowa State Veterinary Diagnostic Laboratory) were compiled. All MICs were determined by use of the broth micro-dilution techniquel in accordance with guidelines described by the Clinical Laboratory Standards Institute.31 The MIC50 and MIC90 were calculated when data were available for ≥ 10 isolates of a particular pathogen. On the basis of those data and results of antimicrobial susceptibilities for respiratory tract pathogens reported in previous surveys,3,4 a plasma florfenicol concentration of 1.0 μg/mL was selected as the target MIC required to effectively treat disease caused by M haemolytica and P multocida in sheep.
Results
Pharmacokinetics
In 1 sheep, the plasma florfenicol concentration peaked twice (18 μg/mL at 0.25 hours and 53.3 μg/mL at 1 hour) after IM administration of formulation B. In another sheep, the t1/2β was exceptionally long (100.7 hours) following SC administration of formulation B, compared with that for the other sheep in the same treatment group. The data from those 2 sheep were included in the plasma florfenicol concentration plots, but were excluded from pharmacokinetic analyses. The mean ± SD plasma florfenicol concentrations over time following IM (Figure 1) and SC (Figure 2) administration of florfenicol formulations A and B were plotted. Comparisons of pharmacokinetic parameters between formulations A and B following IM (Table 1) and SC (Table 2) administration were summarized. The median Cmax(obs) achieved for formulation B was significantly greater than that for formulation A, whereas the median tmax(obs) did not differ between the 2 formulations, regardless of whether the formulations were administered at 20 mg/kg, IM, or 40 mg/kg, SC. The median Thigh(obs) for formulation B was greater than that for formulation A at both doses and administration routes, but that difference was not significant.
Mean ± SD plasma florfenicol concentration over time for 8 healthy adult mixed-breed sheep following IM administration of a single dose (20 mg/kg) of formulation A (florfenicol [300 mg/mL] with the excipient N-methyl-2-pyrrolidone; white circles) and B (florfenicol [300 mg/mL] with the excipient 2-pyrrolidone and triacetin; black circles). The study had a crossover design. Sheep were separated into 2 groups (4 sheep/group) and then randomly allocated to receive either formulation A or B. After a 2-week washout period, each group received the opposite formulation. Notice the y-axis has a logarithmic scale.
Citation: American Journal of Veterinary Research 79, 1; 10.2460/ajvr.79.1.107
Mean ± SD plasma florfenicol concentration over time for 8 healthy adult mixed-breed sheep following IM administration of a single dose (20 mg/kg) of formulation A (florfenicol [300 mg/mL] with the excipient N-methyl-2-pyrrolidone; white circles) and B (florfenicol [300 mg/mL] with the excipient 2-pyrrolidone and triacetin; black circles). The study had a crossover design. Sheep were separated into 2 groups (4 sheep/group) and then randomly allocated to receive either formulation A or B. After a 2-week washout period, each group received the opposite formulation. Notice the y-axis has a logarithmic scale.
Citation: American Journal of Veterinary Research 79, 1; 10.2460/ajvr.79.1.107
Mean ± SD plasma florfenicol concentration over time for 8 healthy adult mixed-breed sheep following IM administration of a single dose (20 mg/kg) of formulation A (florfenicol [300 mg/mL] with the excipient N-methyl-2-pyrrolidone; white circles) and B (florfenicol [300 mg/mL] with the excipient 2-pyrrolidone and triacetin; black circles). The study had a crossover design. Sheep were separated into 2 groups (4 sheep/group) and then randomly allocated to receive either formulation A or B. After a 2-week washout period, each group received the opposite formulation. Notice the y-axis has a logarithmic scale.
Citation: American Journal of Veterinary Research 79, 1; 10.2460/ajvr.79.1.107
Mean ± SD plasma florfenicol concentration over time for 8 healthy adult mixed-breed sheep following SC administration of a single dose (40 mg/kg) of formulation A (white circles) and B (black circles). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 79, 1; 10.2460/ajvr.79.1.107
Mean ± SD plasma florfenicol concentration over time for 8 healthy adult mixed-breed sheep following SC administration of a single dose (40 mg/kg) of formulation A (white circles) and B (black circles). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 79, 1; 10.2460/ajvr.79.1.107
Mean ± SD plasma florfenicol concentration over time for 8 healthy adult mixed-breed sheep following SC administration of a single dose (40 mg/kg) of formulation A (white circles) and B (black circles). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 79, 1; 10.2460/ajvr.79.1.107
Median (range) valves for pharmacokinetic parameters for florfenicol formulations A and B following IM administration of a single dose (20 mg/kg) to each of 8 healthy adult mixed-breed sheep.
| Parameter | Formulation A | Formulation B* | P value |
|---|---|---|---|
| Cmax(obs) (μg/mL) | 3.76 (2.40–6.50) | 7.72 (3.82–10.25) | 0.005 |
| tmax(obs) (h) | 0.99 (0.50–1.53) | 0.78 (0.51–2.03) | 0.96 |
| Thigh(obs) (h) | 8.06 (5.69–20.68) | 13.48 (7.64–15.78) | 0.23 |
| AUClast (μg•h/mL) | 24.88 (21.91–40.51) | 41.53 (31.84–48.25) | 0.002 |
| AUC0–∞ (μg•h/mL) | 36.75 (27.80–63.04) | 47.77 (39.42–52.00) | 0.28 |
| t1/2β (h) | 13.44 (8.27–24.11) | 5.98 (1.50–14.58) | 0.02 |
| λz (h−1) | 0.05 (0.03–0.08) | 0.12 (0.05–0.46) | 0.02 |
Formulation A consisted of florfenicol (concentration, 300 mg/mL) with the excipient N-methyl-2-pyrrolidone, and formulation B consisted of florfenicol (concentration, 300 mg/mL) with the excipient 2-pyrrolidone and triacetin. The study had a crossover design. Sheep were separated into 2 groups (4 sheep/group) and then randomly allocated to receive either formulation A or B. After a 2-week washout period, each group received the opposite formulation. Pharmacokinetic parameters were estimated by noncompartmental methods. None of the parameters were normally distributed; therefore, a Mann-Whitney test was used for all comparisons between the 2 formulations. Values of P < 0.05 were considered significant.
AUC0-∞ = Area under the plasma concentration-time curve from time 0 to infinity. AUClast = Area under the plasma concentration-time curve from time 0 to the last quantifiable measurement.
The plasma florfenicol concentration in 1 sheep peaked twice following administration of formulation B, and the data from that sheep were excluded from the pharmacokinetic analysis. Therefore, only 7 sheep contributed to the median (range) values in this column.
Median (range) values for pharmacokinetic parameters for florfenicol formulations A and B following SC administration of a single dose (40 mg/kg) to each of 8 healthy adult mixed-breed sheep.
| Parameter | Formulation A | Formulation B* | P value |
|---|---|---|---|
| Cmax(obs) (μg/mL) | 2.63 (1.61–4.55) | 4.70 (4.65–9.86) | < 0.001 |
| tmax(obs) (h) | 3.00 (0.29–6.00) | 1.00 (0.76–3.03) | 0.27 |
| Thigh(obs) (h) | 13.78 (8.28–23.81) | 17.50 (12.98–23.94) | 0.12 |
| AUClast (μg•h/mL) | 31.63 (19.32–49.51) | 48.32 (35.59–78.42) | 0.01 |
| AUC0–∞ (μg•h/mL) | 43.48 (22.50–64.06) | 73.58 (54.40–104.80) | 0.004 |
| t1/2β (h) | 12.48 (7.51–19.75) | 16.60 (6.59–33.76) | 0.28 |
| λz (h−1) | 0.06 (0.04–0.092) | 0.04 (0.02–0.11) | 0.28 |
The t1/2β for 1 sheep was exceptionally long (100.7 hours) following SC administration of formulation B, compared with that for the other sheep in the same treatment group, and the data for that sheep were excluded from the pharmacokinetic analysis. Therefore, only 7 sheep contributed to the median (range) values in this column.
See Table 1 for remainder of key.
MIC data
During the 6-year search period, a total of 367 ovine isolates originating from sheep in 13 states were identified and had an MIC for florfenicol determined (Table 3). The MIC range, MIC50, and MIC90 reported for florfenicol were ≤ 0.25 to 2.0, 0.5, and 1 μg/mL, respectively, for M haemolytica isolates and ≤ 0.25 to 1, 0.25, and 0.5 μg/mL, respectively, for P multocida isolates. Those data supported our selection of a target MIC for florfenicol of 1.0 μg/mL for the treatment of bacterial infections in sheep. Respiratory tract bacterial pathogens with a reported MIC90 > 1.0 μg/mL included Campylobacter spp (2.0 μg/mL) and Salmonella spp (2.0 μg/mL).
Summary florfenicol MIC data for ovine bacterial isolates identified between January 1, 2011, and December 31, 2016, at 4 veterinary diagnostic laboratories.
| Pathogen | No. of isolates | MIC50 (μg/mL) | MIC90 (μg/mL) | MIC range (μg/mL) |
|---|---|---|---|---|
| Bibersteinia trehalosi | 16 | 1 | 8 | 0.5 to 8 |
| Campylobacter fetus | 11 | 1 | 2 | 1 to 4 |
| Campylobacter jejuni | 45 | 1 | 2 | 0.5 to 8 |
| Corynebacterium pseudotuberculosis | 15 | 2 | 2 | 0.5 to 2 |
| Escherichia coli | 41 | 4 | 8 | 2 to > 8 |
| Mannheimia haemolytica | 117 | 0.5 | 1 | ≤ 0.25 to 2 |
| Pasteurella multocida | 50 | 0.25 | 0.5 | < 0.25 to 1 |
| Pasteurella spp | 12 | 0.5 | 1 | < 0.25 to 1 |
| Salmonella spp | 38 | 1 | 2 | 1 to > 8 |
| Staphylococcus aureus | 11 | 4 | 8 | 2 to > 8 |
| Trueperella pyogenes | 11 | 0.5 | 1 | ≤ 0.25 to 2 |
The 4 laboratories were the University of California-Davis Veterinary Medical Teaching Hospital Clinical Microbiology Laboratory, South Dakota Animal Disease Research and Diagnostic Laboratory, University of Missouri Veterinary Medical Diagnostic Laboratory, and Iowa State Veterinary Diagnostic Laboratory. The antimicrobial administration history was unavailable for the sheep from which the bacterial isolates were obtained. Data are provided only for the pathogens that had > 10 isolates identified.
Discussion
Results of the present study indicated that some pharmacokinetic parameters were comparable, whereas others differed significantly, for the 2 florfenicol formulations approved by the FDA for parenteral administration to livestock in the United States when they were administered by the IM and SC routes to healthy sheep. Nevertheless, the mean plasma florfenicol concentration achieved following administration of both formulations A (florfenicol [300 mg/mL] with the excipient N-methyl-2-pyrrolidone) and B (florfenicol [300 mg/mL] with the excipient 2-pyrrolidone and triacetin) at a dose of 20 mg/kg, IM, or 40 mg/kg, SC, was > 1 μg/kg (the target MIC of florfenicol required to treat respiratory tract disease caused by M hemolytica and P multocida in sheep selected on the basis of antimicrobial susceptibility data for ovine respiratory tract pathogens isolated at 4 veterinary diagnostic laboratories over a 6-year period from 2011 to 2016 and previous reports3,4) for a period of time; thus, both formulations could be effective for the treatment of respiratory tract disease in sheep, although regional differences in antimicrobial susceptibility of pathogens should always be considered.
In the present study, the mean Cmax(obs) for formulation B was significantly greater than that for formulation A at both doses (20 mg/kg, IM, and 40 mg/kg, SC) evaluated. This could be clinically relevant for the treatment of respiratory tract disease in sheep because florfenicol has concentration-dependent activity against certain organisms.32 Distribution of florfenicol to the central compartment was rapid (within 1 and 3 hours after IM and SC administration, respectively) for both formulations. The Cmax(obs) (median, 3.76 μg/mL; range, 2.40 to 6.50 μg/mL), and tmax(obs) (median, 0.99 hours; range, 0.50 to 1.53 hours) for formulation A following IM administration of the 20-mg/kg dose were similar to the mean ± SD Cmax and tmax reported following IM administration of the same dose to sheep of other studies21,23,25,33 (Table 4). Following SC administration of the 40-mg/kg dose, the median Cmax(obs) for both formulations A (2.63 μg/mL; range, 1.61 to 4.55 μg/mL), and B (4.70 μg/mL; range, 4.65 to 986 μg/mL) for the sheep of the present study was fairly similar to that reported for formulations A (4.69 μg/mL) and B (5.93 μg/mL) in cattle30; however, the median tmax(obs) for the sheep of this study (formulation A, 3 hours; formulation B, 1 hour) was less than that for the cattle of that study30 (formulation A, 6 hours; formulation B, 5 hours).
Summary of pharmacokinetic parameters for florfenicol following IM or SC administration to sheep and cattle as reported in the veterinary literature.
| Pharmacokinetic parameter | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Reference No. | Species | No. of animals | Formulation | Dose (mg/kg) | Route of administration | Cmax | tmax (h) | AUC (μg•h/mL) | t1/2•(h) | ke (h−1) | F (%) |
| 21* | Sheep | 6 | A | 20 | IM | 1.04 ± 0.10 | 1.44 ± 016 | 49.56 ± 5.51† | 2.25 ± 0.20 | — | 65.82 ± 6.71 |
| 23‡ | Sheep | 5 | A | 20 | IM | 4.13 ± 0.29 | 1.45 ± 0.16 | 67.95 ± 0.61 | 10.34 ± 1.11 | 0.07 ± 0.01 | 89.04 |
| 25‡ | Sheep | 5 | A | 20 | IM | 4.86 ± 0.21 | 2.0 ± 0.05 | 50.51 ± 1.51 | 3.01 ± 0.28 | — | — |
| 22‡ | Sheep | 10 | A | 40 | SC | 2.64 ± 1.07 | 2.0 ± 1 | 42.2 ± 13.4 | 34.7 ± 9.6 | 0.02 ± 0.005 | 40.2 ± 16.2§ |
| 30‖ | Cattle | 24 | A | 40 | SC | 4.69 (47.3) | 6.0 (2–10.0) | 141.78 (27) | 51.79 (42) | — | — |
| 30‖ | Cattle | 24 | B | 40 | SC | 5.93 (38.3) | 5.0 (2.0–12.0) | 150.2 (20.9) | 37.67 (27.3) | — | — |
Results reported as mean ± SE.
AUC0-∞.
Results reported as mean ± SD.
Systemic bioavailability from time 0 to infinity.
Results reported as mean (% coefficient of variation) or media (range).
— = Not reported. AUC = Area under the plasma concentration-time curve. F = Systemic availability. ke = Elimination rate constant. See Table 1 for remainder of key.
For the sheep of the present study, the median area under the plasma concentration-time curve from time 0 to infinity and area under the plasma concentration-time curve from time 0 to the last quantifiable measurement for formulation B were significantly greater than those for formulation A, regardless of whether the drug was administered by the IM or SC route. This finding was likely an artifact of flip-fop kinetics and the fact that only a small number of samples were collected during the elimination phase of the concentration-time curve. In the present study, the florfenicol concentration-time curve plateaued during the elimination phase in a manner similar to that reported following florfenicol administration to sheep of other studies.22–24 A persistent plateau in the concentration-time curve also occurs following IV administration of florfenicol, which further supports the occurrence of flip-fop kinetics owing to the continuous absorption and redistribution of florfenicol from the site of injection.24
The median plasma t1/2β of florfenicol was significantly shorter and the median λz, was significantly longer for formulation B, compared with those for formulation A, following IM administration; however, the median t1/2β and λz, did not differ significantly between the 2 formulations following SC administration. The median plasma t1/2β of florfenicol for both formulations A (12.48 hours) and B (16.60 hours) following SC administration was shorter than that (34.7 hours) reported in another study22; however, the median t1/2β of florfenicol for formulations A (1344 hours) and B (5.98 hours) following IM administration was 2 to 5 times that reported following IM administration of the same dose to sheep of 2 other studies21,23 (Table 4). The median t1/2β for both formulations A and B following SC administration to the sheep of this study was substantially shorter than that following SC administration of the same dose (40 mg/kg) of formulations A (51.79 hours) and B (37.67 hours) to cattle.30 The median t1/2β of florfenicol in cattle following IM administration of formulation A at a dose of 20 mg/kg (18.3 hours)11 was 1.4 and 3 times that following IM administration of the same dose of formulations A and B, respectively, to the sheep of the present study. The reasons for the differences in the t1/2β and λz between the sheep of this study and cattle of those other studies11,30 are unknown. Unfortunately, information regarding the metabolism of florfenicol in animals is scarce. Results of a study34 involving calves indicate that florfenicol is excreted in its parent form via urine, which suggests the drug is cleared by the kidneys. However, to our knowledge, the metabolism of florfenicol in sheep has not been elucidated. Some differences in the pharmacokinetic parameters for florfenicol observed among the present study and other studies might be attributable to the use of less sensitive assay methods21 or individual variation among animals.22 Furthermore, although the λz was estimated on the basis of the plasma florfenicol concentration at a minimum of 3 points during the elimination phase, a more optimal estimate would have been obtained had more blood samples been acquired during the elimination phase.
The MICs of florfenicol reported for ovine M haemolytica and P multocida isolates identified at 4 veterinary diagnostic laboratories during the 6-year period from 2011 to 2016 were similar to those reported in multiple studies.3,32,35,36 This finding indicated that florfenicol is active against various bacterial pathogens in vitro and suggested that it may be an effective treatment for sheep with respiratory tract disease or other diseases caused by susceptible organisms. However, the antimicrobial susceptibility pattern for some pathogens can vary among regions, and veterinarians should evaluate local antimicrobial susceptibility trends prior to using florfenicol to optimize the probability of treatment success. Additionally, the antimicrobial administration history was unavailable for the sheep from which the bacterial isolates described in this study were obtained (Table 3), and prior antimicrobial administration could have affected the MICs reported.
Although florfenicol is generally considered to have a time-dependent mechanism of action, it has a concentration-dependent mechanism of action against certain organisms32; therefore, the duration that the plasma florfenicol concentration is greater than the target MIC should be at least 50% of the dosing interval to achieve optimal inhibition of bacterial growth.37 On the basis of that guideline, the veterinary literature suggests that, in sheep, administration of florfenicol at a dose of 20 to 30 mg/kg, IM,23,25 or 40 mg/kg, SC,33 daily for 3 days is necessary to achieve and maintain plasma florfenicol concentrations that will be effective against most bacterial pathogens. That dosing interval is shorter than the FDA-approved dosing interval (once or every 48 hours for SC and IM administration, respectively) for florfenicol in cattle.17,20 Further studies involving diseased sheep are necessary to evaluate the accumulation of and potential adverse effects associated with administration of multiple doses of florfenicol, especially in regard to injection site safety. In the present study, the median Thigh(obs) was > 12 hours for formulation B following administration of both the IM (13.48 hours) and SC (17.50 hours) doses; however, it did not differ significantly from that for formulation A following administration of either dose, and was substantially less than the Thigh(obs) reported for sheep following IM administration of formulation A at a dose of 20 mg/kg in another study23 (24 hours). Because the median Thigh(obs) did not differ between formulations A and B following administration by either the IM or SC route, altering the dosing interval of florfenicol to sheep on the basis of formulation might be unnecessary. Nonetheless, IM administration of formulation A (20 mg/kg) resulted in the shortest median Thigh(obs) (8 hours) in the present study, and the Thigh(obs) should be considered when patients fail to respond to treatment. Practitioners are also encouraged to use regional MIC data to calculate appropriate dosing intervals for florfenicol to optimize treatment efficacy.
In the United States, administration of florfenicol to sheep is considered extralabel drug use, which is permitted as long as it is in accordance with the AMDUCA, and requires practitioners to determine an appropriate withdrawal interval. Results of a study33 in which sheep were administered 40 mg of formulation A/kg, SC, daily for 3 days indicate that treated sheep have florfenicol residues present in adipose tissue for a prolonged period necessitating a withdrawal interval for meat of at least 42 days. To our knowledge, tissue florfenicol residues in sheep following administration of formulation B have not been investigated. In the United States, veterinarians can contact the Food Animal Residue Avoidance Databank38 to obtain scientifically based withdrawal intervals for drugs administered in an extralabel manner in accordance with AMDUCA.
In the present study, the median Cmax(obs) of florfenicol in sheep following administration of formulation B was significantly greater than that following administration formulation A, regardless of whether the 2 formulations were administered at a dose of 20 mg/kg, IM, or 40 mg/kg, SC, and that difference may be clinically relevant and affect treatment outcome. Although the median Thigh(obs) of florfenicol did not differ between the 2 formulations at the doses and administration routes evaluated, it was shortest following administration of formulation A at a dose of 20 mg/kg, IM. The MICs of florfenicol for ovine isolates of M hemolytica and Pasteurella spp, common respiratory tract pathogens, ranged from 0.25 to 1.0 μg/mL. Given the plasma florfenicol concentrations achieved in the sheep of the present study, both florfenicol formulations evaluated may be effective against those and other bacterial pathogens of sheep. However, antimicrobial susceptibility results for bacterial pathogens can vary regionally, and local antimicrobial susceptibility trends should be evaluated prior to use of florfenicol to optimize the probability of treatment success. Even though the median t1/2β was estimated for each of the 4 treatments evaluated in the present study, a multiple-dose study in which plasma florfenicol concentrations are determined more frequently during the elimination phase than in this study is warranted as is a tissue residue study following administration of formulation B to sheep.
Acknowledgments
Supported by the USDA National Research Support Project—7 (Minor Use Animal Drug Program).
The authors declare that there were no conflicts of interest.
ABBREVIATIONS
| λz | Elimination rate constant |
| BRD | Bovine respiratory disease |
| Cmax(obs) | Observed maximum plasma concentration |
| MIC | Minimum inhibitory concentration |
| MIC50 | Minimum antimicrobial concentration that inhibits growth of 50% of organisms |
| MIC90 | Minimum antimicrobial concentration that inhibits growth of 90% of organisms |
| t1/2β | Elimination half-life |
| Thigh(obs) | Observed duration during which the plasma drug concentration was greater than the MIC (1.0 μg/mL) |
| tmax(obs) | Time at which the maximum plasma concentration was observed |
Footnotes
Nuflor injectable solution, Schering-Plough Animal Health, Kenilworth, NJ.
Nuflor Gold injectable solution, Intervet Schering-Plough Animal Health, Roseland, NJ.
Milacath, Mila International Inc, Erlanger, Ky.
0.9% Sodium chloride injection USP, Baxter Healthcare Corp, Deerfield, Ill.
Heparin sodium injection USP (1000 U/mL), APP Pharmaceuticals LLC, Schaumburg, Ill.
Becton Dickinson and Company, Franklin Lakes, NJ.
Waters 2690 Alliance System, Waters Corp, Milford, Mass.
Zorbax SB-C18, 5 μm, 4.6 × 250 and 4.6 × 12.5 mm, Agilent Technologies Inc, Wilmington, Del.
SAS, version 9.4, SAS Institute Inc, Cary, NC.
WinNonLin, version 6.3, Pharsight Corp, Mountain View, Calif.
Prism, version 7.0, GraphPad Software Inc, La Jolla, Calif.
Sensititre, Thermo Fisher Scientific, Oakwood Village, Ohio.
References
1. USDA. Sheep and lamb nonpredator death loss in the United States, 2011. Available at: www.aphis.usda.gov/animal_health/nahms/sheep/downloads/sheep11/Sheep11_is_DeathLoss.pdf. Accessed Aug 2, 2014.
2. USDA. Sheep and lamb nonpredator death loss in the United States, 2015. Available at: www.aphis.usda.gov/animal_health/nahms/sheep/downloads/sheepdeath/SheepDeathLoss2015.pdf. Accessed Jun 24, 2016.
3. Berge AC, Sischo WM, Craigmill AL. Antimicrobial susceptibility patterns of respiratory tract pathogens from sheep and goats. J Am Vet Med Assoc 2006; 229: 1279–1281.
4. Welsh RD, Dye LB, Payton ME, et al. Isolation and antimicrobial susceptibilities of bacterial pathogens from bovine pneumonia: 1994–2002. J Vet Diagn Invest 2004; 16: 426–431.
5. Compendium of Veterinary Products—US edition (online database). 2015 ed. Available at: bayerall.naccvp.com/?u=bayer&p=dvm. Accessed Aug 2, 2014.
6. FDA. Freedom of information summary, supplement to NADA 140–338, Naxcel sterile powder (ceftiofur sodium). Available at: www.fda.gov/downloads/AnimalVeterinary/Products/ApprovedAnimalDrugProducts/FOIADrugSummaries/ucm049843.pdf. Accessed Aug 2, 2014.
7. FDA. Freedom of information summary, supplemental abbreviated new animal drug application, ANADA 200–026, Pennox 343. Available at: www.fda.gov/downloads/AnimalVeterinary/Products/ApprovedAnimalDrugProducts/FOIADrugSummaries/UCM252253.pdf. Accessed on Aug 3, 2014.
8. FDA. Freedom of information summary, supplemental new animal drug application, NADA 065–010, Norocillin. Available at: www.fda.gov/downloads/AnimalVeterinary/Products/ApprovedAnimalDrugProducts/FOIADrugSummaries/UCM218419.pdf. Accessed Aug 3, 2014.
9. FDA. Freedom of information summary, supplemental new animal drug application, NADA 140–929, Micotil 300. Available at: www.fda.gov/downloads/AnimalVeterinary/Products/ApprovedAnimalDrugProducts/FOIADrugSummaries/UCM255919.pdf. Accessed Aug 3, 2014.
10. Graham R, Palmer D, Pratt BC, et al. In vitro activity of florphenicol. Eur J Clin Microbiol Infect Dis 1988; 7: 691–694.
11. Lobell RD, Varma KJ, Johnson JC, et al. Pharmacokinetics of florfenicol following intravenous and intramuscular doses to cattle. J Vet Pharmacol Ther 1994; 17: 253–258.
12. Neu HC, Fu KP. In vitro activity of chloramphenicol and thiamphenicol analogs. Antimicrob Agents Chemother 1980; 18: 311–316.
13. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. 3rd ed. CLSI supplement VET01S. Wayne, Pa: Clinical and Laboratory Standards Institute, 2015; 28, 51, 58, 61, 64, 67, 69.
14. de Craene BA, Deprez P, D'Haese E, et al. Pharmacokinetics of florfenicol in cerebrospinal fluid and plasma of calves. Antimicrob Agents Chemother 1997; 41: 1991–1995.
15. Regnier A, Laroute V, Gautier-Bouchardon A, et al. Florfenicol concentrations in ovine tear fluid following intramuscular and subcutaneous administration and comparison with the minimum inhibitory concentrations against mycoplasmal strains potentially involved in infectious keratoconjunctivitis. Am J Vet Res 2013; 74: 268–274.
16. Gilliam JN, Streeter RN, Papich MG, et al. Pharmacokinetics of florfenicol in serum and synovial fluid after regional intravenous perfusion in the distal portion of the hind limb of adult cows. Am J Vet Res 2008; 69: 997–1004.
17. FDA. Freedom of information summary, supplemental new animal drug application, NADA 141–063, Nuflor injectable solution. Available at: www.fda.gov/downloads/AnimalVeterinary/Products/ApprovedAnimalDrugProducts/FOIADrugSummaries/ucm116745.pdf. Accessed on Aug 2, 2014.
18. Sahin O, Plummer PJ, Jordan DM, et al. Emergence of a tetracycline-resistant Campylobacter jejuni clone associated with outbreaks of ovine abortion in the United States. J Clin Microbiol 2008; 46: 1663–1671.
19. Vandyke S, Wallace L, Sterle SW, et al. Treatment of ovine foot rot: use of florfenicol versus oxytetracycline for treatment of ovine foot rot. Sheep Goat Res J 1999; 15: 54–57.
20. FDA. Freedom of information summary, supplemental new drug application, NADA 141–265, Nuflor Gold. Available at: www.fda.gov/downloads/AnimalVeterinary/Products/ApprovedAnimalDrugProducts/FOIADrugSummaries/UCM198114.pdf. Accessed on Aug 2, 2014.
21. Ali BH, Al-Qarawi AA, Hashaad M. Comparative plasma pharmacokinetics and tolerance of florfenicol following intramuscular and intravenous administration to camels, sheep and goats. Vet Res Commun 2003; 27: 475–483.
22. Lane VM, Villarroel A, Wetzlich SE, et al. Intravenous and subcutaneous pharmacokinetics of florfenicol in sheep (Erratum published in J Vet Pharmacol Ther 2004:27:539). J Vet Pharmacol Ther 2004; 27: 191–196.
23. Shen J, Li X, Jiang H, et al. Bioavailability and pharmacokinetics of florfenicol in healthy sheep (Erratum published in J Vet Pharmacol Ther 2004;27:539). J Vet Pharmacol Ther 2004; 27: 163–168.
24. Palma C, Ramirez J, Benavente A, et al. Pharmacokinetics of florfenicol and florfenicol-amine after intravenous administration in sheep. J Vet Pharmacol Ther 2012; 35: 508–511.
25. El-Sheikh WMA, Shaheen HM, El-Ghoneimy A. Comparative pharmacokinetics of florfenicol after intravenous, intramuscular and subcutaneous injection in sheep. Assiut Vet Med J 2009; 55: 100–109.
26. Lavy E, Ziv G, Soback S, et al. Clinical pharmacology of florfenicol in lactating goats. Acta Vet Scand Suppl 1991; 87: 133–136.
27. Atef M, el-Gendi AY, Amer MM, et al. Pharmacokinetic properties of florfenicol in Egyptian goats. Dtsch Tierarztl Wochenschr 2000; 107: 147–150.
28. Holmes K, Bedenice D, Papich MG. Florfenicol pharmacokinetics in healthy adult alpacas after subcutaneous and intramuscular injection. J Vet Pharmacol Ther 2012; 35: 382–388.
29. Pentecost RL, Niehaus AJ, Werle NA, et al. Pharmacokinetics of florfenicol after intravenous and intramuscular dosing in llamas. Res Vet Sci 2013; 95: 594–599.
30. FDA. Freedom of information summary, original new animal drug application, NADA 141–265, Nuflor Gold injectable solution. Available at: www.fda.gov/downloads/AnimalVeterinary/Products/ApprovedAnimalDrugProducts/OIADrug Summaries/ucm062315.pdf. Accessed on Aug 3, 2014.
31. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard. 4th Edition. CLSI document VET01–A4. Wayne, PA: Clinical and Laboratory Standards Institute; 2013;36–39.
32. Illambas J, Potter T, Sidhu P, et al. Pharmacodynamics of florfenicol for calf pneumonia pathogens. Vet Rec 2013;172:340.
33. Lane VM, Villarroel A, Wetzlich SE, et al. Tissue residues of florfenicol in sheep. J Vet Pharmacol Ther 2008; 31: 178–180.
34. Varma KJ, Adams PE, Powers TE, et al. Pharmacokinetics of florfenicol in veal calves. J Vet Pharmacol Ther 1986; 9: 412–425.
35. Sidhu P, Rassouli A, Illambas J, et al. Pharmacokinetic–pharmacodynamic integration and modelling of florfenicol in calves. J Vet Pharmacol Ther 2014; 37: 231–242.
36. Ural K, Ulutas B, Kirkan S, et al. Florfenicol therapy during natural Mannheimia haemolytica infection in Sakiz sheep. Acta Sci Vet 2011;39:961.
37. Toutain PL, Lees P. Integration and modelling of pharmacokinetic and pharmacodynamic data to optimize dosage regimens in veterinary medicine. J Vet Pharmacol Ther 2004; 27: 467–477.
38. Food Animal Residue Avoidance Databank. Available at: www.farad.org. Accessed Jul 25, 2017.
