Abstract
Objective
To investigate the disposition of enrofloxacin and its active metabolite, ciprofloxacin, in plasma, pulmonary epithelial lining fluid (PELF), peritoneal fluid, and CSF in horses following IV administration of enrofloxacin at doses of 5 mg/kg and 7.5 mg/kg of body weight.
Methods
6 healthy, mature mares were randomly assigned to receive a single dose of enrofloxacin at either 5 mg/kg or 7.5 mg/kg in a crossover design with a washout period of 10 days. Concentrations of enrofloxacin and ciprofloxacin were determined in plasma, PELF, peritoneal fluid, and CSF.
Results
Both doses of enrofloxacin were generally well tolerated. One horse developed focal, self-limiting limb edema. The median maximum concentration extrapolated to time 0 and area under the plasma concentration-versus-time curve from time 0 to the last quantifiable time point (24 hours) for enrofloxacin in plasma were significantly greater when horses were given enrofloxacin at 7.5 mg/kg. Similarly, the median elimination rate constant, half-life of the terminal phase, peak serum concentration (Cmax), area under the plasma concentration-versus-time curve from time 0 to the last quantifiable time point (24 hours), area under the plasma concentration-versus-time curve extrapolated to infinity, and mean residence time for ciprofloxacin in plasma were significantly greater following administration of enrofloxacin at 7.5 mg/kg. There were no significant differences between doses in any of the measured pharmacokinetic variables in PELF.
Conclusions
There was no apparent pharmacokinetic advantage of enrofloxacin at the 7.5-mg/kg dose for susceptible isolates; however, this dose achieved higher concentrations and prolonged persistence in fluid matrices. Further studies are required to evaluate repeated administration at this dose for tolerability and clinical efficacy.
Clinical Relevance
Despite the wide use of enrofloxacin in horses, pharmacokinetic data is limited. This study provides pharmacokinetic data that can be used in a clinical setting.
Enrofloxacin is a second-generation fluoroquinolone antimicrobial that is highly active against a wide array of gram-negative bacteria, including Enterobacterales, which are commonly isolated from horses with a variety of disease syndromes.1,2 Moreover, enrofloxacin is one of the few antimicrobials for which oral administration is well tolerated and practical in adult horses. This is crucial because prolonged antibiotic therapy (> 2 weeks) is typical in cases of equine bronchopneumonia, septic peritonitis, and bacterial meningitis, rendering the oral route of administration advantageous. Additionally, enrofloxacin has a low nephrotoxic potential.3 Therefore, when aminoglycoside antimicrobials are contraindicated in patients with renal impairment, enrofloxacin is often selected to provide empiric gram-negative antimicrobial coverage.
Fluoroquinolones have a unique mechanism of action as the primary target is bacterial DNA gyrase, an enzyme responsible for controlling the supercoiling of bacterial DNA.4 Fluoroquinolones are also moderately lipophilic, another advantage over aminoglycoside drugs, crossing the intact blood-brain barrier as well as penetrating phagocytic cells and the barriers of the respiratory tract.5 An additional advantage of enrofloxacin administration to horses is substantial conversion to the active metabolite ciprofloxacin. In plasma, ciprofloxacin concentrations have been shown to achieve 20% to 35% of the parent drug.6 Although ciprofloxacin is poorly absorbed in horses after oral administration, the conversion of enrofloxacin to ciprofloxacin in horses provides greater antimicrobial activity against gram-negative bacteria.7,8
Enrofloxacin is relatively safe when administered to adult horses; however, it is not routinely administered to foals because of the risk of injury to the articular cartilage of young growing animals.9,10 Previous work has shown that enrofloxacin is well tolerated in most horses even when given at high doses (15 to 25 mg/kg, IV) for up to 21 days.11 Reported adverse effects include sporadic, self-limiting musculoskeletal pathologies, such as aseptic tenosynovitis, tendinitis, and cellulitis. Additionally, enrofloxacin is sometimes associated with the development of antimicrobial-induced colitis in horses. Indeed, one study found that enrofloxacin was the most frequently recorded antimicrobial associated with the development of diarrhea requiring hospitalization in the practice areas of Kentucky, New Jersey, and Florida.12 In the aforementioned study, all instances recorded were following IV administration of enrofloxacin; however, in the authors’ experience, enrofloxacin-induced colitis can occur after either oral or IV administration.
Despite the relatively widespread use of enrofloxacin in equine medicine, there are sparse pharmacokinetic (PK) data in horses, and the dose used in a clinical setting varies among clinicians. The Clinical and Laboratory Standards Institute (CLSI) relied on an analysis of 10 published PK datasets to perform Monte Carlo simulations and establish an approved clinical breakpoint for testing equine isolates for susceptibility. The approved breakpoints reported for horses are 0.12 µg/mL, 0.25 µg/mL, and 0.5 µg/mL for the susceptible, intermediate, and resistant interpretive categories, respectively, at an oral dose of 7.5 mg/kg once daily for Escherichia coli, Pasteurella aeruginosa, Staphylococcus spp, and Streptococcus spp.13 The efficacy of enrofloxacin is dependent upon optimizing antimicrobial exposure at the site of infection. The more recently published PK data are limited to plasma concentrations of enrofloxacin and the active metabolite ciprofloxacin in healthy horses.14 Data published more than 25 years ago described enrofloxacin concentrations in the equine CSF and peritoneal fluid; however, the doses evaluated were low, and the methodology employed in that study, a microbiologic assay, tends to overestimate concentrations of enrofloxacin compared to HPLC.15,16 Additionally, there are no data describing enrofloxacin concentrations in equine pulmonary epithelial lining fluid (PELF). In recent years, studies17–19 from both human and veterinary medicine have demonstrated that the concentration of a drug in PELF is a better predictor of efficacy against extracellular bacterial pathogens than either lung tissue or plasma concentrations for antimicrobials. Optimal dosing of enrofloxacin cannot be achieved without PK and pharmacodynamic (PD) data of enrofloxacin at the target tissues.
In human healthcare, the fluoroquinolones (ciprofloxacin, levofloxacin, and moxifloxacin) are considered critically important antibacterial drugs and remain the treatment of choice for zoonotic infections caused by Salmonella and Campylobacteria in many countries. In 2016, the FDA issued a safety warning that stated fluoroquinolones should be reserved for the treatment of complicated infections, in part because of the high potential for disabling side effects in human patients.20 Enrofloxacin, a strictly veterinary fluoroquinolone, use is banned in poultry medicine and restricted in food producing species in the US due to the development of fluoroquinolone resistance in bacteria isolated from food animals as well as zoonotic bacteria isolated in human infection.21 While horses are primarily considered companion animals in the US, accidental human exposure to resistant Salmonella sp, a fecal/orally transmitted zoonotic pathogen, is a potential threat. The purpose of the research conducted herein was in part fueled by the authors’ desire to provide data that might assist in determining the most appropriate dose to potentially reduce resistance pressures and maximize efficacy. Optimizing antimicrobial dosage produces concentrations of enrofloxacin and the metabolite ciprofloxacin necessary for a high probability of target attainment using a PK-PD parameter of area under the free drug concentration-time curve (fAUC)/minimum inhibitory concentration (MIC) of > 72, representing the area under the curve (AUC) of the unbound (free) drug concentration above the MIC of the isolate for a 24-hour interval.22 Thus, the primary objective of this study was to determine a dose of enrofloxacin that will potentially optimize therapeutic outcome while also reducing selection pressure for resistant bacteria important to equine health. We hypothesized that, when given at a dose of 7.5 mg/kg, IV, enrofloxacin will reach an appropriate PK-PD target in plasma. Pulmonary epithelial lining fluid, peritoneal fluid, and CSF concentrations were evaluated to determine the relationship between plasma concentrations and extracellular fluid concentrations in healthy horses.
Methods
This study and all procedures were approved by the IACUC at the University of Georgia College of Veterinary Medicine (AUP #A2022 04-015-A1).
Experimental animals
Six mature, nonpregnant, nonlactating mares ranging from 8 to 23 years old and weighing 423 to 550 kg from the University of Georgia College of Veterinary Medicine Research Herd at the Oconee County Farm were used for this study. Mares were considered healthy based on a normal physical examination as well as normal renal (plasma creatinine concentration) and hepatic (plasma gamma glutamyl transferase activity) biochemical parameters. A baseline musculoskeletal assessment was performed to identify any blemishes or distal limb lesions, and all horses were assessed for baseline lameness using a modified American Association of Equine Practitioners (AAEP) scale.23 Mares were randomly assigned to receive a single IV dose of enrofloxacin at either 5 mg/kg or 7.5 mg/kg in a crossover design with a washout period of 10 days between doses. The dose of 7.5 mg/kg constitutes off-label use. Investigation of doses higher than 7.5 mg/kg was considered but was not pursued as they are likely impractical, cost prohibitive, and potentially increase the likelihood of toxicity.
Drug administration and plasma collection
An IV catheter was placed aseptically in each jugular vein. One catheter was used for enrofloxacin administration, and, following enrofloxacin administration, the catheter was immediately flushed with 10 mL of heparinized flush and removed. The other IV catheter was maintained for blood sample collection. Enrofloxacin (Baytril 100; Elanco) was diluted to a final volume of 60 mL with 0.9% saline and was administered over a 5-minute period. Blood for plasma drug concentration analysis was collected into heparinized tubes at 0 (immediately prior to drug administration), 5, 15, 30, and 60 minutes and 2, 4, 8, 12, and 24 hours after drug administration. Blood was centrifuged at 300 X g for 5 minutes, and plasma was collected and stored in cryovials at −80 °C until analysis.
Bronchoalveolar lavage
Mares were sedated with xylazine (Bimeda) at a dose of 0.5 mg/kg, IV, and a 10-mm-diameter, 2.4-m bronchoalveolar lavage (BAL) catheter (Jorgensen Laboratories) was passed blindly via a nasal approach until wedged into a bronchus at 2, 4, 8, 12, and 24 hours after drug dosing. The lavage solution consisted of 4 aliquots of 60 mL physiologic saline (0.9% NaCl) solution that was infused and aspirated. The total volume of fluid retrieved was recorded for each procedure. Bronchoalveolar fluid was centrifuged at 200 X g for 10 minutes, and the supernatant fluid removed. Supernatant BAL fluid, which represents the PELF, was frozen at −80 °C until assayed.
Cerebrospinal and peritoneal fluid collection
Cerebrospinal and peritoneal fluid were collected from each horse at 2 and 24 hours after drug dosing. Horses were restrained in stocks for CSF collection, and the procedure was performed using standing sedation as described above for BAL, with the addition of 0.01 mg/kg detomidine (Dormosedan; Zoetis), IV. Lidocaine hydrochloride (Covetrus; 200 mg) was infiltrated SC and into the musculature at the C1 to C2 site to provide local anesthesia before the final aseptic preparation of the skin. Atlantoaxial approach to CSF collection was performed as previously described with an 18-gauge, 3.5-inch spinal needle (McKesson); a total of 2 mL of CSF was collected from the collection site.24 Horses’ heads were kept at withers height until sedative effects had abated. Following CSF collection, peritoneal fluid was collected routinely. Briefly, lidocaine hydrochloride (Covetrus; 200 mg) was infiltrated SC and into musculature on the ventral abdomen to the right of midline and just caudal to the xiphoid process after initial preparation of the site (hair clipped and initial scrub). After final aseptic preparation, abdominocentesis was performed as previously described with a teat cannula, and approximately 2 mL of peritoneal fluid was obtained.25 Flunixin meglumine (Banamine; Merck) was administered at a dose of 1.1 mg/kg, IV, at approximately 12 and 24 hours post drug administration to provide analgesia following invasive sampling procedures.
Monitoring
Horses were observed continuously during drug administration and for 1 hour afterward for assessment of neurologic status, bizarre behavior, vocalization, muscle fasciculation, and signs of apprehension or altered locomotion. Mares were examined twice daily for 48 hours following drug administration to monitor for signs of drug-associated adverse effects (depression, anorexia, diarrhea, musculoskeletal abnormalities, or jugular vein phlebitis). Once daily, all limbs were examined visually and palpated for any swelling, thickening, heat, or pain. Horses were jogged in hand for evaluation of soundness, and any lameness noted was scored using the AAEP scale of 0 to 5. The catheter sites were examined, and signs of swelling, pain, thrombosis, or discharge were documented.
Analysis of enrofloxacin and ciprofloxacin concentrations in plasma, PELF, peritoneal fluid, and CSF
Reverse-phase HPLC with fluorescence detection was used to determine the total (and unbound) concentrations of enrofloxacin and the active metabolite ciprofloxacin in the plasma, pulmonary epithelial lining, and cerebrospinal and peritoneal fluids. The assays for enrofloxacin and ciprofloxacin have been previously validated and published.5 Eighty percent water and 20% acetonitrile with 0.1% trifluoroacetic acid were added as a mobile phase modifier. The injection volumes for all fluids analyzed were 20 μL. The limit of quantitation was 0.05 μg/mL for enrofloxacin and ciprofloxacin in all fluids analyzed. Calibration curves and quality control (QC) samples were prepared fresh for each run from fortified blank (control) tissue fluids. The assay of QC samples met the criteria for acceptance with each run, which was based on a linear calibration curve with a R2 value of at least 0.99 and QC samples that were within 15% of the nominal concentration. The limit of quantification and limit of detection were based on the lowest value on a linear calibration curve and acceptable signal-to-noise ratio as described in other guidelines.26,27
Calculation of enrofloxacin and ciprofloxacin concentrations in PELF
An estimation of the volume of PELF was determined by urea dilution method.28,29 Urea concentrations in plasma (UreaPLASMA) and in BAL fluid (UreaBAL) were determined in 96-well plates by use of a commercial kit (Biochain). Plasma samples (5 μL) were run in duplicates and compared to standards (50 mg/dL) using blank subtraction following the manufacturer’s guidelines. Bronchoalveolar lavage fluid was also assayed in duplicate using a sample volume of 50 μL and compared against a standard curve in a similar manner. For plasma and BAL fluid, the absorbance values were read at 520 nm with a spectrophotometer. The volume of PELF (VPELF) in BAL fluid was derived from the following equation: VPELF = VBAL X (UreaBAL/UreaPLASMA), where VBAL is the volume of recovered BAL fluid. The concentration of enrofloxacin and ciprofloxacin in PELF (Enrofloxacin/CiprofloxacinPELF) was derived from the following relationship: Enrofloxacin/CiprofloxacinPELF = Enrofloxacin/CiprofloxacinBAL X (VBAL/VPELF), where Enrofloxacin/CiprofloxacinBAL is the measured concentration of enrofloxacin or ciprofloxacin in BAL fluid. Data derived from these calculations are provided in Supplementary Material S1.
Pharmacokinetic analysis
Statistical analysis
The normality of the data and equality of variance were assessed by use of the Shapiro-Wilk and Levene tests, respectively. Plasma and PELF PK parameters did not meet assumptions for parametric testing, and comparisons between treatments were done using the Wilcoxon sign-rank test. A value of P < .05 was considered significant.
Results
All horses tolerated both the 5-mg/kg and 7.5-mg/kg doses of enrofloxacin without acute adverse reactions. Physical examination parameters (heart rate, respiratory rate, and temperature) remained within normal limits for all horses. All horses had normal borborygmi, passed normal manure, and did not show any signs of colitis during the study period. There were no changes in behavior or mentation noted during the study period. One horse developed SC swelling over the jugular vein consistent with a catheter site reaction of the blood-sampling catheter at the 24-hour monitoring time point. The site was slightly warm and sensitive to palpation, but the vein filled appropriately, with no evidence of thrombus formation. Topical diclofenac (Surpass; Boehringer Ingelheim Animal Health) was applied to the site twice daily for 72 hours at which time the site was improved, and treatment was discontinued. One horse in the 5-mg/kg dose group developed focal edema associated with the right lateral digital extensor tendon at 24 hours post enrofloxacin administration. The horse walked comfortably but was sensitive to palpation. The edema and sensitivity resolved without any specific treatment by 72 hours post enrofloxacin administration. All 6 horses developed transient, mild, SC, nonpainful swelling at the atlantoaxial centesis and abdominocentesis sites, which were all self-limiting and required no specific treatment.
The results of the PK analysis of drug plasma concentrations are shown in Table 1. Quantifiable enrofloxacin and ciprofloxacin concentrations were present in the plasma of all 6 horses within 5 minutes of drug administration regardless of the dose administered. The median maximum concentration extrapolated to time 0 and the area under the plasma concentration-versus-time curve from time 0 to the last quantifiable time point of 24 hours (AUC0-24) for enrofloxacin in plasma were significantly greater following administration of enrofloxacin at 7.5 mg/kg than 5 mg/kg. Similarly, median λz, terminal half-life, peak serum concentration (Cmax), area under the plasma concentration-versus-time curve from time 0 to the last quantifiable time point (24 hours), area under the plasma concentration-versus-time curve extrapolated to infinity, and mean residence time for ciprofloxacin in plasma were significantly greater following administration of enrofloxacin at 7.5 mg/kg than 5 mg/kg. The plasma concentrations-versus-time profiles of both enrofloxacin and ciprofloxacin are shown in Figure 1.
Pharmacokinetic variables (median and 10th and 90th percentiles) for enrofloxacin and its main metabolite, ciprofloxacin, in plasma after IV administration of enrofloxacin at doses of 5 mg/kg and 7.5 mg/kg to healthy adult mares (n = 6).
| Dose | |||
|---|---|---|---|
| Variable | 5 mg/kg | 7.5 mg/kg | P value |
| Enrofloxacin | |||
| λz (L/h) | 0.086 (0.067–0.097) | 0.102 (0.055–0.106) | .345 |
| t1/2 (h) | 8.23 (5.29–10.3) | 6.78 (4.29–12.7) | .463 |
| Cl (L/h/kg) | 0.196 (0.154–0.461) | 0.228 (0.168–0.280) | .916 |
| AUC0–t (µg·h/mL) | 22.5 (10.3–26.7) | 30.4 (25–34.1) | .046 |
| AUC0–∞ (µg·h/mL) | 25.5 (10.8–32.5) | 33 (26.7–44.6) | .116 |
| Vd (L/kg) | 2.50 (2.12–4.10) | 2.25 (1.7–3.07) | .173 |
| MRT (h) | 10.8 (5.8–14.2) | 8.15 (6–16) | .463 |
| C0 (µg/mL) | 1.70 (0.80–2.10) | 3.15 (2–4.60) | .046 |
| Ebody (%) | 0.34 (0.27–0.80) | 0.39 (0.28–0.49) | .917 |
| Ciprofloxacin | |||
| λz (l/h) | 0.065 (0.024–0.085) | 0.071 (0.062–0.097) | .028 |
| t1/2 (h) | 10.8 (8.16–29.2) | 9.79 (7.15–11.1) | .028 |
| Cmax (µg/mL) | 0.2 (0.14–0.25) | 0.38 (0.22–0.61) | .035 |
| Tmax (h) | 0.75 (0.5–1) | 0.5 (0.5–1) | .317 |
| AUC0–t (µg·h/mL) | 2.4 (2.1–2.5) | 3.7 (2.4–4.2) | .046 |
| AUC0–∞ (µg·h/mL) | 3 (2.7–5.4) | 4.55 (3.1–5.3) | .027 |
| MRT (h) | 15.6 (13.5–40.5) | 13.8 (10.7–16.2) | .075 |
λz = Elimination rate constant. AUC0–∞ = Area under the plasma concentration-versus-time curve extrapolated to infinity. AUC0–t = Area under the plasma concentration-versus-time curve from time 0 to the last quantifiable time point (24 hours). C0 = Maximum concentration extrapolated to time 0. Cl = Systemic plasma clearance. Cmax = Peak serum concentration. Ebody = Global extraction ratio. MRT = Mean residence time. t½ = Half-life of the terminal phase. Tmax = Time to peak plasma concentration. Vd = Apparent volume of distribution based on the area under the curve.
Mean (± SD) concentrations of enrofloxacin and its main metabolite, ciprofloxacin, in the plasma of healthy adult mares (n = 6) after IV administration of enrofloxacin at doses of 5 mg/kg and 7.5 mg/kg.
Citation: American Journal of Veterinary Research 86, 4; 10.2460/ajvr.24.08.0229
The results of the PK analysis of enrofloxacin in PELF are shown in Table 2. There were no significant differences in any of the measured variables. Concentrations of ciprofloxacin were below the limit of quantification of the HPLC assay at most of the time points evaluated regardless of dose administered. The mean concentration-versus-time profiles of enrofloxacin and ciprofloxacin in PELF are shown in Figure 2.
Pharmacokinetic variables (median and 10th and 90th percentiles) for enrofloxacin in pulmonary epithelial lining fluid (PELF) after IV administration of enrofloxacin at doses of 5 mg/kg and 7.5 mg/kg to healthy adult mares (n = 6).
| Enrofloxacin dose | |||
|---|---|---|---|
| Variable | 5 mg/kg | 7.5 mg/kg | P value |
| λz (L/h) | 0.163 (0.072–0.575) | 0.264 (0.089–0.519) | .345 |
| t1/2 (h) | 4.54 (1.21–9.66) | 2.65 (1.34–7.79) | .463 |
| AUC0–t (µg·h/mL) | 11.7 (3.2–28.7) | 15.6 (5.3–37.8) | .833 |
| AUC0–∞ (µg·h/mL) | 12.2 (3.2–37) | 16.4 (6.2–39.8) | .833 |
| Cmax (μg/mL) | 1.71 (1.1–3.44) | 2.16 (0.48–5.19) | .140 |
| Tmax (h) | 3 (2–8) | 2 (2–4) | .493 |
| MRT (h) | 7.85 (4.4–16.2) | 9.55 (5.5–12.8) | .753 |
Cmax = Maximum concentration in PELF.
Mean (± SD) concentrations of enrofloxacin and its main metabolite, ciprofloxacin, in the pulmonary epithelial lining fluid (PELF) of healthy adult mares (n = 6) after IV administration of enrofloxacin at doses of 5 mg/kg and 7.5 mg/kg.
Citation: American Journal of Veterinary Research 86, 4; 10.2460/ajvr.24.08.0229
The mean (± SD) concentrations of enrofloxacin and ciprofloxacin in plasma, PELF, CSF, and peritoneal fluid at 2 and 24 hours after enrofloxacin administration as well as the fluid:plasma ratio at each time point are shown in Table 3. Little-to-no ciprofloxacin was detected in any fluid matrix. Nevertheless, at 24 hours post enrofloxacin administration at a dose of 5 mg/kg, the peritoneal fluid:plasma ratio of ciprofloxacin was 2, suggesting that ciprofloxacin might have a greater ability to penetrate into this fluid matrix than the others evaluated due to its enhanced water solubility.
Mean ± SD concentrations (in μg/mL), as well as fluid:plasma ratios, of enrofloxacin and its main metabolite, ciprofloxacin, in plasma and body fluids at 2 and 24 hours after IV administration of enrofloxacin at doses of 5 mg/kg and 7.5 mg/kg to healthy adult mares (n = 6).
| Time after enrofloxacin administration (h) | ||||||||
|---|---|---|---|---|---|---|---|---|
| 2 | 24 | |||||||
| Dose | 5 mg/kg | Fluid:plasma | 7.5 mg/kg | Fluid:plasma | 5 mg/kg | Fluid:plasma | 7.5 mg/kg | Fluid:plasma |
| Enrofloxacin | ||||||||
| Plasma | 1.90 ± 0.44 | Referent | 2.69 ± 0.57 | Referent | 0.23 ± 0.15 | Referent | 0.30 ± 0.15 | Referent |
| PELF | 1.11 ± 0.46 | 0.582 | 2.90 ± 1.8 | 1.09 | 0.19 ± 0.24 | 0.852 | 0.32 ± 0.26 | 1.08 |
| CSF | 0.69 ± 0.14 | 0.361 | 1.10 ± 0.49 | 0.495 | 0.03 ± 0.05 | 0.147 | 0.13 ± 0.14 | 0.652 |
| PF | 1.03 ± 0.75 | 0.538 | 2.20 ± 0.28 | 0.955 | 0.13 ± 0.11 | 0.582 | 0.21 ± 0.11 | 1.01 |
| Ciprofloxacin | ||||||||
| Plasma | 0.17 ± 0.03 | Referent | 0.28 ± 0.08 | Referent | 0.05 ± 0.01 | Referent | 0.06 ± 0.01 | Referent |
| PELF | 0.00 ± 0.01 | 0 | 0.02 ± 0.03 | 0.071 | 0.00 ± 0.00 | 0 | 0.02 ± 0.03 | 0.0333 |
| CSF | 0.00 ± 0.00 | 0 | 0.03 ± 0.03 | 0.107 | 0.00 ± 0.00 | 0 | 0.00 ± 0.00 | 0 |
| PF | 0.04 ± 0.03 | 0.235 | 0.01 ± 0.01 | 0.036 | 0.10 ± 0.03 | 2.00 | 0.02 ± 0.00 | 0.333 |
PF = Peritoneal fluid.
The results of the calculations for AUC0–24/MIC of enrofloxacin and ciprofloxacin in plasma after administration of 5-mg/kg and 7.5-mg/kg doses are shown in Tables 4 and 5.
Area under the concentration from time 0 to 24 hours (AUC0–24)/minimum inhibitory concentration (MIC) of enrofloxacin and its metabolite, ciprofloxacin, in plasma after administration of a single 5-mg/kg, IV, dose of enrofloxacin.
| MIC values (μg/mL) | |||||||
|---|---|---|---|---|---|---|---|
| Antimicrobial | 0.03 | 0.06 | 0.125 | 0.25 | 0.5 | 1 | |
| AUC0–24 (μg·h/mL) | AUC0–24/MIC | ||||||
| Enrofloxacin | 22.5 | 750 | 375 | 180 | 90 | 45 | 22.5 |
| Ciprofloxacin | 2.4 | 80 | 40 | 19.2 | 9.6 | 4.8 | 2.4 |
| Enrofloxacin + ciprofloxacin | 24.9 | 830 | 415 | 199 | 99.6 | 49.8 | 24.9 |
Area under the concentration from time 0 to 24 hours (AUC0–24)/MIC of enrofloxacin and its metabolite, ciprofloxacin, in plasma after administration of a single 7.5-mg/kg, IV, dose of enrofloxacin.
| MIC values (μg/mL) | |||||||
|---|---|---|---|---|---|---|---|
| Antimicrobial | 0.03 | 0.06 | 0.125 | 0.25 | 0.5 | 1 | |
| AUC0–24 (μg·h/mL) | AUC0–24/MIC | ||||||
| Enrofloxacin | 30.4 | 1,013 | 507 | 243 | 122 | 60.8 | 30.4 |
| Ciprofloxacin | 3.7 | 123 | 61.7 | 29.6 | 14.8 | 7.4 | 3.7 |
| Enrofloxacin + ciprofloxacin | 34.1 | 1,137 | 568 | 273 | 137 | 68.2 | 34.1 |
Discussion
Enrofloxacin is almost exclusively used to provide gram-negative bacterial isolate coverage in critically ill or renally impaired adult horses with polymicrobial infections. Other indications include the need for an oral agent to treat a susceptible bacterial infection for which the FDA approved trimethoprim-sulfadiazine product is an inappropriate therapy. Enrofloxacin has been studied in horses, but the dose recommendations vary. One study15 recommended a dose of 5.5 mg/kg, IV, or 7.5 mg/kg orally once a day, whereas another31 suggested 7.5 mg/kg, IV, or orally once a day may be effective. Within the authors’ practice, the dose of IV enrofloxacin varies from 5 mg/kg to 7.5 mg/kg and is clinician dependent. The approved CLSI breakpoint (provided earlier) for testing bacterial isolates from horses used an oral dose of 7.5 mg/kg once daily (Table E in CLSI Vet01[S]),13 which was the consensus from the experts polled by the CLSI during the analysis (polling data not shown). Due to the wide range of doses reported, standardization of dosing recommendations for enrofloxacin would be helpful. The doses used in this study were chosen based on current clinical prescribing practices (5 mg/kg, IV, to 7.5 mg/kg, IV) and extrapolations from previous data that suggest higher IV doses are needed to reach therapeutic levels for many gram-negative bacterial isolates.11 An fAUC/MIC of > 72 is needed to reach a high probability of clinical cure.32 If the dose does not reach concentrations to exceed this value, there is a risk of therapeutic failure and selection of drug-resistant bacteria.33 However, while the fAUC/MIC target of 72 is provided based on updated CLSI guidelines, the authors caution against broad application as this target is veterinary species and bacterial pathogen dependent. Although PK-PD analysis should always use unbound drug concentrations in plasma, this study provides additional information on the extracellular fluid concentrations achieved in treated horses. Other studies15 that reported enrofloxacin concentrations in the equine CSF and peritoneal fluid used doses of 2.5 mg/kg and 5 mg/kg, which is lower than the doses examined in the current study. In addition, the methodology employed to determine enrofloxacin concentrations in the aforementioned study, namely a microbiologic assay, tends to overestimate concentrations of enrofloxacin compared to HPLC. Microbiologic assays should not be used for PK studies of enrofloxacin.15,16 There are no reports of enrofloxacin and ciprofloxacin concentrations in the PELF of horses despite extensive analysis in cattle.5 This information will inform equine veterinarians using enrofloxacin to treat respiratory infections in horses.
The results presented in our study show that a dose of 7.5 mg/kg, IV, produced plasma enrofloxacin concentrations for fAUC:MIC ratios high enough for most susceptible bacteria. Plasma ciprofloxacin concentrations for fAUC:MIC ratios were not high enough for most bacteria, but enrofloxacin and ciprofloxacin have an additive effect, and the AUC from each drug should be added for PK-PD analysis.8,34
The concentration in PELF for fAUC/MIC ratio was below 72 for both the 5-mg/kg and 7.5-mg/kg dose. As shown in Figure 2, ciprofloxacin was not detected in the PELF.
The tissue:plasma concentration ratios were calculated for the PELF, CSF, and peritoneal fluid at 2 and 24 hours and demonstrated a higher ratio for the 2- and 24-hour time point at the 7.5-mg/kg, IV, dose of enrofloxacin compared to the 5-mg/kg, IV, dose. These results showed that a higher dose of enrofloxacin produces greater penetration into these body fluids, most likely because of a higher concentration gradient. Additionally, not only were the concentrations higher, but the 7.5-mg/kg dose showed that it had greater persistence in these body cavities.
In comparison, the fluid:plasma ratio for ciprofloxacin for PELF and CSF was 0 for both 5 mg/kg and 7.5 mg/kg at 2 and 24 hours. The fluid:plasma ratio for ciprofloxacin for peritoneal fluid was lower than that of enrofloxacin at both 2 and 24 hours. As previously mentioned, there was no accumulation of ciprofloxacin, likely because it is a less lipophilic compound.
There was no statistical difference for global extraction ratio between the 5-mg/kg and 7.5-mg/kg dose of enrofloxacin, supporting that this was not dose dependent. However, at both doses the whole-body extraction ratio was high, indicating that the horses have high clearance of enrofloxacin; however, the organ of clearance (liver or kidney) is unknown for horses. During this study, the horses were sedated multiple times with an α-2 adrenergic agonist (xylazine and detomidine). The effect that these sedatives may have had on enrofloxacin and ciprofloxacin clearance is undetermined without additional study.
There was high between-subject (inter-individual) variability in our study. The urea dilution method was used to estimate the volume of PELF. Although it is a common methodology for determining PELF volume, it is important to understand that this method has limitations. It may result in overestimation of urea concentrations in BAL fluid, leading to a false increase in the volume of PELF.28 Therefore, this method could lead to underestimation of drug concentration in PELF.18,35 In calves, 2 methods for sampling PELF have been compared.5 These methods included collecting BAL fluid and then performing the urea collection method, which is what was used in this current study, and direct sampling via bronchial swab collection. With the direct sampling method, the filter paper’s weight determines the volume of PELF. The results of that study showed that when measuring enrofloxacin, direct sampling was more consistent than BAL, and there was less variation among the calves. It is possible that the BAL and urea dilution method may have contributed to the interhorse variation in the current study.
Tendinopathy is occasionally reported with enrofloxacin administration in adult horses.11 Therefore, each horse had their limbs palpated prior to drug administration and then again at 12, 24, 36, and 48 hours. The horses were jogged in a straight line prior to drug administration and then again at 24 and 48 hours. However, a limitation of this study was accurately evaluating lameness because the horses received 1.1-mg/kg doses of flunixin meglumine at 12 and 24 hours. Four horses were lame at the beginning of the study (AAEP grade 3/5), but their lameness improved during their repeat lameness examinations. This most likely corresponded to the analgesic and anti-inflammatory effects of flunixin meglumine.
One horse developed focal edema over the lateral digital extensor tendon on the right front limb that was reactive on palpation at 48 hours. The horse’s baseline mild lameness was present at this time. The horse received an additional 1.1-mg/kg, IV, dose of flunixin meglumine to manage the mild pain and swelling. The horse did not develop any other musculoskeletal signs, and the swelling resolved by 72 hours without any further treatment. It was the authors’ opinion that this was related to trauma that could have occurred while the horse was stalled rather than an adverse effect of drug administration. No other horses showed any evidence of musculoskeletal disease related to enrofloxacin administration.
Antibiotic-associated diarrhea is a possible serious complication related to antibiotic use in horses. In this study, none of the horses developed clinical signs of colitis. However, horses only received single doses of enrofloxacin, and enrofloxacin was not combined with any other antibiotic. Intravenous administration of ciprofloxacin to horses reliably produced mild-to-severe gastrointestinal complications in horses; these complications appear to be avoided or reduced when exposure to ciprofloxacin follows enrofloxacin metabolism.7
Other limitations of this study included small sample size, IV route only, and a single-dose study. The PK of orally administered enrofloxacin in horses have been reported in multiple other studies.14,15,36 While it would have been advantageous to look at various oral doses, this would have greatly expanded the scope and cost of our study (inclusion of more animals and/or time to provide additional washout periods), and due to both financial and available animal limitations the oral route was not investigated in this study. Additionally, protein binding was not evaluated due to budgetary constraints. However, plasma protein binding for fluoroquinolones in horses is stable and ranges from 21% to 28%.37
In conclusion, there is no apparent PK advantage to the higher dose when targeting isolates classified as susceptible using CLSI breakpoints. A single 7.5-mg/kg, IV, dose of enrofloxacin was well tolerated in the horses. Finally, administration of enrofloxacin at the 7.5-mg/kg IV dose resulted in higher concentrations in plasma and the other matrix sampled as well as greater persistence in the peritoneal fluid, PELF and CSF fluid.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors acknowledge Rachel Anders, Natalia Rodriguez, and Roya Shizard.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
Funded by the University of Georgia for Love of The Horse Fund.
ORCID
M. A. Larson https://orcid.org/0009-0008-3793-0152
References
- 1.↑
Lizondo M, Pons M, Gallardo M, Estelrich J. Physicochemical properties of enrofloxacin. J Pharm Biomed Anal. 1997;15(12):1845–1849. doi:10.1016/S0731-7085(96)02033-X
- 2.↑
Byrne BA, Tell LA, Martin K, Payne M. Chapter 45: Rational Antimicrobial Therapy. 1545–1553. In: Smith BP, Van Metre DC, Pusterla N, eds. Large Animal Internal Medicine. 6th ed. Elsevier; 2020.
- 3.↑
Foster JD, Abouraya M, Papich MG, Muma NA. Population pharmacokinetic analysis of enrofloxacin and its active metabolite ciprofloxacin after intravenous injection to cats with reduced kidney function. J Vet Intern Med. 2023;37(6):2230–2240. doi:10.1111/jvim.16866
- 4.↑
Martinez M, McDermott P, Walker R. Pharmacology of the fluoroquinolones: a perspective for the use in domestic animals. Vet J. 2006;172(1):10–28. doi:10.1016/j.tvjl.2005.07.010
- 5.↑
Foster DM, Sylvester HJ, Papich MG. Comparison of direct sampling and bronchoalveolar lavage for determining active drug concentrations in the pulmonary epithelial lining fluid of calves injected with enrofloxacin or tilmicosin. J Vet Pharmacol Ther. 2017;40(6):e45–e53. doi:10.1111/jvp.12412
- 6.↑
Kaartinen L, Panu S, Pyörälä S. Pharmacokinetics of enrofloxacin in horses after single intravenous and intramuscular administration. Equine Vet J. 1997;29(5):378–381. doi:10.1111/j.2042-3306.1997.tb03143.x
- 7.↑
Yamarik TA, Wilson WD, Wiebe VJ, Pusterla N, Edman J, Papich MG. Pharmacokinetics and toxicity of ciprofloxacin in adult horses. J Vet Pharmacol Ther. 2010;33(6):587–594. doi:10.1111/j.1365-2885.2010.01167.x
- 8.↑
Blondeau JM, Borsos S, Blondeau LD, Blondeau BJ. In vitro killing of Escherichia coli, Staphylococcus pseudintermedius and Pseudomonas aeruginosa by enrofloxacin in combination with its active metabolite ciprofloxacin using clinically relevant drug concentrations in the dog and cat. Vet Microbiol. 2012;155(2–4):284–290. doi:10.1016/j.vetmic.2011.08.015
- 9.↑
Davenport CL, Boston RC, Richardson DW. Effects of enrofloxacin and magnesium deficiency on matrix metabolism in equine articular cartilage. Am J Vet Res. 2001;62(2):160–166. doi:10.2460/ajvr.2001.62.160
- 10.↑
Vivrette SL, Bostian A, Bermingham E, Papich MG. Quinolone-induced arthropathy in neonatal foals. In: Proceedings of the 47th Annual Convention of the American Association of Equine Practitioners. American Association of Equine Practitioners; 2001:376–377.
- 11.↑
Bertone AL, Tremaine WH, Macoris DG, et al. Effect of long-term administration of an injectable enrofloxacin solution on physical and musculoskeletal variables in adult horses. J Am Vet Med Assoc. 2000;217(10):1514–1521. doi:10.2460/javma.2000.217.1514
- 12.↑
Barr BS, Waldridge BM, Morresey PR, et al. Antimicrobial-associated diarrhea in three equine referral practices. Equine Vet J. 2013;45(2):154–158. doi:10.1111/j.2042-3306.2012.00595.x
- 13.↑
Clinical Laboratory and Standards Institute. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals. 7th ed. Clinical and Laboratory Standards Institute; 2024.
- 14.↑
Haines GR, Brown MP, Gronwall RR, Merritt KA. Serum concentrations and pharmacokinetics of enrofloxacin after intravenous and intragastric administration to mares. Can J Vet Res. 2000;64(3):171–177.
- 15.↑
Giguère S, Sweeney RW, Bélanger M. Pharmacokinetics of enrofloxacin in adult horses and concentration of the drug in serum, body fluids, and endometrial tissues after repeated intragastrically administered doses. Am J Vet Res. 1996;57(7):1025–1030. doi:10.2460/ajvr.1996.57.07.1025
- 16.↑
Küng K, Riond JL, Wolffram S, Wanner M. Comparison of an HPLC and bioassay method to determine antimicrobial concentrations after intravenous and oral administration of enrofloxacin in four dogs. Res Vet Sci. 1993;54(2):247–248. doi:10.1016/0034-5288(93)90065-N
- 17.↑
Wise R, Baldwin DR, Andrews JM, Honeybourne D. Comparative pharmacokinetic disposition of fluoroquinolones in the lung. J Antimicrob Chemother. 1991;28(suppl C):65–71. doi:10.1093/jac/28.suppl_C.65
- 18.↑
Giguère S, Huang R, Malinski TJ, Dorr PM, Tessman RK, Somerville BA. Disposition of gamithromycin in plasma, pulmonary epithelial lining fluid, bronchoalveolar cells, and lung tissue in cattle. Am J Vet Res. 2011;72(3):326–330. doi:10.2460/ajvr.72.3.326
- 19.↑
Menge M, Rose M, Bohland C, et al. Pharmacokinetics of tildipirosin in bovine plasma, lung tissue, and bronchial fluid (from live, nonanesthetized cattle). J Vet Pharmacol Ther. 2012;35(6):550–559. doi:10.1111/j.1365-2885.2011.01349.x
- 20.↑
FDA advises restricting use of fluoroquinolones for certain infections. US FDA. May 12, 2016. Accessed: September 25 2024. https://www.fda.gov/drugs/drug-safety-and-availability/fda-drug-safety-communication-fda-advises-restricting-fluoroquinolone-antibiotic-use-certain
- 21.↑
Jensen LB, Angulo FJ, Mølbak K, Wegener HC. In: Guardabassi L, Jensen LB, Kruse H, eds. Human health risks associated with antimicrobial use in animals. Guide to Antimicrobial Use in Animals. Blackwell; 2008:13–26.
- 22.↑
Clinical Laboratory and Standards Institute. Development of Quality Control Ranges, Breakpoints, and Interpretive Categories for Antimicrobial Agents Used in Veterinary Medicine. 4th ed. Clinical and Laboratory Standards Institute; 2021.
- 23.↑
Pluim M, Martens A, Vanderperren K, et al. Short- and long-term follow-up of 150 sports horses diagnosed with tendinopathy or desmopathy by ultrasonographic examination and treated with high-power laser therapy. Res Vet Sci. 2018;119:232–238. doi:10.1016/j.rvsc.2018.06.003
- 24.↑
Pease A, Behan A, Bohart G. Ultrasound-guided cervical centesis to obtain cerebrospinal fluid in the standing horse. Vet Radiol Ultrasound. 2012;53(1):92–95. doi:10.1111/j.1740-8261.2011.01855.x
- 25.↑
Radcliffe RM, Hill JA, Liu SY, Cook VL, Hurcombe SDA, Diver TJ. Abdominocentesis techniques in horses. J Vet Emerg Crit Care (San Antonio). 2022;32(suppl 1):72–80. doi:10.1111/vec.13118
- 26.↑
Validation of analytical procedures: text and methodology. International Conference on Harmonisation of Technical Requirements for Requirements for Registration of Pharmaceuticals for Human Use. Accessed June 23 2024. https://database.ich.org/sites/default/files/Q2(R1)%20Guideline.pdf
- 27.↑
USP. General chapter. <1225> Validation of Compendial Procedures. United States Pharmacopeia; 2023.
- 28.↑
Marcy TW, Merrill WW, Rankin JA, Reynolds HY. Limitations of using urea to quantify epithelial lining fluid recovered by bronchoalveolar lavage. Am Rev Respir Dis. 1987;135(6):1276–1280. doi:10.1164/arrd.1987.135.6.1276
- 29.↑
Rennard SI, Basset G, Lecossier D, et al. Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution. J Appl Physiol (1985). 1986;60(2):532–538. doi:10.1152/jappl.1986.60.2.532
- 30.↑
Toutain PL, Bousquet-Mélou A. Plasma clearance. J Vet Pharmacol Ther. 2004;27(6):415–425. doi:10.1111/j.1365-2885.2004.00605.x
- 31.↑
Boeckh S, Buchanan C, Boeckh A, et al. Pharmacokinetics of the bovine formulation of enrofloxacin (Baytril 100) in horses. Vet Ther. 2001;2(2):129–134.
- 32.↑
Papich MG, Gunnett LA, Lubbers BV. Revision of fluoroquinolone breakpoints used for interpretation of antimicrobial susceptibility testing of canine bacterial isolates. Am J Vet Res. 2023;84(11):ajvr.23.07.0159. doi:10.2460/ajvr.23.07.0159
- 33.↑
Martinez MN, Papich MG, Drusano GL. Dosing regimen matters: the importance of early intervention and rapid attainment of the pharmacokinetic/pharmacodynamic target. Antimicrob Agents Chemother. 2012;56(6):2795–2805. doi:10.1128/AAC.05360-11
- 34.↑
Lautzenhiser SJ, Fialkowski JP, Bjorling D, Rosin E. In vitro antibacterial activity of enrofloxacin and ciprofloxacin in combination against Escherichia coli and staphylococcal clinical isolates from dogs. Res Vet Sci. 2001;70(3):239–241. doi:10.1053/rvsc.2001.0466
- 35.↑
Baldwin DR, Honeybourne D, Wise R. Pulmonary disposition of antimicrobial agents: methodological considerations. Antimicrob Agents Chemother. 1992;36(6):1171–1175. doi:10.1128/AAC.36.6.1171
- 36.↑
Ellerbrock RE, Curcio BR, Zhong L, et al. Pharmacokinetics of intravenous and oral administration of enrofloxacin to the late-term pregnant and non-pregnant mares. Equine Vet J. 2020;52(3):464–470. doi:10.1111/evj.13175
- 37.↑
Mercer M. Quinolones, including fluoroquinolones, use in animals. Merck Veterinary Manual. Updated September 2022. Accessed June 20 2024. https://www.merckvetmanual.com/pharmacology/antibacterial-agents/quinolones-including-fluoroquinolones-use-in-animals
