Equine bacterial pneumonia is often caused by polymicrobial infections and requires long-term antimicrobial treatment.1–3 Oral antimicrobial medications are crucial for managing the condition and should ideally have a high volume of distribution, good pulmonary penetration, broad-spectrum activity, and reduced administration frequency.3 However, oral antimicrobials for horses often have limitations in spectrum of activity, volume of distribution, sufficient bioavailability, or resistance against common respiratory pathogens.4–7
Chloramphenicol, a time-dependent, bacteriostatic antimicrobial, binds to the 50S subunit of the 70S bacterial ribosome, demonstrating broad-spectrum activity against aerobic and anaerobic bacteria.8 Studies9–12 in various species show that chloramphenicol has a high volume of distribution and wide tissue penetration. Oral formulations, including commercial tablets and compounded suspension for equine use are commonly available, affordable, and already frequently prescribed by equine veterinary practitioners.
Chloramphenicol could be considered among the options for long-term treatment of complicated polymicrobial lung infections, involving not easily treatable anaerobic bacteria or opportunistic bacterial strains that show resistance to narrow-spectrum antibiotics.
Despite its properties, recent pharmacokinetic studies in healthy adult horses show highly variable individual oral bioavailability.13–15 Reported serum concentrations often fall below the recommended susceptibility breakpoint for most bacteria or are not maintained above the MIC for at least half of the dosing interval, which is necessary for a time-dependent efficacy.13–15 A susceptibility breakpoint of ≤ 8 µg/mL is recommended for most airway pathogens, with lower breakpoints of ≤ 4 μg/mL or ≤ 2 µg/mL for selected bacteria, such as certain anaerobes and Streptococcus species, respectively.13,14,16
When treating bacterial pneumonia, achieving adequate antimicrobial concentrations in the lower airways, particularly in the pulmonary epithelial lining fluid (PELF), is crucial.17,18 Plasma pharmacokinetics do not necessarily reflect pulmonary pharmacokinetics; thus, measuring drug concentrations in PELF and airway cells is preferred to predict intrapulmonary activity.4,17,19–24 The pulmonary distribution and pharmacokinetics of chloramphenicol in adult horses have not previously been reported. However, minocycline, another lipophilic oral antimicrobial, shows superior concentrations in PELF compared to plasma in adult horses.20 Therefore, determining if therapeutic pulmonary concentrations can be achieved and sustained is necessary to support chloramphenicol use in equine pneumonia treatment.
The primary objective of this study was to describe the pulmonary disposition and pharmacokinetics of a single dose of chloramphenicol administered to healthy, fasted adult horses orally (commercial tablets or compounded suspension) and intragastrically (compounded suspension). We hypothesized that for all formulations and routes of administration, chloramphenicol would be detected in the PELF and in the bronchoalveolar lavage (BAL) fluid (BALF) cells after drug administration. We also hypothesized that maximum drug concentration and total drug exposure would be higher in the PELF than in plasma, regardless of formulation and route of administration, and that only PELF concentrations would remain within a clinically significant MIC range for relevant equine pathogens (2 to 8 µg/mL) for at least 4 hours (50% to 75% of a 6- to 8-hour dosing interval).
A secondary objective was to assess the impact of the type of formulation (tablet vs suspension) and route of administration (oral vs intragastric) on plasma and pulmonary pharmacokinetics. We hypothesized that bioavailability, maximum concentration, and total drug exposure in PELF and plasma would not differ according to formulation but would be higher and less variable after intragastric compared to oral administration.
Methods
Animals
Six university-owned healthy adult (9- to 16-year-old) mixed-breed geldings, ranging in body weight from 510 to 660 kg, were included. Sample size was calculated by power analysis (G*power), estimating α = 0.05, β = 0.8, and a large size effect based on the Cohen index of 0.8. Horses were considered healthy based on a history of no illness within the previous 6 months, physical examination, rebreathing examination, and CBC and blood biochemistry. Horses were housed in individual stalls for a minimum of 12 hours before and throughout each experimental condition. During washout periods, horses were maintained on pastures. Horses had free access to water and were kept on their standard feeding regimen, except when fasted for experimental purposes. Physical examinations were performed before and then every 12 hours during each experimental period. Horses were monitored hourly during each experimental condition and then daily during the washout periods. All procedures were reviewed and approved by the Auburn University IACUC (protocols No. 2021-3919 and 2023-5281).
Experimental design
Two prospective experiments (experiments 1 and 2) were conducted with a minimum washout of 21 days between experiments.
Experiment 1 was designed to provide complementary IV pharmacokinetic data in a subset of the horses (IV protocol). Three of the 6 study horses were randomly selected for administration of a single 25-mg/kg dose of IV chloramphenicol sodium succinate (chloromycetin; 100 mg/mL), while they remained on a standard feeding regimen. Experiment 2 included all 6 horses and was designed as a 3-treatment, Latin-square randomized, crossover study with a minimum 7-day washout period between each treatment. Horses were fasted for 12 hours before and 1 hour after administration of a single 50-mg/kg dose chloramphenicol according to the following protocols: oral chloramphenicol commercial tablets (Viceton; 1 g/tablet), named the tablet PO (TPO) protocol; oral chloramphenicol compounded suspension (Wedgewood pharmacy, 500 mg/mL), named the suspension PO (SPO) protocol; and intragastric chloramphenicol compounded suspension (Wedgewood pharmacy, 500 mg/mL), named the suspension intragastric (SIG) protocol.
In both experiments, blood samples were collected immediately before (time 0) and 5, 10, 15, 20, and 30 minutes and 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours after drug administration. Bronchoalveolar lavage was performed 1, 4, and 8 hours after drug administration for determination of chloramphenicol concentrations in the PELF and BALF cells. These time points corresponded to the predicted time of maximum concentration (1 hour), to 50% to 75% of a 6- to 8-hour dosing interval (4 hours), and to the end of an 8-hour dosing interval. Feed was withheld for 40 minutes after BAL to allow for recovery from sedation.
Drug administration
In experiment 1, chloramphenicol was administered as a single, nondiluted injection via a dedicated jugular catheter, different from the catheter used for blood sample collection. In experiment 2, for the TPO protocol, the calculated number of tablets was split into two 60-cc catheter tip oral syringes and allowed to dissolve with 45 mL of water each to form a slurry for 8 hours before administration. As chloramphenicol remains stable for over 24 hours when in aqueous solution,25 the operator was not directly exposed to the tablets when crushing them. For SPO and SIG protocols, the calculated amount of compounded suspension was drawn up into a 60-cc catheter tip syringe immediately before experiments. A 10-mL aliquot of compounded suspension from each bottle was frozen at the end of the experimental period and stored at −80 °C for measurement of drug concentration. For oral administration (SPO and TPO protocols), tablet slurry or compounded suspension was directly administered to the horse mouth via the catheter tip syringe. For the SIG protocol, the suspension was administered to nonsedated, physically restrained horses, via standard nasogastric intubation. The tube was flushed with 1 L of water to ensure complete delivery of the drug to the stomach.
Blood sampling and processing
In both experiments, serial blood samples were collected via a dedicated jugular catheter. Samples were then immediately placed into sodium heparin tubes on ice and subsequently centrifuged (400 X g for 10 minutes at 4 °C). Plasma was separated and stored at −80 °C until analysis.
Bronchoalveolar lavage procedure and BALF processing
The BAL procedure is described in Supplementary Material S1. The total recovered BALF was pooled, and the volume was recorded. A 5-mL aliquot was stored in EDTA for determination of total nucleated cell count and cytology. The remainder was immediately centrifuged (2,654 X g for 10 minutes) to separate the BAL cells from the supernatant. Aliquots of supernatant, containing the PELF, were separated, and stored at −80 °C until analysis. The remainder of the supernatant was discarded, and the BAL cell pellet was rinsed by centrifugation (2,654 X g for 10 minutes) using PBS. The supernatant was discarded, and the pellet was resuspended in PBS and stored at −80 °C until analysis.
Measurement of chloramphenicol concentrations via liquid chromatography–tandem mass spectrometry
Sample analysis was performed according to previous methods.13,14 A detailed description is available for reproducibility in Supplementary Material S1.
Determination of chloramphenicol concentrations in PELF and BAL cells
where ChlorPellet represents the chloramphenicol concentration measured by LC-MS-MS in the cell pellet and VolCell stands for the mean volume of horse BAL cells, previously determined to be 1.20 μL/106 BAL cells.7,21
Retrospective review of chloramphenicol MIC
Information regarding the susceptibility of equine bacterial isolates and MIC of chloramphenicol was obtained by retrospective evaluation of the Auburn University Large Animal Hospital clinical database. The results of all microbiological reports available for equine hospitalized patients over a 3-year period (2019 to 2022) were extracted from the clinical database (VetView) into a spreadsheet (Microsoft Excel). Reports of bacterial growth with tested susceptibility to chloramphenicol were identified. For each susceptible bacterial isolate, the MIC determined by broth microdilution (VITEK-2) was recorded and reported as descriptive statistics. For Streptococcus spp, zone diameters obtained with disk diffusion were interpreted via the human zone diameter standards (correlating MIC breakpoints of ≤ 4 susceptible, 8 intermediate, and ≥ 16 resistant). For Actinobacillus spp, zone diameters were extrapolated from Pasteurella human standards (susceptible-only correlating MIC breakpoint of ≤ 2), since no equine-specific chloramphenicol standards were available.
Pharmacokinetic analysis
Plasma chloramphenicol concentration versus time data were analyzed based on noncompartmental pharmacokinetic analysis using a commercially available software (Microsoft Excel PK Solver). For the IV data, initial plasma concentration was calculated while for all other routes of administration (SIG, SPO, and TPO), the value for maximum plasma concentration (Cmax) as well as the time at which it was observed (Tmax), were directly obtained from the data. The AUC to the last time point (AUCall) and AUC to infinity as well as the AUC under the moment curve were calculated by use of the linear trapezoidal method. The terminal rate constant and subsequently the terminal half-life were determined via calculation of the terminal slope using the last 3 points of the plasma concentration versus time curve. For the IV route, apparent volume of distribution (Vd) and Vd at steady state, systemic clearance (Cl), and mean residence time were also reported. For all other routes of administration (SIG, SPO, and TPO), Vd and Cl were calculated as the ratio of absolute bioavailability (F) and are reported as Vd/F and Cl/F. Using the IV data, the F for each treatment (TPO, SPO, and SIG) was calculated as (AUC#/AUCIV) X (DOSEIV/DOSE#), where # represents the treatment-specific AUC and DOSE. Relative bioavailability was calculated for oral versus intragastric suspension protocols as AUCSPO/AUCSIG and for oral suspension versus oral tablets as AUCSPO/AUCTPO. For PELF, the maximum observed concentration (PELF Cmax_obs) was directly obtained from each horse data. To estimate and compare plasma and pulmonary drug exposure, the AUC including only 1- to 8-hour sampling points was calculated for both plasma (AUC1_8plasma) and PELF (AUC1_8PELF).
Statistical analysis
For all protocols (IV, SPO, SIG, and TPO), concentrations achieved in the BAL cells were too low to be clinically significant. Thus, descriptive statistics (median and ranges) were calculated, but no statistical analysis was performed. For the IV protocol, plasma pharmacokinetic variables and PELF concentrations are reported as means and SD but are not included in any statistical comparisons because of the insufficient sample size. Similarly, no statistical analysis was performed on relative bioavailability, and only descriptive data are reported.
The remainder of the data was analyzed using a commercially available statistical software package (GraphPad Prism 9.5.1). Data distribution was assessed using the Kolmogorov-Smirnov and Shapiro-Wilk tests for normality. Normally distributed data are reported as mean ± SD. Data are otherwise presented as median (ranges). Additionally, the coefficient of variation was calculated as the SD/mean. Mean (or median) Cmax, Tmax, AUCall, PELF Cmax_obs, and AUC1_8PELF were compared between intragastric and oral routes (SPO vs SIG) and between the 2 different oral formulations (SPO vs TPO), using a paired t test or Wilcoxon signed rank test. For each protocol, a 1-tailed paired t test (or Wilcoxon signed rank test) was used to compare mean (or median) plasma Cmax with mean (or median) PELF Cmax_obs and mean (or median) AUC1_8plasma with mean (or median) AUC1_8PELF. The level of statistical significance was set at P < .05. The percentage difference of plasma Cmax/PELF Cmax_obs and AUC1_8plasma/AUC1_8PELF was also calculated and reported descriptively.
Results
Horses
No adverse reactions to chloramphenicol were observed. One horse included in experiment 1 developed complications (fever and tachypnea) following repeated BAL procedures during the first treatment of experiment 2. Complications were resolved with supportive care and nonsteroidal anti-inflammatory therapy. The horse was removed from the study; thus, data for experiment 2 are available only for 5 horses. All other horses tolerated repeated BAL procedures without complications. Physical parameters remained normal, and no clinical abnormalities were observed during all experimental periods. Descriptive statistics (median, ranges) for BALF cytology are reported in Supplementary Table S1. Recovered BALF volumes are reported in Supplementary Table S2.
Retrospective review of chloramphenicol MIC
Isolates tested for susceptibility to chloramphenicol included 26 Streptococcus spp, 8 Staphylococcus aureus, 6 Escherichia coli, 5 Enterobacter cloacae, 4 Klebsiella spp (3 Klebsiella pneumoniae and 1 Klebsiella aerogenes), 2 Enterococcus spp, 1 Salmonella spp, and 1 Actinobacillus equuli. All Streptococcus spp isolates were susceptible based on described standards (≤ 4 µg/mL). Most Streptococcus spp isolates were β-hemolytic and included Streptococcus equi zooepidemicus (14/26), Streptococcus dysgalactiae equisimilis (2/26), Streptococcus equi equi (2/26), Streptococcus canis (1/26), and Streptococcus pseudoporcinus (1/26). Other Streptococcus species included Streptococcus viridans (3/26), Streptococcus gallolyticus (2/26), and Streptococcus caballi (1/26). All isolates of Staphylococcus aureus were susceptible, 75% (6/8) with MIC ≤ 4 μg/mL and 25% (2/8) with MIC ≤ 8 μg/mL. Among Escherichia coli isolates, 50% (3/6) were susceptible, 17% (1/6) had MIC ≤ 2 μg/mL, and 33% (2/6) had MIC ≤ 4 μg/mL. Among Enterobacter cloacae isolates, 40% (2/5) were susceptible and had MIC ≤ 4 μg/mL. Among Klebsiella spp isolates, 75% (3/4) were susceptible and had MIC ≤ 2 μg/mL. Only 1 of the 2 Enterococcus spp isolates was susceptible, with MIC ≤ 4 μg/mL. Both Salmonella spp and A equuli isolates were susceptible, with MIC ≤ 4 μg/mL and ≤ 2 μg/mL, respectively.
Pulmonary disposition and pharmacokinetics
In both experiments, chloramphenicol concentrations within a clinically relevant range of 2 to 8 µg/mL were detected in the PELF. Pulmonary epithelial lining fluid and corresponding plasma concentrations over time for each treatment protocol in relation to susceptibility breakpoints for clinically relevant bacteria are summarized in Figure 1. Individual calculated PELF apparent concentrations are reported in Supplementary Table S2. Estimated chloramphenicol concentrations in BALF cells were considered too low to be of clinical relevance (Table 1).
Chloramphenicol concentrations (µg/mL) after administration of a single dose of compounded suspension PO (SPO; A) or intragastrically (SIG; B) at 50 mg/kg, commercial base tablets PO (TPO; C) at 50 mg/kg, and IV solution (D) at 25 mg/kg. In panels A to D, the side-by-side box plots compare calculated pulmonary epithelial lining fluid (PELF; left side) and plasma (right side) concentrations measured 1 hour (gray bars), 4 hours (white bars), and 8 hours (oblique dashed pattern bars) after chloramphenicol administration. The lower and upper limits of the box represent the 25th and 75th percentiles, the whiskers delimit the ranges, and the median and the mean are represented by the horizontal line and by the dot within the box, respectively. The dotted horizontal lines across each graph define the clinically relevant susceptibility breakpoint range (2 to 8 µg/mL) for bacteria commonly detected in equine pneumonia. Note the different scales for the 2 segments of the y-axis.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.08.0223
Median (range) of chloramphenicol concentrations in bronchoalveolar lavage cells 1, 4, and 8 hours after administration of a single dose at 50 mg/kg of tablets PO (TPO) or suspension PO (SPO) or intragastrically (SIG) or at 25 mg/kg IV.
| Time (hour) | SIG (ng/cell) | SPO (ng/cell) | TPO (ng/cell) | IV (ng/cell) |
|---|---|---|---|---|
| 1 | 0.125 (0.004–0.803) | 0.004 (0.001–0.006) | 0.009 (0.003–0.016) | 0.100 (0.06–0.120) |
| 4 | 0.087 (0.026–0.230) | 0.003 (0.002–0.007) | 0.004 (0.003–0.354) | 0.04 (0.03–0.04) |
| 8 | 0.036 (0.013–0.047) | 0.002 (0.001–0.005) | 0.002 (0.001–0.003) | 0.02 (0.02–0.04) |
Comparison between plasma and pulmonary pharmacokinetics
Relevant plasma and PELF pharmacokinetic variables are summarized in Table 2. Regardless of formulation and route of administration, AUC1_8PELF was higher than AUC1_8plasma (SPO, P = .02; TPO, P = .03; SIG, P = .002). The mean percent difference between AUC1_8plasma and AUC1_8PELF was 24 ± 17%, 13 ± 7%, and 23 ± 18%, respectively, for SPO, SIG, and TPO protocols. The PELF Cmax_obs was higher than plasma Cmax for the SPO (P = .03) and SIG (P = .01) protocols. For the TPO protocol, PELF Cmax_obs and plasma Cmax did not differ (P = .06). In terms of average percent differences, the plasma Cmax was 27 ± 13%, 20 ± 17%, and 22 ± 23% of the PELF Cmax_obs for SPO, SIG, and TPO protocols, respectively. Time-matched plasma and PELF chloramphenicol concentrations at 1, 4, and 8 hours for each treatment protocol, in relation to clinically relevant bacteria susceptibility breakpoints are shown in Figure 1. At 4 hours postadministration (50% to 75% of a 6- to 8-hour dosing interval), plasma concentrations are equal to or below 2 µg/mL, regardless of route of administration and type of formulation. Mean PELF concentrations remain above 8 µg/mL, at 8.23 ± 5.87 µg/mL, 12.98 ± 4.77 µg/mL, and 9.68 ± 8.63 µg/mL, respectively, for the SPO, SIG, and TPO protocols. However, considering individual data (Supplementary Table S2), PELF concentrations remain above 8 µg/mL in all horses only in the SIG protocol. In the SPO and TPO protocols, PELF concentrations were above 4 µg/mL at 4 hours in 4/5 horses but above 8 µg/mL only in 2/5 horses. In 1 horse, PELF concentrations at 4 hours were already below 2 µg/mL.
Mean ± SD (coefficient of variation) for relevant plasma and pulmonary epithelial lining fluid (PELF) pharmacokinetic variables after administration of a single dose at 50 mg/kg TPO, SPO, or SIG or 25 mg/kg IV.
| Variable | SIG | SPO | TPO | IV |
|---|---|---|---|---|
| λ (h−1) | 0.27 ± 0.07 (26%) | 0.19 ± 0.07 (35%) | 0.25 ± 0.07 (30%) | 0.26 ± 0.02 (7%) |
| t1/2λz (h) | 2.73 ± 0.80 (29%) | 4.00 ± 1.56 (39%) | 2.97 ± 0.81 (27%) | 2.72 ± 0.20 (8%) |
| Tmax (h) | 2.4 ± 1.34 (56%) | 2.90 ± 1.43 (49%) | 3.60 ± 1.52 (42%) | |
| Cmax (μg/mL) | 5.73 ± 2.99a,b (52%) | 2.52 ± 1.40a,b (56%) | 3.72 ± 2.34 (63%) | |
| C0 (μg/mL) | 114.37± 4.28 (4%) | |||
| AUCall (μg/mL·h) | 16.24 ± 3.10a (19%) | 12.89 ± 3.62a (28%) | 14.08 ± 2.53 (18%) | 44.58 ± 6.41 (14%) |
| AUC0-∞ (μg/mL·h) | 16.31 ± 3.07 (19%) | 13.75 ± 4.94 (36%) | 14.25 ± 2.67 (19%) | 44.59 ± 6.40 (14%) |
| AUCMC (μg/mL·h2) | 62.25 ± 15.48 (25%) | 111.10 ± 78.84 (71%) | 77.17 ± 35.38 (46%) | 33.19 ± 0.66 (2%) |
| MRT0-∞ (h) | 3.95 ± 1.26 (32%) | 7.46 ± 2.79 (37%) | 5.39 ± 2.13 (40%) | 0.75 ± 0.10 (14%) |
| Vd ([mg/kg]/[μg/mL]) | 2.25 ± 0.53 (24%) | |||
| Vdss ([mg/kg]/[μg/mL]) | 0.44 ± 0.13 (30%) | |||
| Cl ([mg/kg]/[μg/mL]/h) | 0.57 ± 0.09 (16%) | |||
| Vd/F ([mg/kg]/[μg/mL]) | 12.58 ± 4.74 (38%) | 21.41 ± 5.36 (25%) | 15.12 ± 3.51 (23%) | |
| Cl/F ([mg/kg]/[μg/mL]/h) | 1.17 ± 0.71 (22%) | 4.01 ± 1.39 (35%) | 3.61 ± 0.66 (18%) | |
| F (%) | 18 | 14 | 16 | |
| AUC1–8plasma (μg/mL·h) | 11.95 ± 3.20b (27%) | 8.19 ± 2.68b (32%) | 10.54 ± 2.84b (27%) | 6.77 ± 0.47 (7%) |
| AUC1–8PELF (μg/mL·h) | 103.43 ± 36.26a,b (35%) | 51.41 ± 37.16a,b (72%) | 76.86 ± 58.19b (75%) | 35.17 ± 8.87 (25%) |
| Cmax_obsPELF (μg/mL) | 39.31 ± 27.04a,b (69%) | 12.92 ± 10.23a,b (79%) | 32.16 ± 34.34 (106%) | 18.81 ± 9.71 (52%) |
AUC0-∞ = AUC to infinity. AUC1_8PELF = PELF AUC including only 1- to 8-hour sampling points. AUC1_8plasma = Plasma AUC including only 1- to 8-hour sampling points. AUCall = AUC to the last time point (24 hours). AUCMC = Area under the moment curve. C0 = Initial plasma concentration (IV). Cl = Systemic clearance. Cmax = Maximum plasma concentration. Cmax_obsPELF = PELF maximum observed concentration. F = Absolute bioavailability. MRT0-∞ = Mean residence time to inifinity. t1/2λz = Terminal half-life. Tmax = Time to maximum plasma concentration. Vd = Apparent volume of distribution. Vddss = Apparent volume of distribution at steady state. λz = Terminal rate constant.
Impact of route and formulation on pharmacokinetics
Plasma chloramphenicol concentrations versus time data for all treatment protocols are presented in Figure 2. The SIG administration resulted in higher Cmax (P = .03), AUCall (P = .02), PELF Cmax_obs (P = .04), and AUC1_8PELF (P = .03) values compared to SPO administration of the same formulation. The Tmax did not differ between SIG and SPO administration (P = .23). The relative bioavailability of SPO versus SIG administration was 79%. No differences were observed for Cmax (P = .21), Tmax (P = .37), AUCall (P = .55), PELF Cmax_obs (P = .2), or AUC1_8PELF (P = .33), between SPO and TPO administration. The relative bioavailability of SPO versus TPO administration was 91%. Measured drug concentrations for reserved aliquots from each bottle of compounded suspension were 476 and 435 mg/mL (manufacturer reported concentration was 500 mg/mL).
Plasma chloramphenicol concentrations (log µg/mL; y-axis) versus time data (hours; x-axis) after administration SPO (dotted line and squares) or SIG (dashed line and dots) and TPO (full line and triangles) at 50 mg/kg. The insert graph (top right; dotted/dashed line and triangles) represents plasma chloramphenicol concentrations (log µg/mL; y-axis) versus time data (hours; x-axis) after administration of a single IV dose at 25 mg/kg. Each data point represents the mean and whiskers represent positive standard errors.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.08.0223
Discussion
This study is the first to describe the pulmonary pharmacokinetics of chloramphenicol in adult horses, comparing different formulations and routes of administration. Obtaining data on chloramphenicol pulmonary disposition could assist veterinarians in making judicious, case-by-case decisions about its appropriateness for treating equine pneumonia, according to antimicrobial stewardship. As hypothesized, chloramphenicol concentrations were detected in PELF after a single dose, averaging 20% higher than in plasma. However, only in a subset of horses, chloramphenicol concentrations in PELF remained above the MIC range for equine respiratory pathogens (2 to 8 µg/mL) for at least 4 hours, regardless of the formulation and administration route.
Chloramphenicol, known for its high lipid solubility and low plasma protein binding (approx 30% in horses), diffuses readily through biological membranes, achieving therapeutic concentrations in various equine tissues and body cavity fluids.27,28 This study is the first to demonstrate that chloramphenicol may achieve PELF concentrations above the MIC for some bacteria, indicating effective bronchial-blood barrier crossing and ability to reach the airway lumen where most pathogens establish respiratory infection in adult horses.3 In our study, chloramphenicol was also distributed to the PELF, with drug exposure (AUC1–8) and maximum observed concentrations that were 13% to 24% and 20% to 27% higher than in plasma, respectively. This behavior has been noted in other species administered phenicol derivatives and in horses administered other lipophilic antimicrobials, such as tetracyclines.20,29,30 Drug concentrations in the PELF are more predictive of antimicrobial efficacy in treating lower respiratory tract infections than plasma or lung tissue concentrations and can be estimated by different techniques.31 When using BAL as the sampling technique, the urea dilution method used in this study remains the preferred method to estimate drug concentrations in the PELF of horses.4,5,19,29,32 In our study, after intragastric administration, PELF chloramphenicol concentrations remained > 8 µg/mL for at least 4 hours in all horses. However, after oral administration, PELF chloramphenicol concentrations at 4 hours remained > 4 µg/mL in only 4/5 horses, suggesting that, in most horses, administration every 6 hours could target equine respiratory extracellular pathogens with an MIC ≤ 2 and 4 µg/mL.13–15 Based on the retrospective review of our hospital database and previous literature,33 only some bacterial isolates could be susceptible to this therapeutic regimen, mostly including strains of aerobic gram positives (Streptococcus spp and 75% of S aureus) and some aerobic/facultative anaerobic intestinal opportunistic gram negatives (A equuli, 75% of Klebsiella spp, and 50% of E coli). Notably, PELF concentrations were < 2 µg/mL at 4 hours after oral tablets and suspension administration in 1/5 horses. Because intragastric administration is not typically feasible in clinical settings, veterinary practitioners should take into consideration that clinically relevant pulmonary concentrations might not be reached in a subset of horses, even for very susceptible isolates. Additionally, considering its broad spectrum, chloramphenicol should not be used indiscriminately to treat any susceptible infection. Firstline, narrow-spectrum antimicrobials such as penicillins, aminoglycosides, and metronidazole should be preferred to target gram-positive aerobes, gram-negative aerobes, and anaerobes, respectively. Chloramphenicol use should be reserved for susceptible isolates (with MIC values ≤ 2 μg/mL for most horses) or polymicrobial infections that demonstrate resistance to narrow-spectrum antimicrobials.
Contrary to the PELF, chloramphenicol concentrations in BAL cells were considerably lower than MIC at all measured time points. In vitro, chloramphenicol has been reported to accumulate in cells to a moderate extent.34,35 However, the discrepancy between in vitro and in vivo intracellular accumulation has been observed with other lipophilic antimicrobials, such as tetracyclines.20,32,34,36 Even though highly lipophilic molecules should easily penetrate the cell membrane, factors such as limited retention due to outward transport or intracellular degradation, as well as technical limitations like inadvertent cell lysis during BALF processing, may contribute to low intracellular concentrations.34 Intracellular concentrations reflect effectiveness in treating pathogens capable of intracellular survival.36 While most common equine airway pathogens, particularly in adult horses, are extracellular, further investigation is required to clarify chloramphenicol's efficacy against obligate or facultative intracellular bacteria.37
In the present study, plasma pharmacokinetics and pulmonary disposition, including PELF Cmax_obs and AUC1_8PELF, were similar when oral compounded suspension was compared to commercial tablets, with a relative bioavailability of 91%. We studied a compounded chloramphenicol product prepared by an approved compounding company from a commercially available FDA-approved product, because FDA-approved chloramphenicol suspension is not available on the market. Equine veterinary practitioners tend to choose compounded suspension (or paste), instead of commercial tablets, when prescribing treatment with chloramphenicol, for various reasons. First, considering the rare but serious complication of aplastic anemia reported in humans, crushing chloramphenicol tablets for administration poses a higher risk of incidental operator exposure than using a compounded suspension.38 Additionally, tablets for veterinary use have variable and often limited availability on the market. Chloramphenicol concentrations measured in saved aliquots of suspension were consistent with the manufacturer's label. However, we did not assess potency or stability over time. Furthermore, the volume administered to each horse was calculated based on the intended suspension concentration (500 mg/mL) and not the actual concentrations (476 and 435 mg/mL), likely causing slight underdosing. Considering the generally poor and variable oral bioavailability in horses, it is unlikely that 5% to 10% difference in formulation concentration contributed to interindividual variability, and this nevertheless reflects what is likely to occur in clinical practice where compounded suspension volumes are estimated based on intended and not actual concentrations. Compounded formulations have intrinsic variability, and pharmacokinetic properties, including solubility, may differ between compounded and FDA-approved products; thus, the present results might not be replicable with a different formulation. Furthermore, one should consider the possibility of our study being unable to detect a difference between tablets and suspension due to low power. Slightly higher plasma Cmax and AUC to infinity were previously reported for horses administered a different compounded oral suspension. Higher interindividual variability was also observed, resulting in lower relative bioavailability of the compounded formulation (78.1%) than reported here.14 Regardless of formulation, the F of oral chloramphenicol in our study (14% to 18%; Table 2) appeared low and comparable to previous studies (18% to 40%).14,26 Veterinarians should consider the risks of promoting antimicrobial resistance in the environment and intestinal dysbiosis when using oral antimicrobials. A low F means a proportionally higher amount of unabsorbed drug potentially increasing selective pressure on gastrointestinal microbiota and the environment through fecal dispersion.39
To our knowledge, this is the first study comparing intragastric and oral routes of administration of chloramphenicol to horses. As hypothesized, intragastric administration provided higher Cmax and drug exposure (AUC) in both plasma and PELF, with lower interindividual variability. Oral administration achieved PELF concentrations ≥ 8 µg/mL for at least 4 hours only in 2 horses, whereas 4 horses achieved PELF concentrations of 4 μg/mL at the 4-hour mark, suggesting that only bacteria with MIC values ≤ 2 or possibly 4 μg/mL may be targeted in most horses with oral dosing every 6 hours. Horse behavior and tolerance to administration by mouth might have contributed to the observed pharmacokinetic differences between intragastric and oral administration. Given the observed similarities between these routes for Tmax and plasma concentrations over time, transmucosal oral absorption seems unlikely. Transmucosal absorption bypasses first hepatic metabolism and its thorough assessment would require measurement of chloramphenicol metabolites, which was not performed in this study.40
Similar to what has been previously reported,14 a double peak phenomenon (DPP) was observed in plasma chloramphenicol concentration over time data for oral and intragastric treatments (Figure 2). In both our study and previous studies,41,42 horses were fasted before drug administration, and the second peak coincides with refeeding, suggesting that increases in gastric peristalsis initiated by ingesta entering the stomach might promote a secondary phase of absorption. Accordingly, DDP is not observed when chloramphenicol is administered to horses that are not fasted.15 It is also possible that decreased peristalsis induced by sedation for BAL procedures contributed to the DDP observed in the present study, although it remains undetermined to which extent. Enterohepatic recycling, another recognized cause for DPP, is considered unlikely as DPP was not observed after IV administration.42
Despite the study being adequately powered, marked interindividual variability was observed across all plasma and pulmonary pharmacokinetic variables, except after IV administration (Table 2). High interindividual variability with unpredictable and sometimes poor bioavailability has been previously reported13–15 in horses after oral administration of chloramphenicol and other drugs. Thus, caution should be used when extrapolating to make clinical recommendations, as some horses do not achieve clinically relevant pulmonary concentrations. Interindividual variability depends on factors that cannot always be modified, such as breed, body weight, level of exercise, time, and type of feed.41 In this study, we attempted to control feed-related variability by fasting horses before drug administration; however, this is not replicable in clinical settings. Even though feeding did not impact chloramphenicol plasma pharmacokinetics in a previous study,15 additional studies in fed horses are required to exclude any impact of feeding on pulmonary pharmacokinetics.
Limitations of the study include using a small number of age-matched healthy horses belonging to the same herd. Results may differ for other equine populations or different age ranges and systemic or lung disease could impact pulmonary drug delivery and penetration. Even though chloramphenicol reaches concentrations exceeding the in vitro MIC for some equine respiratory pathogens, this does not directly translate into clinical effectiveness. Chloramphenicol should be chosen considering antimicrobial stewardship on a case-by-case basis and not relying exclusively on susceptibility and pharmacokinetics. One should also consider the risk of associated toxicities such as myelosuppression and dysbiosis in both horses and people. Additionally, only a single dose was administered to fasted horses, and chloramphenicol is known to progressively accumulate due to potent inhibition of cytochrome P450 enzyme (predominantly CYP2C19 and CYP3A4, moderately CYP2D6 in human liver microsomes). This leads to higher serum concentrations after multiple doses and concurrent decreased clearance of other drugs such as phenytoin in humans and methadone in dogs.13,43,44 In horses, the occurrence of drug-drug interactions after multidose chloramphenicol administration remains to be investigated. Our ability to measure PELF drug exposure (AUC) was also limited by the fact that only 3 sampling points over time were used. Furthermore, chloramphenicol and urea concentrations were quantified only in a single randomly selected PELF aliquot instead of averaging multiple aliquot analyses and intravial variability could impact the robustness of quantification. Similarly to what has been done previously,20 we used a commercially available ELISA kit to measure urea concentration, instead of biochemistry or methods based on LC-MS-MS, which could demonstrate greater accuracy. While on average plasma urea concentrations fell within clinically acceptable values, one should consider possible technique-related variability as an important limitation of this study, potentially impacting estimated PELF chloramphenicol concentrations. In summary, additional studies on chloramphenicol pulmonary concentrations at steady state after multidose administration in fed and ill horses are required to provide recommendations for clinical use in the treatment of equine pneumonia.
In conclusion, in a subset of horses, chloramphenicol compounded suspension or commercial tablets administered orally at a dose of 50 mg/kg resulted in PELF concentrations at 4 hours above the recommended susceptibility breakpoints for some of the bacteria commonly encountered in equine pneumonia (2 and 4 µg/mL). One should however consider that, due to high interindividual variability, in certain horses, pulmonary concentrations might not reach clinically relevant threshold. Additional multidose studies in fed horses are needed to assess the impact of feeding and accumulation on chloramphenicol bioavailability and pulmonary pharmacokinetics. When extrapolating clinical recommendations, practitioners should prioritize following antimicrobial stewardship, rather than exclusively considering susceptibility and pharmacokinetic data. Additionally, veterinarians should consider that pharmacokinetic properties may differ between compounded and FDA-approved products and should adhere to compounding regulations.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors acknowledge Jessica Brown for her help with horse handling, Qiao Zhong for her help with ELISAs, Dan McKemie and Sandy Kim for their help with LC-MS-MS, and Erik Hoffmeister for his advice in statistical analysis.
Disclosures
The authors have nothing to disclose.
ChatGPT and Grammarly were used to screen the manuscript for grammar errors and misspellings and to occasionally rephrase unclear sentences.
Funding
The study was funded by the Birmingham Racing Commission (G00015299).
ORCID
L. Dedecker https://orcid.org/0000-0002-8195-0934
S. Ceriotti https://orcid.org/0000-0002-6905-0538
References
- 1.↑
Pusterla N, Watson J, Wilson W. Diagnostic approach to infectious respiratory disorders. Clin Techn Equine Pract. 2006;5:174–186 doi:10.1053/j.ctep.2006.03.012
- 2.
Traub-Dargatz J, Dargatz D. Antibacterial drug resistance and equine practice. Equine Vet. Educ. 2009;21:49–56.
- 3.↑
Reuss SM, Giguère S. Update on bacterial pneumonia and pleuropneumonia in the adult horse. Vet Clin North Am Equine Pract. 2015;31(1):105–120. doi:10.1016/j.cveq.2014.11.002
- 4.↑
Winther L, Hansen S, Baptiste K, Friis C. Antimicrobial disposition in pulmonary epithelial lining fluid of horses, part II. Doxycycline. J Vet Pharmacol Ther. 2011;34(3):285–289. doi:10.1111/j.1365-2885.2010.01229.x
- 5.↑
Winther L, Guardabassi L, Baptiste K, Friis C. Antimicrobial disposition in pulmonary epithelial lining fluid of horses. Part I. Sulfadiazine and trimethoprim. J Vet Pharmacol Ther. 2011;34(3):277–284. doi:10.1111/j.1365-2885.2010.01228.x
- 6.
Ensink JM, Bosch G, Van Duijkeren E. Clinical efficacy of prophylactic administration of trimethoprim/sulfadiazine in a Streptococcus equi subsp. zooepidemicus infection model in ponies. J Vet Pharmacol Ther. 2005;28(1):45–49. doi:10.1111/j.1365-2885.2004.00624.x
- 7.↑
Davis JL, Gardner SY, Jones SL, Schwabenton BA, Papich MG. Pharmacokinetics of azithromycin in foals after i.v. and oral dose and disposition into phagocytes. J Vet Pharmacol Ther. 2002;25(2):99–104. doi:10.1046/j.1365-2885.2002.00387.x
- 8.↑
Kasten MJ. Clindamycin, metronidazole, and chloramphenicol. Mayo Clinic Proc. 1999;74(8):825–833. doi:10.4065/74.8.825
- 9.↑
Ambrose PJ. Clinical pharmacokinetics of chloramphenicol and chloramphenicol succinate. Clin Pharmacokinet. 1984;9:222–238. doi:10.2165/00003088-198409030-00004
- 10.
Haggett EF, Wilson WD. Overview of the use of antimicrobials for the treatment of bacterial infections in horses. Equine Vet Educ. 2008;20(8):433–448. doi:10.2746/095777308X338893
- 11.
Watson AD, McDonald PJ. Distribution of chloramphenicol in some tissues and extravascular fluids of dogs after oral administration. Am J Vet Res. 1976;37(5):557–559.
- 12.↑
Watson A. Chloramphenicol 2. Clinical pharmacology in dogs and cats. Aust Vet J. 1991;68(1):2–5. doi:10.1111/j.1751-0813.1991.tb09827.x
- 13.↑
Estell KE, Knych HK, Patel T, Edman JM, Magdesian KG. Pharmacokinetics of multiple doses of chloramphenicol in fed adult horses. Vet J. 2020;257:105446. doi:10.1016/j.tvjl.2020.105446
- 14.↑
Patel T, Magdesian KG, Estell KE, Edman JM, Knych HK. Pharmacokinetics of chloramphenicol base in horses and comparison to compounded formulations. J Vet Pharmacol Ther. 2019;42(6):609–616. doi:10.1111/jvp.12777
- 15.↑
McElligott EM, Sommardahl CS, Cox SK. Pharmacokinetics of chloramphenicol base after oral administration in adult horses. J Am Vet Med Assoc. 2017;251(1):90–94. doi:10.2460/javma.251.1.90
- 16.↑
Adamson PJ, Wilson WD, Hirsh DC, Baggot JD, Martin LD. Susceptibility of equine bacterial isolates to antimicrobial agents. Am J Vet Res. 1985;46(2):447–450.
- 17.↑
Cazzola M, Blasi F, Terzano C, Matera MG, Marsico SA. Delivering antibacterials to the lungs: considerations for optimizing outcomes. Am J Respir Med. 2002;1(4):261–272. doi:10.1007/BF03256617
- 18.↑
Drusano GL. Infection site concentrations: their therapeutic importance and the macrolide and macrolide-like class of antibiotics. Pharmacotherapy. 2005;25(12):150S–158S. doi:10.1592/phco.2005.25.12part2.150S
- 19.↑
Winther L. Antimicrobial drug concentrations and sampling techniques in the equine lung. Vet J. 2012;193(2):326–335. doi:10.1016/j.tvjl.2012.02.010
- 20.↑
Echeverria KO. Pulmonary disposition and pharmacokinetics of minocycline in the adult horse. University of Illinois at Urbana-Champaign. 2017. Accessed February 13, 2024. https://hdl.handle.net/2142/97319
- 21.↑
Jacks S, Giguère S, Gronwall RR, Brown MP, Merritt KA. Pharmacokinetics of azithromycin and concentration in body fluids and bronchoalveolar cells in foals. Am J Vet Res. 2001;62(12):1870–1875. doi:10.2460/ajvr.2001.62.1870
- 22.
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
- 23.
Rodvold KA, Danziger LH, Gotfried MH. Steady-state plasma and bronchopulmonary concentrations of intravenous levofloxacin and azithromycin in healthy adults. Antimicrob Agents Chemother. 2003;47(8):2450–2457. doi:10.1128/AAC.47.8.2450-2457.2003
- 24.↑
Giguère S, Tessman RK. Rational dosing of antimicrobial agents for bovine respiratory disease: the use of plasma versus tissue concentrations in predicting efficacy. Intern J Appl Res Vet Med. 2011;9(4):342–355.
- 25.↑
Ehrlich J, Bartz QR, Smith RM, Joslyn DA, Burkholder PR. Chloromycetin, a new antibiotic from a soil actinomycete. Science. 1947;106(2757):417–417. doi:10.1126/science.106.2757.417
- 26.↑
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
- 27.↑
Sisodia CS, Kromer LL, Gupta VS, Lerner DJ, Taksos L. A pharmacological study of chloramphenicol. Can J Comp Med. 1975;39(2):216–223.
- 28.↑
Gronwall R, Brown MP, Merritt AM, Stone HW. Body fluid concentrations and pharmacokinetics of chloramphenicol given to mares intravenously or by repeated gavage. Am J Vet Res. 1986;47(12):2591–2595.
- 29.↑
Womble A, Giguère S, Lee EA. Pharmacokinetics of oral doxycycline and concentrations in body fluids and bronchoalveolar cells of foals. J Vet Pharmacol Ther. 2007;30(3):187–193. doi:10.1111/j.1365-2885.2007.00857.x
- 30.↑
Foster DM, Martin LG, Papich MG. Comparison of active drug concentrations in the pulmonary epithelial lining fluid and interstitial fluid of calves injected with enrofloxacin, florfenicol, ceftiofur, or tulathromycin. PLoS One. 2016;11(2):e0149100. doi:10.1371/journal.pone.0149100
- 31.↑
Kiem S, Schentag JJ. Interpretation of antibiotic concentration ratios measured in epithelial lining fluid. Antimicrob Agents Chemother. 2008;52(1):24–36. doi:10.1128/AAC.00133-06
- 32.↑
Giguère S, Burton AJ, Berghaus LJ, Haspel AD. Comparative pharmacokinetics of minocycline in foals and adult horses. J Vet Pharmacol Ther. 2017;40(4):335–341. doi:10.1111/jvp.12366
- 33.↑
Isgren CM, Williams NJ, Fletcher OD, et al. Antimicrobial resistance in clinical bacterial isolates from horses in the UK. Equine Vet J. 2022;54(2):390–414. doi:10.1111/evj.13437
- 34.↑
Tulkens PM. Intracellular distribution and activity of antibiotics. Eur J Clin Microbiol Infect Dis. 1991;10(2):100–106. doi:10.1007/BF01964420
- 35.↑
Jacobs RF, Wilson CB. Intracellular penetration and antimicrobial activity of antibiotics. J Antimicrob Chemother. 1983;12(suppl C):13–20. doi:10.1093/jac/12.suppl_c.13
- 36.↑
Bongers S, Hellebrekers P, Leenen LPH, Koenderman L, Hietbrink F. Intracellular penetration and effects of antibiotics on Staphylococcus aureus inside human neutrophils: a comprehensive review. Antibiotics. 2019;8(2):54. doi:10.3390/antibiotics8020054
- 37.↑
Sweeney CR, Holcombe SJ, Barningham SC, Beech J. Aerobic and anaerobic bacterial isolates from horses with pneumonia or pleuropneumonia and antimicrobial susceptibility patterns of the aerobes. J Am Vet Med Assoc. 1991;198(5):839–842.
- 38.↑
Rheingold JJ, Spurling CL. Chloramphenicol and aplastic anemia. J Am Med Assoc. 1952;119(14):1301–1304. doi:10.1001/jama.1952.02930310037008
- 39.↑
Álvarez Narváez S, Huber L, Giguère S, Hart KA, Berghaus RD, Sanchez S, Cohen ND. Epidemiology and molecular basis of multidrug resistance in Rhodococcus equi. Microbiol Mol Biol Rev. 2021;85(2):e00011-21. 10.1128/mmbr.00011-21.
- 40.↑
Zhang H, Zhang J, Streisand JB. Oral mucosal drug delivery: clinical pharmacokinetics and therapeutic applications. Clin Pharmacokinet. 2002;41(9):661–680. doi:10.2165/00003088-200241090-00003
- 41.↑
Song Y, Day CM, Afinjuomo F, Tan JQE, Page SW, Garg S. Advanced strategies of drug delivery via oral, topical, and parenteral administration routes: where do equine medications stand? Pharmaceutics. 2023;15(1):186. doi:10.3390/pharmaceutics15010186
- 42.↑
Davies NM, Takemoto JK, Brocks DR, Yáñez JA. Multiple peaking phenomena in pharmacokinetic disposition. Clin Pharmacokinet. 2010;49(6):351–377. doi:10.2165/11319320-000000000-00000
- 43.↑
Park JY, Kim KA, Kim SL. Chloramphenicol is a potent inhibitor of cytochrome P450 isoforms CYP2C19 and CYP3A4 in human liver microsomes. Antimicrob Agents Chemother. 2003;47(11):3464–3469. doi:10.1128/AAC.47.11.3464-3469.
- 44.↑
KuKanich B, Kukanich K. Chloramphenicol significantly affects the pharmacokinetics of Ooral methadone in greyhound dogs. Vet Anesth Analg. 2015;42(6):597–607.
