Bacterial pneumonia is an important cause of illness in adult horses.1 Owners can incur substantial financial losses as a result of pneumonia, especially in affected competition or race horses for which transport-associated pneumonia is not uncommon.2,3 Although many bacterial pathogens can be associated with pneumonia, Streptococcus equi subsp zooepidemicus is the bacterial pathogen most commonly recovered from transtracheal washes of adult horses.4,5,a Antimicrobial treatment for bacterial pneumonia, particularly when culture and susceptibility results are unavailable, should allow for broad-spectrum coverage as well as good susceptibility for common pathogens (eg, S equi subsp zooepidemicus) at the site of infection.4,5 Prompt and effective treatment carries a favorable prognosis for return to competition in horses with uncomplicated pneumonia.6,7
Orally administered antimicrobials typically are the mainstay for treatment of pneumonia in adult horses because of their relative ease of administration and, often, a reduction in the financial burden to the owner. Broad-spectrum antimicrobials available for oral administration to adult horses are limited to chloramphenicol, potentiated sulfonamides (sulfadiazine or sulfamethoxazole, combined with trimethoprim), and doxycycline. Although chloramphenicol and potentiated sulfonamides are widely used, there are drawbacks to both drugs. Chloramphenicol is expensive, must be administered 4 times/d, and is associated with risks to humans.1,8 In horses, potentiated sulfonamides do not achieve equal concentrations of both drugs in the lungs,9 and their clinical utility for treating infections of the lower respiratory tract (lung parenchyma, bronchioles, and alveoli) is limited because of their lack of in vivo activity against S equi subsp zooepidemicus.9–12 Doxycycline has good distribution to the lungs and a broad spectrum of activity against many common bacterial respiratory pathogens including Streptococcus species13; however, a market shortage of doxycycline resulted in marked increases in price.14 Although the cost and availability of doxycycline have improved recently, there still remains a clinical need for additional orally administered antimicrobials for treatment options in adult horses.
Minocycline, a tetracycline derivative, is a time-dependent bacteriostatic antimicrobial. As such, its efficacy is influenced by the duration of time that the drug concentration remains above the MIC. Minocycline is available for extralabel oral administration to horses,15 and it represents an attractive addition to the antimicrobial options because of its broad spectrum of antimicrobial activity and excellent tissue penetration,16,17 relatively low cost, and potential anti-inflammatory properties.18 Unfortunately, information on minocycline oral bioavailability and disposition into the lungs of adult horses is limited. This information is important to enable clinicians to more accurately predict the efficacy of this drug for treating bacterial pneumonia.
Intragastric administration of 5 doses of minocycline (4 mg/kg) resulted in minocycline concentrations in synovial fluid and CSF of adult horses adequate to treat infections caused by susceptible (MIC < 0.25 μg/mL) bacterial organisms.15 In another study,19 administration of a single dose of minocycline (2.2 mg/kg, IV) resulted in minocycline concentrations in lung tissue homogenates that exceeded plasma concentrations at 2 hours after drug administration.
Although these findings suggest drug distribution into the lungs, tissue homogenate concentrations are often inaccurate because they do not allow for differentiation between free (active) drug and drug bound to biological material.20–22 Measurement of drug concentrations in the PELF and BAL cells is thought to provide superior information regarding intrapulmonary antimicrobial acvitity.19 Measurement of antimicrobial concentrations in the PELF and BAL cells has been described for many animals, including adult horses and foals.23–25
The objectives of the study reported here were to determine the pharmacokinetics of minocycline after a single dose administered IV and intragastrically and to determine the pharmacokinetics and pulmonary disposition of minocycline after intragastric administration of multiple doses to adult horses. Our hypotheses were that minocycline would be present in the PELF and BAL cells at concentrations exceeding those in plasma within 3 hours after IV or intragastric administration and that achievable trough concentrations in the PELF and BAL cells after intragastric administration of multiple doses of minocycline would exceed a target concentration of 0.25 μg/mL.
Materials and Methods
Animals
Seven adult horses (3 nonpregnant mares and 4 geldings) were used for the study. Horses were between 10 and 30 years of age, and body weight was between 484 and 552 kg. Horses were included in the study on the basis of no abnormalities detected during physical and rebreathing examinations and no abnormal results for a routine plasma biochemical analysisb performed prior to study onset. Horses were housed individually in stalls beginning 24 hours before and continuing through the end of each experimental period; horses were housed in pastures between experimental periods. During the experimental periods, horses were fed 2 flakes of grass and alfalfa hay twice daily and had ad libitum access to water. Physical examinations were performed every 12 hours for 48 hours after completion of an experimental period to monitor horses for drug reactions. The study was approved by the University of Illinois Institutional Animal Care and Use Committee (protocol No. 14261).
Experimental design
Two experiments were conducted during the study. There was a washout period of 21 days between the 2 experiments.
Experiment 1 involved IV or intragastric administration of a single dose of minocycline by use of a randomized crossover experimental design. A single dose of minocycline hydrochloride was administered IV (2.2 mg/kg) or intragastrically by use of nasogastric intubation (4 mg/kg) to each horse; there was a 7-day washout period, and each horse then received a single dose of minocycline by the other route of administration. Hay was provided in the afternoon of the day preceding drug administration and again the next morning 2 hours after IV or intragastric drug administration.
Horses were instrumented by placement of a 14-gauge extended-use over-the-needle catheterc into the left jugular vein for the purpose of collecting serial blood samples. A similar 14-gauge catheterc was placed in the right jugular vein and used for IV administration of minocycline. For IV administration, minocycline hydrochloride powderd was dissolved by use of aseptic conditions in sterile watere to form a solution with a concentration of 5 mg/mL. The solution was filtered through a 25-mm syringe filter with a 0.2-μm disc filter membrane,f placed in light-protective sterile bottles, and stored out of direct light until administration (all solutions were administered to horses within 2 hours after preparation). Minocycline was administered IV over a 5-minute period; the catheter in the right jugular vein was removed immediately after drug administration. Heart and respiratory rates were monitored every minute during drug administration and every 2.5 minutes for 15 minutes after drug administration. For intragastric administration, minocycline hydrochloride powderd was suspended in 500 mL of tap water and administered by use of nasogastric intubation immediately after preparation of the suspension. The nasogastric tube was flushed with 1,500 mL of tap water after drug administration, and the nasogastric tube then was immediately removed.
Blood samples (10 mL/sample) were collected immediately before drug administration (time 0) and 0.08, 0.17, 0.25, 0.33, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, and 48 hours after drug administration. The catheter in the left jugular vein was removed 24 hours after drug administration, and samples at 36 and 48 hours were collected by direct venipuncture of the left jugular vein. Bronchoalveolar lavage was performed 2, 4, 8, and 24 hours after drug administration.
Experiment 2 involved intragastric administration of multiple doses of minocycline. Minocycline was administered intragastrically by use of nasogastric intubation (4 mg/kg, q 12 h, for a total of 5 doses). Horses were maintained on a standard morning and afternoon feeding schedule, with the morning feeding provided 2 hours after the morning drug administration and the afternoon feeding provided approximately 4 hours before the evening drug administration. Blood samples were collected via direct venipuncture of the left jugular vein immediately before administration of the first dose (time 0) and 0.5, 1, 1.5, 2, 6, 12, 14, 24, 26, 36, 38, 48, 48.5, 49, 49.5, 50, 54, 60, 72, and 96 hours after administration of the first dose of minocycline. Bronchoalveolar lavage was performed 24, 36, 48, 54, 60, and 72 hours after administration of the first dose of minocycline. Collection of blood samples at 12, 24, 36, and 48 hours and collection of BAL fluid at 24, 36, and 48 hours were performed immediately before drug administration and were defined as trough concentrations for the purposes of the study.
Processing was identical for all blood samples of both experiments. Blood was placed into sodium heparin tubesg and centrifuged (2,500 × g for 10 minutes at 4°C) within 15 minutes after sample collection. Plasma was harvested, placed in aliquots, and frozen at −80°C until batch analysis.
BAL procedure and processing of BAL fluid
Before each BAL was performed, horses were sedated by IV administration of 150 mg of xylazine hydrochlorideh; the xylazine was injected into the jugular vein opposite that used for collection of blood samples. The BAL fluid was collected through the biopsy channel of a sterilized 3-m (9.9-mm outside diameter and 3.7-mm channel) endoscope.i Collection of BAL fluid was alternated between the right and left lungs, the order of which was randomly assigned (lottery method) before the initial BAL. Instillation of a dilute lidocaine solution (5 mL of 2% lidocaine diluted in 20 mL of saline [0.9% NaCl] solution) was used for passage of the endoscope into the trachea and past the level of the carina. Once the endoscope was wedged in the most distal bronchus, 300 mL of sterile isotonic saline solutionj was infused in 60-mL aliquots through the endoscope biopsy channel, which was followed by gentle aspiration into sterile 60-mL syringes immediately after infusion of the last aliquot. The total volume of fluid recovered and side for the lung from which samples were collected (right or left) were recorded, and the individual samples then were pooled for immediate sample processing. A 5-mL sample of the pooled BAL fluid was removed for subsequent determination of total nucleated cell count, and the remainder of the pooled BAL fluid was centrifuged (2,500 × g for 10 minutes at 4°C) for separation of BAL cells from BAL supernatant containing the PELF. The cell pellet was washed with PBS solution and resuspended in 500 μL of a solution of acetonitrile–0.2% formic acid. The BAL supernatant and cells were stored at −80°C until batch analysis.
Minocycline analysis via LC-MS-MS
Plasma, BAL cell, and BAL supernatant samples were thawed immediately before preparation for LC-MS-MS analysis. For metabolite extraction from plasma samples, 50 μL of plasma was mixed with 70 μL of methanol and spiked with 2 μL of internal standard (demeclocyclinek; 13.8 μg/mL); samples were mixed in a vortex device and then centrifuged. For metabolite extraction from BAL supernatant, 50 μL of supernatant was mixed with 70 μL of methanol and spiked with 1 μL of internal standard, which was followed by mixing in a vortex device. The liquid was then loaded into a sample vial for LC-MS-MS injection. For BAL cell samples, cell pellets were subjected to controlled sonicationl at the following settings: 4°C, amplitude 95, 10 minutes of processing time, pulse-on time of 30 seconds, and pulse-off time of 45 seconds. Cell extracts were subjected to centrifugation (21,100 × g for 10 minutes at 4°C). The supernatant was rapidly dried and then resuspended in 150 μL of 80% methanol, spiked with internal standard, and mixed in a vortex device. The liquid was then loaded into a sample vial for LC-MS-MS injection.
The LC-MS-MS analysis was performed in accordance with standard laboratory proceduresm by use of a hybrid triple-quadrupole linear accelerator trap mass spectrometern designed for LC-MS-MS analysiso and specialized software for data acquisition and analysis.p Liquid chromatography separation was performed with a column of 4.6 × 50 mm and inside diameter of 5 μm. Mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile) were used. Flow rate was 0.35 mL/min. Linear gradient was as follows: 100% phase A for 0 to 2 minutes, transition from 100% phase A to 100% phase B from 2 to 7 minutes, 100% phase B for 7 to 10.5 minutes, transition back to 100% phase A from 10.5 to 11 minutes, and 100% phase A from 11 to 15.5 minutes. Autosampler temperature was 15°C, and injection volume was 15 μL. Mass spectra were acquired by use of positive electrospray ionization with the ion spray voltage at +5,500 V. Source temperature was 450°C; curtain gas, ion source gas 1, and ion source gas 2 were at 220,632, 344,738, and 448,159 Pa, respectively. Multiple reaction monitoring was used for quantitation of minocycline (m/z, 458.2 → 441.1), with demeclocycline as the internal standard (m/z, 465.2 → 448.2). Aliquots of drug-free equine plasma, BAL fluid, and BAL cell suspensions were provided for generation of standard calibration curves prior to sample analysis. Extraction efficiency for minocycline and the internal standard and within- and between-run accuracy and precision for minocycline detection in plasma, BAL supernatant, and BAL cell suspensions were performed.
Determination of minocycline concentration in PELF and BAL cells
Estimation of the volume of PELF was performed via the urea dilution method, as described elsewhere.18,21 Urea concentrations within BAL fluid and time-matched plasma samples were measured by use of a commercially available quantitative colorimetric urea determination kit.q The kit involved use of the improved Jung method, which utilized a chromogenic reagent that specifically formed a colored complex with urea. Intensity of the color (measured at 520 nm) was directly proportional to the urea concentration in the sample. The concentration of urea in the BAL fluid and plasma was used to calculate the volume of PELF as follows:
where VolBAL represented the volume of BAL fluid measured at time of collection, and UreaBAL and UreaPlasma represented the concentration of urea in BAL fluid and plasma, respectively.
Once the volume of PELF was known, concentrations of minocycline in PELF were calculated as follows:
where MinoBAL represented the concentration of minocycline measured in BAL fluid by use of LC-MS-MS.
Pharmacokinetic and statistical analysis
For each horse, plasma minocycline concentration-versus-time data were analyzed with noncompartmental pharmacokinetics by use of commercially available software.r Values for Cmax and Tmax were obtained directly from the data. Values for λz were determined by linear regression of the logarithmic plasma concentration-versus-time curve by use of a minimum of 4 data points. Half-life of the terminal phase was calculated as ln 2/λz. The AUC and AUMC were calculated by use of the trapezoidal rule, with extrapolation to infinity by use of the plasma concentration at the last measurable time point. Mean residence time was calculated as AUMC/AUC. Bioavailability was calculated as (AUCIntragastric/AUCIV) × (doseIV/doseIntragastric), where AUCIntragastric and AUCIV were the AUCs for the intragastric and IV administrations, respectively, and doseIV and doseIntragastric were the doses for the IV and intragastric administrations, respectively. Apparent volume of distribution based on the AUC was calculated as doseIV/(AUC × λz), apparent volume of distribution at steady state was calculated as (doseIV/AUC)/(AUMC/AUC), and systemic clearance was calculated as doseIV/AUC.
Normality of the data and equality of variances were assessed by use of the Shapiro-Wilk and Levene tests, respectively. A paired t test was used for comparisons of Cmax in PELF and BAL cells after intragastric and IV administration of minocycline and for comparison of AUC to determine steady state. Comparison of Cmax among sample types (plasma, PELF, and BAL cells) was performed by use of a Friedman repeated-measures ANOVA. When necessary, multiple pairwise comparisons were performed by use of the Student-Newman-Keuls method. Comparison of minocycline trough concentrations in plasma and PELF was performed by use of a linear mixed-effects model, with horse included as a random effect and the sample type, time, and time-by-sample type interaction included as fixed nominal effects. Model fit was assessed by use of Akaike information criterion values. Significance was set at P < 0.05.
Results
Horses
The same horses were used for experiments 1 and 2, except for 1 gelding that was lost to the study for an unrelated reason after experiment 1 and was replaced with another gelding for experiment 2. All horses tolerated IV and intragastric administration of minocycline and BAL procedures well for both experiments, and adverse clinical signs were not evident during drug administration. One horse developed a mild fever (rectal temperature, 39.4°C) at 36 hours of experiment 2. All other physical examination findings were within anticipated limits, and a single dose of flunixin meglumine (1.1 mg/kg, IV) was administered to that horse. The BAL at 36 hours was not performed, but the drug dose was administered at 36 hours. Results for all subsequent physical examinations of that horse were within anticipated limits through 96 hours after drug administration, and the horse was included in the remainder of the study. Mean BAL total WBC counts increased over the course of both experiments, which corresponded to relative decreases in the percentage of macrophages and increases in the percentage of neutrophils identified (Table 1).
Mean ± SD total WBC count and percentage of various cells measured in BAL fluid obtained from 6 horses during 2 experiments.
| Time (h)* | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Experiment | Variable | 2 | 4 | 8 | 24 | 36 | 48 | 54 | 60 | 72 |
| 1 | ||||||||||
| No. of WBCs | 154 ± 95 | 197 ± 97 | 171 ± 54 | 180 ± 50 | — | — | — | — | — | |
| Macrophages (%) | 37 ± 4 | 37 ± 13 | 37 ± 15 | 32 ± 9 | — | — | — | — | — | |
| Lymphocytes (%) | 54 ± 7 | 53 ± 17 | 53 ± 16 | 49 ± 7 | — | — | — | — | — | |
| Neutrophils (%) | 5 ± 2 | 9 ± 7 | 17 ± 24 | 16 ± 7 | — | — | — | — | — | |
| Eosinophils (%) | 0.3 ± 0.5 | 0.1 ± 0.2 | 0.2 ± 0.3 | 0.3 ± 0.4 | — | — | — | — | — | |
| Mast cells (%) | 3.0 ± 4.0 | 3.0 ± 4.0 | 3.0 ± 2.0 | 3.0 ± 3.0 | — | — | — | — | — | |
| 2 | No. of WBCs | — | — | — | 230 ± 94 | 267 ± 86 | 336 ± 150 | 516 ± 548 | 874 ± 1,048 | 493 ± 342 |
| Macrophages (%) | — | — | — | 44 ± 13 | 45 ± 6 | 32 ± 17 | 29 ± 10 | 28 ± 15 | 24 ± 9 | |
| Lymphocytes (%) | — | — | — | 47 ± 13 | 47 ± 11 | 43 ± 13 | 48 ± 18 | 37 ± 15 | 38 ± 19 | |
| Neutrophils (%) | — | — | — | 8 ± 5 | 6 ± 5 | 23 ± 24 | 21 ± 26 | 35 ± 29 | 37 ± 27 | |
| Eosinophils (%) | — | — | — | 0.1 ± 0.2 | 0.1 ± 0.3 | 0.1 ± 0.2 | 0.2 ± 0.3 | 0.2 ± 0.5 | 0.1 ± 0.2 | |
| Mast cells (%) | — | — | — | 2.0 ± 1.0 | 1.0 ± 0.6 | 2.0 ± 0.6 | 1.0 ± 2.0 | 0.7 ± 0.8 | 1.0 ± 1.0 | |
For experiment 1, a single dose of minocycline was administered IV (2.2 mg/kg) or intragastrically (4 mg/kg) to 6 horses; time 0 was the time of drug administration, and there was a 7-day washout period between treatments. For experiment 2, minocycline was administered intragastrically (4 mg/kg, q 12 h, for 5 doses) to 6 horses; time 0 was the time of the administration of the first dose.
— = Not determined.
For experiment 1, mean plasma concentration-time curves for minocycline after IV and intragastric administration were constructed (Figure 1). Mean ± SD Cmax and oral bioavailability and median Tmax after intragastric administration of minocycline were 1.584 ± 0.9 μg/mL, 48 ± 15%, and 1.0 hour (range, 0.5 to 1.0 hour), respectively (Table 2).
Mean ± SEM values for the concentration-time curve after intragastric (4 mg/kg; black squares) and IV (2.2 mg/kg; white circles) administration of a single dose of minocycline to 6 horses; there was a 7-day washout period between treatments. The AUC was 14 ± 5.2 μg•h/mL after IV administration and 12 ± 5.1 μg•h/mL after intragastric administration.
Citation: American Journal of Veterinary Research 78, 11; 10.2460/ajvr.78.11.1319
Mean ± SD values for plasma pharmacokinetic variables after IV (2.2 mg/kg) or intragastric (4 mg/kg) administration of a single dose of minocycline to 6 adult horses in experiment 1.
| Variable | IV | Intragastric |
|---|---|---|
| λz (h−1) | 0.067 ± 0.008 | NA |
| t1/2λz (h) | 10.5 ± 1.3 | NA |
| Cinitial (μg/mL) | 3.862 ± 1.378 | NA |
| Vdarea (L/kg) | 2.73 ± 1.24 | NA |
| Vdss (L/kg) | 2.16 ± 0.84 | NA |
| CL (mL/h/kg) | 176.2 ± 65.2 | NA |
| AUC0–24h (μg•h/mL) | 13.5 ± 5.0 | 10.8 ± 4.0 |
| AUC0–∞ (μg•h/mL) | 14.0 ± 5.2 | 11.9 ± 5.1 |
| AUMC0–∞ (μg•h2/mL) | 170.3 ± 60.6 | NA |
| MRT (h) | 12.2 ± 0.9 | NA |
| C12h (μg/mL) | 0.294 ± 0.197 | 0.248 ± 0.073 |
| Cmax (μg/mL) | NA | 1.584 ± 0.899 |
| Tmax (h)* | NA | 1.0 (0.5–1.0) |
| F (%) | NA | 48.2 ± 15.2 |
Value reported is median (range).
AUC0–24h = The AUC from time 0 to 24 hours. AUC0–∞ = The AUC extrapolated from time 0 to infinity. AUMC0–∞ = The AUMC extrapolated from time 0 to infinity. C12h = Plasma concentration 12 hours after drug administration. Cinitial = Initial plasma concentration measured at 5 minutes after drug administration. CL = Systemic clearance. F = Oral bioavailability. MRT = Mean residence time. NA = Not applicable. t1/2λz = Half-life of the terminal phase. Vdarea = Apparent volume of distribution as determined on the basis of AUC. Vdss = Apparent volume of distribution at steady state.
Mean ± SD urea dilution factor for minocycline concentrations in PELF was 61.15 ± 42.2 for experiments 1 and 2. Concentrations of minocycline detected in PELF and BAL cells after a single dose of minocycline administered IV or intragastrically were summarized (Table 3). Mean ± SD Cmax and median Tmax in PELF after intragastric and IV administration of minocycline were 5.032 ± 1.26 μg/mL and 3 hours (range, 2.0 to 4.0 hours), respectively, and 8.65 ± 8.29 μg/mL and 2.0 hours (range, 2.0 to 4.0 hours), respectively. Mean ± SD Cmax and median Tmax in BAL cells after intragastric and IV administration of minocycline were 0.006 ± 0.008 μg/mL and 2 hours (range, 2.0 to 8.0 hours), respectively, and 0.007 ± 0.007 μg/mL and 6.0 hours (range, 2.0 to 8.0 hours), respectively. Values for Cmax did not differ significantly between intragastric and IV administration of minocycline for PELF (P = 0.314) or BAL cells (P = 0.799). The Cmax in PELF was significantly (P < 0.001) greater than the Cmax in plasma and BAL cells for both intragastric and IV administration. The Cmax in BAL cells was significantly (P < 0.001) lower than the Cmax in plasma and PELF for both IV and intragastric administration of minocycline.
Mean ± SD minocycline concentrations (μg/mL) in PELF and BAL cells after IV (2.2 mg/kg) or intragastric (4 mg/kg) administration of a single dose of minocycline to 6 adult horses in experiment 1.
| PELF | BAL cells | |||
|---|---|---|---|---|
| Time (h) | IV | Intragastric | IV | Intragastric |
| 2 | 8.53 ± 8.40 | 4.48 ± 1.60 | 0.005 ± 0.007 | 0.005 ± 0.008 |
| 4 | 3.31 ± 1.30 | 3.96 ± 1.40 | 0.003 ± 0.005 | 0.003 ± 0.003 |
| 8 | 2.29 ± 1.90 | 1.21 ± 0.71 | 0.003 ± 0.002 | 0.001 ± 0.001 |
| 24 | 0.70 ± 0.40 | 0.72 ± 0.50 | 0.001 ± 0.001 | 0.001 ± 0.001 |
For experiment 2, plasma pharmacokinetic variables after intragastric administration of the first and fifth doses of minocycline were summarized (Table 4). Plasma minocycline concentrations for experiment 2 were graphed (Figure 2). The mean ± SD AUC from time 0 extrapolated to infinity after intragastric administration of the first dose of minocycline (15.2 ± 5.5 μg•h/mL) was not significantly (P = 0.613) different from that after intragastric administration of the fifth dose (between 48 and 60 hours; 13.9 ± 6.4 μg•h/mL), which indicated that steady state was reached. At steady state, mean plasma Cmax was 2.3 ± 1.3 μg/mL, mean terminal half-life was 11.8 ± 0.5 hours, and median Tmax was 1.3 hours (range, 1.0 to 1.5 hours).
Mean ± SEM minocycline concentrations in plasma during intragastric administration of multiple doses (4 mg/kg, q 12 h, for a total of 5 doses) to achieve a steady state in 6 horses. Time points in circles represent trough concentrations obtained immediately before minocycline administration. Time 0 was the administration of the first dose of minocycline.
Citation: American Journal of Veterinary Research 78, 11; 10.2460/ajvr.78.11.1319
Mean ± SD values for plasma pharmacokinetic variables after intragastric administration of minocycline (4 mg/kg, q 12 h, for 5 doses) to 6 adult horses.
| Variable | First dose | Fifth dose |
|---|---|---|
| Cmax (μg/mL) | 1.73 ± 1.05 | 2.33 ± 1.27 |
| Tmax (h)* | 1.0 (1.0–1.5) | 49.3 (49.0–49.5) |
| AUC0–∞ (μg•h/mL) | 15.20 ± 5.50 | 39.90 ± 15.74 |
| AUC0–12h (μg•h/mL) | 9.11 ± 3.84 | NA |
| AUC0–SS (μg•h/mL) | NA | 38.55 ± 15.07 |
| t1/2λz (h) | NA | 11.80 ± 0.53 |
Time of administration of the first dose was designated as time 0.
AUC0–12h = The AUC from time 0 to 12 hours. AUC0–SS = The AUC from time 0 to steady state (12 hours after the fifth dose).
See Tables 2 and 3 for remainder of key.
Minocycline was detected in PELF and BAL cells at all measurement points, but concentrations in PELF were much higher than those measured in BAL cells, and changes in concentrations in PELF or BAL cells were not detected during administration to achieve plasma steady state (Figure 3). Time-matched pharmacokinetic variables for PELF, BAL cells, and plasma after intragastric administration of 5 doses of minocycline were summarized (Table 5). The Cmax of minocycline in PELF was significantly greater than that in both plasma and BAL cells, and the Cmax in BAL cells was significantly lower than that in both plasma and PELF. Minocycline concentrations measured in PELF were significantly (P < 0.001) greater than plasma concentrations at all trough time points (Figure 4). Mean trough concentrations in plasma were all > 0.55 μg/mL; for each horse, the minimum trough concentration in plasma was > 0.25 μg/mL. Median concentrations in PELF at all time points were > 2.0 μg/mL; for each horse, the minimum concentration in PELF was > 0.68 μg/mL.
Box-and-whisker plots of minocycline concentrations in PELF (A) and BAL cells (B) obtained from 6 horses before intragastric administration (24, 36, and 48 hours) and at 6, 12, and 24 hours (time points 54, 60, and 72 hours, respectively) after administration of the final of 5 doses of minocycline (experiment 2). Time 0 was the administration of the first dose of minocycline. Each box represents the interquartile range, the horizontal line in each box represents the median, and the whiskers represent the range (minimum to maximum) of drug concentrations. Notice that the scale on the y-axis differs between panels.
Citation: American Journal of Veterinary Research 78, 11; 10.2460/ajvr.78.11.1319
Trough concentration of minocycline in plasma (circles) and PELF (squares) obtained from 6 horses receiving 5 doses of minocycline via intragastric administration. Time 0 was the administration of the first dose of minocycline. Values reported are mean and range (minimum to maximum) for plasma and median and range (minimum to maximum) for PELF. The target concentration of 0.25 μg/mL is indicated (dashed horizontal line). *Within a time point, values differ significantly (P < 0.001) between plasma and PELF.
Citation: American Journal of Veterinary Research 78, 11; 10.2460/ajvr.78.11.1319
Median (interquartile range) values for plasma, PELF, and BAL cell pharmacokinetic variables after oral administration of minocycline (4 mg/kg, q 12 h, for 5 doses) to 6 adult horses.
| Variable | Plasma* | PELF | BAL cells |
|---|---|---|---|
| Cmax (μg/mL) | 0.980 (0.620–1.140)a | 5.23 (2.990–17.600)b | 0.007 (0.004–0.017)c |
| Tmax (h) | 6.0 (6.0–6.0) | 9.0 (4.5–12.0) | 3.0 (0–6.0) |
| AUC48h–96h (μg•h/mL) | 15.40 (9.73–17.70)a | 66.34 (42.46–220.66)b | 0.067 (0.031–0.182)c |
Time of administration of the first dose was designated as time 0.
Only plasma samples obtained at the time of BAL collection were considered for calculation of variables.
AUC48h–96h = The AUC from 48 hours to 96 hours.
Within a row, values with different superscript letters differ significantly (P < 0.05; Friedman repeated-measures ANOVA and, when necessary, multiple pairwise comparisons by use of the Student-Newman-Keuls method).
Discussion
In the present study, oral bioavailability (48%) differed considerably among adult horses (35% to 74.5%). Although minocycline was detected in the PELF and BAL cells within 3 hours after IV or intragastric administration, only concentrations in the PELF exceeded those in plasma. As predicted, trough concentrations in PELF exceeded the target concentration of 0.25 μg/mL at all measurement points. Contrary to our hypothesis, minocycline concentrations in BAL cells at all measurement points were well below concentrations detected in plasma and PELF as well as the target concentration of 0.25 μg/mL.
Oral bioavailability of minocycline differs considerably across species.26–28 Estimated oral bioavailability of minocycline was 23% in fed adult horses of 1 study.15 Recently, investigators determined that mean ± SD oral bioavailability of minocycline was 32.0 ± 18.0% in adult horses and 57.8 ± 19.3% in foals.29 In experiment 1 of the study reported here, bioavailability was greater than that reported for adult horses in both of those aforementioned studies15,29 and greatly exceeded estimates of bioavailability after oral administration of doxycycline to adult horses.13,27,30 In the present study and those of other investigators,15,29 minocycline was administered as a single dose (4 mg/kg) via nasogastric intubation, but it is not known whether differences in feeding schedules between the present study (hay fed twice daily) and those other studies15,29 (hay fed ad libitum) may have influenced drug absorption and thus measurement of bioavailability.
Feeding can decrease oral bioavailability for a variety of drugs.31–34 Although the impact of feeding on minocycline bioavailability has not been investigated in adult horses, significant reductions in Cmax of minocycline have been identified in fed versus nonfed dogs,34 and Cmax of doxycycline was significantly reduced in horses when the drug was administered concurrently with feed as opposed to 2 hours after feeding.33 During experiment 2 of the present study, horses were fed twice daily in accordance with a standard feeding schedule. Because of constraints associated with collection of BAL samples, horses were fed 2 hours after the morning drug administration and 3 to 4 hours before the evening drug administration. Interestingly, plasma concentrations of minocycline measured 2 hours after morning drug administration were consistently higher than those measured 2 hours after evening drug administration, which suggested that feeding may have impacted drug bioavailability.
In experiments 1 and 2 of the study reported here, minocycline concentrations in PELF exceeded concentrations in plasma at all time points, with a mean Cmax in PELF that was 5 times as high as the Cmax in plasma at steady state. Penetration into the PELF is influenced by drug lipophilicity and protein binding.35 The lipophilic properties of group 2 tetracyclines (minocycline and doxycycline)16,18,26 may explain the high minocycline concentrations in PELF in the present study as well as similar findings reported for minocycline concentrations in foals29 and for doxycycline concentrations in adult horses and foals.30,33
Plasma protein binding influences a drug's ability to penetrate tissue because only the unbound fraction is available to cross biological membranes. The percentage of plasma protein binding of minocycline was not determined in the present study but has been reported to be approximately 68% in adult horses,19 which is similar to percentages in dogs and humans16,26 and lower than estimated protein binding of doxycycline.30 Binding to protein within the PELF can further influence the amount of active drug that is available,24 particularly during inflammatory conditions that result in changes in protein concentrations within the PELF. Protein concentrations in PELF are much lower than those in plasma of healthy humans and humans with pulmonary disease,23 and in healthy adult horses, the percentage of protein binding of doxycycline in the PELF is much lower than the percentage of protein binding in the plasma.35
Pulmonary pharmacokinetic analysis in the study reported here was based on measurement of minocycline concentrations in the PELF and BAL cells by use of BAL fluid accompanied by volume correction with the urea dilution method. The PELF represents secretions on the interior alveolar wall and within the small bronchi and is often a fluid where bacterial organisms reside.35 Thus, measurement of drug concentrations in the PELF (and alveolar cells) is thought to provide superior information on intrapulmonary antimicrobial activity, compared with measurement of drug concentrations in plasma or tissue homogenate.20,35,36
Whereas BAL with urea dilution has been widely described in horses and other species,9,29,30,35–40 potential limitations associated with urea dilution that may result in overestimation or underestimation of active drug concentrations within the PELF include cell lysis during collection of BAL fluid, blood contamination, increased BAL fluid dwell time, and measurement of both protein bound and unbound drug.20,24,41,42 Standardization of the BAL technique, as was done in the present study, is believed to minimize some of these limitations.24 Direct collection of PELF samples by use of a bronchial swab technique has also been described for large animals and offers the advantage of not requiring volume correction.42 This technique measures drug concentrations at the level of the trachea or bronchi and thus may not be directly comparable to BAL-derived measurements for samples from the bronchioles and alveoli.24,41
Bronchoalveolar lavage in standing sedated horses is a repeatable and relatively noninvasive procedure that typically causes little iatrogenic damage.28,43,44 The possibility exists that multiple BALs within the same horse could influence drug concentrations in PELF because of residual fluid remaining in a lung from a previous BAL or the potential that multiple BALs could cause inflammation within the lungs. Visual evidence of residual fluid was not detected during the bronchoscopic-guided BALs. However, cytologic examination of BAL samples identified increases in cell count and neutrophil numbers, which suggested some degree of inflammation associated with the procedure. Inflammation is thought to improve pulmonary drug absorption by facilitating drug transport into the PELF.24,28 Whether the degree of transient inflammation associated with the BALs was sufficient to influence minocycline concentrations in PELF in the present study is unknown.
In the study reported here, minocycline Cmax in BAL cells represented only 0.4% of minocycline Cmax in plasma and 0.1% of minocycline Cmax in PELF after single and multiple orally administered doses. This finding was surprising because we expected to detect higher intracellular concentrations. High intracellular concentrations have been reported for tetracyclines,33,45,46 with cellular-to-intracellular concentration ratios in polymorphonuclear neutrophilic leukocytes of 13 and 17 for humans46 and adult horses,33 respectively. Minocycline concentrations in PELF and BAL cells for foals29 were similar to those in the present study. Although it is possible that intracellular drug concentration in BAL cells is less than that of peripheral cells, given that the drug must first penetrate into the lungs, drug concentrations achieved in BAL cells of foals administered doxycycline were similar to serum concentrations.30 The exact reason for decreased minocycline concentrations in BAL cells in the study reported here is unknown. Lysis of BAL cells during sample processing can result in loss of drug for analysis, and it is possible that lysis may have occurred during preparation of the cell pellet for analysis. If poor minocycline activity truly exists in BAL cells, it would be less likely to be of clinical concern in adult horses because bacterial pathogens typically associated with pneumonia are extracellular and would thus be susceptible to the high minocycline concentrations in the PELF.
Tetracyclines have good antimicrobial activity against many Streptococcus spp.15 To accurately predict the efficacy of an antimicrobial for the treatment of bacterial pneumonia, it is important to have knowledge of specific MICs for common bacterial pathogens in relation to achievable concentrations in PELF. An MIC < 0.25 μg/mL is often used to predict efficacy of doxycycline and minocycline for the treatment of pneumonia and other bacterial infections in horses5; therefore, it was chosen as the target drug concentration in the PELF and BAL cells for the present study. Information on MIC values for minocycline against S equi subsp zooepidemicus in horses is limited. The minocycline MIC that inhibits 50% of all isolates is reported to be between 0.06 and 0.12 μg/mL,15,46 whereas the minocycline MIC that inhibits 90% of all isolates is reported to be between 0.12 and 8 μg/mL.15,43 Geographic differences in antimicrobial susceptibility may exist between those studies; however, those results suggest that minocycline concentrations achievable in the PELF would be sufficient to treat most S equi subsp zooepidemicus–induced bacterial pneumonias.
Results of the present study suggested that minocycline was distributed into the PELF and BAL cells of healthy adult horses, but only minocycline concentrations in PELF would have been sufficient for the treatment of bacterial pneumonia. To better predict drug bioavailability and pulmonary disposition in clinically affected horses, the impact of feeding should be investigated, as should the pulmonary distribution of minocycline in horses with pulmonary disease.
Acknowledgments
Supported by the Illinois Equine Industry Research and Promotion Board, University of Illinois Companion Animal Memorial Research Fund, and American Quarter Horse Association.
Presented in part in abstract form at the American College of Veterinary Internal Medicine Forum, Denver, June 2016.
ABBREVIATIONS
| AUC | Area under the concentration-time curve |
| AUMC | Area under the first moment of the concentration-time curve |
| BAL | Bronchoalveolar lavage |
| Cmax | Maximum plasma concentration |
| λz | Rate constant of the terminal phase |
| LC-MS-MS | Liquid chromatography–mass spectrometry–mass spectrometry |
| MIC | Minimum inhibitory concentration |
| PELF | Pulmonary epithelial lining fluid |
| Tmax | Time to achieve maximum plasma concentration |
Footnotes
Foreman JH, Hungerford LL, Folz SD. Transport stress-induced pneumonia: a model in young horses (abstr), in Proceedings. 6th Int Conf Equine Infect Dis 1992;6:313.
Beckman Coulter AU680, Beckman Coulter Inc, Indianapolis, Ind.
Mila International Inc, Erlanger, Ky.
Fagron Inc, Saint Paul, Minn.
Hospira Inc, Lake Forest, Ill.
Supor membrane, Pall Animal Health, Port Washington, N Y.
Covidien Ltd, Dublin, Ireland.
AnaSed 100 injection, Lloyd, Shenandoah, Iowa.
Olympus Corporation of the Americas, Center Valley, Pa.
Abbott Laboratories, North Chicago, Ill.
Sigma-Aldrich Corp, Atlanta, Ga.
QSonica LLC, Newton, Conn.
Metabolomics Laboratory, Roy J. Carver Biotechnology Center, University of Illinois, Urbana, Ill.
5500 QTrap LC/MS/MS system, Sciex, Framingham Mass.
1200 series HPLC system, Agilent Technologies, Santa Clara, Calif.
Analyst software, version 1.6.2, AB Sciex LP, Concord, ON, Canada.
BioChain urea assay kit Z5030016, Biochain Institute Inc, Newark, Calif.
PK Solutions, version 2.0, Summit Research Services, Montrose, Colo.
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