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    Mean ± SD clarithromycin concentrations measured by use of an HPLC method (black diamonds) or clarithromycin activity measured by use of a microbiologic assay (gray squares) in serum samples obtained from 6 foals after IV administration of a single dose of clarithromycin (7.5 mg/kg). Time 0 = Time of IV administration.

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    Mean ± SD clarithromycin activity measured by use of a microbiologic assay in serum samples obtained from 6 foals administered 6 doses of clarithromycin (7.5 mg/kg) IG at 0, 24, 36, 48, 60, and 72 hours.

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Pharmacokinetics of clarithromycin and concentrations in body fluids and bronchoalveolar cells of foals

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  • 1 Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610-0136.
  • | 2 Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610-0136.
  • | 3 Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610-0136.
  • | 4 Departments of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610-0136.

Abstract

Objective—To determine pharmacokinetics of clarithromycin and concentrations in body fluids and bronchoalveolar (BAL) cells of foals.

Animals—6 healthy 2-to 3-week-old foals.

Procedures—In a crossover design, clarithromycin (7.5 mg/kg) was administered to each foal via IV and intragastric (IG) routes. After the initial IG administration, 5 additional doses were administered IG at 12-hour intervals. Concentrations of clarithromycin and its 14-hydroxy metabolite were measured in serum by use of high-performance liquid chromatography. A microbiologic assay was used to measure clarithromycin activity in serum, urine, peritoneal fluid, synovial fluid, CSF, pulmonary epithelial lining fluid (PELF), and BAL cells.

Results—After IV administration, elimination half-life (5.4 hours) and mean ± SD body clearance (1.27 ± 0.25 L/h/kg) and apparent volume of distribution at steady state (10.4 ± 2.1 L/kg) were determined for clarithromycin. The metabolite was detected in all 6 foals by 1 hour after clarithromycin administration. Oral bioavailability of clarithromycin was 57.3 ± 12.0%. Maximum serum concentration of clarithromycin after multiple IG administrations was 0.88 ± 0.19 μg/mL. After IG administration of multiple doses, clarithromycin concentrations in peritoneal fluid, CSF, and synovial fluid were similar to or lower than concentrations in serum, whereas concentrations in urine, PELF, and BAL cells were significantly higher than concentrations in serum.

Conclusions and Clinical Relevance—Oral administration of clarithromycin at 7.5 mg/kg every 12 hours maintains concentrations in serum, PELF, and BAL cells that are higher than the minimum inhibitory concentration (0.12 μg/mL) for Rhodococcus equiisolates for the entire 12-hour dosing interval.

Abstract

Objective—To determine pharmacokinetics of clarithromycin and concentrations in body fluids and bronchoalveolar (BAL) cells of foals.

Animals—6 healthy 2-to 3-week-old foals.

Procedures—In a crossover design, clarithromycin (7.5 mg/kg) was administered to each foal via IV and intragastric (IG) routes. After the initial IG administration, 5 additional doses were administered IG at 12-hour intervals. Concentrations of clarithromycin and its 14-hydroxy metabolite were measured in serum by use of high-performance liquid chromatography. A microbiologic assay was used to measure clarithromycin activity in serum, urine, peritoneal fluid, synovial fluid, CSF, pulmonary epithelial lining fluid (PELF), and BAL cells.

Results—After IV administration, elimination half-life (5.4 hours) and mean ± SD body clearance (1.27 ± 0.25 L/h/kg) and apparent volume of distribution at steady state (10.4 ± 2.1 L/kg) were determined for clarithromycin. The metabolite was detected in all 6 foals by 1 hour after clarithromycin administration. Oral bioavailability of clarithromycin was 57.3 ± 12.0%. Maximum serum concentration of clarithromycin after multiple IG administrations was 0.88 ± 0.19 μg/mL. After IG administration of multiple doses, clarithromycin concentrations in peritoneal fluid, CSF, and synovial fluid were similar to or lower than concentrations in serum, whereas concentrations in urine, PELF, and BAL cells were significantly higher than concentrations in serum.

Conclusions and Clinical Relevance—Oral administration of clarithromycin at 7.5 mg/kg every 12 hours maintains concentrations in serum, PELF, and BAL cells that are higher than the minimum inhibitory concentration (0.12 μg/mL) for Rhodococcus equiisolates for the entire 12-hour dosing interval.

Clarithromycin is a semisynthetic macrolide antimicrobial agent chemically derived from erythromycin A. It differs from erythromycin A in that clarithromycin has an O-methyl ether substitution at position 6 of the macrolide ring. This change provides greater stability in gastric acid, which results in enhanced absorption after oral administration. In humans, this structural difference also results in a longer t1/2, a larger volume of distribution, as well as improved uptake by tissues and phagocytic cells, compared with erythromycin.1,2

Clarithromycin undergoes extensive hepatic metabolism in humans and is primarily metabolized to 14-hydroxy-clarithromycin.3 This metabolite is responsible for approximately 50% of the total biological activity of clarithromycin and has an additive or synergistic effect with the parent compound.4,5

Macrolide antimicrobial agents are commonly used in combination with rifampin for treatment of foals with Rhodococcus equi infections. Rhodococcus equi, a gram-positive, facultative, intracellular pathogen that survives in macrophages, is a common cause of pneumonia in foals between 3 weeks and 5 months of age. Historically, combined treatment with erythromycin and rifampin has dramatically improved the survival rate of affected foals.6 However, evidence indicates that clarithromycin may be superior to erythromycin for the treatment of foals with pneumonia attributable to R equi. Clarithromycin is more active against R equi in vitro than is erythromycin or azithromycin.7 In addition, in a retrospective study8 of foals examined and treated at a referral institution, the combination of clarithromycin and rifampin was found to be superior to the combination of azithromycin and rifampin or erythromycin and rifampin for the treatment of foals with pneumonia caused by R equi.

In a preliminary study,9 investigators confirmed that therapeutic concentrations are achieved in serum after oral administration of clarithromycin to foals. However, only a single dose was orally administered, which precluded accurate determination of steady-state drug concentrations and calculation of important pharmacokinetic variables such as oral bioavailability, clearance, and apparent volume of distribution. In addition, concentrations of the drug in body fluids, PELF, and BAL cells were not measured. Recent evidence suggests that the concentration of macrolides at the site of infection may be a better indicator of clinical efficacy than are serum concentrations alone.10 Finally, the methods used to measure drug concentrations in the aforementioned preliminary study9 did not allow detection of metabolites, such as 14-hydroxyclarithromycin, that may influence efficacy.

The objectives of the study reported here were to determine the pharmacokinetics and oral bioavailability of clarithromycin in foals as well as to measure drug concentrations in body fluids and BAL cells after IG administration of a multiple-dose regimen of clarithromycin. An additional objective was to determine whether clarithromycin is converted to the 14-hydroxy metabolite in foals.

Materials and Methods

Animals—Four male and 2 female foals (5 Thoroughbreds and 1 Quarter Horse) between 2 and 3 weeks of age and weighing between 71 and 100 kg were selected for use in the study. The foals were considered healthy on the basis of the medical history and results of physical examination, a CBC, and plasma biochemical analysis. The foals were allowed to remain with their dams. Each dam and foal were housed in a separate stall during the experiment. Horses were provided ad libitum access to grass hay and water. The study was approved by the Institutional Animal Care and Use Committee of the University of Florida.

Experimental design—Clarithromycin (7.5 mg/kg) was administered via the IV and IG routes in accordance with a 2-treatment, 2-period crossover design. A washout period of at least 10 days was allowed between administrations for the 2 dosing routes. For IV administration, purified clarithromycin powdera (potency of 0.977 μg/mg) was dissolved in sterile water to achieve a concentration of 100 mg/mL and administered to each foal as a single bolus through a catheter inserted into the left jugular vein. Blood samples were obtained from a catheter inserted in the right jugular vein before administration (time 0) and 3, 6, 10, 20, 30, 60, and 90 minutes and 2, 3, 4, 6, 8, 12, and 24 hours after the drug was administered.

For IG administration, clarithromycin tablets (250-mg tablets)b were dissolved in 50 mL of water and administered to each foal by use of a nasogastric tube. For the first 24 hours, blood samples were collected at the time points described for IV administration. Beginning 24 hours after the initial administration, 5 additional doses of clarithromycin were administered IG at 12-hour intervals (ie, 24, 36, 48, 60, and 72 hours). Blood samples were collected immediately before administration of each additional dose and 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 12 hours after doses 2, 4, and 6.

Bronchoalveolar lavage was performed and samples were collected 2 and 12 hours after the last IG administration. Foals were sedated by IV administration of xylazine hydrochloride (1.0 mg/kg) and butorphanol tartrate (0.07 mg/kg). A 10-mm-diameter, 1.8-m bronchoscopec was passed via the nasal passages into the left or right lung until it became wedged in a fourth-to sixth-generation bronchus.

The lavage solution consisted of 4 aliquots of 50 mL of physiologic saline (0.9% NaCl) solution, which was infused and aspirated immediately. The bronchoscope was alternately passed into the left or right lung to prevent the effect of repeated BAL lavages on differential cell counts. Total nucleated cell count in BAL lavage fluid was determined by use of a hemacytometer. The BAL fluid was centrifuged at 200 × g for 10 minutes. Then, BAL cells were washed, resuspended in 1 mL of PBS solution, vortexed, and frozen at −80°C until assayed. Supernatant BAL lavage fluid was also frozen at −80°C until assayed.

Immediately after collection of BAL fluid, general anesthesia was induced by IV administration of diazepam (0.1 mg/kg) and ketamine (2.5 mg/kg), and samples of synovial fluid, peritoneal fluid, CSF, and urine were collected aseptically. Samples of synovial fluid were obtained from an intercarpal or radiocarpal joint by use of a 20-gauge needle. Samples of CSF were collected from the atlantooccipital space by use of a 3.5-inch, 20-gauge spinal needle. Abdominal fluid was collected by use of an 18-gauge needle. A flexible 8-F Foley catheter was used to collect urine directly from the bladder. Samples were centrifuged, and supernatants were stored at −80°C until analysis.

Preparation of BAL cells—Cell pellets were thawed, vortexed vigorously, and sonicated for 2 minutes to ensure complete cell lysis. The resulting suspension was centrifuged at 500 × g for 10 minutes. Supernatant was harvested and used for determination of intracellular concentrations of clarithromycin.

Drug analysis by use of HPLC—Serum samples were processed through a 2-step extraction procedure and then analyzed by use of HPLC. Samples (500 μL) of serum were thawed, mixed with an equal volume of internal standard (roxithromycind; diluted in 10mM NaH2PO4 buffer [pH, 3.0] to achieve a final concentration of 4 μg/mL), and acidified with 2N HCl (final pH, 3.0). Each acidified sample was mixed briefly and transferred quantitatively onto a solidphase extraction columne that had been conditioned with 5 mL of methanol and 10mM phosphate buffer (pH, 3). Following loading of samples, each column was rinsed with 5 mL of 10mM phosphate buffer (pH, 3.0) prior to elution of drugs with 5 mL of alkalinized methanol (mixture of methanol:1N NaOH, 99:1). Eluates were collected and evaporated to dryness in a vacuum concentrator at ambient temperature. Dried samples were reconstituted in 4N NaOH (500 μL) by incubation at 25°C for 30 minutes with intermittent vortexing. Then, 3 mL of hexane:ethyl acetate (50:50) was added, and samples were mixed vigorously. Aqueous and organic layers were separated by centrifugation (4,000 × g for 4 minutes), and a portion of the organic layer was removed and evaporated to dryness.

Dried samples were reconstituted in the mobile phase and analyzed immediately by use of HPLCf with electrochemical detection. Samples were injected and separated on a reverse-phase columng by use of a filtered (0.2 μm), degassed mobile phase containing a mixture (55:45 [vol:vol]) of 1mM sodium phosphate (pH, 7.0):acetonitrile (final adjusted pH, 7.5) at a flow rate of 1 mL/min. Concentrations of the macrolide antimicrobials were measured by amperometric detection by use of an electrochemical detectorh with a platinum electrode set at +1,100 mV potential (full scale, 1 nA).

Peak areas for all 3 compounds had a linear relationship (r ≥ 0.99) for drug concentrations in the range of 0.25 to 5.00 μg/mL (14-hydroxy-clarithromycini) and 0.50 to 5.00 μg/mL (clarithromycinj and roxithromycin). Each sample was assayed in duplicate, and drug concentrations were estimated by comparison of peak areas against linear standard curves for each analyte. Thus, 0.5 μg/mL was used as the lowest limit of quantification for clarithromycin.

Mean retention time was 3.8, 9.0, and 11.0 minutes for 14-hydroxy-clarithromycin, clarithromycin, and roxithromycin, respectively. In spiked serum samples, drug extraction yields of clarithromycin (mean ± SD, 76 ± 3.2%) and the internal standard roxithromycin (77 ± 3.9%) were highly correlated (r = 0.98). In contrast, extraction yield for 14-hydroxy-clarithromycin was greater but more variable (92 ± 11.4%) and was poorly correlated (r = 0.78) with the internal standard. For that reason, exact concentrations of the metabolite were not reported. Within-day variability was ± 2.8% for 12 plasma samples containing 3.6μM roxithromycin and ± 2.0% for 12 plasma samples containing 4.8μM clarithromycin. Between-day relative SD values were ± 5.2% and ± 5.8% for 10 plasma samples containing 2.4μM roxithromycin and 3μM clarithromycin, respectively.

Measurement of clarithromycin activity by use of a microbiologic assay—Clarithromycin activity was determined in serum, synovial fluid, peritoneal fluid, CSF, BAL fluid, and BAL cells by use of an agar well diffusion microbiologic assay with Micrococcus luteusk as the assay organism. One milliliter of a bacterial suspension was grown overnight in trypticase soy broth and adjusted to an optical density of 0.5 at 550 nm. This suspension was added to tempered neomycin assay agarl and distributed evenly over the assay plates. Plates were allowed to solidify for 45 minutes, and 0.5-mm wells were punched and filled with 50 μL of samples or clarithromycinj standard ranging in concentration from 0.02 to 5.0 μg/mL. Known amounts of purified clarithromycin were added to equine serum, synovial fluid, and urine and used to generate standard curves for each type of matrix. The BAL cells, BAL fluid, CSF, and peritoneal fluid were assayed with standards diluted in PBS solution.

Agar plates were incubated for 36 hours at 30°C. Zones of bacterial inhibition were measured to the nearest millimeter by use of Vernier calipers. Each sample or standard was assayed in triplicate, and mean values for 3 measurements of the zone diameters were determined. The lower limit of quantification of the assay was 0.02 μg/mL for serum, BAL cells, and body fluid samples. Negative control samples did not cause bacterial inhibition, which indicated no antibacterial activity of equine serum, body fluids, or BAL cell supernatants. Plots of zone diameters versus standard clarithromycin concentrations were linear between 0.02 and 5 μg/mL (r values ranged between 0.993 and 0.998). Both intraday and interday coefficients of variation for repeated assay of samples were < 5% and < 10% at concentrations > 0.1 and < 0.1 μg/mL, respectively.

Determination of clarithromycin concentrations in PELF and BAL cells—Pulmonary distribution of clarithromycin was determined as reported elsewhere.11 Estimation of the volume of PELF was accomplished by use of a urea dilution method.12,13 Serum concentrations of urea nitrogen were determined by use of an enzymatic methodm on a chemistry analyzer.n For measurement of urea concentration in BAL fluid, the proportion of reagents to specimen was changed from 300 μL of reagent/3 μL of serum to 225 μL of reagent/50 μL of BAL fluid. The volume of PELF in BAL fluid was derived by use of the following equation:

article image

where VPELF is the volume of PELF and VBALis the volume of recovered BAL fluid. The clarithromycin activity in PELF was derived by use of the following equation: clarithromycin activity in PELF = measured clarithromycin activity in BAL fluid × (VBAL/VPELF)

Clarithromycin activity in BAL cells was calculated by use of the following equation:

Clarithromycin activity in BAL cells = clarithromycin activity in the BAL cell pellet supernatant/VBALC

where VBALC is the mean volume of foal BAL cells. A VBALC of 1.20 μL/106 cells was used for calculations on the basis of results of another study14 in foals.

Pharmacokinetic analysis—For each foal, the plasma concentration–versus–time data were analyzed in accordance with noncompartmental pharmacokinetics by use of computer software.o The value for Kel was determined by linear regression of the terminal phase of the logarithmic plasma concentration–versus–time curve by use of a minimum of 3 datum points. The value for t1/2 was calculated as 0.693/Kel. Pharmacokinetic values were calculated as reported elsewhere.15 The AUC and AUMC were calculated in accordance with the trapezoidal rule with extrapolation to infinity by use of Cmin/Kel, where Cmin was the final measurable plasma clarithromycin activity. Mean residence time was calculated as AUMC/AUC. Apparent volume of distribution based on the AUC was calculated as follows: dose/(AUC × Kel). The Vdsswas calculated as follows: (dose/AUC)/(AUMC/AUC). Systemic clearance was calculated as dose/AUC. Bioavailability was calculated as follows: (AUCIG/AUCIV) × (doseIV/doseIG), where AUCIG and AUCIV were the AUC for the IG and IV administrations, respectively.

Statistical analysis—In preliminary analyses, a Mann-Whitney U test was used to assess the effect of the order of route of administration (IV administration first vs IG administration first) on each pharmacokinetic variable and the drug activity. Because there were no significant effects, data from the 6 foals were pooled. Pharmacokinetic-derived data were reported as mean ± SD unless otherwise specified. Paired t tests were used to compare differences in Kel between IV and IG administration; paired t tests were also used to detect significant differences between Cmax 0–24h and Cmax 72–84h. The Friedman repeated-measures ANOVA on ranks was used to compare clarithromycin activity among sample types (serum, synovial fluid, peritoneal fluid, CSF, urine, PELF, and BAL cells). When indicated, multiple pairwise comparisons were conducted by use of the Student-Newman-Keuls test. For each analysis, differences were considered significant at values of P < 0.05.

Results

One foal developed transient tachypnea and profuse sweating 5 minutes after IV administration of the clarithromycin bolus. One foal developed diarrhea after the third IG administration, and another foal developed diarrhea after the last IG administration. In both of these foals, the diarrhea resolved without treatment within 36 hours after onset.

After IV administration of 7.5 mg of clarithromycin/kg, serum drug concentrations were similar when measured by use of HPLC or the microbiologic assay, although HPLC-based measurements typically were higher immediately after drug administration (Figure 1). Pharmacokinetic variables were calculated from data obtained by use of the microbiologic assay because the high limit of quantification of the HPLC method did not allow accurate evaluation of the terminal elimination phase of the drug. The harmonic mean t1/2 of clarithromycin was 5.4 hours, mean ± SD body clearance was 1.27 ± 0.25 L/h/kg, and mean Vdss was 10.4 ± 2.1 L/kg (Table 1). Initial detection of 14-hydroxy-clarithromycin was at 0.5 hours after IV administration in 3 foals and by 1 hour after administration in all 6 foals. Mean time to maximum concentration of 14-hydroxy-clarithromycin after IV administration was 1.7 ± 1.2 hours.

Table 1—

Mean ± SD values for pharmacokinetic variables after IV administration of a single dose of clarithromycin (7.5 mg/kg) or IG administration of 6 doses of clarithromycin (7.5 mg/kg at 0, 24, 36, 48, 60, and 72 hours) to 6 foals.

VariableIVIG
Kel (/h)0.129±0.0220.141±0.050
AUC ([μg•h]/mL)6.2±1.53.4±1.1
AUMC ([μg•h2]/mL)51.1±16.224.4±9.7
MRT (h)8.3±1.07.1±1.7
t1/2(h)5.4*NA
Vdarea (L/kg)9.9±1.8NA
Vdss (L/kg)10.4±2.1NA
Clearance (L/h/kg)1.27±0.25NA
Tmax (h)NA1.6±0.4
Cmax 0–24h (μg/mL)NA0.52±0.17
Cmax 72–84h (μg/mL)NA0.88±0.19
C84h(μg/mL)NA0.20±0.06
F (%)NA57.3±12.0

Value reported is the harmonic mean

MRT = Mean residence time. NA = Not applicable. Vdarea = Apparent volume of distribution based on the AUC. Tmax = Time to maximum serum concentration of clarithromycin. C84h = Minimum serum concentration of clarithromycin 12 hours after administration of the last dose. F = Oral bioavailability.

Figure 1—
Figure 1—

Mean ± SD clarithromycin concentrations measured by use of an HPLC method (black diamonds) or clarithromycin activity measured by use of a microbiologic assay (gray squares) in serum samples obtained from 6 foals after IV administration of a single dose of clarithromycin (7.5 mg/kg). Time 0 = Time of IV administration.

Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1681

After IG administration, quantifiable clarithromycin activity was detected in 4 of 6 foals at 10 minutes and in all 6 foals at 15 minutes. Mean ± SD time to maximum serum clarithromycin activity was 1.6 ± 0.4 hours, and mean oral bioavailability was 57.3 ± 12.0% (Table 1). Mean maximum serum clarithromycin activity after IG administration of multiple doses (Cmax 72–84h, 0.88 ± 0.19 μg/mL) was significantly (P = 0.011) higher than that achieved after IG administration of the first dose (Cmax 0–24h, 0.52 ± 0.17 μg/mL; Figure 2). Differences between Kel after IV and IG administration did not differ significantly (P = 0.617).

Figure 2—
Figure 2—

Mean ± SD clarithromycin activity measured by use of a microbiologic assay in serum samples obtained from 6 foals administered 6 doses of clarithromycin (7.5 mg/kg) IG at 0, 24, 36, 48, 60, and 72 hours.

Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1681

After IG administration of multiple doses, clarithromycin activity in peritoneal fluid, CSF, and synovial fluid was similar to or lower than clarithromycin activity in serum, whereas clarithromycin activity in urine, PELF, and BAL cells was significantly higher than concurrent values in serum (Table 2). Initial detection of 14-hydroxy-clarithromycin was at 0.5 hours after IG administration in 2 foals and by 2 hours after administration in all 6 foals with a mean time to maximum concentration of 14-hydroxy-clarithromycin of 1.7 ± 1.2 hours.

Table 2—

Mean ± SD clarithromycin activity in body fluids and BAL cells of 6 foals after IG administration of the last of six 7.5 mg/kg doses.

SampleTime after clarithromycin administration (h)
 212
Serum (μg/mL)0.83±0.18a0.20±0.06a
Synovial fluid (μg/mL)27±0.06b0.08±0.02a
Peritoneal fluid (μg/mL)*0.43±0.32b0.11±0.06a
Urine (μg/mL)15.58±11.66c2.53±0.82b
CSF (μg/mL)0.22±0.09b0.13±0.09a
PELF (μg/mL)76.23±59.43c21.36±20.53c
BAL cells (μg/mL)269.00±232.24d117.27±107.84d

Represents results for 3 foals at 2 hours and 5 foals at 12 hours.

Represents micrograms of drug activity per milliliter of BAL cell volume.

Within a column, values with different superscript letters differ significantly (P<0.05).

Discussion

Clarithromycin undergoes extensive metabolism in humans. Of the 8 metabolites that have been identified, 14-hydroxy-clarithromycin is the most abundant and the only one with substantial antimicrobial activity.3,4 Metabolism of clarithromycin is unique in humans because it is the only 14-membered macrolide to have 14-hydroxylation. The 14-hydroxy metabolite of clarithromycin is also produced in monkeys but not in rats,3 mice,16 or desert tortoises.17 The study reported here confirms the production of 14-hydroxy-clarithromycin in foals with maximum concentrations detected approximately 1.7 hours after IV or IG administration. In humans, peak concentrations of 14-hydroxy-clarithromycin of approximately 1.3 μg/mL have been detected 3 hours after oral administration of a dose of 7.5 mg/kg.18 Unfortunately, exact concentrations of 14-hydroxy-clarithromycin could not be determined in our study because of the inability to find an internal standard that has parallel recovery efficiency.

The microbiologic assay used to calculate pharmacokinetic variables in the study reported here only allows an approximation of drug disposition because it does not differentiate between clarithromycin and its 14-hydroxy metabolite. However, in a clinical situation, total antimicrobial activity measured by the microbiologic assay is adequate to determine a dosage regimen. Oral bioavailability of clarithromycin in our study (57%) is similar to that reported in humans19 (55%) but lower than the value of 70% to 75% reported in dogs.20 The oral bioavailability of clarithromycin in our study is similar to that of azithromycin (38% to 56%) and much higher than that of erythromycin (14%) in foals.14,21,22 Clarithromycin t1/2in our study (5.4 hours) was slightly longer than that reported20 after oral administration to dogs (3.9 hours). The t1/2 values reported2 in humans range from 3 to 5 hours for clarithromycin and 4 to 9 hours for 14-hydroxy-clarithromycin. The t1/2 of clarithromycin in the study reported here is longer than that for erythromycin (1 hour) but considerably shorter than that for azithromycin (16 to 20 hours) in foals.14,21,23–25

The optimal dosing of antimicrobial agents is dependent not only on the pharmacokinetics, but also on the pharmacodynamics of the drug. The pharmacodynamic properties of a drug address the relationship between drug concentration and antimicrobial activity. Much confusion exists over the pharmacodynamics of macrolides and azalides because their serum concentration–time pattern is low, relative to the MICs of pathogens for which they typically are used. An important factor in determining the efficacy of many macrolides in animals with infections experimentally induced by use of extracellular bacteria is T > MIC.2 In mice with experimentally induced Streptococcus pneumoniae infection, serum T > MIC for at least 60% of the dosage interval for clarithromycin was the best predictor of efficacy.26 In mice with experimentally induced pneumococcal pneumonia, serum T > MIC of 50% to 70%, a quotient of 3 to 7 for maximum serum concentration divided by MIC, and a quotient of 40 to 100 for AUC for the first 24 hours divided by MIC were all comparable in predicting efficacy.27 Analysis of data suggests that traditional pharmacodynamic variables based on serum concentrations of macrolides may not be the best information to use when considering treatment of animals with pulmonary infections and infections caused by facultative intracellular pathogens such as R equi.10

Although drug concentration in plasma is clearly a driving force for penetration to the site of infection, the actual drug-concentration time pattern at a peripheral site may differ substantially from that of plasma.10 Macrolides cross cellular membranes primarily by passive diffusion.28 They are potent weak bases that become ion-trapped within acidic intracellular compartments, such as lysosomes and phagosomes. Results of in vitro and in vivo studies29,30 support the notion that WBCs act as carriers for the delivery of macrolides to the site of infection. However, the theory of WBC delivery of macrolides does not explain the extremely high concentrations of these drugs in PELF that were detected in healthy subjects in which trafficking of WBCs to the PELF should have been minimal.1,31 High concentrations of macrolides in PELF have long been proposed as a key factor in their efficacy against respiratory pathogens in humans. The preferential activity of clarithromycin in the lungs was reported32 in mice infected with S pneumoniae isolates with efflux-mediated macrolide resistance. Consistent killing of bacteria was observed in lungs, whereas no effect of drug was evident in experimentally infected thigh muscle.32 These differences in bacterial activity between sites were explained by the higher concentrations of macrolide in PELF than in serum.

In the study reported here, administration of clarithromycin at a rate of 7.5 mg/kg every 12 hours resulted in serum concentrations above the MIC90 for R equi isolates (0.12 μg/mL)7 throughout the entire dosing interval, a quotient of 7 for mean maximum concentration divided by MIC90, and a quotient of 57 for mean AUC for the first 24 hours divided by MIC90. Because serum concentrations alone should not be used to determine the likelihood of clinical efficacy in the treatment of foals with pneumonia attributable to R equi, clarithromycin concentrations were also measured in PELF and BAL cells.

Concentrations of clarithromycin in PELF and BAL cells in our study considerably exceeded the MIC90 of R equi isolates obtained from foals with pneumonia. Clarithromycin concentrations in PELF and BAL cells in our study were also considerably higher than concentrations reported after multiple daily administrations of azithromycin to foals. In the study reported here, clarithromycin concentrations in BAL cells and PELF had decreased considerably by 12 hours after administration. This is in contrast to azithromycin concentrations in PELF and BAL cells, which do not decrease for at least 48 hours after administration to foals.14 Collectively, these findings in foals are consistent with results of studies13,31,33 in humans that reveal much higher peak clarithromycin concentrations in PELF and BAL cells, compared with concentrations of azithromycin, but much longer persistence of azithromycin than clarithromycin at these sites. After oral administration of a single dose to humans, clarithromycin is no longer detectable in PELF by 24 hours and in BAL cells by 48 hours.13 The release of azithromycin from cells is much slower than that of erythromycin and clarithromycin, which results in sustained concentrations of azithromycin in tissues for days after discontinuation of treatment.28 In the study reported here, clarithromycin concentrations in peritoneal fluid, synovial fluid, and CSF were significantly lower than PELF concentrations, which indicated preferential diffusion of clarithromycin into pulmonary fluid.

Adverse effects in humans receiving clarithromycin are rare and usually related to the gastrointestinal tract, with diarrhea, nausea, and abdominal pain being reported most commonly.34 Two of 6 foals in the study reported here developed mild, self-limiting diarrhea. The incidence of diarrhea in the foals of our study was similar to that reported in a retrospective study8 in which 5 of 18 (28%) foals with pneumonia attributable to infection with R equi were treated with clarithromycin and rifampin and developed diarrhea. This is similar to the incidence of diarrhea reported8,35 for foals being treated with erythromycin and rifampin (17% to 36%). In contrast, the incidence of adverse effects on the gastrointestinal tract of humans is significantly lower during treatment with clarithromycin (4%) than during treatment with erythromycin (19%).36

ABBREVIATIONS

t1/2

Elimination half-life

PELF

Pulmonary epithelial lining fluid

BAL

Bronchoalveolar

IG

Intragastric

HPLC

High-performance liquid chromatography

Kel

Elimination rate constant

AUC

Area under the concentration-time curve

AUMC

Area under the first moment of the concentration-time curve

Vdss

Apparent volume of distribution at steady state

Cmax 0–24h

Maximum serum concentration of clarithromycin after the first dose

Cmax 72–84h

Maximum serum concentration of clarithromycin after repeated doses

MIC

Minimum inhibitory concentration

T > MIC

Length of time that serum concentrations exceed the MIC of the pathogen

MIC90

MIC that would inhibit 90% of isolates

a.

Clarithromycin injection, courtesy of Franks Pharmacy, Ocala, Fla

b.

Biaxin, Abbott Laboratories, Chicago, Ill

c.

Pentax, Welch Allen, Orangeburg, NY

d.

Roxithromycin, Sigma Chemical Co, St Louis, Mo

e.

Varian Bond Elut C18, 500 mg, Varian Inc, Palo Alto, Calif

f.

Beckman System Gold, Beckman Coulter Inc, Fullerton, Calif

g.

Supelco Discovery C18, 150 × 5.6 mm, 5-μm particle size, Sigma-Aldrich, St Louis, Mo

h.

LC-4C, BAS, West Lafayette, Ind.

i.

14-hydroxy-clarithromycin, courtesy of Abbott Laboratories, Abbott Park, Ill

j.

Clarithromycin, US Pharmacopeia, Rockville, Md

k.

ATCC 9341, American Type Culture Collection, Rockville, Md

l.

Neomycin assay agar, Fischer Scientific Inc, Pittsburgh, Pa

m.

BUN reagent, Labsco Laboratory Supply Co, Louisville, Ky

n.

Hitachi 911 analyzer, Boehringer Mannheim Inc, Indianapolis, Ind

o.

PK Solutions 2.0, Summit Research Services, Montrose, Colo.

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Contributor Notes

Supported by the Florida Thoroughbred Breeders' and Owners' Association.

Address correspondence to Dr. Giguère.