Materials and Methods

Animal selection—Twenty-three Thoroughbred mares and geldings were evaluated for inclusion in this study. All horses were obtained from the research herd maintained by the Louisiana State University Equine Health Studies Program and were returned to the herd at the conclusion of the study. This study conformed to the university guidelines for animal use and was approved by the Louisiana State University Institutional Animal Care and Use Committee.

Horses were from 3 to 10 years old at the time of evaluation and were free of obvious lameness (< grade 1/5 on all limbs, as determined with the American Association of Equine Practitioners grading system). Horses were determined to be healthy on the basis of results of routine physical examination, CBC, and serum biochemistry analysis. History of previous clenbuterol administration was unknown for all horses in the study.

An echocardiogram was performed during initial evaluation, and horses were excluded if major alterations in echocardiographic measurements of left ventricular size or function were detected.^19,20 Thoracic auscultation during use of a breathing bag and endoscopic examination of the upper portion of the airway and trachea were performed. Horses were excluded if there was any clinical evidence of inflammation of the lower portion of the airway, specifically if abnormal lung sounds were detected on auscultation or the tracheal mucus score was > 1 on endoscopic examination.²¹ Histologic examination of semimembranosus muscle biopsy specimens was performed at an outside laboratory,^a and horses were excluded if severe lesions were reported. Twelve horses met all selection criteria and were randomly assigned to either the control (n = 6) or clenbuterol (6) group.

Study design—Horses were housed in individual box stalls for 48 hours after initial evaluation and biopsy to allow for observation of signs of pain or other adverse events associated with the biopsy site. Horses were then turned out on pasture with the research herd until test results were available to determine qualification for inclusion in the study. Horses that met all requirements for inclusion in the study began the treatment protocol ≥ 14 days after initial evaluation. During the initial evaluation, monitoring periods, and treatment protocol, a physical examination was performed once daily. Study horses were fed a diet of high-quality Bermuda grass hay (3% of total body weight daily [eg, 13.5 kg for a 450-kg horse]) and a commercial 10% crude protein pelleted equine feed^b (0.8% total body weight daily [eg, 3.6 kg for a 450-kg horse]) while in stall confinement. Water was available ad libitum.

During the treatment period, horses were also monitored for signs of clenbuterol toxicosis, including muscle fasciculation or stiffness, signs of anxiety, tachycardia, and excessive sweating. Horses in the clenbuterol group (n = 6) received clenbuterol^c (72.5 μg/mL, PO, q 12 h [at 8 am and 8 pm] for 14 days). The dose was incrementally increased during the treatment period to minimize adverse effects, according to the following schedule: 0.8 μg/kg every 12 hours for 3 days, then 1.6 μg/kg every 12 hours for 3 days, then 2.4 μg/kg every 12 hours for 3 days, and then 3.2 μg/kg every 12 hours for 5 days. This schedule follows label administration recommendations. Horses in the control group (n = 6) received saline solution, PO, every 12 hours for 14 days. The volumes of saline solution administered to the control group each day were equal to the volumes of clenbuterol solution administered to the treatment group.

On day 1 of the treatment period, horses also were exercised on a high-speed treadmill^d for 15 minutes at a walk, trot, and fast canter. Treadmill exercise was performed once daily on weekdays (5 d/wk) for 14 days. Skin sutures were removed from the biopsy site prior to the beginning of treadmill exercise. Speeds for the walk (mean ± SD, 3.93 ± 0.31 miles/h) and trot (mean ± SD, 9.11 ± 0.60 miles/h) were determined subjectively on the basis of each horse's stride length and perceived level of exertion. Fast canter speed (mean ± SD, 19.66 ± 1.27 miles/h) was determined on the first day of exercise as the maximum speed at which the horse could keep pace with the treadmill for 2 minutes. Exercise intensity was gradually increased during the first week of the treatment period by increasing the time spent at a canter from 2 minutes on the first day to 5 minutes by day 8.

During initial evaluation (day 0) and on days 3, 6, 9, and 12 of treatment, blood was collected from the jugular vein into sterile evacuated tubes containing no additives,^e sterile evacuated tubes containing lithium heparin,^f and sterile evacuated tubes containing K₂EDTA.^g During the treatment period, samples were obtained immediately prior to the morning treatment administration, which was approximately 12 hours after the previous evening's treatment. Serum and plasma were separated within 1 hour after collection and stored at −70°C until batched analyses were performed. Echocardiography was repeated 7 days after beginning treatment (day 8). Venous blood collection, echocardiography, and biopsy of the semimembranosus muscle were also repeated 12 hours after completion of the treatment protocol (day 15).

Determination of serum clenbuterol concentration—Determination of serum clenbuterol concentrations was performed by the Louisiana Equine Medication Surveillance Laboratory on thawed serum via gas chromatography following extraction with sodium hydroxide, petroleum ether, and methylboronic acid. A standard curve was generated by use of metoprololol as an internal standard and known concentrations of clenbuterol (0.01 to 5.00 ng/mL) to determine the limits of detection for this method. Thereafter, for each batch of samples tested, known concentrations of 0.25, 0.50, 1.00, 2.50, 5.00, and 6.00 ng/mL were used for clenbuterol (linear regression). The minimum acceptable value of R² for the resulting linear regression equation was set at 0.990. Samples were analyzed in batches (20 samples), and quality assurance samples (blanks, positive, and negative controls and standard curves by means of fortified blank serum samples) were analyzed with each batch.

Determination of muscle enzyme activities—Creatine kinase and AST assays were performed on thawed (lithium) heparinized plasma by the Louisiana State University Clinical Pathology Laboratory with an analyzer.^h Samples of K₂EDTA plasma for cTnI analysis were shipped overnight on dry ice to the Clinical Pathology Laboratory at the University of Florida. Assays were performed on thawed K₂EDTA plasma with a diagnostic test instrumentⁱ by use of cardiac panel cartridges.

Echocardiography—Echocardiograms were performed with a 3.5-mHz cardiac probe with a depth of 30 cm attached to a portable ultrasonography machine.^j Hair was clipped in the right axillary region, and ultrasonography coupling gel was applied to improve image quality. Then, 2-D and M-mode analyses were performed to obtain the following standard measurements of cardiac function and contractility: measurements of LVIDs and LVIDd, IVSs and IVSd, LVPWs and LVPWd, FS, EF, and diameters of the AR and PA. All echocardiograms were performed with horses at rest.

Muscle biopsy procedure—Horses were sedated with detomidine hydrochloride^k (10 μg/kg, IV) and butorphanol tartrate^l (0.01 mg/kg, IV) for biopsy procedures. Lidocaine^m (2%) was also instilled SC over the biopsy site for local anesthesia. Pretreatment biopsy specimens were obtained from the semimembranosus muscle approximately 5 cm ventral to the tuber ischii on the right side. Posttreatment biopsy specimens were obtained from the same muscle group on the contralateral side, 12 hours after the final treatment administration.

Briefly, a 1-inch (2.5-cm) cube of muscle was surgically excised through a 2.5-inch (6.4-cm) incision. The skin and subcutaneous tissues were closed in 2 layers with a simple continuous pattern by use of 2-0 nylonⁿ and 3-0 polyglactin 910,^o respectively. Horses were administered 2 g of phenylbutazone^p IV following each biopsy for analgesia and to minimize local inflammatory response.

A portion of the pretreatment biopsy specimen was wrapped immediately in a saline solution–moistened gauze sponge, placed in a watertight container surrounded by ice packs, and analyzed histologically at an outside reference laboratory to rule out preexisting myopathy. The remaining portions of the pretreatment biopsy specimens as well as all posttreatment biopsy specimens were fixed in zinc formalin and embedded in paraffin. For each horse, 2 paraffin blocks were prepared at each sampling point. From each available paraffin block, one 5-μm-thick section was stained with H&E, and 2 sequential 5-μm-thick sections were cut for immunohistochemical staining.

Histologic examination—Muscle specimens stained with H&E were examined at 400× magnification by a single investigator (DBP) unaware of group assignments. Slide-mounted specimens were evaluated for evidence of muscle damage by use of a scoring system (Appendix), which included grades 0 to 3 for the presence of central nuclei, contraction band necrosis, intracytoplasmic granules, myocyte atrophy, swollen myocytes, and Zenker necrosis and grades 0 to 1 for the absence or presence of myocyte fragmentation, where myofibrils are observed to break apart from the overlying sarcolemma.

Immunohistochemical analysis—The first of the 2 sequential muscle sections was evaluated for apoptosis by use of a polyclonal rabbit anti–caspase 3 antibody^q with an avidin-biotin peroxidase complex system.^r Briefly, sections were deparaffinized and rehydrated routinely with xylene and graded ethanol. Endogenous peroxidase activity was quenched by incubation in 100 μL of 3% hydrogen peroxide for 10 minutes, followed by protein blockade with 100 μL of goat serum for 30 minutes. Sections were incubated in 100 μL of a 1:1,500 dilution of the primary anti–caspase 3 antibody for 30 minutes, followed by application of 100 μL biotinylated anti-rabbit secondary antibody for 30 minutes, 100 μL of avidin and biotinylated horseradish peroxidase complex for 30 minutes, and 100 μL of Nova Red peroxidase enzyme substrate^s for 8 minutes. Specimens were rinsed in Tris buffer between each step. Counterstaining was performed with hematoxylin (100 μL for 5 minutes), and specimens were cleared and dehydrated prior to permanent mounting.

The corresponding sequential muscle section from each block was used as a negative control by substituting deionized water for the primary anti–caspase 3 antibody in the first step of the procedure. Sections of equine laminar tissue from a patient with laminitis were used as positive controls²²; these samples were stored for approximately 12 months prior to use. Positive staining for caspase 3 was defined as bright red stain uptake in either the nucleus or cytoplasm of myocytes. Normal nuclei and sarcoplasm were various shades of purple. Muscle sections from treated and control horses were examined for apoptosis by a single investigator (JAT) unaware of group assignments. The entire section was classified as either positive or negative for apoptosis on the basis of presence or absence of positive (red) cytoplasmic or perinuclear staining for active caspase 3. In addition, staining characteristics of the nuclei were recorded for 500 nuclei/specimen, and the percentage of positively staining nuclei was recorded.

Statistical analysis—The response variables CK activity, AST activity, cTnI concentration, IVSd, IVSs, LVIDd, LVIDs, LVPWd, LVPWs, EF, FS, and diameters of AR and PA were compared between treated horses and controls and across time periods by means of a Mann-Whitney U test and Friedman test for repeated nonnormally distributed data, respectively. An adjusted level of significance at P < 0.01 was used to reduce type I error. These response variables were summarized as median and range values. Percentage apoptotic nuclei and serum clenbuterol concentrations were summarized as mean ± SD values.

Histologic scores were considered as pseudometric data and were analyzed via repeated-measures ANOVA with a split-plot arrangement of treatments. Post hoc comparisons of main effects were performed by use of a Tukey test, and pairwise t tests of least squares means were performed for post hoc comparisons of interaction effects. The presence of apoptosis and myocyte fragmentation was compared via Cochran-Mantel-Haenszel stratified analysis. Values of P < 0.05 were considered significant for all histologic data. All statistical analyses were performed with statistical software.^t

Results

Treatment groups—Of 23 horses initially evaluated, 12 met the inclusion criteria. Of the 11 horses excluded, 1 was excluded because of reduced FS, and 10 were excluded on the basis of abnormal muscle biopsy results; 1 of these horses also had echocardiographic evidence of cardiac insufficiency. Horses in the control group (n = 6) ranged in age from 3 to 10 years and included 2 mares and 4 geldings. The clenbuterol group (n = 6) was comprised of 6 geldings ranging from 4 to 7 years of age.

Serum clenbuterol concentrations—The limit of detection for clenbuterol concentration determined via gas chromatography was 0.25 ng/mL (250 pg/mL). Clenbuterol was not detectable in the serum of any horses prior to treatment (day 0) or in the serum of saline solution–treated controls at any time point. For horses treated with clenbuterol, mean ± SD serum clenbuterol concentration on day 3 was 635.2 ± 173.9 pg/mL. On the morning of day 6, mean serum clenbuterol concentration was 1,142.8 ± 292.1 pg/mL, and on day 9, mean serum clenbuterol concentration was 1,874.5 ± 526.4 pg/mL. For days 12 and 15, mean serum clenbuterol concentrations were 2,232.3 ± 717.7 pg/mL and 2,819.0 ± 759.2 pg/mL, respectively.

Muscle enzyme activities—There were no significant differences in CK or AST activities between clenbuterol-treated horses and controls or across time periods for either treated horses or controls.

Concentration of cTnI was increased in 1 horse (5 ng/mL) in the clenbuterol group on day 6. For this horse at all other time points and for all other horses at all time points, cTnI concentration was < 0.05 ng/mL. As a group, there was no significant difference in cTnI concentration between treated horses and controls or across time periods for either treated horses or controls.

Echocardiography—There were no significant differences between treated horses and controls or across time periods for either group for all echocardiographic variables examined (IVSd, IVSs, LVIDd, LVIDs, LVPWd, LVPWs, EF, FS, and diameters of AR and PA). The measured echocardiographic variables were summarized (Table 1).

Pretreatment muscle biopsy specimen classification—Of the 22 specimens sent to an outside laboratory^a for histologic evaluation, 9 were classified as normal, 3 were classified as suspicious for vitamin E deficiency, 4 were classified as having a nonspecific myopathy, 5 were classified as consistent with polysaccharide storage myopathy, and 1 was classified as consistent with recurrent exertional rhabdomyolysis.

Histologic scoring—Ten horses had both pretreatment and posttreatment muscle biopsy specimens available for sectioning. Posttreatment biopsy specimens were unavailable for 1 horse from each group.

The score for contraction bands was significantly higher in clenbuterol-treated horses after treatment, compared with pretreatment scores (P < 0.05) and scores for control horses at both time points (P < 0.01). Muscle atrophy scores were significantly lower in the control group before treatment, compared with clenbuterol-treated horses before (P < 0.01) and after (P < 0.05) treatment. Mean atrophy score was also significantly (P < 0.05) lower in control horses after treatment, compared with clenbuterol-treated horses before treatment. However, the posttreatment atrophy scores of clenbuterol-treated horses were not significantly different from those of control horses. For the criteria of central nuclei, there was a significant interaction of time but not group on histologic score. Specifically, scores were significantly lower in the control group before treatment, compared with both the control and clenbuterol groups after treatment. For all other criteria evaluated, there was no significant difference in histologic scores between groups at either time or within groups over time.

Immunohistochemical analysis—Ten horses had both pretreatment and posttreatment muscle biopsy specimens available for sectioning. Posttreatment biopsy specimens were unavailable for 1 horse from each group. No pretreatment biopsy specimens were classified as positive for cellular apoptosis. Three posttreatment specimens from the control group and 2 posttreatment specimens from the clenbuterol group had cytoplasmic staining (Figure 1). There was no significant interaction of group or time on the presence of apoptosis.

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Photomicrographs of sections of semimembranosus muscle in a horse treated with clenbuterol. Notice positive-staining (red) apoptotic cells (A) and nuclei (B). Caspase 3 immunohistochemical Nova Red stain; bar = 100 μm.

Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.875

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Mean ± SD percentage of positive Nova Red–staining nuclei in the pretreatment biopsy specimens was 24.6 ± 7.56% for the control group and 19.0 ± 7.91% for the clenbuterol group. After treatment, mean percentage of positively staining nuclei for the control group was 18.7 ± 5.63%, compared with 25.6 ± 7.85% for the clenbuterol group.

Adverse events—Colic was the most frequently encountered adverse event, occurring in 3 horses. One horse in the clenbuterol group developed a large-colon impaction and colon displacement on day 7 of treatment, necessitating early removal from the study. This horse was removed from final data analysis; therefore, complete data were only available for 5 horses in the clenbuterol group.

One horse in the clenbuterol group developed a large-colon impaction during the last week of the study, and 1 horse in the control group developed colic during the observation period after study completion. Both cases resolved within 24 hours with administration of flunixin meglumine and orally administered fluids. One horse in the control group developed diarrhea at the beginning of the study, which resolved within 48 hours after beginning treatment with di-trioctahedral smectite,^u bismuth subsalicylate, and metronidazole. One horse in the clenbuterol group developed incisional dehiscence 7 days after pretreatment biopsy, but the site healed well by second intention after daily lavage, and the horse was able to begin treadmill exercise on schedule with no lameness.

Tachycardia, muscle tremors, or other signs of clenbuterol toxicosis were not recorded for any horse at any time point. Subjectively, horses in the clenbuterol group sweated more readily during treadmill exercise, and their perspiration did not decrease with conditioning, as it did in the control horses. Exercise tolerance was not specifically assessed, and all horses were able to complete the prescribed exercise protocol at all times.

Discussion

Oral clenbuterol administration for 14 days, at doses up to 3.2 μg/kg every 12 hours, did not cause measurable adverse effects on equine skeletal or cardiac muscle. This is contrary to other studies in horses that have revealed negative effects of low to moderate doses of clenbuterol on endurance^14,23 and echocardiographic variables²⁴ and induced measurable effects on lean muscle mass and fat mass,^17,25,26 although those studies either used a different route of administration or identified changes after a much longer treatment period. The route and time course of administration of clenbuterol used in the present study were different from all prior reports.

For the selected indicators of skeletal muscle damage in the present study, there was no association of clenbuterol or exercise conditioning with muscle injury. The finding that serum CK activity did not increase with clenbuterol administration, particularly at higher doses, was unexpected and inconsistent with data reported by the manufacturer via product labeling that warns of increased serum CK activity observed in some cases. However, publicly available information does not specify to what extent CK activity was reportedly increased or at what time point following clenbuterol administration that CK activity increased. The sampling time in the present study was chosen to correspond with the time that clenbuterol induces maximal myocyte necrosis in rats.¹³ Blood samples were collected 15 to 20 hours after exercise, when CK activities may have been returning to reference range after potential exercise-induced injury. However, increases in serum AST activity would still be expected under these circumstances. Fillies in race training are reported to have a higher prevalence of increased CK and AST activity than are colts²⁷; the absence of females in the clenbuterol group may have further reduced the probability of detecting alterations in these variables. In many of the studies^23–25 that found adverse effects of clenbuterol, the study population was composed exclusively of mares.

The semimembranosus muscle was chosen for evaluation because it is the optimal site to detect common equine myopathies in routine biopsy specimens.²⁸ Unfortunately, this muscle is normally composed of 70% to 75% glycolytic (type IIX) fibers,²⁹ which are not the primary fiber types affected by clenbuterol in other species,¹⁸ thereby limiting the probability of detecting an effect of clenbuterol. Comparable clenbuterol-susceptible muscle groups (soleus, cardiac, and anterior tibialis) are not easily accessible for biopsy in horses, which made this comparison unavailable for the present study. In murine studies,^{10,13,15,30,31} a substantial difference in response to clenbuterol has been reported between skeletal muscles composed of predominantly fast-twitch fibers and those composed of predominantly slow-twitch fibers and cardiac muscle. Additionally, histologic evidence of myotoxicosis appears to be attenuated after 8 days in other species,¹³ so the 14-day treatment period used in the present study may have further reduced the potential for detecting a histologic difference between groups.

Nuclear staining is not generally observed in immunoassays for caspase 3, which detect active caspase 3 in the cytosol. In both equine laminae²² and rat myocytes,⁹ caspase 3–positive cells are described as up-taking stain throughout the entire cytosol. Because of this characteristic staining pattern of caspase 3, along with the observation that nuclear staining occurred at a relatively constant rate independent of time or treatment assignment, nuclear staining was interpreted as artifact (Figure 1).

Although long-term use of clenbuterol ultimately increases lean muscle mass, at least 1 previous study²⁶ has detected an initial loss of total body mass in horses subjected to simultaneous clenbuterol administration and treadmill exercise. However, although muscle changes (fiber-type switching) were detected in the present study, we did not investigate whether this seemingly paradoxical loss of total body mass was attributable to initial loss of muscle mass, fat mass, or another of several factors related to training of unconditioned horses. Similarly, most horses in the present study lost weight during the treatment period. However, weight loss in the population of the present study was not significantly different between treated and control horses, supporting a greater role for external factors such as increased physical activity with controlled treadmill exercise. Delineation of clenbuterol's effects on body weight, muscle mass, and changes in muscle composition in horses will require additional experimentation with a larger number of animals as well as earlier and more frequent biopsy collection from muscle groups more likely to manifest signs of toxic damage.

The present study also failed to find an association between exercise conditioning or clenbuterol administration and an increase in plasma cTnI concentration or alterations in echocardiographic measurements at rest. Results of the present study agree with those of a previous study²⁴ that did not find an effect of clenbuterol (2.4 μg/kg daily for 8 weeks) on resting echocardiographic variables or plasma concentration of cTnI. However, Sleeper et al²⁴ found that substantial changes in postexercise echocardiographic variables in horses occur with clenbuterol treatment, regardless of exercise conditioning. Possibly because of limited data that were due to small sample size, this phenomenon was not observed in the present study.

Cardiac troponin I concentration is used in clinical practice as a noninvasive indicator of myocardial damage.³² Concentration of cTnI increased beyond the reference range in only 1 horse at 1 time point, but group changes were not significant. Given that echocardiography also failed to detect significant changes in measured variables, we were unable to evaluate the sensitivity of cTnI concentration for measuring cardiac injury in the study population. Increased activity of the cardiac isoenzyme of CK may also be used as a marker of cardiac injury. Although total CK activities for horses in the present study were within the reference interval, the relative activity of the cardiac isoenzyme versus the skeletal and brain isoenzymes was not specifically evaluated and may have been abnormal or different between groups.

One proposed mechanism for clenbuterol-associated cardiotoxicosis is that of relative hypoxia, in which the myocardial oxygen demand is increased beyond achievable levels by the positive inotropic and chronotropic effects of clenbuterol.¹⁴ Because treadmill speeds were arbitrarily assigned on the basis of perceived exertion, rather than a quantitative measure of exercise intensity such as heart rate or oxygen consumption, it is possible that the horses in the present study were not exercised sufficiently to induce hypoxic necrosis. Intravenous administration of clenbuterol temporarily reduces aerobic capacity in horses.¹⁴ In contrast, previous studies^17,24,26 revealed that exercise does not alter or potentiate other effects of clenbuterol. The present study neither confirmed nor refuted previous data regarding the effects of clenbuterol on aerobic capacity or endurance because these effects were not specifically assessed. However, because any changes induced by clenbuterol appear to be subclinical in the absence of exercise, future investigation into the effects of clenbuterol on cardiac function should incorporate a standardized exercise test with evaluation of heart rate and rhythm before, during, and after exercise as well as consistent postexercise echocardiographic evaluation. Determination of standardized measurements of exercise tolerance and anaerobic threshold may also be informative.

Other than the 3 episodes of colic, no adverse effects were associated with clenbuterol administration in the present study. The gradually increasing dosage schedule, as recommended by the manufacturer, was intended to reduce the occurrence of clinically apparent adverse effects. Downregulation of β₂-adrenoreceptors occurs after only 10 days of clenbuterol administration and results in a 35% and 45% decrease in receptor expression in skeletal muscle and lung, respectively.³³ Previous investigators^17,23–25 reported that horses in experimental studies were unable to tolerate dosages of clenbuterol > 2.4 μg/kg every 12 hours. The absence of clinical signs of toxicosis in the study population was attributed to receptor tachyphylaxis or drug tolerance. In contrast, in 3 cases of clenbuterol toxicosis,² the magnitude of the overdose resulted in maximal activation of β₂ adrenoreceptors as well as nonspecific activation of other β adrenoreceptors, inducing multisystemic effects and marked evidence of muscle injury. The colic episodes (1 each in treatment group, control group, and horses excluded from the study) may have been related to the changes in environment and exercise regimen, considering that all horses had been sedentary on pasture for several weeks prior to the study. The increasing dosages used in this study to successfully limit adverse clinical events may also have limited responses in the experimental variables measured and resulted in an inability to detect significant differences.

Overall, the small sample size was not sufficient for acceptable statistical power (estimated) and was therefore detrimental to the detection of differences between groups. One barrier to obtaining a larger number of subjects was the unexpected high prevalence of abnormal muscle biopsy results during the screening process. Approximately half of the horses examined were excluded from enrollment in the study on the basis of abnormal muscle biopsy results. Twelve of 23 horses screened had ≥ 1 histopathologic abnormality, and 10 of these horses had conditions severe enough to require exclusion. Horses with preexisting myopathy were excluded from the present study because increases in serum CK and AST activities are commonly detected in horses with conditions such as polysaccharide storage myopathy and recurrent exertional rhabdomyolysis.^27,28 Previous studies^{14,17,23–26} examining the effects of clenbuterol in horses did not consider this factor. Nevertheless, the high percentage (10/22 [45%]) of horses affected with some form of histologically apparent myopathy was an unexpected finding, and further investigation is needed to clarify the relationship between these myopathies and poor performance or response to clenbuterol in horses with and without myopathy. The effect of an inherent metabolic disorder of skeletal muscle on the potential for clenbuterol-induced myopathy is unknown, but one may speculate that its myotoxicity would be enhanced. For the horses examined in the present study, history of clenbuterol administration during their racing careers could not be confirmed.

Confirmation of serum clenbuterol concentrations in all treatment-group animals excluded the possibility that the lack of effects seen were caused by inadequate drug administration. Previous reports on clenbuterol pharmacokinetics have not delineated the effects of gradually increasing doses on serum clenbuterol concentration. Two reports^34,35 examining oral administration of 1.6 μg of clenbuterol/kg every 12 hours concluded that the 12-hour serum trough concentration in adult horses was 508.1 ± 249.4 pg/mL and 648.0 ± 189.7 pg/mL after 2 and 7 days, respectively. The values obtained in the present study on day 6 (1,142.8 ± 292.1 pg/mL) following 2 days of oral clenbuterol administration at 1.6 μg/kg were higher than previously reported data. The reason for this difference was unclear; however, it could be related to the elimination half-life or other animal-related differences such as ingestion or absorption factors. In a previous study,³⁵ the plasma concentrations of clenbuterol following 7 days of oral administration decreased monoexponentially with a median elimination half-life of 12.9 hours, a clearance of 94.0 mL/h/kg, and volume of distribution of 1,574.7 mL/kg, with the last oral administration baseline plasma concentration of 537.5 ± 268.4 pg/mL, increasing to 1,302.6 ± 925.0 pg/mL at 15 minutes. The dose of clenbuterol in the present study was held constant (at 3.2 μg/kg, PO, q 12 h) from day 10 to 14; therefore, the increase in serum clenbuterol concentration from day 12 to 15 could be attributable to the dose administered.

The dosage regimen used in the present study was consistent with current recommendations for the use of clenbuterol in the treatment of inflammatory airway disease in young, athletic horses, assuming that clinical signs resolve after a single course of treatment. Results of this study suggested that orally administered clenbuterol, at doses up to 3.2 μg/kg for ≤ 2 weeks, does not induce skeletal or cardiac muscle damage in horses with no preexisting myopathy. This finding is important, given the high prevalence and potential economic impact of inflammatory airway disease in performance horses. However, considering that prolonged or repeated clenbuterol administration is a common practice in the performance horse industry, precise knowledge of the nature and extent of potential clenbuterol-induced injury is important to protect the welfare of equine athletes. Additionally, the authors' clinical experience with fatal clenbuterol toxicosis in horses administered a compounded clenbuterol product underscores the concept that extralabel use of this medication is not benign. Further research is needed to assess the effects and potential for long-term skeletal and cardiac muscle damage associated with treatment periods > 14 days and in horses with preexisting myopathy.