Clenbuterol is the only FDA-approved medication for horses with reversible bronchospasm. A β2-adrenoceptor agonist, clenbuterol is administered as an oral preparation with a wide dosage range (0.8 to 3.2 μg/kg, q 12 h for up to 30 days). It has efficacy in the treatment of RAO1–3 and is also used to treat IAD,4 a disease that affects up to one-third of racehorses in training.5–7 However, in asthmatic humans as well as in other mammals with experimentally induced broncho-constriction, chronic treatment with β2-adrenoceptor agonists results in tolerance or tachyphylaxis (desensitization) to the effects of the drug.8 This can be a positive attribute in that adverse effects (eg, sweating, tachycardia, and agitation) attributable to administration of β2-adrenoceptor agonists decrease over the first few weeks of use; however, it also is associated with a decrease in efficacy of the bronchodilatory effects of the treatment and a worsening of asthma control.9 Minimal data are available regarding the effect of long-term treatment with β2-receptor agonists on bronchodilation in horses, although small-scale studiesa,b in horses with RAO and IAD have indicated little to no effect.
Clenbuterol can increase dynamic compliance and decrease pulmonary resistance in ponies bronchoconstricted by IV administration of histamine.10 Clenbuterol has a dose-dependent effect, but it is not effective in all horses. At the commonly administered dose of 0.8 μg/kg, only 25% of horses with RAO have a clinical response, which increases to 75% at an administered dose of 3.2 μg/kg.2 Similar to horses with RAO, horses with IAD also have hyperreactivity to inhaled histamine, which leads to bronchoconstriction.11–13 In addition to bronchodilation, clenbuterol has anti-inflammatory effects and decreases mucus production by goblet cells in many species, including horses.14,15 However, the ability of clenbuterol to protect against bronchoconstriction resulting from natural (hay dust or other inhaled triggers) and investigational (histamine and methacholine) stimuli is unclear.
In contrast to those of other mammals, sweat glands of horses contain β2-adrenoceptors16 that have rapid desensitization to β2-adrenoceptor agonists, which leads to subnormal sweat production in vitro.17–21 Although iatrogenic causes of anhidrosis or hypohidrosis have not been investigated, naturally occurring anhidrosis (an idiopathic syndrome of reduced sweat production) causes clinically relevant exercise intolerance.22 Therefore, the potential for reduced sweating in response to clenbuterol may be of clinical importance. To our knowledge, there are no published data on the effects of prolonged administration of clenbuterol on sweating in horses.
The purpose of the study reported here was to determine whether clenbuterol is bronchoprotective against the effects of inhaled histamine. We hypothesized that tachyphylaxis would develop within 21 days, which would result in histamine reactivity near or below baseline values. We also hypothesized that long-term administration of clenbuterol would lead to hypohidrosis in horses as a result of tachyphylaxis, causing a decrease in sweating to below baseline amounts.
Materials and Methods
Animals—Eight adult (mean age, 9.5 years; range, 6 to 16 years) Thoroughbreds with IAD diagnosed by use of the American College of Veterinary Internal Medicine consensus guidelines4 were included in a randomized, blinded crossover study. All of the horses were retired from racing. Five horses had ≥ 5% neutrophils (range, 2% to 18%), of which 2 also had ≥ 0.1% eosinophils (range, 0% to 4%) or ≥ 2% mast cells (range, 0% to 6%) in BAL fluid, and 1 horse had increased eosinophils and mast cells only. Additionally, 5 horses were hyperreactive to inhaled histamine, with a PC35 value < 6 mg of histamine/mL. The horses were housed in box stalls at the University of Pennsylvania New Bolton Center, maintained on straw bedding, and fed good-quality hay and small amounts of concentrate daily. All animal testing was performed with the approval of the Institutional Animal Care and Use Committee of the University of Pennsylvania.
Experimental design—Horses were randomly assigned (paper lottery) to receive clenbuterolc (0.8 μg/kg, PO, q 12 h for 21 days) or a placebo (equal volume of clear corn syrup, PO, q 12 h for 21 days). After a washout period of ≥ 30 days, which was selected on the basis of a study23 on the disposition and elimination of clenbuterol, the alternate treatment was administered.
Airway reactivity testing—Flowmetric plethysmographyd with histamine bronchoprovocation was used to assess pulmonary function and airway reactivity, as described elsewhere.24,25 Horses were lightly sedated with xylazine hydrochloridee (0.025 mg/kg, IV) and noninvasively instrumented with a close-fitting mask with a pneumotachograph and abdominal and rib inductance bands that measured volume changes of the thorax over time. A differential pressure transducer and analogue-to-digital converter allowed comparison of thoracic flow and nasal airflow (Δflow) to determine total airway impedance. Measurements of Δflow were obtained during expiration during 3 minutes of tidal breathing, and a mean Δflow value was calculated for each breath. The highest and lowest values were automatically discarded by the software, and rare aberrant measurements associated with movement were manually excluded. Most horses had a respiratory rate of 8 to 16 breaths/min, which resulted in a minimum of 24 respiratory cycles/measurement period. Saline (0.9% NaCl) solutionf was then administered via a nebulizer for 2 minutes (negative control procedure), followed by administration via nebulizer of serial increases in the concentration of histamineg (2, 4, 8, 16, and 32 mg/mL), with 3 minutes of recording after administration of each nebulized dose of histamine. The concentration of histamine at which there was a 35% increase in Δflow (ie, PC35) was calculated, with higher values of PC35 corresponding to decreased airway reactivity to histamine. Baseline values of PC35 were obtained in all horses on day 0. All investigators were not aware of the treatment (clenbuterol or placebo) administered to each horse. Horses were tested for 6 weeks (every 7 days during the 21-day treatment period and every 7 days for the first 3 weeks after treatment). Tachyphylaxis was defined as a decrease in PC35 to at or below the baseline value.
Assessment of sweating—To assess sweating, 0.1 mL of epinephrineh was injected ID at increasing 10-fold concentrations (1:1,000,000 to 1:1,000), with saline solution used as a negative control injection, as described.18,26 Injections were performed in the skin of the neck, which had not been clipped of hair but was marked with tape at each injection point; specific sites were varied between test periods. After 15 minutes, a score of 0 to 4 (0 = not sweating at any concentration of epinephrine; 4 = sweating at all concentrations of epinephrine) was assigned. Tests were performed on day 0 (baseline) and at 10-day intervals throughout the 6-week period (21 days of treatment and the first 3 weeks after treatment). A diminished ability to sweat was defined as a score of < 4, with no sweating at the administration site of the saline solution.
Statistical analysis—The PC35 values were calculated by use of linear interpolation for each horse at each time point via the software associated with the flowmetric plethysmography system.d Each horse underwent 6 airway reactivity tests and 5 sweat tests for each treatment (clenbuterol and placebo). The PC35 values from the 6 airway reactivity tests were plotted to determine changes in lung reactivity in response to treatment over time. Paired values for clenbuterol versus the placebo for all flowmetric plethysmography and histamine bronchoprovocation and sweat tests were compared by use of a Wilcoxon matched-pairs signed rank testi; values of P < 0.05 were considered significant.
Results
Airway reactivity testing—When horses received clenbuterol, they had a significant (P = 0.04) increase in peak PC35 from the baseline value (mean change, 11.5 mg/mL; range, 3.7 to 25.5 mg/mL; Figure 1), compared with when horses received the placebo (mean change, −1.56 mg/mL; range, −21.1 to 3.6 mg/mL; Figure 2). Peak effect was at day 14 of clenbuterol treatment, with PC35 exceeding 100% of the baseline value in 5 of 8 horses. Tachyphylaxis was evident by day 21; 7 of 8 horses had a PC35 lower than the baseline value (mean change, −0.48 mg/mL; range, −1.7 to 1.1 mg/mL) by the end of the clenbuterol treatment period. All PC35 values returned to approximately baseline values during the washout period. One horse began to develop severe nasal edema in response to inhaled histamine during the second part of the study, and these data for that horse were excluded.
Graph of PC35 for 8 horses when receiving clenbuterol (0.8 μg/kg, PO, q 12 h) for 21 days. Treatment was followed by a washout period of ≥ 30 days, the first 14 of which are shown (gray-shaded area). Day 0 represents baseline values. Each symbol represents results for 1 horse; solid lines represent horses that had an increase of ≥ 100% over the baseline PC35 during the 21 days of treatment, whereas dotted lines represent horses with an increase of < 100% over the baseline PC35.
Citation: American Journal of Veterinary Research 73, 1; 10.2460/ajvr.73.1.140
Graph of PC35 for 8 horses when receiving a placebo (clear corn syrup) for 21 days. The symbols correspond to the same horses as in Figure 1. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 1; 10.2460/ajvr.73.1.140
Assessment of sweating—We did not detect evidence of reduced sweating during the clenbuterol treatment period, with 8 of 8 horses having a sweat score of 4 at every time point when receiving clenbuterol. All 8 horses also had a sweat score of 4 at every time point when receiving the placebo, except for the last day of testing, when 7 horses had a score of 4 and 1 had a score of 2 (no sweat in response to the 2 lowest [1:100,000 and 1:1,000,000] concentrations of epinephrine administered ID). There was no significant difference in sweat score between treatments.
Discussion
Analysis of results of the study reported here revealed that treatment with clenbuterol (0.8 μg/kg, PO, q 12 h) resulted in a significant bronchoprotective effect. Five of the 8 horses had a ≥ 100% increase in PC35, which peaked at day 14 of administration. This degree of efficacy is slightly higher than that reported in another study2 in which 25% of horses with RAO had clinical evidence of bronchodilation at the same dosage of clenbuterol. This difference in efficacy may be attributable to superior sensitivity of pulmonary function testing, compared with clinical examination for detecting bronchoconstriction; the greater reversibility of bronchoconstriction in horses with IAD, compared with those with RAO-induced airway remodeling; or differences in volume or composition of airway secretions between the 2 diseases.
All horses with a bronchoprotective effect developed tachyphylaxis by day 21 of treatment; 7 of 8 horses had PC35 values that were actually lower than the baseline value, which indicated that pulmonary function may have worsened during the treatment period. This is consistent with data in humans that indicated prolonged administration of β2-receptor agonists results in lung function worse than at baseline.9 However, there is significant interhorse and intrahorse variability in responsiveness to inhaled histamine as measured via flowmetric plethysmography,24 which resulted in considerable variability in the present study. An advantage of the crossover design of the study is that the effect of interhorse variability was nullified, although the intrahorse variation was evident, especially in 2 horses that had a large increase in reactivity to histamine (decreased PC35) when receiving the placebo during the first 2 weeks of the study. This was likely attributable to fluctuations of airway reactivity associated with exposure to environmental triggers from confinement in a box stall bedded with straw and feeding of dry hay.
Clenbuterol is commonly administered twice daily to racehorses throughout an entire racing season despite a lack of a specific diagnosis or evidence of beneficial effect. Clenbuterol is anabolically active in humans and cattle27,28 as well as horses. Although its leptin- and adiponectin-mediated repartitioning effects29 cause an increase in muscle mass, this potential benefit is offset by a negative ergonomic effect characterized by decreases in aerobic capacity, time to fatigue,30 cardiac function,31 and maximal oxygen consumption.32 Nonetheless, the use of β2-receptor agonists is common in human and equine athletes with lower airway disease, despite these adverse effects and the rapid development of desensitization or tachyphylaxis in response to the effects of this class of drug.
Development of tolerance to β2-adrenoceptor agonists appears to be a multifactorial phenomenon. Long-term administration of β2-adrenoceptor agonists to rats causes a decrease in the density of bronchial epithelial β2-receptors as a result of endosomal sequestration of receptors as well as decreased transcription and translation of mRNA.33 In addition to receptor downregulation, there is also functional receptor desensitization through the β2-adrenoceptor–G-protein–adenylate cyclase pathway. Specifically, intracellular signal transduction is prevented by uncoupling of Gs-protein by members of the G-protein receptor–coupled kinase superfamily34 and β-arrestin and subsequently by cAMP-dependent protein kinase, which further reduces second-messenger signaling.35 It has been reported36,37 that IV administration of clenbuterol for 12 days to adult horses results in a reduced density and responsiveness of β2-adrenoceptors on lymphocytes, which suggests that these effects are conserved across species.
In humans, it is widely acknowledged that β2-adrenoceptor agonists attenuate bronchoconstriction attributable to direct and indirect stimuli.38–42 The bronchoprotective effect of clenbuterol has not been as extensively evaluated in horses, although studies in ponies have revealed that IV administration of clenbuterol has no protective effect against inhaled histamine43 but has some effect against histamine administered IV.10 Similarly, no significant effect on histamine reactivity was detected after administration of aerosolized salbutamol for 10 days to horses with IADb; however, on the basis of data in humans, it is possible that bronchodilator tolerance had already developed, and thus the bronchoprotective window may have been missed. In the treatment of humans with asthma, concurrent use of a corticosteroid attenuates the development of tachyphylaxis to bronchodilators, and it is now widely recommended that daily administration of β2-receptor agonists should be used only in conjunction with inhaled corticosteroids to prevent rapid desensitization.44,45 Although no studies have been performed to evaluate the effect of this drug combination on lung function in horses with IAD and RAO, the decrease in lymphocyte β2-receptor density and function in horses receiving clenbuterol is reversed and prevented by administration of dexamethasone.46
Clinically normal horses treated with clenbuterol do not develop bronchodilation in response to clenbuterol43 because there is minimal smooth muscle tone in the small airways. The present study revealed that clenbuterol lessened the bronchoconstrictive effects of inhaled histamine in some horses with IAD and therefore is bronchoprotective in a subset of horses that respond to clenbuterol. It is possible that use of a higher dose of clenbuterol may have increased the percentage of horses that responded in the present study because in horses with RAO, a dosage of 3.2 μg/kg increases the percentage of responders to 75%.2 However, because 0.8 μg/kg is the dosage most commonly prescribed and administered in a clinical setting, this regimen was chosen to maximize the clinical relevance of the study. In the present study, response to histamine also may have been blunted slightly by the use of xylazine, which causes bronchodilation and increases dynamic compliance,47 although use of this sedative for flowmetric plethysmography and histamine bronchoprovocation has been validated in horses with IAD.25
In contrast to other mammalian species that sweat in response to cholinergic stimulation, sweating in horses is mediated by β2-adrenoceptors.16,48,49 In an experimental setting, there is a gradual decrease in sweat production over the course of prolonged infusion of epinephrine,50 and several studies17–21 have had similar findings of apparent desensitization of sweat gland β2-adrenoceptors both in vivo and ex vivo, whereby cultured equine sweat glands have a rapid and homologous desensitization to epinephrine. It is likely that anhidrosis, a naturally occurring syndrome of reduced sweat production, is attributable to gradual failure of the secretory process of the sweat glands that apparently is caused by desensitization and downregulation of the β2-adrenoceptors in response to endogenous catecholamines.22 This process occurs most commonly in horses that live in hot, humid climates where there is continual stimulation of sweating,22 although the reason that only some horses are affected is not clear. It has been hypothesized that clenbuterol affects thermoregulation, which may be a component of the reduced aerobic capacity of horses receiving high doses of clenbuterol for extended periods.32 Given these findings and anecdotal reports of a diminished ability to sweat in horses being treated with the β2-adrenoceptor agonist clenbuterol, we anticipated a reduction in sweating in horses receiving prolonged administration of clenbuterol.
Diagnostic testing for anhidrosis is usually performed by use of ID administration of β-adrenoceptor agonists. In other studies,18,26 investigators have found that clinically normal horses sweat when administered a 1:1,000,000 concentration of epinephrine and that failure to sweat at this concentration is diagnostic for hypohidrosis or anhidrosis. Terbutaline and salbutamol (β2-adrenoceptor–specific adrenergic agonists) are also used for ID administration during sweat testing,48,51,52 although epinephrine stimulates sweating as effectively as does terbutaline,48 and there is no clear mechanism that would make one test more sensitive or specific than the other. Alternatively, a method to better assess subtle changes in sweating involves collection of sweat with absorbent pads, which are evaluated quantitatively by change in weight.52
Analysis of data for the sweat test after prolonged administration of clenbuterol in the present study failed to reveal a reduction in sweating in response to ID administration of doses of epinephrine as low as 1:1,000,000. The lack of significant findings in this experiment may have been associated with inadequate stimulation of the sweat gland β2-adrenoceptors and thus a lack of subsequent downregulation or may have been a result of insufficient sensitivity of the sweat test itself. It is possible that a higher dose of clenbuterol would have induced a detectable tachyphylactic response. This is suggested by data from another study32 in which investigators found that at a dosage of 3.2 μg/kg, adverse effects (including extreme sweating) were initially observed but subsided after 10 days, although no determination was made as to whether sweating became subnormal. The protocol for the sweat test used in the present study has been used to identify horses with clinically relevant hypohidrosis or anhidrosis18,26 and is the method used in the Widener Hospital for Large Animals at the University of Pennsylvania School of Veterinary Medicine to evaluate horses with suspected sweating deficiencies. We had hoped to compare the magnitude of response to epinephrine administered ID between iatrogenically induced hypohidrosis and naturally occurring anhidrosis. Sweating at even the lowest dose of epinephrine in all horses in the present study leads us to conclude that the effect of clenbuterol at 0.8 μg/kg every 12 hours for 21 days is not likely to have a clinically important effect on sweating. Addition of lower concentrations of epinephrine administered ID may have resulted in a dose-response curve that could be used to identify subclinical changes in sweat gland function, but the clinical relevance of decreased sweating at extremely low epinephrine concentrations is unknown.
Administration of clenbuterol at a standard dosage resulted in a decrease in the bronchoconstrictive response to inhaled histamine, compared with the response when horses were administered a placebo treatment. Tachyphylaxis was detected in 7 of 8 horses, whereby the response to histamine had returned to the baseline value or lower by day 21 of clenbuterol treatment. These data are consistent with results of experiments conducted in humans that found a bronchoprotective effect of β2-adrenoceptor agonists against a variety of stimuli, which wanes over time because of tachyphylaxis and often results in worsening of lung function.8 We were not able to identify an effect of long-term administration of clenbuterol on sweating as assessed via a semiquantitative intradermal epinephrine test; this may have been attributable to inadequate sensitivity of this test or a lack of drug effect on the dermal β2-adrenoceptors in horses. Further investigation of the molecular effects of long-term administration of clenbuterol on β2-adrenoceptors of the bronchial epithelium and skin would be useful in elucidating the specific mechanisms of these findings. On the basis of results of an in vitro study46 in horses and extensive clinical data in humans,45 it is likely that extended use of clenbuterol should be combined with inhaled corticosteroids, although the safety and efficacy of this approach have not been validated in horses. However, analysis of results of the present study suggests that clenbuterol alone is less effective as a bronchodilator after treatment for 14 consecutive days, and courses of administration should not exceed 2 weeks.
ABBREVIATIONS
| Δflow | Difference between thoracic airflow and nasal airflow |
| IAD | Inflammatory airway disease |
| PC35 | Concentration of histamine required to increase total airway impedance by 35% |
| RAO | Recurrent airway obstruction |
Bayly WM, Mitten L, Hines MT, et al. Repeated doses of aerosolized albuterol sulfate in equidae with recurrent airway obstruction (abstr), in Proceedings. 18th Vet Comp Respir Soc Symp 2000;A48.
Mazan MR, Lascola K, Gazzola K, et al. Salbutamol inhalation does not modify airway hyper-reactivity in the horse (abstr), in Proceedings. 51st Annu Conv Am Assoc Equine Pract 2005;P2609.1205. Available at: www.ivis.org. Accessed Dec 7, 2005.
Ventipulmin, Boehringer Ingelheim Vetmedica, St Joseph, Mo.
Open Pleth, Ambulatory Monitoring Inc, Ardsley, NY.
AnaSed, Akorn Inc, Decatur, Ill.
Hospira Inc, Lake Forest, Ill.
Histamine diphosphate monohydrate, MP Biomed, Solon, Ohio.
Epinephrine HCl, IMS Ltd, South El Monte, Calif.
Stata, version 10.0, StataCorp, College Station, Tex.
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