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Keywords:

  • horse;
  • sildenafil;
  • EIPH;
  • exercise;
  • pulmonary arterial pressure

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Manufacturers’ addresses
  10. References

Reasons for performing study: Sildenafil, a phosphodiesterase-5 inhibitor vasodilator, increases cGMP concentrations by inhibiting enzymatic degradation. Marketed to treat erectile dysfunction in men, it also reduces pulmonary arterial pressure (PAP). Because it reduces PAP, sildenafil may enhance performance and/or prevent exercise induced-pulmonary haemorrhage (EIPH).

Objective: To determine if sildenafil citrate administration altered commonly measured indices of performance or reduced EIPH in exercised horses.

Methods: Thirteen athletically conditioned Thoroughbred horses (2 mares and 11 geldings, age 3–12 years) were administered sildenafil citrate or placebo in 2 crossover design exercise testing studies. In a step-wise test to exhaustion, inspired/expired gas analysis, blood lactate, heart rate, runtime and bronchoalveolar lavage (BAL) cytology were measured. In a 13 m/s test to exhaustion, blood lactate, heart rate, runtime, BAL cytology and pulmonary arterial pressure were measured. Data were analysed with paired and unpaired t tests, one-way ANOVA and Tukey's pair-wise multiple comparison and Friedman repeated measure analysis of variance on ranks.

Results: The administration of sildenafil did not alter mean inspired/expired gas measurements, plasma lactate concentrations or acute pulmonary haemorrhage in either exercise test or pulmonary arterial pressure measurement in the 13 m/s trial. Heart rates in both stress tests were significantly different at submaximal speeds and during the early recovery period. Run time was not affected by sildenafil administration in the step-wise trial (P = 0.622) or in the 13 m/s trial (P = 0.059).

Conclusions: Sildenafil did not alleviate pulmonary haemorrhage or enhance performance-related indices in these trials. Sildenafil administration altered cardiovascular adaptation to intense exercise as evidenced by altered heart rates at submaximal speeds and post exercise. The effect of these alterations on other performance perimeters was not evident.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Manufacturers’ addresses
  10. References

Sildenafil citrate is marketed for the treatment of erectile dysfunction in men. Sildenafil inhibits the enzyme, phosphodiesterase-5 (PDE-5), that breaks down cyclic guanidine monophosphate (cGMP). Cyclic guanidine monophosphate concentration increases following soluble and particulate enzyme activation by the vasoactive mediators, nitric oxide (NO) and natriatic proteins, causing vascular relaxation and blood pressure reduction. While the drug is marketed for its vasodilatory effect on penile vessels, a peripheral vasodilatory effect unrelated to dose rate and without an effect on heart rate, and a vasodilatory effect in the PDE-5 rich tissue of the lungs has been demonstrated (Zusman et al. 1999; Weimann et al. 2000; Mahmud et al. 2001; Schermuly et al. 2004). Other studies indicate that it may also reduce pain induced by noxious stimuli and enhance the anti-nociceptive effect of pain-relieving drugs via an incompletely defined pathway mediated through cGMP and NO (Mixcotal-Zecuatl et al. 2000; Jain et al. 2001; Ambriz-Tututi et al. 2005).

Sildenafil is not marketed for veterinary use, has no recognised therapeutic use in equine veterinary medicine, but has been found in post race samples of horses competing in Pari-Mutuelly sanctioned races. The New York Thoroughbred Horseman's Association has stated that, in 2000, sildenafil was the 8th most frequent drug found in Thoroughbred and harness racing samples. Sildenafil has been classified as a Class 3 substance by the Association of Racing Commissioners International (ARCI) and, as such, is considered to have a moderate potential for influencing performance.

Numerous studies have demonstrated that sildenafil reduces pulmonary vascular resistance for different forms of hypertension in man and animals (Weimann et al. 2000; Wilkens et al. 2001; Ghofrani et al. 2002a,b 2003; Sastry et al. 2002; Schermuly et al. 2004). Mean pulmonary arterial pressure (PAP) in men with stable ischaemic heart disease was decreased by 27 and 19% during rest and ergometric cycle exercise, respectively, while accomplishing the same workload as maintained during baseline measurements (Jackson et al. 1999).

In a randomised, double-blind, placebo-controlled crossover study, Ghofrani et al. (2004) reported the effects of sildenafil administration to healthy mountaineers at rest and performing cycle exercise under 2 conditions, an acute hypoxia-induced vascular hypertensive state equivalent to low altitude hypoxia and acclimatised for 6 days at high altitude. Systolic PAP was significantly lower after sildenafil administration at rest at low and high altitude and during maximal exercise at low and high altitude. At exhaustion, cardiac output and exercise tolerance were higher at both altitudes in these hypoxic participants given sildenafil.

Other studies have demonstrated PAP reduction when sildenafil was administered to normal subjects in hypoxia at rest (Zhao et al. 2001) or exercised under altitude-induced hypoxia (Richalet et al. 2005). Sildenafil administration had no effect on PAP at rest or after exercise in normoxic healthy, nonacclimatised subjects, but produced a significant PAP reduction in subjects in a hypoxic state (Ricart et al. 2005).

The effect of sildenafil on haemodynamics in healthy, normoxic subjects has been studied. Webb et al. (1998) reported that a single oral dose of sildenafil in normal subjects produced a transient drop in blood pressure that averaged 10 mmHg and persisted for 4 h after drug administration. Systolic and diastolic blood pressures significantly decreased in healthy individuals after i.v. (Jackson et al. 1999) and oral sildenafil (Mahmud et al. 2001; Vardi et al. 2002) with no difference in heart rate and no linear dose-effect relationship for either route of administration.

While the pharmacokinetics and effect on cGMP of sildenafil administration to horses has been described (Colahan et al. 2006), the effects of sildenafil administration on PAP, on the performance related physiological parameters, or the prevalence of exercise-induced pulmonary haemorrhage (EIPH) in exercising horses are unknown. The current study was undertaken to determine the effect of sildenafil citrate administration in horses on PAP, the prevalence of EIPH and select physiological parameters in step-wise and sustained maximal exertion (13 m/s) exercise tests to exhaustion.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Manufacturers’ addresses
  10. References

The Institutional Animal Care and Use Committee approved the use of the animals in this project and the methods employed.

Horses

Thirteen Thoroughbred horses (11 geldings, 2 mares; 3–12 years old; mean ± s.d. weight, 509 ± 43 kg) were used. Horses were examined for abnormalities including locomotion disturbances and upper airway abnormalities. Routine preventive medicine procedures including control of internal parasites, vaccination for infectious diseases and monitoring of general health on a daily basis were conducted.

Housing and conditioning regimen

Horses were kept in pairs at pasture except during exercise training or testing and sample collection. All horses were exercised 3 days/week for a minimum of 8 weeks prior to the study. Exercise conditioning and trials was conducted on a high-speed treadmill1 operated by experienced technicians. The training regimen was designed to enable the horses to gallop one mile (1.6 km) in 2 min without undue stress as indicated by heart rate recovery to pre-exercise rate in 40 min after a 2 mile gallop.

Blood sampling

For the step-wise to exhaustion trial, blood samples were collected via catheters in the left jugular vein over 8 h. Samples taken 24–72 h post administration were obtained by venipuncture. Prior to sample collection from a catheter, 10 ml of saline and blood was withdrawn from the extension tubing and disposed of as waste. Fresh blood was collected via syringe and dispensed into 10 ml partially evacuated collection tubes2 and centrifuged to obtain plasma. Blood samples for the analysis of sildenafil citrate were collected into lithium heparin containing tubes before drug administration and at 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 24 and 48 h. Samples for plasma lactate analysis were taken into potassium oxalate/sodium fluoride containing tubes pre-exercise, at the end of the warm-up period, at 1 min intervals before the treadmill speed was increased, and at failure. Blood samples for lactate analysis were immediately placed in ice after collection, and were centrifuged and plasma harvested within 20 min. All plasma was stored at −20°C until analysis.

For the 13 m/s to exhaustion exercise test blood samples were collected via catheters (14 gauge × 14 cm) in the left jugular vein just prior to exercise and at 1 h post drug for sildenafil assay. Samples for plasma lactate analysis were taken into potassium oxalate/sodium fluoride containing tubes pre-exercise, at the end of the warm-up period, at 1 min intervals during the test, and at failure.

Bronchoalveolar lavage (BAL)

One veterinarian collected all BAL samples. Pre-exercise BAL was obtained on all horses one week prior to the initiations of exercise trials and 24 h after each exercise trial. To collect the samples, the horses were sedated with 2 ml xylazine and a 2 m, flexible fibreoptic endoscope passed through the nostril and into a sub-segmental bronchus. Lidocaine (10 ml) was flushed into the airway for anaesthesia. Four 50 ml aliquots of saline were infused into the bronchi and then aspirated. The lavage recovered aspirate volume was measured. Two treatment-blinded clinical pathologists of the Clinical Pathology laboratory at the Veterinary Medical Centre conducted quantitative and qualitative cytological examinations. For data analysis, acute and chronic haemorrhage was ranked and analysed separately.

Drug preparation and administration

Sildenafil citrate tablets (Viagra 300 or 600 mg)3 needed to provide the appropriate dose (5.0 mg/kg bwt) were ground with mortar and pestle then mixed with 60 ml of water. The solution was drawn into a 60 ml syringe and then injected into a nasogastric tube to administer to each horse. The tubing was flushed with 120 ml water to ensure total administration.

Exercise tests

All training was suspended 5 days prior to the exercise trials. Two exercise trials of different design were used. In the first, the horses were exercised to exhaustion at incrementally increasing speeds for a step-wise trial and, in the second, they were exercised to exhaustion at 13 m/s. For both studies 12 horses were randomly divided into 2 treatment groups of equal number and were assigned to either placebo or drug treatment groups in a crossover design, with each horse as its own control. For the step-wise trials, a 14–21 day and for the 13 m/s trial, a 30 day recovery interval separated the drug and placebo trials for each horse. Exercise trials were conducted 1 h after drug or placebo administration.

On the day of the step-wise trial, horses were aseptically prepared and a jugular catheter placed and a telemetric ECG monitor4 with electrodes under a saddle on both sides of the withers and under the girth at the level of the elbow and transponder attached to the saddle. Following a warm-up period at 4 m/s on the flat, the treadmill was raised to a 10% incline and the speed increased to 8 m/s. Incrementally, the speed was increased by 1 m/s every minute until exhaustion. This occurred when the horse could not stay in place on the treadmill despite humane urging by the treadmill operator and was recorded as the end of run-time. The treadmill was immediately lowered to 0% incline. To cool down, the horses were trotted (4 m/s) for 2 min on the treadmill, then exited the treadmill and were hand-walked.

For the 13 m/s trial, horses were aseptically prepared and a jugular catheter placed for blood sampling. A 7 French introducer catheter5 was inserted in the jugular vein caudal to the collection catheter. A saline filled 7 French, 120 cm micro-tip pressure transducer catheter with lumen6 was connected to the cardiac monitor, then threaded through the introducer. The transducer catheter was advanced through the jugular vein and the right ventricle and 8 cm into the pulmonary artery. Proper placement of the pressure transducer catheter was confirmed by pressure recording on the cardiac monitor7. The transducer was balanced and calibrated with a mercury manometer before introduction.

The 13 m/s trial consisted of a 2 min walk (2 m/s), a 2 min trot (4 m/s), and a 2 min canter (8 m/s) at no incline. Then, the treadmill was inclined to 5% and the speed increased to 13 m/s. This speed and incline was maintained until the horse failed to stay in place on the treadmill despite humane encouragement. This was recorded as the end of runtime. The treadmill was immediately lowered to 0% incline. To cool down, the horses were trotted (4 m/s) for 2 min on the treadmill and then exited the treadmill for hand-walking.

Sildenafil assay

Plasma concentrations of sildenafil citrate and desmethylsildenafil were quantified using a high-performance liquid chromatography system with UV detection at 230 nm (Rudy and Kahler 2002; Colahan et al. 2006). The limits of quantification and detection were determined to be 2 and 1 ng/ml, respectively. Plasma lactate concentration was measured by a colorimetric, enzymatic method8. Absorbance of lactate samples was read on a spectrophotometer9.

Statistical analysis

Comparisons between treatments were made using statistical software10 to conduct paired t tests for total runtime and heart rate, oxygen consumption, carbon dioxide production, lactate and mean pulmonary arterial pressure at each time point. One-way repeated measures analysis of variance was used to compare pre- and post exercise treatments for bronchoalveolar white blood cell counts. When significance was found amongst the treatment groups, a Tukey pair-wise multiple comparison test was employed. Qualitative haemorrhage information in the BAL analyses was assigned rank. Acute and chronic haemorrhage ranking was analysed separately. Pre-exercise, post exercise placebo and post exercise sildenafil treatments for acute or chronic pulmonary haemorrhage were compared using the Friedman repeated measures analysis of variance on ranks. When significance was found amongst the treatment groups, the Tukey pair-wise multiple comparison test was used. Significance level for treatment comparisons was P<0.05. Desired power is 0.800. Data are reported as mean ± s.d.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Manufacturers’ addresses
  10. References

Drug concentration

During the step-wise exercise test, immediately prior to exercise at 1 h post drug administration, mean plasma sildenafil was 172.3 ± 94.5 ng/ml and mean plasma desmethylsildenafil concentration was 76.9 ± 62.7 ng/ml. The peak sildenafil and desmethylsildenafil plasma concentrations occurred at 3 h at 229.8 ± 102.0 ng/ml and 126.2 ± 69.9 ng/ml, respectively (Colahan et al. 2006). During the 13 m/s exercise test, immediately prior to exercise at 1 h post drug administration, mean plasma sildenafil was 106.8 ± 100.9 ng/ml and mean plasma desmethylsildenafil concentration was 47.8 ± 51.3 ng/ml.

Inspired/expired gas (VO2, VCO2)

Mean VO2 and VCO2 increased with treadmill speed as expected in the step-wise trial (P<0.001), but there was no effect of sildenafil administration at any time. Inspired/expired gas analysis was not measured during the 13 m/s exercise trial.

Runtime

In the step-wise exercise trial, mean run times measured from 8 m/s to failure were 239.8 ± 27.8 s and 242.3 ± 28.6 s for drug and placebo treatments respectively, and were not significantly different (P = 0.622, with power of test with α= 0.050:0.050). In the 13 m/s exercise trial, runtime was shorter for sildenafil-treated horses (164.2 ± 44.6 s) compared to placebo-treated horses (184.0 ± 47.5 s), and approached significance (P = 0.059, with power of test with α= 0.050:0.384).

Heart rate

In the step-wise trial, heart rate was significantly higher for sildenafil-treated horses vs. placebo-treated horses during exercise at 9 m/s (203.8 ± 8.8 vs. 200.8 ± 9.8 beats/min; P = 0.021, with power of test with α= 0.050:0.625), and at 2 min (140.5 ± 20.1 vs. 133.1 ± 20.9 beats/min; P<0.001, with power of test with α= 0.050:1.000) and 5 min (101.8 ± 14.6 vs. 99.0 ± 17.7 beats/min; P = 0.033 with power of test with α= 0.050:0.518) post failure. No difference between treatment groups was seen at 8 m/s (198.9 ± 9.9 vs. 195.6 ± 9.5 beats/min, P = 0.055, with power of test with α= 0.050:0.405) or 10 m/s (208.3 ± 9.2 vs. 206.3 ± 10.0 beats/min, P = 0.052, with power of test with α= 0.050:0.413).

In the 13 m/s trial, heart rate was higher in sildenafil-treated horses at end of 8 m/s (170.8 ± 22.4 vs. 161.6 ± 20.7 beats/min; P = 0.048 with power of test with α= 0.050:0.431) and at 2 min post exercise (137.0 ± 20.0 vs. 125.8 ± 10.8 beats/min; P = 0.007, with power of test with α= 0.050:0.833). Heart rate was not higher in sildenafil-treated horses at end of 2 m/s (103.9 ± 22.3 vs. 104.8 ± 20.1 beats/min; P = 0.986, with power of test with α= 0.050: 0.050) and at end of 4 (138.9 ± 21.0 vs. 135.5 ± 16.4 beats/min; P = 0.339, with power of test with α= 0.050:0.050), at failure (209.2 ± 8.8 vs. 206.3 ± 6.6 beats/min; P = 0.145, with power of test with α= 0.050:0.188) or at 1 min post exercise (137.0 ± 20.0 vs. 125.8 ± 10.8 beats/min; P = 0.007, with power of test with α= 0.050:0.833).

Plasma lactate concentration

Mean plasma lactate concentrations increased with increasing treadmill speed (P<0.001), but lactate concentrations did not differ between drug and placebo treatments any time during exercise during either exercise test.

Pulmonary arterial pressure (Fig 1)

image

Figure 1. Pulmonary arterial pressure measured during 13 m/s exercise trials, following the administration of sildenafil (5 mg/kg bwt) or placebo. Results presented as mean ± s.d.

Download figure to PowerPoint

Mean PAP did not differ between sildenafil- and placebo-treated horses at any time during the 13 m/s trial. Maximal PAP at 13 m/s ranged from 75.9–148.7 mmHg (mean 115.2 ± 22.6 mmHg) for placebo horses and 78.9–131.9 mmHg (mean 108.7 ± 35.6 mmHg). Five of 12 placebo horses and 4 of 12 sildenafil horses had PAP greater than 90 mmHg at 8 m/s. While PAP during exercise was greater than pre-exercise (P<0.001) and PAP at 13 m/s was greater than at 2 (P = 0.034) and 4 m/s (P = 0.027), it was not greater at 13 m/s than at 8 m/s (P = 0.091). Pressure at 8 m/s was significantly greater than at 2 m/s (P = 0.044).

WBC in BAL

In the step-wise trial, post exercise BAL WBC count was higher in placebo-treated horses (P = 0.044), but not drug-treated horses (P = 0.48), compared to pre-exercise. There was no difference in BAL WBC counts between drug and placebo treatments (Table 1) (P = 0.718).

Table 1. White blood cell count in BAL
 Step-wise13 m/s
  1. Total WBC count (cells/µl ± s.d.) in BAL fluid before and 24 h after each of 2 different exercise trails both involving 2 exercise tests to exhaustion one each under the influence of sildenafil or placebo.

Pre-exercise135.8 ± 67.6134.9 ± 55.6
Post exercise (placebo)251.4 ± 153.0341.0 ± 195.9
Post exercise (sildenafil)243.0 ± 149.0328.4 ± 109.9

In the 13 m/s trial, post exercise BAL WBC counts in both placebo- and sildenafil-treated horses were higher than pre-exercise (P = 0.001 and P = 0.005). There was no difference in BAL WBC counts between drug and placebo treatments (P = 0.891).

Haemorrhage in BAL

No horse had signs of acute pulmonary haemorrhage prior to the exercise trials (Table 2). In both exercise trials, both sildenafil and placebo groups had more acute haemorrhage in post exercise trial BAL than pre-exercise trial BAL (P<0.001 for both step-wise and 13/s). No effect of sildenafil administration on acute pulmonary haemorrhage was seen (step-wise P = 0.41, power of performed test with α= 0.050: 0.999 and 13 m/s P = 0.741, power of performed test with α= 0.050: 0.999). Two horses had signs of chronic pulmonary haemorrhage prior to the exercise trials (Table 3). In the step-wise trial, there was significantly greater evidence of chronic haemorrhage in drug-treated, but not placebo-treated horses compared to the pre-exercise BALs. No difference was noted in chronic haemorrhage in sildenafil and placebo-treated post exercise BAL cytology in either exercise trial (step-wise P = 0.298 and 13 m/s P = 0.939).

Table 2. Ranked evidence of recent haemorrhage based on presence of erythrocytes In epithelial lining fluid obtained by BAL before and after each of 2 different exercise trials both involving 2 exercise tests to exhaustion one each under the influence of sildenafil or placebo
HorsePre-exerciseStep-wise sildenafilStep-wise placebo13 m/s sildenafil13 m/s placebo
  1. 0 = no haemorrhage; 1 = very mild haemorrhage; 2 = mild haemorrhage; 3 = moderate haemorrhage.

Pride02203
Chip02022
CJ02232
Cali00012
Bart03220
Sky002  
Skip00202
Fan03322
Will02232
Buddy02233
Secret02212
Kee02000
Spot0  22
Table 3. Ranked evidence of haemorrhage occurring 10 days or more previously based on presence of haemosiderophages In epithelial lining fluid obtained by BAL before and after each of 2 different exercise trials both involving 2 exercise tests to exhaustion one each under the influence of sildenafil or placebo
HorsePre-exerciseStep-wise sildenafilStep-wise placebo13 m/s sildenafil13 m/s placebo
  1. 0 = no haemorrhage; 1 = very mild haemorrhage; 2 = mild haemorrhage; 3 = moderate haemorrhage.

Pride00200
Chip02000
CJ00220
Cali02202
Bart00200
Sky020  
Skip23220
Fan22301
Will02000
Buddy02200
Secret03000
Kee03303
Spot0  00

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Manufacturers’ addresses
  10. References

Sildenafil concentrations

At a higher oral dose rate (5.0 mg/kg bwt) than recommended for man (approximately 0.5–1.0 mg/kg bwt), the plasma concentrations produced in these horses were similar to those reported in man and other species (Nichols et al. 2002). Preliminary administration at a lower dose rate (1.5 mg/kg bwt) produced mean plasma sildenafil concentrations varying by only 10 ng/ml between 4 and 6 h, but 12% of the horses developed plasma concentrations at or below the limit of quantification for 2 h following administration (Colahan et al. 2006). A higher 5 mg/kg bwt dose was selected to produce plasma concentrations comparable to those found to be therapeutic in other species and overcome the considerable variability in plasma concentrations seen following oral administration.

Had a preventative effect for EIPH been identified, the development of sildenafil as a therapeutic agent would have required further studies to develop dose/effect data. Under current racing rules, sildenafil is an illegal drug. The 1 h interval between drug administration and exercise provided plasma blood concentrations in a therapeutic range, a plausible time of illegal administration prior to a race and concurrence with other studies (Mahmud et al. 2001). The dose rate and timing of administration were maintained for both exercise trials based on the assumption that plasma concentrations at the time of exercise would be similar and therapeutic plasma concentration would be obtained in all horses. Furthermore, given evidence of absorption and measureable plasma sildenafil concentrations, the peripheral hypotensive effects of sildenafil are not dose related (Zusman et al. 1999).

Sildenafil administration did not produce differences between treatments in oxygen consumption or CO2 production in the step-wise exercise trial or in BAL WBC counts or plasma lactate concentrations recorded during either exercise trial.

At similar time points, mean heart rate was higher in both exercise trials following sildenafil administration compared to placebo during submaximal exercise and during recovery (step-wise: 8 m/s and 2 min post exercise; 13 m/s: 9 m/s, 2 min and 5 min post exercise). It is interesting that heart rate was higher at these times under the influence of sildenafil before and after maximal exertion, but an effect on heart rate was not identifiable during maximal exertion. This may be explained by peripheral sildenafil-induced vasodilation that adds to or alters the vasodilatory effect induced by maximal exercise (Evans 1994; Zusman et al. 1999; Mahmud et al. 2001). Alteration of the vasodilatory response of intense exercise in which blood flow is directed to working muscles (Evans, 1994) by a more generalised vasodilatory effect induced by sildenafil administration would impose a demand for increased cardiac output and hence increased heart rate. This accommodation to an increased peripheral vascular demand for perfusion would disappear as heart rate becomes maximal. A direct cardiac effect of sildenafil was not seen in resting heart rates recorded before exercise and is reported not to occur (Zusman et al. 1999). During the prolonged period of maximal exertion imposed by the 13 m/s trial, the added cardiac work required by sildenafil induced peripheral vasodilation could have had a deleterious effect on performance as is indicated by the 20 s shorter runtime in the sildenafil treatment group. However, since this effect did not reach significance (P = 0.059), but had a low statistical power (0.384 desired power 0.800) and was only noted under one of the 2 exercise regimens, it will have to be confirmed in subsequent studies.

The use of BAL for assessing the severity of EIPH has been effective used in field studies (Lester et al. 1999) and found to be a reliable and reproducible technique in treadmill laboratory conditions (Kindig et al. 2001a). Use of BAL permits a delay in conducting the examination useful in undertaking multiple exercise trials each day and provides for sensitivity in detection of haemorrhage (Couetil and Hinchcliff 2004).

Haemosiderophages in BAL fluid are an indication of an episode of pleural haemorrhage approximately 10 days or more prior to sampling, and require 3 weeks or longer to be cleared from the bronchi (McKane and Slocombe 1999; Couetil and Hinchcliff 2004). The pre-exercise BAL was preceded by exercise at conditioning intensity only with no periods of intense exercise, but haemosiderophages in the pre-exercise test BAL fluids of 2 horses is evidence of pulmonary haemorrhage during conditioning. To condition the horses and mimic the condition of horses in race training, the horses in this study maintained a regular training regimen prior to and between exercise tests. Also, the second step-wise trial followed the first for some horses by <3 weeks. The high prevalence of haemosiderophages in low to moderate numbers in BAL fluid after the exercise trials may be explained by haemorrhage that occurred during training or the previous exercise test. The presence of haemosideraophages and the increased WBC counts in the BAL fluids obtained post exercise trial are indications that the trials caused haemorrhage and inflammation, but were not helpful in detecting a sildenafil effect because the time of the occurrence the haemorrhage and persistence of the subsequent inflammatory response are unknown. The observation of erythrocytes in BAL fluid is an indication of haemorrhage within no more than 5 days (McKane and Slocombe 1999). No training was undertaken 5 days prior to exercise testing. All horses in the study demonstrated mild haemorrhage after at least one exercise trial. No horse experienced more than moderate haemorrhage.

Despite <20 years research, the role of PAP as a contributory factor of EIPH is incompletely defined (Couetil and Hinchcliff 2004). It is documented that pulmonary capillary transmural pressures of exercising horses exceed those of other species (Couetil and Hinchcliff 2004). Yet, while many studies in horses have suggested a positive correlation between pulmonary vascular pressures and EIPH, horses suffering EIPH do not have higher PAP during exercise than unaffected horses and the reduction of pulmonary arterial pressure with NO does not reduce EIPH severity (Manohar and Goetz 1996; Kindig et al. 2001b). Manohar and Goetz (1996) compared consistently EIPH-positive to consistently negative horses and found, after 12 weeks of maximal treadmill exercise training, no significant differences in PAP. Furthermore, Lester et al. (1999) found no significant relationship between peak PAP and red cell counts in field-tested galloping Thoroughbred horses. Alternative factors such as locomotory impact-induced trauma have been proposed (Schroter et al. 1998; Takahashi et al. 2001; Newton et al. 2005).

The administration of clenbuterol (Manohar et al. 2000) or nitric oxide inhalation (Kindig et al. 2001b) does not prevent equine EIPH. Frusemide is routinely administered to prevent EIPH, but, because frusemide may have a performance-enhancing effect or compromise by urine dilution the detection of prohibited substances in post race samples, alternative prophylactic treatments for EIPH have been sought. However, frusemide has been found to be an effective prophylaxis for EIPH (Hinchcliff et al. 2009). The administration of vasoactive agents such NO or, theoretically, sildenafil to reduce pulmonary arterial pressure may be counterproductive in preventing EIPH by altering pulmonary vascular responses that may be protective (Kindig et al. 2001b).

In this study oral sildenafil was not found to influence PAP in horses at maximal exertion and did not reduce signs of pulmonary haemorrhage post exercise. No indication for the use of sildenafil as a preventative treatment for EIPH was found. In addition, the alteration of heart rate during exercise by sildenafil indicates a potential to alter performance and supports its classification as a prohibited drug in racehorses.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Manufacturers’ addresses
  10. References

Funding for this project was provided by State Division of Pari-Mutuel Wagering Contracts #01-00006-00, #02-00026-00 and #03-00037-00.

Conflicts of interest

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Manufacturers’ addresses
  10. References

Dr Colham has received research grants from the Racing Medication and Testing Consortium, a non-profit organisation. The remaining authors have no potential conflicts.

Manufacturers’ addresses

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Manufacturers’ addresses
  10. References

1 Equine Dynamics Inc., Lexington, Kentucky, USA, modified to plans drawn by Bruce Richards, West Danby, New York, USA.

2 Becton Dickinson, Franklin Lakes, New Jersey, USA.

3 Pfizer Inc., New York, New York, USA.

4 Hewlett Packard, Houston, Texas, USA.

5 Arrow International, Reading, Pennsylvania, USA.

6 Millar Instruments, Inc., Houston, Texas, USA.

7 Datascope, Montvale, New Jersey, USA.

8 Sigma Chemical Co, Sigma Diagnostics, St Louis, Missouri, USA.

9 Thermos Supertonic, Madison, Wisconsin, USA.

10 SPSS, Inc., Chicago, Illinois, USA.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Manufacturers’ addresses
  10. References
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