Prophylactic bosentan does not improve exercise capacity or lower pulmonary artery systolic pressure at high altitude

Respiratory Physiology & Neurobiology 165 (2009) 123–130

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Prophylactic bosentan does not improve exercise capacity or lower pulmonary artery systolic pressure at high altitude夽 Roger D. Seheult a,b , Katja Ruh a,b , Gary P. Foster a,c , James D. Anholm a,b,∗ a

VA Loma Linda Healthcare System, Loma Linda, CA, United States Department of Medicine, Division of Pulmonary & Critical Care Medicine, Loma Linda University, Loma Linda, CA, United States c Department of Medicine, Division of Cardiology, Loma Linda University, Loma Linda, CA, United States b

a r t i c l e

i n f o

Article history: Accepted 6 October 2008 Keywords: Echocardiography Endothelin Hypoxia Hypertension Lung Bosentan

a b s t r a c t Hypoxic pulmonary vasoconstriction in response to high altitude ascent may contribute to decreased exercise capacity. Endothelin receptor antagonists reduce pulmonary artery pressure and improve exercise capacity in patients with pulmonary arterial hypertension, but their effects on exercise capacity at altitude are unknown. We studied the efficacy of bosentan started 5 days prior to ascent on exercise capacity and pulmonary artery systolic pressure (PASP) at 3800 m altitude. Eight healthy subjects completed a doubleblinded, randomized, placebo-controlled, crossover study. The end-points were time to complete a cycle ergometer time trial, PASP, and hemoglobin oxygen saturation (SpO2 ). The time to complete the time trial at altitude in subjects on placebo and bosentan was 527 ± 159 and 525 ± 156 s respectively (P = 0.90). PASP was not different on bosentan compared with placebo. Mean SpO2 during the altitude time trial was lower in subjects taking bosentan compared to placebo (78 ± 6 vs. 85 ± 8% respectively, P = 0.03). Bosentan initiated 5 days prior to ascent to high altitude did not improve exercise capacity or reduce PASP, and worsened SpO2 during high intensity exercise at altitude. Published by Elsevier B.V.

1. Introduction Rapid ascent to high altitude causes hypoxic pulmonary vasoconstriction (HPV) resulting in elevated pulmonary artery pressure (Hultgren et al., 1964; Bartsch et al., 2005). Other consequences of ascent to altitude include increased right ventricular afterload, reduction in arterial oxygen content, dyspnea, fatigue, exercise intolerance, and, in some individuals, high altitude pulmonary edema (HAPE) (Hackett and Roach, 2001). Given these potentially serious consequences and the increasing number of sojourners traveling to high altitude destinations improved understanding of altitude-induced pulmonary hypertension and its complications remains important. Serum endothelin (ET)-1 levels are closely associated with the acute increase in pulmonary artery pressure at high altitude (Goerre et al., 1995; Maggiorini et al., 2001; Berger et al., 2007) and may play a role in the development of HAPE (Morganti et

夽 Clinical Trial Registration Information: NCT00432978; URL: http://clinicaltrials.gov/show/NCT00432978. ∗ Corresponding author at: 11201 Benton St, Loma Linda, CA 92357, United States. Tel.: +1 909 583 6098; fax: +1 909 777 3214. E-mail addresses: [email protected] (R.D. Seheult), [email protected] (J.D. Anholm). 1569-9048/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.resp.2008.10.005

al., 1995; Sartori et al., 1999). A prior study has shown that the non-selective endothelin receptor antagonist bosentan reduced pulmonary artery systolic pressure (PASP) when taken after acute ascent from sea level to 4559 m and improved resting hemoglobin oxygen saturation during the first day at altitude (Modesti et al., 2006). Another study found that during hypoxic exercise bosentan administration improved hemoglobin oxygen saturation and improved ventilation–perfusion matching (Loeckinger et al., 2006). These findings suggest that bosentan might decrease PASP and improve exercise capacity at high altitude. The factors limiting exercise capacity at high altitude are not fully known, but limitations in oxygen transport to the working muscles are a major factor at least below 4000 m (Calbet et al., 2003). In addition to a reduction in arterial saturation, HPV leading to excessive right ventricular afterload may limit exercise capacity by reducing stroke volume and cardiac output (Ghofrani et al., 2004; Faoro et al., 2007). Recent studies by Ghofrani et al. (2004), and Richalet et al. (2005), found both a reduction in PASP and improved arterial oxygenation after taking sildenafil. These changes were accompanied by a significant improvement in exercise capacity suggesting that HPV may contribute to impaired exercise capacity at altitude even in subjects without HAPE (Ghofrani et al., 2004; Richalet et al., 2005). We hypothesized that bosentan, a non-selective endothelin receptor antagonist, would improve pulmonary hemodynamics, exercise capacity, and hemoglobin oxygen

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Fig. 1. General outline of study design. Long vertical dotted lines represent ascent to high altitude. Low altitude testing (SL testing) was performed 6–18 h prior to ascent. High altitude testing consisted of daily Lake Louise Questionnaire, echocardiography 24 and 36 h after ascent, and exercise testing. LLQ = Lake Louise Questionnaire for acute mountain sickness; Echo = echocardiography; TT = cycling time-trial. Time-line is not drawn to scale.

saturation at high altitude. To test this hypothesis we conducted a double-blinded, block-randomized, placebo-controlled, crossover trial with bosentan initiated 5 days prior to ascent to 3800 m. 2. Methods 2.1. Subjects Eight active, non-smoking volunteers (four men, four women) completed this trial. These non-acclimatized volunteers aged 23–46 were experienced cyclists or runners. Exclusion criteria were known heart, lung, or liver disease, current medications including nitrates, glyburide, cyclosporine A, pregnancy, and the inability to measure the velocity of the tricuspid regurgitation (TR) jet on transthoracic echocardiography. The age, height, and weight for the subjects were 37.1 ± 8.0 years, 173 ± 12 cm, and 69.6 ± 11.8 kg (mean ± S.D.). Before randomization and ascent to high altitude subjects completed baseline testing including a maximal oxygen consumption test (V˙ O2 peak ) (3.62 ± 0.95 l/min or 52.2 ± 10.6 ml/(kg min)) at a mean peak power output of 311 ± 65 watts, echocardiography, serum aspartate and alanine aminotransferases, and a complete blood count. Females required a negative serum qualitative beta-human chorionic gonadotropin pregnancy test to participate. Twelve subjects were enrolled and signed the informed consent. One subject with a history of HAPE in the past completed the study procedures but the data are not reported due to the known differences in pulmonary artery pressure responsiveness to hypoxia and exercise among HAPE susceptible individuals (Dehnert et al., 2005). Three subjects were unable to complete the study due to scheduling difficulties or illness. The remaining eight subjects completed randomization and all procedures and the results are based on their data. Due to winter storms the high altitude laboratory became inaccessible after completion of the study protocol in eight subjects. The Institutional Review Board of the VA Loma Linda Health Care System (VALLHCS) approved the protocol. All subjects gave written informed consent to participate in the study. The subjects were under the direct observation of investigators and were monitored for symptoms of HAPE and high altitude cerebral edema (HACE). Each day at altitude subjects completed the Lake Louise Questionnaire of AMS (Roach et al., 1993). Evacuation, oxygen, and medications for the treatment of AMS and HAPE were available at all times. 2.2. General study design Subjects were block randomized to receive either bosentan 125 mg twice daily (Actelion Pharmaceuticals US, Inc., South San

Francisco, CA, USA) or matching placebo in a crossover study design. Medication or placebo was started 5 days before ascent and continued for 2 days at altitude. An independent research pharmacist performed block randomization. Measurements including heart rate, pulse oximetry, the Lake Louise Questionnaire, echocardiography, blood pressure, and duration of a cycle ergometer time trial were performed within 24 h prior to each ascent and at high altitude. All subjects taken by motor vehicle from low altitude (Loma Linda, California, USA; altitude 370 m) to the University of California-Barcroft Laboratory (White Mountains, California; altitude 3800 m) in less than 8 h (day 0) except during one trip when weather necessitated a 12 h stay at the Crooked Creek Research Facility (3094 m) before ascending to the Barcroft Laboratory. Barometric pressure at the Barcroft Laboratory during testing was 483–485 mmHg. After the first night at altitude we recorded heart rate and oxygen saturation, and administered the Lake Louise AMS Questionnaire. Echocardiography was performed approximately 24 h after ascent (day 1). After a second night at altitude, approximately 36 h after ascent (day 2), heart rate, oxygen saturation, echocardiogram and a cycle ergometer time trial were performed. The time between successive trips to high altitude for any given subject was 2–4 weeks (Fig. 1). 2.3. Screening After informed consent all subjects completed a detailed medical health questionnaire which included their experience at high altitude and exercise training history. Maximal oxygen consumption (V˙ O2 peak ) and exercise capacity were determined during a continuous progressive exercise test to volitional exhaustion on an upright electronically braked cycle ergometer (Excalibur; Lode BV, Groningen, Netherlands). Expired respiratory gases were collected continuously and analyzed using a metabolic cart (V˙ max model 229; SensorMedics; Yorba Linda, California). Serum samples were drawn and analyzed at the VALLHCS clinical laboratory. 2.4. Doppler echocardiography To assess PASP, echocardiography was performed with an Agilent Sonos (Agilent 5500; Agilent, Andover, Massachusetts) ultrasound machine with a phased array s3 transthoracic transducer (Allemann et al., 2000). All echocardiographic studies were performed by standard techniques using the same instrument and by the same echocardiographer (GF). PASP was calculated by Doppler echocardiography based on the velocity of the tricuspid regurgitation (TR) jet using the modified Bernoulli equation:

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PASP (mmHg) = 4v2 + right atrial pressure (mmHg), where v is the peak TR velocity (m/sec) at end expiration and 4v2 is the trans-tricuspid gradient (mmHg). All echocardiographic data were digitally recorded for later analysis. Calculations were based on multiple TR velocity measurements in nearly all cases (median number of TR velocity measurements = 6). Calculation of the TR velocity and other echocardiographic measurements was performed at the end of the study by a separate sonographer who was blinded to medication assignment and the performance of the subjects. Right atrial (RA) pressure was estimated in the supine position by measuring the height between the ultrasound imaged collapse of the basilic vein in the upright extended right arm and the center of the RA determined by echocardiographic triangulation. This measurement was converted into mmHg (1 cm H2 O = 0.736 mmHg). RA estimates were not made in the upright cycle position or supine on day 1 at altitude. 2.5. Cycle ergometer warm-up and time trial To assess exercise performance while minimizing the variability observed in open-ended cycling endurance tests, subjects performed a cycling task designed to simulate a time trial. Time trials of this nature elicit maximal V˙ O2 , are more reproducible than cycling to exhaustion, and reasonably simulate race conditions (Foster et al., 1993; Jeukendrup et al., 1996). When set in the linear mode, the Lode cycle ergometer becomes pedal rate dependent, such that increased pedal rates result in increased work according to the formula: W = L·(RPM)2 , where L is a constant linear factor and RPM is the pedaling rate in revolutions per minute. The linear factor was chosen so that each subject’s preferred pedaling rate (observed during constant workload exercise) would cause them to work at the same wattage that elicited 55% of the maximal power output on their V˙ O2 peak test. The subject’s linear factor was not changed for the duration of the study. Each subject completed a 100 kJ time trial estimated to require 5–10 min to complete. Each subject performed two practice time trials prior to randomization to become familiar with the protocol. Each time trial at sea level and at altitude was preceded by a 20 min standardized warm-up period adjusted in intensity to each subject’s power output (Fig. 2). This type of warmup protocol has been used successfully before by others (Foster et al., 1993). Echocardiography was performed at rest and during the warm-up at 40% maximal power output. For each trip to altitude, subjects completed a time trial within 24 h before ascent and 36 h after arrival to high altitude. To simulate real-world race conditions, the subject’s instantaneous power, heart rate, cadence, total work, and total time were visible to the subjects throughout the standardized warmup and time trial (Foster et al., 1993). Heart rate was measured by a Polar heart rate monitor (Polar Vantage NV, Polar Electro Inc., Lake Success, New York). At altitude hemoglobin oxygen saturation (pulse oximeter attached to either a fingertip or earlobe) was measured at rest, during the standardized warm-up, and during the

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Fig. 2. Subjects completed a 20 min standardized warm-up prior to the time trial. Echocardiography was performed at rest and during 40% maximal power output.

time trial (Radical Docking Station RDS-1 signal extraction pulse oximeter, Masimo Corporation, Irvine, California). Subjects began the time trial in a “gate-like” manner starting from rest. Subjects were informed of their progress at 10 kJ intervals and given strong verbal encouragement to complete the time trial as quickly as possible. All subjects were highly motivated and were given a small performance-driven monetary reward. 2.6. Statistical analysis Based on a randomized, crossover design, we estimated a sample size of 8–12 subjects was required for an 80% power to detect a 5 mmHg change in PASP or 15 s improvement in exercise capacity with an ˛ value of 0.05. Statistical analysis was performed using Systat software (Systat Software Inc., Richmond, California). Data from the subject with a history of HAPE did not alter the significance of any of the analyses, thus the data are included here. Continuous data were analyzed with two within factors repeated-measures analysis of variance with factors for altitude and drug (placebo vs. bosentan). Categorical data were analyzed using McNemar test for comparing the change in Lake Louise scores and AMS incidence observed between the bosentan and placebo groups. Unless otherwise noted all data are presented as mean ± 1 S.D. A P-value < 0.05 was considered significant. 3. Results 3.1. Pulmonary hemodynamics and resting hemoglobin oxygen saturation PASP at 36 h after arrival to high altitude (day 2) was not different in subjects taking placebo or bosentan (Table 1). The increase in PASP from sea level to altitude on day 2 in resting, supine subjects taking placebo vs. bosentan was 8.2 ± 8.9 and 15.0 ± 8.9 mmHg respectively, (P = 0.12). In both groups after ascent, the trans-tricuspid gradient increased on day 1 (P = 0.01) but then declined on day 2 (P = 0.02, Table 1 and Fig. 3). Bosentan had no

Table 1 Supine resting hemodynamic data. Variable

TR velocity, m/s Right atrial pressure, mmHg PASP, mmHg

Sea level

High altitude

Day 0

Day 1

Placebo

Bosentan

Placebo

2.48 ± 0.20 5.7 ± 1.7 30.4 ± 4.7

2.44 ± 0.17 4.8 ± 3.0 28.8 ± 5.0

3.27 ± 0.40 – –

Day 2 Bosentan 3.26 ± 0.52 – –

Placebo

Bosentan

2.80 ± 0.32* 6.9 ± 2.3 38.6 ± 6.6

2.91 ± 0.26* 9.7 ± 4.2 43.9 ± 7.0

Values are means ± S.D.; n = 8 subjects. All values measured in the supine position. All comparisons between sea level and high altitude were statistically significant, P < 0.01. No comparisons between placebo and bosentan were statistically significant. * Effect of duration of time at altitude (day 2 TR velocity vs. day 1), P = 0.017.

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Table 2 Upright rest and exercise hemodynamic data. Variable

Sea level

High altitude

Placebo

Bosentan

Rest Submaximal exercise HR, beats/min Systolic BP, mmHg Diastolic BP, mmHg TR velocity, m/s Trans-tricuspid gradient, mmHg SaO2 , % Time-trial Duration, s Peak HR, beats/min Average HR Average watts, W Average SaO2 , %

62 131 77 2.30 21.3 98

± ± ± ± ± ±

14 14 6 0.16 2.9 1

Exercise

Rest

126 ± 14 – – 2.70 ± 0.23 29.3 ± 5.0 –

64 123 72 2.52 25.9 97

408 ± 183 ± 170 ± 264 ± –

± ± ± ± ± ±

8 7 4 0.31 6.1 1

114 11 9 79

Placebo Exercise

Rest

131 ± 15 – – 2.82 ± 0.45 32.5 ± 10.0 –

74 138 81 2.84 32.6 93

414 ± 185 ± 172 ± 263 ± –

120 11 9 86

± ± ± ± ± ±

Bosentan

15 16 5 0.31 6.9 3

Exercise

Rest

126 ± – – 3.20 ± 41.3 ± 87 ±

78 129 79 3.01 36.8 92

527 178 167 207 85

± ± ± ± ±

15

0.26 6.7 3 159 13 9 68 8

Exercise ± ± ± ± ± ±

11 8 5 0.37 8.8 2

129 ± – – 3.21 ± 41.4 ± 87 ±

0.24 6.1 3

± ± ± ± ±

156 14 10 73 6*

525 179 167 209 78

9

Values are mean ± S.D.; n = 8 subjects. All values measured while upright on bicycle ergometer. SaO2 and blood pressure were not measured during sea level exercise. Within each treatment condition values during exercise were significantly different from those at rest, P < 0.05. * SaO2 during the time-trial was lower on bosentan than placebo (P < 0.01); no other comparisons between bosentan and placebo conditions were significant.

effect on the trans-tricuspid gradient at any time point. On day 2, the estimated RA pressure was higher than at sea level but was unaffected by bosentan administration (P = 0.43, Table 1). Bosentan did not affect the resting hemoglobin oxygen saturation at altitude (Table 2). 3.2. Exercise capacity, pulmonary hemodynamics, and hemoglobin oxygen saturation during the time trial

groups. Upright TR velocity increased during exercise and at altitude when compared to sea level but was unaffected by bosentan (Table 2). Hemoglobin oxygen saturation at rest on days 1 and 2 (Table 2) and during the warm-up period (Table 2) was not different between placebo and bosentan. During the exercise time trial the hemoglobin oxygen saturation declined in all subjects but the mean hemoglobin oxygen saturation was lower in subjects on bosentan than in subjects on placebo (78 ± 6% on bosentan vs. 85 ± 8 on placebo; P < 0.05, Fig. 4 and Table 2).

All subjects completed the subjectively difficult 100 kJ time trial at altitude. Bosentan administration did not affect exercise performance compared to placebo during the altitude time trial (527 ± 159 s on placebo and 525 ± 156 s on bosentan; P = 0.69; Table 2). Exercise performance in the time trial decreased at altitude in both the placebo and bosentan groups compared to sea level, (P < 0.001) but the decrement in performance (28.7 ± 10.8% on placebo and 26.7 ± 9.2% on bosentan) was the same in both

Fig. 3. Supine trans-tricuspid pressure gradient (mmHg) measured at low altitude (day 0) (370 m), at 24 h (day 1), and at 36 h (day 2) after arrival to high altitude (3800 m). Diamonds () represent subjects on placebo, circles (䊉) represent subjects on bosentan. Bosentan and placebo responses were not significantly different. *P = 0.01, compared with day 0. + P = 0.02, compared with day 1.

Fig. 4. Mean hemoglobin oxygen saturation during the time trials completed at high altitude (top). Error bars represent ±1 S.D. Diamonds () represent subjects on placebo, circles (䊉) represent subjects on bosentan. The mean difference between hemoglobin oxygen saturation (mean ) on bosentan and placebo are plotted with the 95% confidence intervals (CI) throughout the time trial (bottom).

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3.3. Clinical effects of high altitude Bosentan administration did not affect the Lake Louise score, incidence of AMS, blood pressure, or heart rate. No cases of HAPE or HACE occurred. 4. Discussion This study demonstrates for the first time that bosentan does not reduce resting or exercise trans-tricuspid pressure gradient or improve exercise capacity when initiated 5 days prior to ascent and continued throughout the time at altitude. Bosentan was associated with lower hemoglobin oxygen saturation during high intensity exercise. Since sildenafil improves exercise performance at high altitude (Ghofrani et al., 2004; Richalet et al., 2005; Hsu et al., 2006) we hypothesized that bosentan working via the endothelin pathway would also decrease pulmonary artery pressure and improve exercise performance in healthy subjects at high altitude as it does in patients with pulmonary arterial hypertension (Rubin et al., 2002). The study results do not support our initial hypothesis. Instead, the findings appear to be mediated by complex, time-dependent interactions between the pulmonary and renal effects of bosentan during hypoxia (Modesti et al., 2006). Bosentan reduced PASP when given after ascent at altitude in the study by Modesti et al. (2006), but this reduction disappeared after 3 days at altitude. When bosentan was initiated 5 days before ascent and continued throughout the time at altitude in the present study the drug did not reduce PASP. This discrepancy may be explained in part by the interaction of endothelin in the pulmonary vasculature and the renal system (Hildebrandt et al., 2000; Modesti et al., 2006). Ascent to high altitude causes an increase in urinary volume (Maresh et al., 2004; Loeppky et al., 2005) mediated in part by the endothelin antagonism of arginine vasopressin (Modesti et al., 1998; Ge et al., 2005). This physiologic ETB receptor-modulated diuretic mechanism may be important in preventing AMS and HAPE (Zeidel et al., 1989; Oishi et al., 1991; Tomita et al., 1993; Modesti et al., 2006) Endothelin excreted in the urine is produced locally in the kidneys and is independent of systemic endothelin production (Serneri et al., 1995; Modesti et al., 1998). Modesti et al. (2006) found that subjects given bosentan at altitude had a significantly lower 24-h urine volume on days 1 and 2 after ascent when compared to subjects on placebo. The early reduction in PASP and later urinary free water retention caused by initiation of bosentan after ascent prompted Modesti and others to propose a therapeutic window for bosentan’s use at altitude to avoid fluid retention and possible interstitial pulmonary edema (Modesti et al., 2006; Rubin, 2006). It is possible that in our study the delayed fluid retention properties of bosentan (both an ETA and an ETB receptor antagonist) negated any potential reduction in PASP because bosentan was initiated 5 days prior to ascent. In Modesti’s study the PASP difference between bosentan and placebo disappeared by day 3 (Modesti et al., 2006). Data collection for this study was already underway when the results of Modesti’s study were published. Although the timing of bosentan administration was initially planned to ensure stable bosentan blood levels, the study design allowed us to address questions raised in Modesti’s study. We found that bosentan administration 5 days before altitude ascent does not reduce PASP or trans-tricuspid gradient, does not improve exercise performance and worsens SaO2 during high-intensity exercise. 4.1. Pulmonary hemodynamics In previous studies, administration of bosentan to subjects shortly after ascent to high altitude (real or simulated) resulted

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in a lower pulmonary artery pressure and improved saturation at rest (Modesti et al., 2006) and during exercise (Loeckinger et al., 2006). The improved exercise oxygen saturation was due to improved ventilation–perfusion matching from blunted HPV (Loeckinger et al., 2006). In the current study, where bosentan was initiated prior to ascent, subjects had neither a lower PASP nor did their resting hemoglobin oxygen saturation improve. The trans-tricuspid gradient (Fig. 3) was highest on day 1 with a reduction thereafter on day 2 in both groups, consistent with previously published data (Modesti et al., 2006). In none of the testing conditions did bosentan significantly lower the trans-tricuspid gradient or PASP. Indeed, it is notable that seven of eight subjects taking bosentan had a larger increase in resting PASP after ascent to high altitude on day 2 when compared to placebo (P = 0.12). While the estimation of right atrial pressure was higher on day 2 at altitude in both groups compared to sea level (P < 0.001), there was no effect from bosentan (P = 0.43; Table 1), although the study was not powered to detect a difference in right atrial pressure. Right atrial pressure, however, may not reflect fluid accumulation in subjects on bosentan as peripheral edema occurs in up to 20% of patients on chronic bosentan treatment for pulmonary hypertension, despite demonstration of a decreased RA pressure (Channick et al., 2001). 4.2. Exercise capacity This is the first study to investigate the effects of bosentan on exercise capacity at high altitude. Our results show that bosentan initiated 5 days prior to ascent to high altitude does not improve exercise capacity. The factors limiting exercise capacity at high altitude are not fully known but relate to various components of the oxygen transport system (Wagner, 1996). In the present study bosentan did not lower trans-tricuspid gradient at altitude in the upright position at rest or during submaximal work. It is unknown exactly how pulmonary artery pressure affects exercise capacity. It is conceivable that elevated right ventricular afterload from hypoxic pulmonary vasoconstriction may limit cardiac output and thus exercise capacity (Faoro et al., 2007). The improvement in exercise capacity associated with a reduction of pulmonary artery pressure at altitude with sildenafil supports this idea (Ghofrani et al., 2004; Richalet et al., 2005; Hsu et al., 2006). Sildenafil also improves arterial oxygenation which may contribute to the exercise changes. Faoro et al. (2007) studied subjects under both acute and chronic hypoxic conditions. With acute hypoxia sildenafil lowered PASP, improved arterial oxygenation, and increased maximal oxygen uptake. During chronic hypoxia sildenafil lowered PASP but did not alter arterial oxygenation or exercise capacity. They conclude that the improvement in oxygenation is more important to improved exercise capacity with acute hypoxia than is any reduction in PASP (Faoro et al., 2007). Therefore, it is not surprising that bosentan did not improve exercise capacity in our study given that it did not lower PASP and was associated with lower hemoglobin oxygen saturation during the high intensity time trial. In the study by Hsu et al. (2006), 40% of their subjects accounted for nearly all of the performance improvement found on sildenafil. With bosentan it was impossible to identify any such group of responders vs. non-responders. What may be surprising is that subjects on bosentan did not have a lower exercise capacity at altitude given the lower hemoglobin oxygen saturation during the time trial. This is likely explained by many complicating biological and psychological factors responsible for determining exercise capacity. The finding of equivalent exercise performance despite differences in hemoglobin oxygen saturation as seen in the current study is not new (West et al., 1962; Schoene,

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1984). Multiple adaptive factors such as change in cardiac output and oxygen extraction by working muscles could contribute to the results we observed. 4.3. Hemoglobin oxygen saturation Pulse oximetry during high intensity exercise is notoriously inaccurate although oxygen desaturation during exercise at altitude is well known (West et al., 1962). The crossover design makes it likely that the relative differences between bosentan and placebo are real, nevertheless confirmation of these results with direct blood measurements are needed. Several studies have examined the cause of hemoglobin oxygen desaturation during high intensity exercise at altitude. In normal subjects, ventilation–perfusion mismatch and diffusion disequilibrium increase during exercise at altitude (Gale et al., 1985; Torre-Bueno et al., 1985; Hammond et al., 1986a,b; Wagner et al., 1986). Ventilation–perfusion inequalities that increase during exercise at altitude may be caused by non-uniform vasoconstriction or the development of interstitial pulmonary edema (Wagner et al., 1986). Wagner and co-workers found a correlation between pulmonary artery pressure and ventilation–perfusion mismatch (Wagner et al., 1986) again suggesting interstitial pulmonary edema or non-uniform pulmonary vasoconstriction as a mechanism for the ventilation–perfusion mismatch. Short duration, high-intensity exercise at low altitude causes disruption in the blood–gas barrier associated with high pulmonary artery pressures in a similar manner to the blood–gas barrier disruption thought to occur in high altitude pulmonary edema (West et al., 1995; Hopkins et al., 1997). In a subsequent study, Hopkins et al. (1998) found that sustained submaximal exercise at low altitude did not alter the blood–gas barrier of the lung. Severe, prolonged exercise at altitude, however, produces radiographic changes of early pulmonary edema that may impair exercise capacity (Anholm et al., 1999). Schaffartzik et al. (1992) found that the ventilation–perfusion inequality and its persistence during recovery 20 min after hypoxic exercise was consistent with pulmonary edema as evidenced by acidosis, greater pulmonary blood flow, and presumed higher pulmonary artery pressures. Finally, using high intensity exercise in hypobaric hypoxia, Eldridge et al. (2006) found bronchoalveolar lavage fluid protein and red blood cell concentrations similar to those found in early HAPE. Their findings support the concept that intense exercise, especially with hypoxia, causes disruption of the pulmonary blood–gas barrier. While the preceding evidence helps explain why humans have lower hemoglobin oxygen saturation at altitude during exercise, the cause for even lower hemoglobin oxygen saturation in the subjects taking bosentan is speculative. Some have suggested that due to bosentan’s fluid retention effect on the kidney, administration before ascent to altitude may result in more pulmonary alveolar fluid or interstitial pulmonary edema and worsen ventilation–perfusion relationships (Modesti et al., 2006; Rubin, 2006). While there was no difference in hemoglobin oxygen saturation during the preceding warm-up period and at the start of the time trial, the decline in oxygen saturation in subjects taking bosentan increased as the time trial progressed (P < 0.05), consistent with worsening ventilation–perfusion mismatching and possibly interstitial pulmonary edema formation during high intensity exercise at altitude (Fig. 4). Other potential mechanisms by which bosentan may have contributed to the exercise oxygen desaturation include intrapulmonary shunting and hypoventilation. Bosentan may have altered ventilatory drive leading to relative hypoventilation compared with placebo although recent evidence indicates this is unlikely (Gujic et al., 2007).

4.4. Clinical effects of high altitude Similar to the results found by Modesti et al. (2006), we found no difference in the incidence of AMS on either day 1 or day 2. Diuresis after ascent to altitude is an important adaptation to altitude and a decreased urinary volume is associated with increased AMS symptoms (Loeppky et al., 2005). If bosentan initiated 5 days prior to ascent increased fluid retention, it did not increase the incidence of AMS in our study, however, the study was not powered to detect the effects of bosentan on AMS. 4.5. Limitations of the study The primary goal in this study was to evaluate the effects of bosentan on pulmonary artery pressure, exercise capacity, and hemoglobin oxygen saturation at altitude. The explanation of our unexpected results is based in part on Modesti’s work which was published after our data collection had begun (Modesti et al., 2006). As such we did not measure body weight, urinary output, or free water clearance. We did not evaluate lung water by chest radiography or other techniques. These data might help establish the etiology of our results, but they do not change the overall conclusions of our study. The accuracy of pulse oximetry saturation measurements during high-intensity exercise remains controversial with evidence for (Martin et al., 1992; Benoit et al., 1997) and against (Smyth et al., 1986; Norton et al., 1992) its reliability. Despite the limitations of this technique, the relative difference of pulse oximetry saturation measurements between bosentan and placebo trials in this study is unlikely to be artifactual but should be confirmed with arterial blood gases. The method for estimating right atrial pressures is new, but unpublished invasive right heart catheterization data from our laboratory indicate this method accurately approximates RA pressures. At present no reliable methods to estimate RA pressure exist. Published methods assessing the inferior vena cava are few with questionable study design requiring extrapolation of results in excessively wide ranges of values (Simonson and Schiller, 1988; Kircher et al., 1990; Capomolla et al., 2000). The trans-tricuspid gradient results, which are unaffected by RA pressure (Fig. 3), likewise indicate no effect from bosentan treatment. It is possible that our finding of no change in exercise capacity is due to the relatively small number of subjects. The crossover study design, however, increased the power to detect an effect if one existed. Given our results, even doubling the number of subjects would not lead to a difference in exercise capacity in the two treatment arms. Although a simulated time trial is a reliable, validated measure of exercise performance (Foster et al., 1993; Jeukendrup et al., 1996) it is possible that other exercise testing protocols may have shown different results. Finally, even though all subjects had at least 2 weeks between trips to altitude, we cannot completely exclude a carry-over effect. 4.6. ETA and ETB receptors and future study Bosentan inhibits both ETA and ETB receptors (Channick et al., 2001). The ETA receptors are located on smooth muscle cells and are responsible, in part, for smooth muscle proliferation and constriction. ETB receptors, however, are located on both smooth muscle cells and on endothelial cells and can cause vasoconstriction or vasodilation respectively depending on their location (Soma et al., 1999). Transgenic rats lacking pulmonary vascular ETB receptors have enhanced pulmonary vasoconstrictor responses to both acute and chronic hypoxia (Ivy et al., 2001, 2002). These transgenic rats had reduced endothelial nitric oxide synthase and decreased

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nitric oxide (NO) production suggesting that the ETB receptors help modulate pulmonary vascular tone during hypoxia by their participation in the regulation of NO production (Ivy et al., 2001, 2002). Despite the preceding, Modesti et al. (2006) found a decrease in PASP using the mixed endothelin antagonist bosentan when the drug was given acutely at altitude. The effects of the ETB receptor are not limited to the lungs (Serneri et al., 1995). The renal ETB receptor modulates both an increase in urinary volume and an inhibition of sodium reabsorption (Ge et al., 2005, 2006). Stimulation of renal ETB receptors causes a diuretic effect via NO synthase 1 (Nakano et al., 2008). Renal ETB receptor inhibition by bosentan leading to fluid retention may well be responsible for the negative findings in the current study. It is possible that selective ETA receptor antagonists would yield very different results from those found with bosentan. 5. Conclusions Bosentan initiated 5 days prior to altitude ascent did not reduce pulmonary artery pressure or improve exercise capacity in healthy subjects. Bosentan administration resulted in lower hemoglobin oxygen saturation during high intensity exercise at altitude, consistent with increased fluid retention and interstitial pulmonary edema formation. Bosentan’s dichotomous effects on pulmonary artery pressure appear to be related to the timing of its administration relative to ascent and the complex interplay between its effects on pulmonary and renal physiology. Additional studies altering the timing of bosentan administration are needed to improve our understanding of bosentan’s effect on pulmonary artery pressure and exercise performance at altitude. Acknowledgments We would like to thank the study subjects for their participation, Barcroft Laboratory, White Mountain Research Station for providing food, lodging and laboratory space, the VA Loma Linda Health Care system for providing transportation to Barcroft Laboratory, and Khaled Bahjri, M.D., M.P.H. for statistical support. We would also like to thank the following for their assistance in conducting the study: Gaja Andzel, M.D., Vince Cacho, David Choe, M.D., Adam Dunn, M.D., Mike Plunkett, and Aaron Wagner. This work was supported by a research grant from Actelion Pharmaceuticals US, Inc. References Allemann, Y., Sartori, C., Lepori, M., Pierre, S., Melot, C., Naeije, R., Scherrer, U., Maggiorini, M., 2000. Echocardiographic and invasive measurements of pulmonary artery pressure correlate closely at high altitude. Am. J. Physiol. Heart Circ. Physiol. 279, H2013–H2016. Anholm, J.D., Milne, E.N.C., Stark, P., Bourne, J.C., Friedman, P., 1999. Radiographic evidence of interstitial pulmonary edema after exercise at altitude. J. Appl. Physiol. 86, 503–509. Bartsch, P., Mairbaurl, H., Maggiorini, M., Swenson, E.R., 2005. Physiological aspects of high-altitude pulmonary edema. J. Appl. Physiol. 98, 1101–1110. Benoit, H., Costes, F., Feasson, L., Lacour, J.R., Roche, F., Denis, C., Geyssant, A., Barthelemy, J.C., 1997. Accuracy of pulse oximetry during intense exercise under severe hypoxic conditions. Eur. J. Appl. Physiol. Occup. Physiol. 76, 260–263. Berger, M.M., Bartsch, P., Luks, A., Bailey, D., Castell, C., Schendler, G., Menold, E., Faoro, V., Mairbaurl, H., Swenson, E., Dehnert, C., 2007. Indirect markers of pulmonary endothelial function correlate with pulmonary artery pressure at high altitude [abstract]. Adv. Exp. Med. Biol. 618, 322–323. Calbet, J.A., Boushel, R., Radegran, G., Sondergaard, H., Wagner, P.D., Saltin, B., 2003. Determinants of maximal oxygen uptake in severe acute hypoxia. Am. J. Physiol. Regul. Integr. Comp. Physiol. 284, R291–R303. Capomolla, S., Febo, O., Caporotondi, A., Guazzotti, G., Gnemmi, M., Rossi, A., Pinna, G., Maestri, R., Cobelli, F., 2000. Non-invasive estimation of right atrial pressure by combined Doppler echocardiographic measurements of the inferior vena cava in patients with congestive heart failure. Ital. Heart J. 1, 684–690.

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