Simultaneous LC-MS Analysis of Plasma Concentrations of Sildenafil, Tadalafil, Bosentan, Ambrisentan, and Macitentan in Patients with Pulmonary Arterial Hypertension

ORIGINAL ARTICLES

Department of Pharmacy Practice & Science1, School of Pharmaceutical Sciences, University of Shizuoka, Department of Clinical Pharmacology & Therapeutics2, Hamamatsu University School of Medicine, Japan

Simultaneous LC-MS analysis of plasma concentrations of sildenafil, tadalafil, bosentan, ambrisentan, and macitentan in patients with pulmonary arterial hypertension

S. TANAKA1, S. UCHIDA1,*, A. HAKAMATA2, S. MIYAKAWA2, K. ODAGIRI2, N. INUI2, H. WATANABE2, N. NAMIKI1

Received February 13, 2020, accepted March 27, 2020 *Corresponding author: Shinya Uchida, Department of Pharmacy Practice & Science, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan [email protected] Pharmazie 75: 236-239 (2020) doi: 10.1691/ph.2020.0021

Phosphodiesterase-5 (PDE-5) inhibitors and endothelin receptor antagonists (ERAs) are standard therapies for pulmonary arterial hypertension (PAH). The inter-individual variability of these pharmacokinetics is reported remarkably large, and therapeutic drug monitoring (TDM) can be useful to improve the likelihood of the desired therapeutic and safety outcomes. This study aimed to develop a LC-MS method to determine the concentrations of five PAH drugs (PDE-5 inhibitors: sildenafil and tadalafil, ERAs: bosentan, macitentan, and ambrisentan) from plasma samples using a simple process followed by a single mass spectrometric run, and to validate this approach through pharmacokinetic analyses in patients. A solid extraction method was used for sample preparation of the drugs from human plasma. The total run time for a single injection was within 10 min. The calibration curves for all drugs were linear, and the lower limits of quantitation were 1 (sildenafil), 2 (tadalafil), 5 (ambrisentan), and 10 ng/mL (bosentan, macitentan). The accuracy and precision values suggested that the assay had high accuracy and reliability. To prove the utility of this method, the plasma concentrations of the five PAH drugs were determined after their oral administration to nine PAH patients.

1. Introduction fatigue, weakness, angina and syncope. The United States National Pulmonary arterial hypertension (PAH) is a proliferative vasculop- Prospective Registry for Primary Pulmonary Hypertension in 1991 athy of the small pulmonary muscular arterioles. It is character- reported the median survival of patients with idiopathic PAH to be ized by vasoconstriction, hyperplasia, hypertrophy, fibrosis, and only 2.8 years without treatment (D’Alonzo et al. 1991). thrombosis that involves layers of the vascular wall (Galiè et al. Currently, various drugs that focus on symptom relief and 2016; Runo and Loyd 2003). The symptoms of PAH are related improving prognosis including prostanoids, phosphodies- to progressive right ventricular dysfunction. Initial symptoms are terase-5 (PDE-5) inhibitors, and endothelin receptor antagonists typically induced by exertion. They include shortness of breath, (ERAs) are available as treatment and PAH-related hospitaliza- tions and death may be decreasing (Galiè et al. 2005; Sastry et al. 2004; Galiè et al. 2009; Giaid et al. 1993; Stewart et al. 1991; Channick et al. 2001; Galiè et al. 2008). According to the European Society of Cardiology and the European Respiratory Society guidelines for the diagnosis and treatment of pulmonary hypertension, a combination therapy of PDE-5 inhibitors including sildenafil and tadalafil, and ERAs such as bosentan, ambrisentan, and macitentan (Fig. 1) is the standard therapy for advanced PAH (Galiè et al. 2016). In 2014, it was reported that initial combination therapy with ambrisentan and tadalafil resulted in a significantly lower risk of clinical failure than ambrisentan or tadalafil monotherapy among PAH patients who have not received previous treatment (Galiè et al. 2015); the percentage of death in the combination therapy group during the 192-week follow-up period was only 4%. Therapeutic drug monitoring (TDM) is a strategy used to guide dosing of therapeutic agents using measured drug concentrations to improve the likelihood of the desired therapeutic and safety outcomes. It is also performed to detect and monitor important drug interactions that can alter drug pharmacokinetics in clinical settings. The inter-individual variability of PDE-5 inhibitor and ERA pharmacokinetics is remarkably large even in monothera- pies, and is further amplified in combination therapies due to drug interactions (Markert et al. 2014; Venitzet al. 2012). Thus, TDM of Fig. 1: Structures of (a) sildenafil, (b) tadalafil, (c) bosentan, (d) ambrisentan, and PDE-5 inhibitors and ERAs can be useful in predicting therapeutic (e) macitentan. 236 Pharmazie 75 (2020) ORIGINAL ARTICLES outcome as well as in preventing potentially adverse drug reactions min after optimizing chromatographic conditions (Fig. 2). The and interactions. However, the effective concentrations of these total run time for a sample was 10 min, suggesting that our method PDE-5 inhibitors and ERAs in the plasma are still indeterminate, is rapid and acceptable in clinical settings. and their therapeutic ranges have not been established. Thus, it is important to develop a convenient quantification method for TDM Table 1: Calibration curves, linearity, and sensitivity of the assay on of these drugs in plasma to assess both the effectiveness of combi- human plasma nation therapy and the interactions among these drugs. Retention time Calibration ange Correlation coefficient LLOQ Although some methods to determine the plasma concentrations of Drugs PAH drugs have been reported, these approaches cannot simultane- (min) (ng/mL) (R2) (ng/mL) ously analyze PAH drugs used clinically (Al-Ghazawi et al. 2007; Sildenafil 3.5 1–1000 0.997 ± 0.002 1 Ma et al. 2013; Parekh et al. 2012; Nirogi et al. 2012). Enderle et Tadalafil 2.4 2–2000 0.993 ± 0.006 2 Bosentan 2.1 10–10000 0.996 ± 0.003 10 al (2017) have developed a method involving the quantification of Ambrisentan 1.2 5–1000 0.986 ± 0.009 5 five PAH drugs (PDE-5 inhibitors: sildenafil and tadalafil, ERAs: Macitentan 8.4 10–10000 0.995 ± 0.004 10 bosentan, macitentan, and ambrisentan) and their active metabo- Calibration curves were generated using six different concentrations of each drug within the cal- lites in human plasma using liquid chromatography/tandem mass ibration range, and the data were analyzed by weighted linear regression (weighting factor, 1/x2). spectrometry (LC-MS/MS). The lower limit of quantitation (LLOQ) was defined as the lowest concentration within the linear This study aimed to develop and validate a liquid chromatog- range of the calibration curves that gave an acceptable accuracy between 80% and 120%, and a precision of < 20%. raphy-mass spectrometry (LC-MS) method that is more easily learned and retained than LC-MS/MS in the clinical settings to determine the concentrations of five PAH drugs (sildenafil, tada- Table 2: Precision, accuracy, recovery, and stability in the quality lafil, bosentan, macitentan, and ambrisentan) in human plasma control samples of plasma using a simple process followed by a single mass spectrometric Nominal concentration Accuracy Precision Recovery Stability Drugs run. In addition, we applied this method for determining the (ng/mL) (%) (%) (%) (%) plasma concentrations in patient treated with PAH drugs. Sildenafil 1 99.2 0.8 102 -

5 102 4.8 - 100

2. Investigations, results and discussion 10 105 3.7 - -

2.1. Validation of the assay 100 98.5 4.1 94.1 -

The LC-MS method for TDM of 5 PAH drugs, i.e. sildenafil, 500 96.6 4.4 - 103 tadalafil, bosentan, ambrisentan, and macitentan were developed and validated in this study. Our primary purpose was to develop a 1000 95.1 4.8 - - general assay that could simultaneously detect all five PAH drugs after simplified solid phase extraction procedure from plasma. Tadalafil 2 102 1.4 86.8 - Good sample preparation is essential for ensuring accuracy and reliability in LC-MS analysis. The six peaks of drugs were well 10 88.4 6.1 - 93.6 separated, and their retention times ranged between 1.2 and 8.4 20 99.1 7.9 - - 200 109 2.9 83.4 -

1000 105 4.2 - 103

2000 96.2 2.9 - -

Bosentan 10 99.4 1.1 100 -

50 99.8 4.9 - 100

100 105 5.0 - -

1000 99.7 4.2 102 -

5000 96.1 4.7 - 101

10000 95.6 5.2 - -

Ambrisentan 5 98.6 1.3 80.0 -

10 99.4 3.3 103

50 111 7.1 - -

100 108 5.2 72.3 -

500 97.5 3.7 - 97.7

1000 85.2 7.7 - -

Macitentan 10 97.5 1.1 104 -

50 92.4 6.7 - 100

100 94.0 5.1 - -

1000 103 4.6 103 -

5000 106 4.9 - 95.4

10000 98.5 5.2 - -

The interday accuracy and precision were assessed for each QC sample from five different determinations on three different days. The recovery was assessed for QC samples at two different concentrations (low and medium). The stability for a duration of 1 week at 4 °C was analyzed at two Fig. 2: LC-MS chromatograms of PAH drugs in human plasma. concentrations (low and high). Pharmazie 75 (2020) 237 ORIGINAL ARTICLES

Table 3: Precision of recovery and matrix effect in the quality control We aimed to develop and validate LC-MS method that is more easily samples of plasma learned and retained than LC-MS/MS in the clinical settings to

Drugs Nominal concentration Precision of recovery Matrix effect (%) determine the concentrations of five PAH drugs (sildenafil, tadalafil, (ng/ml) (%) bosentan, macitentan, and ambrisentan) in human plasma using a Mean Precision simple process followed by a single mass spectrometric run. Further- Sildenafil 50 1.60 105 2.80 more, the range of the calibration curves was in agreement with Tadalafil 100 11.9 92.2 3.50 previous reports. Bosentan 500 9.56 94.4 6.21 In conclusion, we developed and validated a LC-MS method of 5 PAH drugs (sildenafil, tadalafil, bosentan, ambrisentan, and Ambrisentan 50 5.44 118 2.21 macitentan), and applied this method for TDM in PAH patients. Macitentan 500 8.44 97.7 4.28 This approach is useful and applicable for TDM in laboratory and

The Precision of recovery and matrix effect were assessed for QC samples at medium concentration clinical settings to assess PAH treatment in patients. of calibration curve (n=3). 2.2. Clinical applications The calibration curves for the drugs in the plasma were linear in To prove the utility of this method, the plasma concentrations of the following concentration ranges: 1–1000 ng/mL for sildenafil, five PAH drugs were determined after oral administration to nine 2–2000 ng/mL for tadalafil, 5–1000 ng/mL for ambrisentan, and patients with PAH. The characteristics of the patients and the plasma 10–10000 ng/mL for bosentan and macitentan. The correlation concentrations of the drugs are shown in Table 4. All concentrations coefficient (R2) for each curve was higher than 0.986 (Table 1). were above the LLOQ for each drug and were comparable to previ- The accuracy and precision values ranged from 85.2% to 111% ously reported values (Yokoyama et al. 2014; Enderle et al. 2015). and from 0.8% to 7.9%, respectively (Table 2). The guidance from As described in Table 4, patient #8-9 was not trough level (3 or U.S. Food and Drug Administration proposed that the accuracy of 5 h after dosing) while patients #1-7 was trough (12 or 24 h after ±15% and precision (CV values) within ≤ 15% were acceptable, dosing). Thus, this result showed that our method was applicable while an accuracy of ±20% and a precision of ≤ 20% at the LLOQ to a wide range of plasma concentrations of PAH drugs from peak were also acceptable. Our assay satisfied these criteria, suggesting levels to trough levels. Our method enables TDM in patients treated of high accuracy and reliability. with those PAH drugs (Pulido et al. 2013; Galiè et al. 2013). The recovery from plasma was adequate (greater than 83.4%), except for ambrisentan (80% and 72.3% for low and middle concentration, respectively). The precision of recovery ranged 3. Experimental from 1.6% to 11.9% (Table 3). The 1-week storage stability at 3.1. Drugs 4 °C of the plasma samples using two concentrations (the low and Sildenafil, tadalafil, bosentan, ambrisentan, macitentan, and homo-sildenafil (internal the high QC concentrations) of the drugs were examined, and the standard, IS) were purchased from Toronto Research Chemicals (Toronto, Canada). analyses were considered stable in the plasma when the initial High-performance liquid chromatography (HPLC) grade methanol, acetonitrile, and formic acid were the products of Sigma-Aldrich (St. Louis, MO, USA). Ammonium concentrations were in the range of 93% to 103% (Table 2). There- acetate solution was obtained from Wako Pure Chemical Industries (Tokyo, Japan). fore, the plasma sample is stable at normal storage and shipping Milli-Q water, reagent-grade deionized water, and the Milli-Q water system were conditions in hospitals and laboratories. acquired from Millipore (Billerica, MA, USA). The matrix effect was expressed as the ratio of the mean peak area of an analyte spiked after extraction to the mean peak area 3.2. Standard solutions and sample preparation of the same analyte standard multiplied by 100. The matrix Standard solutions of sildenafil (100 μg/mL), tadalafil (200 μg/mL), bosentan (1 mg/ effect ranged from 92.2% to 118% for plasma (Table 3). Thus, mL), ambrisentan (100 μg/mL) and macitentan (1 mg/mL) were prepared in methanol in no significant matrix interference was observed, suggesting that polypropylene tubes. An Oasis® HLB 96-well μElution Plate (Waters Co., Milford, MA, this protocol provides a fast and feasible method for excluding USA) was used for the extraction of drugs (sildenafil, tadalafil, bosentan, ambrisentan, and interfering phospholipid-based matrix effects in mass spec- macitentan) from human plasma. The plate was conditioned with 200 μL of methanol and equilibration with 200 μL of distilled water. The plasma samples (300 μL) were spiked trometry. with an internal standard (100 μL of 100 ng/mL homosildenafil in cold acetonitrile), and A previous study has reported the simultaneous determination of mixed with 1000 μL of 0.1% formic acid. After centrifugation at 1000 g for 10 min, the four PAH drugs (sildenafil, tadalafil, bosentan, and ambrisentan) supernatant fluid was applied to the plate. After rinsing with 200 μL distilled water solu- in patients using LC-MS/MS (Yokoyama et al. 2014). Enderle tion containing 5% methanol, the analytes were eluted with 100 μL of acetonitrile. The eluate (10 μL) was injected into the chromatographic system for analysis. et al. have developed a method involving the quantification of five PAH drugs (PDE-5 inhibitors: sildenafil and tadalafil, ERAs: bosentan, macitentan, and ambrisentan) and their active metab- 3.3. Chromatographic conditions and mass spectrometry olites in human plasma using LC-MS/MS (Enderle et al. 2017, Plasma concentration of 5 PAH drugs was measured by liquid chromatography 2015). (Alliance 2695 system, Waters Co.) with mass spectrometer (Micromass ZQ Mass

Table 4: Characteristics of patients with PAH and the plasma concentrations of PAH drugs

Patient Gender Age (years) Classification Time after dosing Concentration in plasma (ng/mL) No. (hr) Sildenafil Tadalafil Bosentan Ambrisentan Macitentan (20 mg; TID) (20 mg; SID) (62.5 mg; BID) (5 mg; SID) (10 mg; SID) 1 Male 47 IPAH 12 1.52 – 73.5 – – 2 Female 68 APAH 12 4.74 – 103 – – 3 Female 51 IPAH 12 1.22 – 94.4 – – 4 Female 38 APAH 12 3.31 – 155 – – 5 Female 45 IPAH 12 (sildenafil)/ 12.6 – – 287 – 24(ambrisentan) 6 Female 69 IPAH 24 – 183 – 81.3 – 7 Female 40 APAH 24 – 173 – 62.5 – 8 Male 35 APAH 5 – 472 – – 183 9 Female 33 APAH 3 155 – – – 89.3

IPAH, idiopathic pulmonary arterial hypertension. APAH, associated pulmonary arterial hypertension. SID, once a day. BID, two times a day. TID, three times a day.

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Table 5: Analytical parameters for LC-MS Channick RN, Simonneau G, Sitbon O, Robbins IM, Frost A, Tapson VF, Badesch DB, Roux S, Rainisio M, Bodin F, Rubin LJ (2001) Effects of the dual endo- Drugs Polarity Target ion (m/z) Collision Energy (eV) thelinreceptor antagonist bosentan in patients with pulmonary hypertension: a randomised placebo-controlled study. Lancet. 358: 1119–1123. Sildenafil + 475.2 60 D’Alonzo GE, Barst RJ, Ayres SM, Bergofsky EH, Brundage BH, Detre KM, Fishman AP, Goldring RM, Groves BM, Kernis JT. Levy PS, Pietra GG, Reid LM, Reeves Tadalafil + 390 30 JT, Rich S, Vreim CE, Williams GW, Wu M (1991) Survival in patients with primary pulmonary hypertension. Results from a national prospective registry. Ann Bosentan + 552.2 40 Intern Med. 115: 343–349. Enderle Y, Meid AD, Friedrich J, Grünig E, Wilkens H, Haefeli WE, Burhenne J Ambrisentan + 379.4 10 (2015) Dried blood spot technique for the monitoring of ambrisentan, bosentan, sildenafil, and tadalafil in patients with pulmonary arterial Hypertension. Anal Macitentan + 589 30 Chem 87: 12112–12120. Enderle Y, Witt L, Wilkens H, Grünig E, Haefeli WE, Burhenne J (2017) Simulta- Homosildenafil (IS) + 489.6 60 neous quantification of endothelin receptor antagonists and phosphodiesterase 5 inhibitors currently used in pulmonary arterial hypertension. J Pharm Biomed Anal 143: 291–298. Galiè N, Ghofrani HA, Torbicki A, Barst RJ, Rubin LJ, Badesch D, Fleming T, Spectrometer; Waters Co.). The MS parameters were as follows: capillary voltage, Parpia T, Burgess G, Branzi A, Grimminger F, Kurzyna M, Simonneau G (2005) 4000 V; nebulizer, 40 psi; dry gas, 10 L/min; dry temperature, 350 °C. The analysis Sildenafil citrate therapy for pulmonary arterial hypertension. N Engl J Med. 353: was performed on Symmetry C18 column (150 mm × 2.1 mm i.d., 5 μm) (Waters 2148–2157. Co.) maintained at 40 °C. The mobile phase consisted of 5 mM ammonium acetate/ Galiè N, Olschewski H, Oudiz RJ, Torres F, Frost A, Ghofrani HA, Badesch DB, acetonitrile (50:50) solution, and the flow rate was 0.3 mL/min. Samples of 10 μL McGoon MD, McLaughlin VV, Roecker EB, Gerber MJ, Dufton C, Wiens BL, were injected, and total run time was 10 min. Rubin LJ (2008) Ambrisentan for the treatment of pulmonary arterial hyperten- PAH drugs were characterized with an electrospray ionization interface operating in sion: results of the ambrisentan in pulmonary arterial hypertension, randomized, the positive ion mode, and data acquisition and quantitation were carried out using the double-blind, placebo-controlled, multicenter, efficacy (ARIES) study 1 and 2. MassLynx software (version 4.1). Table 5 shows the operating parameters of LC-MS Circulation 117: 3010–3019. and mass transitions. Galiè N, Brundage BH, Ghofrani HA, Oudiz RJ, Simonneau G, Safdar Z, Shapiro S, White RJ, Chan M, Beardsworth A, Frumkin L, Barst RJ (2009) Tadalafil therapy for pulmonary arterial hypertension. Circulation 119: 2894–2903. 3.4. Validation of the assay Galiè N, Simonneau G (2013) [The Fifth World Symposium on Pulmonary Hyperten- sion], J Am Coll Cardiol 62: D1–D3. The linearity, precision, and accuracy were evaluated to validate the assay for plasma Galiè N, Barberà JA, Frost AE, Ghofrani HA, Hoeper MM, McLaughlin VV, Peacock samples. Six different concentrations of each drug (Table 1) were used for calibration 2 AJ, Simonneau G, Vachiery JL, Grünig E, Oudiz RJ, Vonk-Noordegraaf A, White curves weighted linear regression (weighting factor, 1/x ). Concentrations of quality RJ, Blair C, Gillies H, Miller KL, Harris JH, Langley J, Rubin LJ (2015) Initial use control (QC) samples in plasma were selected as following; sildenafil: 1, 3, 500, and of ambrisentan plus tadalafil in pulmonary arterial hypertension. N Engl J Med. 1000 ng/mL; tadalafil: 2, 6, 1000, and 2000 ng/mL; bosentan and macitentan: 10, 373: 834–844. 30, 5000, and 10000 ng/mL; and ambrisentan: 5, 15, 500, and 1000 ng/mL. Five Galiè N, Humbert M, Vachiery JL, Gibbs S, Lang I, Torbicki A, Simonneau G, different concentrations of QC samples were used for assessing the interday accuracy Peacock A, Vonk Noordegraaf A, Beghetti M, Ghofrani A, Gomez Sanchez MA, (the percentage of spiking concentration) and precision (the coefficient of variation, Hansmann G, Klepetko W, Lancellotti P, Matucci M, McDonagh T, Pierard LA, CV) on three different days. Trindade PT, Zompatori M, Hoeper M (2016) 2015 ESC/ERS Guidelines for the The lower limit of quantitation (LLOQ) was considered as the lowest concentration diagnosis and treatment of pulmonary hypertension. Eur Heart J 37: 67–119. within the linear range of the calibration curves. Recovery at was assessed at low and Giaid A, Yanagisawa M, Langleben D, Michel RP, Levy R, Shennib H, Kimura S, medium concentrations of the calibration curves were evaluated with the peak areas of Masaki T, Duguid WP, Stewart DJ (1993) Expression of endothelin-1 in the lungs extracted QC samples to corresponding spiked samples. The stability of the five PAH of patients with pulmonary hypertension. N Engl J Med 328: 1732–1739. drugs in human plasma for a duration of 1 week was analyzed at the low and the high Ma B, Shang X, Zhang Q, Li J, Liu Y, Cao X, Xu Q (2013) Rapid analysis of tada- QC drug concentrations at 4 °C. lafil in human blood plasma and seminal plasma by liquid chromatography/tandem mass spectrometry. J Pharm Biomed Anal 77: 149–157. Markert C, Burhenne J, Weiss J, Mikus G, Haefeli WE (2014) CYP2C9 polymor- 3.5. Assessment of precision of recovery and the matrix effect phism is not a major determinant of bosentan exposure in healthy volunteers. Clin The precision of recovery and the matrix effect were assessed for QC samples of Pharmacol Ther 95: 250−251. plasma and urine at middle concentrations for three determinations (Table 4). The Nirogi R, Kandikere V, Komarneni P, Aleti R, Padala N, Kalaikadhiban I (2012) LC– matrix effect was expressed as the ratio of the mean peak area of an analyte spiked ESI-MS/MS method for quantification of ambrisentan in plasma and application to after extraction to the mean peak area of the same analyte standard multiplied by 100. rat pharmacokinetic study. Biomed Chromatogr 26: 1150–1156. Parekh JM, Shah DK, Sanyal M, Yadav M, Shrivastav PS (2012) Development of an SPE-LC–MS/MS method for simultaneous quantification of bosentan and its 3.6. Application of the assay active metabolite hydroxybosentan in human plasma to support a bioequivalence study. J Pharm Biomed Anal 70: 462–470. The clinical applicability of the present method was evaluated by analyzing drug Pulido T, Adzerikho I, Channick RN, Delcroix M, Galiè N, Ghofrani HA, Jansa P, plasma concentrations in nine patients whose ages ranged between 33 and 68 years. Jing ZC, Le Brun FO, Mehta S, Mittelholzer CM, Perchenet L, Sastry BK, Sitbon All the subjects provided written informed consent before the start of the study. The O, Souza R, Torbicki A, Zeng X, Rubin LJ, Simonneau G (2013) SERAPHIN study protocol was approved by the Ethics Committee of the Hamamatsu University Investigators. Macitentan and morbidity and mortality in pulmonary arterial hyper- School of Medicine. The study was registered at the UMIN Clinical Trials Registry tension. N Engl J Med 369: 809–818. (UMIN000002566, UMIN000005464, UMIN000022593). Patients received stable Runo JR, Loyd JE (2003) Primary pulmonary hypertension. Lancet 361: 1533–1544. daily doses of sildenafil, tadalafil, bosentan, ambrisentan, or macitentan. Blood Sastry BK, Narasimhan C, Reddy NK, Raju BS (2004) Clinical efficacy of sildenafil samples (7 mL) were collected after dosing. The EDTA-anticoagulated plasma of in primary pulmonary hypertension: a randomized, placebo-controlled, double- patients were stored at −80 °C until analysis. blind, crossover study. J Am Coll Cardiol 43: 1149–1153. Stewart DJ, Levy RD, Cernacek P, Langleben D (1991) Increased plasma endo- Acknowledgements: The authors are grateful to Ms. Miki Ozawa and Ms. Miki Shino- thelin-1 in pulmonary hypertension: marker or mediator of disease?. Ann Intern hara for their excellent technical assistance. Med 114: 464–469. Venitz J, Zack J, Gillies H, Allard M, Regnault J, Dufton C (2012) Clinical pharma- Conflict of interest: None declared. cokinetics and drug-drug interactions of endothelin receptor antagonists in pulmonary arterial hypertension. J Clin Pharmacol 52: 1784−1805. 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