Evaluation of the combination of endothelin receptor antagonists (ERA) and phosphodiesterase-5 inhibitors for the treatment of pulmonary arterial hypertension (PAH) in pathologic human pulmonary arteries in an ex-vivo organ bath model

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Abstract

Purpose

Medical combination therapy of pulmonary arterial hypertension (PAH) may alleviate the drawbacks of monotherapy by avoiding drug tolerance and by increasing effectiveness, as shown by the combination of ambrisentan and tadalafil (AMBITION trial). The present ex-vivo study evaluated the combination of the endothelin receptor antagonists (ERA) macitentan and bosentan with the phosphodiesterase-5 (PDE-5) inhibitor vardenafil in pulmonary arteries from patients suffering from terminal lung disease as a model of PAH.

Methods

Segments of the pulmonary vessels were excised from resected lungs of patients requiring lung transplantation (LTX). Contraction of pulmonary arteries (PA) was elicited by consecutive dose-response curves of endothelin-1 (ET-1) followed by norepinephrine (NE) to allow inhibition by different pathways. Forces were measured isometrically in an organ bath in the presence and absence of ERA and PDE-5 inhibitors and their combination.

Results

PA of 38 patients were examined between October 2016 and November 2019. Bosentan (1E-7 M) and macitentan (1E-8 M, 3E-8 M, 1E-7 M) inhibited ET-1 induced contractions, whereas vardenafil (1E-6 M, 3E-6 M, 1E-5 M) inhibited only the NE induced part of the contractions. Vardenafil enhanced bosentan-induced inhibition of vasoconstriction in a dose-dependent fashion. Combination effects exceeded single bosentan at 3E-6 M and 1E-5 M vardenafil, and they exceeded single vardenafil at the lower vardenafil concentrations. Macitentan showed a more pronounced inhibition than bosentan regardless of the lower concentrations. Accordingly, combination effects with vardenafil resembled those of macitentan alone.

Conclusions

Macitentan and bosentan were potent antagonists of vasoconstriction in PA of LTX patients. The benefit of drug combinations was demonstrated at selected concentrations only owing to a narrow therapeutic range of vardenafil in this ex-vivo model. These results suggest the utility of drug combinations other than the established pair of ambrisentan and tadalafil in PAH treatment but also make a case for a further assessment of vasodilator properties of drugs complementing ERA.

Introduction

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pressure (mean pulmonary artery pressure (mPAP) ≥ 25 mmHg) in the lung arteries [1]. Compensatory right ventricular overload may then result in right heart failure [2]. Proliferation of smooth muscle cells, fibroblasts, and endothelial cells, fibrosis and increased vasoconstriction contribute to the increase in vascular resistance [3,4]. Prognosis is poor in the absence of treatment with a mean 5-year-survival rate of 34%, whereas an early therapy onset reduces right heart failure and improves the prognosis [1,5,6]. PAH therapy may include both immediate effects such as vasodilation and improved oxygen supply, and long-term effects targeted at the suppression of excess cell proliferation. Medical therapy options include different vasodilators (ERA and PDE-5 inhibitors) and calcium-antagonists and additionally supportive and preventive therapy such as anticoagulation, diuretics and oxygen [7]. A specific PAH therapy for WHO functional class II and III includes the endothelin receptor antagonists (ERA) ambrisentan, bosentan and macitentan, furthermore the phosphodiesterase-5 (PDE-5) inhibitors tadalafil and sildenafil and in off-label use vardenafil as well as the soluble guanylyl cyclase stimulator Riociguat [1,[7], [8], [9]].

Bosentan is a non-selective type A and B ERA with a higher affinity to type A receptors, which has been in clinical use since 2002 [7]. Bosentan therapy is effective for PAH treatment and is not associated with an increase of mortality or of adverse events [10]. Vasodilator effects of bosentan on isolated human pulmonary arteries and veins were reported previously [11]. Macitentan is also a dual ERA and has been available since 2014 [12]. This compound has a lower liver toxicity and was shown to reduce morbidity and mortality of PAH patients [13]. Besides sildenafil and tadalafil, the two PDE-5 inhibitors currently approved for PAH treatment, vardenafil is another option of this substance class. Although a clinical trial showed its safety and efficacy in PAH treatment, it is currently approved for the treatment of erectile dysfunction only [14]. Vardenafil's short lifetime dose does not permit a once-per-day regimen in PAH treatment. Nevertheless, the compound is of interest as it shows the most potent vasodilator effect on isolated human pulmonary arteries [15]. Thus this work focuses on Vardenafil although it is currently used as an off label therapy option [8].

Long-term treatment of PAH with approved medications provided as monotherapy is often limited by a lack of drug efficacy in low to medium-risk patients or a lack of effect in high-risk patients [7]. Current guidelines advise the combination of drugs using different mechanisms of action in case of an insufficient therapeutic effect of monotherapy [13,16,17]. The AMBITION trial established clinical benefits of a combination of the ETA-specific ERA ambrisentan and the PDE-5 inhibitor tadalafil [18]. We have demonstrated the superiority of other combinations of ERA and PDE-5 inhibitors using readily available pulmonary vessels of patients without elevated pulmonary blood pressure in an ex-vivo model [11,12,15,19,20]. However, the vascular changes which contribute to the elevated pulmonary pressure in PAH patients may stem from, or result in, alterations of vasoconstrictor and vasodilator pathways. Therefore the purpose of the present study was to confirm these combination vasodilator effects of the ERA macitentan and bosentan as well as of the PDE-5 inhibitor vardenafil in pulmonary vessels of patients who underwent lung transplantation (LTX) owing to end-stage lung disease. In contrast to former studies which were aimed at investigating impacts of medication on patients, our work focuses on effects of vasodilative medication directly on pulmonary arteries. Experiments were intended to investigate, whether PDE-5 inhibition-mediated vasodilation, in addition to antiproliferation, is likely to be salutary in diseased pulmonary arteries. Observation of the substance class focused on the most potent vasodilator of the clinically available PDE-5 inhibitors.

Section snippets

Study design

Study patients were recruited among patients scheduled for LTX at the University Hospital Munich, Campus Groβhadern from October 2016 to November 2019. Patients were eligible if they were at least 18 years of age and able to provide written informed consent. Exclusion criteria included preoperative chemo- or radiotherapy, tumour infiltration and known infectious diseases such as HIV or Hepatitis C. Pulmonary arteries (segment length approx. 3 cm, internal diameter up to 10 mm) were dissected

Study patients

Pulmonary arteries of 51 patients were suitable for organ bath experiments. Of these, 13 patients had to be excluded from analysis owing to inadequate contractile function. The demographic data of the remaining 38 study patients are shown in Table 1. The study group included three patients with proven PAH (mPAP> 25 mmHg). A total of 36 patients had invasive measurements of the mean pulmonary artery pressures with a mPAP of 24.1 ± 10.1 mmHg (range 14–63 mmHg).

Bosentan and vardenafil

Three series of experiments were

Discussion

With PAH being incurable, the main goal of PAH therapy is to reduce the risk status of patients and to demote their WHO functional class, resulting in a better quality of life [7]. Most of the previous studies targeting PAH treatment were clinical-pharmacological studies or animal experiments [4]. The lack of human ex-vivo studies prompted us to examine the effects of drugs already being prescribed in a human ex-vivo model.

Thus, vardenafil was the strongest vasodilator in our previous

Conclusions

The non-selective ETA/ETB receptor antagonists macitentan and bosentan were potent in regulating vasoconstriction in pathologic human pulmonary arteries, which is the key problem of PAH. Showing better effects than single vardenafil administration, both ERA have a major role in PAH treatment. These data confirm our previous results in non-pathologic human pulmonary arteries. Dual ERA and vardenafil administration was superior to either single drug only in one particular combination owing to the

Ethical approval

All procedures performed in studies involving human participants were accordance with the ethical standards of the ethics committees of the University of Regensburg (file no. 11-101-0133) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants prior to study enrollment.

Author credit roles

Englert, Leonie: Writing- Original Draft, Writing- Review and Editing.

Stadlbauer, Christian: Data Curation.

Spaeth, Mirjam: Investigation.

Hofman, Hans-Stefan: Supervision, Project administration, Conceptualization.

Schneider, Christian: Resources.

Hatz, Rudolf: Resources.

Preissler, Gerhard: Resources.

Michel, Sebastian: Resources.

Golovchenko, Svitlana: Supervision, Resources.

Ried, Michael: Supervision, Project administration, Conceptualization.

Hoenicka, Markus: Methodology, Conceptualization,

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

Bosentan and Macitentan were provided by the company “Actelion Pharmaceuticals Deutschland GmbH” (cooperation contract: “MED-2013-249-Hoenicka”), all other substances were bought by our team.

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