br Conclusion This study demonstrates the presence of functi
Conclusion This study demonstrates the presence of functionally viable ET receptors in the surgically extracted tissue from patients with CTEPH. The ET receptor was associated with smooth muscle cells, mainly the contractile phenotype of SMC that surrounds the recanalised G-1 and can be seen within the more organised chronic thrombus. Both the ETA and ETB receptors were found with the ETA receptor showing more expression. This shows a potential role for ET receptors to influence both compartments in CTEPH and generates questions about how ET antagonists might influence the pathological development of the chronic thrombus in CTEPH.
Conflicts of interest statement Funded in part by an unrestricted educational grant to JP-Z from Actelion Pharmaceuticals, Basle, Switzerland. KKS has received educational support from Actelion, Bayer and GlaxoSmithKline to attend conferences. MS, RVMR, REK, GH, KKS, DPJ, MG and APD have no conflicts of interest.
Acknowledgements We acknowledge the support of the referring UK centres for PH; the Pulmonary Hypertension Association-UK, Wellcome Trust award WT107715/Z/15/Z, Programmes in Translational Medicines and Therapeutics (085686) and in Metabolic and Cardiovascular Disease (096822/Z/11/Z), the British Heart Foundation PG/09/050/27734, and the NIHR Cambridge Biomedical Research Centre. We also acknowledge the support of the Cambridge NIHR BRC Cell Phenotyping Hub and the Papworth Hospital Research Tissue Bank. This report presents independent research funded by the NIHR. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.
The endothelin peptides (ETs) consist of three structurally similar subtypes ET-1, ET-2 and ET-3 each consisting of 21 amino acids. They produce their effects by binding to the two endothelin receptor subtypes ET and ET, and have been implicated in the pathophysiology of various human diseases., , , Endothelin receptor antagonists have been evaluated extensively for the treatment of pulmonary arterial hypertension (PAH) and other hypertensive conditions, cancer, and fibrogenic diseases of the kidney., , , , , , The FDA approved agents and the compounds in clinical trials are either ET-selective antagonists with ET/ET >100 or dual ET/ET receptor antagonists. The role of ET receptor has not been fully elucidated and very few ET selective antagonists have been developed for evaluation in clinical trials., The three clinically available endothelin receptor antagonists, bosentan (Tracleer®), ambrisentan (Letairis®) and macitentan (Opsumit®) have been approved for the treatment of PAH (). Sitaxsentan (Thelin®) was also approved for PAH in 2006 but was withdrawn in 2010 due to fatal hepatotoxicity., , , , Many other compounds are under phase II or phase III clinical trials and include clazosentan for aneurysmal subarachnoid hemorrhage, darusentan for coronary artery disease, tezosentan for acute heart failure, and atrasentan for hormone refractory prostate cancer metastatic to bone and for diabetic neuropathy treatment ()., , , More recently, clinical trials have been conducted to study the efficacy of the combination of some endothelin receptor antagonists with other cardiovascular drugs in improving the therapeutic outcome for PAH and cancer patients., Heterocyclic quinolone derivative in the carboxylic acid series with the -propoxy substituent at position 6 has been reported to be a potent and selective ET receptor antagonist (, )., It exhibited an ET and ET receptor IC values of 4 nM and 2870 nM, respectively with ET-selectivity of 717 in receptor membrane preparations using a radioligand assay. It also antagonized [I]ET-1 with an IC value of 0.11nM in monkey renal cells (CCL-81) expressing the ET receptor. Based on the previous structure activity relationship studies reported by Patel et al., target compounds were designed to expand the SAR on the oxygen atom at position 6 of the quinolone core by substitution of alicyclic rings of varying sizes and introduction of a polar 2-aminoethyl group. The carboxylic acid at position 3 of the benzyl substituent was replaced with an isosteric tetrazole ring to obtain a parallel series of eleven compounds. The R substituents in the tetrazole series included those previously explored in the carboxylic acid series as well as the new R substituents being explored in this project for the carboxylic acid series. Endothelin receptor antagonist activities and selectivity against ET and ET receptors were evaluated using a whole cell based GeneBLAzer® FRET assay. This is a functional assay which uses HEK293T cells containing a mammalian-optimized β-lactamase reporter gene combined with a FRET-enabled substrate under the control of the Nuclear Factor of Activated T-cell (NFAT) response element. This assay has been validated for EC concentrations of ET-1., The FRET assay was chosen as an alternative to the radioligand [I] ET-1 binding assay to avoid the generation of radioactive waste and also the need for a secondary functional assay to identify whether the compounds are agonists or antagonists. To our knowledge, this is the first reported use of a FRET assay for the evaluation of endothelin receptor antagonist activity. All 22 compounds belonging to the tetrazole series and carboxylic acid series were evaluated by this assay for direct comparison of their activities and ET/ET selectivity.