Antiproliferative effect of selexipag active metabolite MRE-269 on pulmonary arterial smooth muscle cells from patients with chronic thromboembolic pulmonary hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a group 4 pulmonary hypertension (PH) characterized by nonresolving thromboembolism in the central pulmonary artery and vascular occlusion in the proximal and distal pulmonary artery. Medical therapy is chosen for patients who are ineligible for pulmonary endarterectomy or balloon pulmonary angioplasty or who have symptomatic residual PH after surgery or intervention. Selexipag, an oral prostacyclin receptor agonist and potent vasodilator, was approved for CTEPH in Japan in 2021. To evaluate the pharmacological effect of selexipag on vascular occlusion in CTEPH, we examined how its active metabolite MRE-269 affects platelet-derived growth factor-stimulated pulmonary arterial smooth muscle cells (PASMCs) from CTEPH patients. MRE-269 showed a more potent antiproliferative effect on PASMCs from CTEPH patients than on those from normal subjects. DNA-binding protein inhibitor (ID) genes ID1 and ID3 were found by RNA sequencing and real-time quantitative polymerase chain reaction to be expressed at lower levels in PASMCs from CTEPH patients than in those from normal subjects and were upregulated by MRE-269 treatment. ID1 and ID3 upregulation by MRE-269 was blocked by co-incubation with a prostacyclin receptor antagonist, and ID1 knockdown by small interfering RNA transfection attenuated the antiproliferative effect of MRE-269. ID signaling may be involved in the antiproliferative effect of MRE-269 on PASMCs. This is the first study to demonstrate the pharmacological effects on PASMCs from CTEPH patients of a drug approved for the treatment of CTEPH. Both the vasodilatory and the antiproliferative effect of MRE-269 may contribute to the efficacy of selexipag in CTEPH.

[Pharmacological characteristics and clinical study results of Selexipag (Uptravi® tablets), a selective prostacyclin receptor agonist].

Selexipag (Uptravi® tablets) is a novel prostacyclin receptor (IP receptor) agonist designed and synthesized at Nippon Shinyaku Co., Ltd., and approved for the treatment of pulmonary arterial hypertension (PAH). Selexipag is converted to MRE-269 in vivo, and the plasma concentration of MRE-269 is maintained at a therapeutic level for a long time. MRE-269 has selective IP receptor agonist activity and exerts vasodilatory and anti-proliferative effects on pulmonary arterial smooth muscle cells. In a study to investigate its vasodilatory effect in isolated rat pulmonary arteries, MRE-269 showed potent vasodilatory effects not only in extralobar but also in small intralobar pulmonary arteries. In a Sugen 5416/hypoxia rat model of PAH, selexipag significantly improved pulmonary artery obstruction, decreased right ventricular systolic pressure, decreased right ventricular hypertrophy and improved survival rate. In a phase II clinical trial for treatment with PAH conducted in Europe, selexipag showed good tolerability with promising efficacy. In an open-label phase II study in 37 patients with PAH in Japan, selexipag significantly decreased pulmonary vascular resistance compared with baseline. In the GRIPHON (Prostacyclin (PGI2) Receptor agonist In Pulmonary arterial HypertensiON) study in 1156 patients with PAH, the largest outcome study ever conducted in PAH, the selexipag treatment group showed a significant reduction in the risk of the primary composite endpoint of death or a complication related to PAH compared with placebo. Selexipag has been shown in clinical trials to prevent the progression of PAH, and is expected to contribute to the treatment of patients with PAH.

The selective PGI2 receptor agonist selexipag ameliorates Sugen 5416/hypoxia-induced pulmonary arterial hypertension in rats.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by a progressive increase in pulmonary artery pressure due to an increase in vessel tone and occlusion of vessels. The endogenous vasodilator prostacyclin and its analogs are used as therapeutic agents for PAH. However, their pharmacological effects on occlusive vascular remodeling have not been elucidated yet. Selexipag is a recently approved, orally available and selective prostacyclin receptor agonist with a non-prostanoid structure. In this study, we investigated the pharmacological effects of selexipag on the pathology of chronic severe PAH in Sprague-Dawley and Fischer rat models in which PAH was induced by a combination of injection with the vascular endothelial growth factor receptor antagonist Sugen 5416 and exposure to hypoxia (SuHx). Oral administration of selexipag for three weeks significantly improved right ventricular systolic pressure and right ventricular (RV) hypertrophy in Sprague-Dawley SuHx rats. Selexipag attenuated the proportion of lung vessels with occlusive lesions and the medial wall thickness of lung arteries, corresponding to decreased numbers of Ki-67-positive cells and a reduced expression of collagen type 1 in remodeled vessels. Administration of selexipag to Fischer rats with SuHx-induced PAH reduced RV hypertrophy and mortality caused by RV failure. These effects were probably based on the potent prostacyclin receptor agonistic effect of selexipag on pulmonary vessels. Selexipag has been approved and is used in the clinical treatment of PAH worldwide. It is thought that these beneficial effects of prostacyclin receptor agonists on multiple aspects of PAH pathology contribute to the clinical outcomes in patients with PAH.

A comparison of vasodilation mode among selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide]), its active metabolite MRE-269 and various prostacyclin receptor agonists in rat, porcine and human pulmonary arteries.

Selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N- (methylsulfonyl)acetamide]) is a novel, orally available non-prostanoid prostacyclin receptor (IP receptor) agonist that has recently been approved for the treatment of pulmonary arterial hypertension (PAH). We examined the effect of the active metabolite of selexipag, MRE-269, and IP receptor agonists that are currently available as PAH therapeutic drugs on the relaxation of rat, porcine and human pulmonary artery. cAMP formation in human pulmonary artery smooth muscle cells was induced by all test compounds (MRE-269, epoprostenol, iloprost, treprostinil and beraprost sodium) and suppressed by IP receptor antagonists (CAY10441 and 2-[4-(1H-indol-4-yloxymethyl)-benzyloxycarbonylamino]-3-phenyl-propionic acid). MRE-269 induced endothelium-independent vasodilation of rat extralobar pulmonary artery (EPA). In contrast, endothelial denudation or the addition of a nitric oxide synthase inhibitor markedly attenuated the vasodilation of EPA induced by epoprostenol, treprostinil and beraprost sodium but not iloprost. The vasorelaxant effects of MRE-269 on rat small intralobar pulmonary artery (SIPA) and EPA were the same, while the other IP receptor agonists induced less vasodilation in SIPA than in EPA. Furthermore, a prostaglandin E receptor 3 antagonist enhanced the vasodilation induced by all IP receptor agonists tested except MRE-269. We also investigated the relaxation induced by IP receptor agonists in pulmonary arteries from non-rodent species and found similar vasodilation modes in porcine and human as in rat preparations. These results suggest that MRE-269, in contrast to other IP receptor agonists, works as a selective IP receptor agonist, thus leading to pronounced vasorelaxation of rat, porcine and human pulmonary artery.

Antiproliferative and apoptotic effects of norethisterone on endometriotic stromal cells in vitro.

OBJECTIVES: Low-dose estrogen-progestin (LEP) agents are often used for relieving endometriosis-associated chronic pelvic pain, but a direct effect of LEP on endometriotic lesions remains to be demonstrated. The objective of this study is to investigate the antiproliferative and apoptotic effects of the synthetic progestin norethisterone (NET) against human endometriotic stromal cells (ESCs). STUDY DESIGN: Ovarian endometrioma specimens were obtained at laparoscopy from 19 patients with endometriosis, and ESCs were isolated. The antiproliferative effect of compounds against cultured ESCs was evaluated by measuring the inhibition of [(3)H]thymidine incorporation. The ability of compounds to induce apoptosis in the cultured cells was evaluated by the measurement of caspase 3/7 activity and by nuclear staining. The cytotoxicity of compounds was evaluated by measuring the leakage of lactate dehydrogenase (LDH) into the supernatant of the cell culture. RESULTS: NET and progesterone (P4) at concentrations of greater than 10nM significantly inhibited [(3)H]thymidine incorporation in a dose-dependent manner. Co-treatment with 17ß-estradiol at 0.1 ng/mL did not affect the inhibition of [(3)H]thymidine incorporation by NET. At concentrations greater than 100 nM, NET significantly increased caspase 3/7 activity and the numbers of apoptotic cells, whereas P4 did not. Treatment of ESCs for 24h with NET or P4 at concentrations of up to 1000 nM did not cause LDH leakage. CONCLUSIONS: NET inhibits the proliferation of ESCs and induces their apoptosis. These results suggest a possible direct effect of NET on endometriotic lesions in patients with endometriosis.

A long-acting and highly selective prostacyclin receptor agonist prodrug, 2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide (NS-304), ameliorates rat pulmonary hypertension with unique relaxant responses of its active form, {4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}acetic acid (MRE-269), on rat pulmonary artery.

2-{4-[(5,6-Diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide (NS-304) is an orally available, long-acting nonprostanoid prostacyclin receptor (IP receptor) agonist prodrug. In a rat model of pulmonary hypertension induced by monocrotaline (MCT), NS-304 ameliorated vascular endothelial dysfunction, pulmonary arterial wall hypertrophy, and right ventricular hypertrophy, and it elevated right ventricular systolic pressure and improved survival. {4-[(5,6-Diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}acetic acid (MRE-269), the active form of NS-304, is much more selective for the IP receptor than are the prostacyclin analogs beraprost and iloprost, which also have high affinity for the EP(3) receptor. To investigate the effect of receptor selectivity on vasodilation of the pulmonary artery, we assessed the relaxant response to these IP agonists in rats. MRE-269 induced vasodilation equally in large pulmonary arteries (LPA) and small pulmonary arteries (SPA), whereas beraprost and iloprost induced less vasodilation in SPA than in LPA. An EP(3) agonist, sulprostone, induced SPA and LPA vasoconstriction, and an EP(3) antagonist attenuated the vasoconstriction. Beraprost showed EP(3) agonism and induced LPA and SPA vasoconstriction, whereas the EP(3) antagonist inhibited this vasoconstriction and enhanced beraprost- and iloprost-induced SPA vasodilation. These findings suggest that the EP(3) agonism of beraprost and iloprost interfered with the SPA vasodilation resulting from their IP receptor agonism. Endothelium removal markedly attenuated the vasodilation induced by beraprost, but not that induced by MRE-269 or iloprost. Moreover, the vasodilation induced by beraprost and iloprost, but not that induced by MRE-269, was more strongly attenuated in LPA from MCT-treated rats than from normal rats. NS-304 is a promising alternative medication for pulmonary arterial hypertension with prospects for good patient compliance.

Effect of adrenomedullin on adrenergic vasoconstriction in mesenteric resistance arteries of the rat.

Adrenomedullin (AM) is a hypotensive peptide that belongs to a family of peptides structurally related to calcitonin gene-related peptide (CGRP). The present study examined the effect of AM on adrenergic nerve-mediated vasoconstriction in rat perfused mesenteric vascular beds without endothelium. Perfusion of AM at 0.1 nM but not 10 nM increased vasoconstrictor responses to periarterial nerve stimulation (PNS) (1-4 Hz), while AM at 10 nM significantly attenuated vasoconstriction induced by bolus injection of norepinephrine (NE). In preparations treated with capsaicin (a CGRP depletor), pressor responses to both PNS and NE injection were markedly attenuated by AM. Perfusion of CGRP(8-37) (a CGRP-receptor antagonist) significantly potentiated the PNS- but not the NE-induced vasoconstriction. Combined perfusion of CGRP(8-37) and AM had no effect on the PNS-induced response and antagonized the inhibitory effect of AM on the NE-induced response. AM(2-52) (an AM-receptor antagonist) did not influence the effect of AM. These findings suggest that AM facilitates adrenergic vasoconstriction by inhibiting neurotransmission of CGRP-containing nerves, which counteract adrenergic nerve-mediated vasoconstriction.