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.

FHIT, a Novel Modifier Gene in Pulmonary Arterial Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by progressive narrowing of pulmonary arteries, resulting in right heart failure and death. BMPR2 (bone morphogenetic protein receptor type 2) mutations account for most familial PAH forms whereas reduced BMPR2 is present in many idiopathic PAH forms, suggesting dysfunctional BMPR2 signaling to be a key feature of PAH. Modulating BMPR2 signaling is therapeutically promising, yet how BMPR2 is downregulated in PAH is unclear. OBJECTIVES: We intended to identify and pharmaceutically target BMPR2 modifier genes to improve PAH. METHODS: We combined siRNA high-throughput screening of >20,000 genes with a multicohort analysis of publicly available PAH RNA expression data to identify clinically relevant BMPR2 modifiers. After confirming gene dysregulation in tissue from patients with PAH, we determined the functional roles of BMPR2 modifiers in vitro and tested the repurposed drug enzastaurin for its propensity to improve experimental pulmonary hypertension (PH). MEASUREMENTS AND MAIN RESULTS: We discovered FHIT (fragile histidine triad) as a novel BMPR2 modifier. BMPR2 and FHIT expression were reduced in patients with PAH. FHIT reductions were associated with endothelial and smooth muscle cell dysfunction, rescued by enzastaurin through a dual mechanism: upregulation of FHIT as well as miR17-5 repression. Fhit-/- mice had exaggerated hypoxic PH and failed to recover in normoxia. Enzastaurin reversed PH in the Sugen5416/hypoxia/normoxia rat model, by improving right ventricular systolic pressure, right ventricular hypertrophy, cardiac fibrosis, and vascular remodeling. CONCLUSIONS: This study highlights the importance of the novel BMPR2 modifier FHIT in PH and the clinical value of the repurposed drug enzastaurin as a potential novel therapeutic strategy to improve 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 antitumor effects of azacitidine against the human myelodysplastic syndrome cell line SKM-1.

BACKGROUND: The myelodysplastic syndromes (MDS) are a group of stem cell disorders characterized by dysplasia of one or more hematopoietic cell lineages and a risk of progression to acute myeloid leukemia. The cytidine analog azacitidine (Vidaza), a hypomethylating agent, improves survival in patients with MDS, but its mechanism of action is not well understood. MATERIALS AND METHODS: The effects of azacitidine on the MDS-derived cell line SKM-1 were investigated by DNA methylation assay, cell proliferation assay, and a subcutaneous xenograft mouse model. RESULTS: Azacitidine and decitabine induced hypomethylation of the tumor suppressor gene cyclin-dependent kinase 4 inhibitor B (CDKN2B) in SKM-1 cells, whereas the deoxycytidine analog cytarabine did not. Azacitidine and decitabine also inhibited SKM-1 cell growth in vitro. In the mouse xenograft model, azacitidine significantly suppressed tumor growth. CONCLUSION: Inhibition of DNA methyltransferase by azacitidine contributes to its antiproliferative and antitumor effects against SKM-1 cells and may explain its clinical efficacy in MDS.