Selexipag Active Metabolite ACT-333679 Displays Strong Anticontractile and Antiremodeling Effects but Lowβ-Arrestin Recruitment and Desensitization Potential

Abstract

Prostacyclin (PGI 2 ) receptor (IP receptor) agonists, which are indicated for the treatment of pulmonary arterial hypertension (PAH), increase cytosolic cAMP levels and thereby inhibit pulmonary vasoconstriction, pulmonary arterial smooth muscle cell (PASMC) proliferation, and extracellular matrix synthesis. Selexipag (Uptravi, 2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]-butoxy}-N-(methylsulfonyl)acetamide) is the first nonprostanoid IP receptor agonist, it is available orally and was recently approved for the treatment of PAH. In this study we show that the active metabolite of selexipag and the main contributor to clinical efficacy ACT-333679 (previously known as MRE-269) behaved as a full agonist in multiple PAH-relevant receptor-distal-or downstream-cellular assays with a maximal efficacy (E max ) comparable to that of the prototypic PGI 2 analog iloprost. In PASMC, ACT-333679 potently induced cellular relaxation (EC 50 4.3 nM) and inhibited cell proliferation (IC 50 4.0 nM) as well as extracellular matrix synthesis (IC 50 8.3 nM). In contrast, ACT-333679 displayed partial agonism in receptor-proximal-or upstream-cAMP accumulation assays (E max 56%) when compared with iloprost and the PGI 2 analogs beraprost and treprostinil (E max ∼100%). Partial agonism of ACT-333679 also resulted in limited b-arrestin recruitment (E max 40%) and lack of sustained IP receptor internalization, whereas all tested PGI 2 analogs behaved as full agonists in these desensitization-related assays. In line with these in vitro findings, selexipag, but not treprostinil, displayed sustained efficacy in rat models of pulmonary and systemic hypertension. Thus, the partial agonism of ACT-333679 allows for full efficacy in amplified receptor-distal PAH-relevant readouts while causing limited activity in desensitization-related receptorproximal readouts.

Effects of selexipag and its active metabolite in contrasting the profibrotic myofibroblast activity in cultured scleroderma skin fibroblasts

“…In accordance with these observations, the results of the present in vitro study showed that in cultured SSc skin fibroblasts/myofibroblasts, ACT-333679 induced the same effects as selexipag, but at a concentration three times lower than that of the parent compound (selexipag 3 μM and 0.3 μM, ACT-333679 1 μM and 0.1 μM). The concentrations of selexipag and especially ACT-333679 were in accordance with several in vitro studies [ 15 , 24 – 26 ]. Moreover, the lowest ACT-333679 concentration (0.1 μM) is in range with the values of its bioavailability which were well-tolerated by the HSs and were indicated by the area under the curve during a dose interval of administration (AUC t ), as reported in several studies [ 25 , 26 ].…”

“…The fibroblasts isolated from the biopsy sample from each patient with SSc were independently cultured up to 80% of confluence and then maintained in serum-free medium for 4 h. After starvation, a portion of these cells was maintained in growth medium at 5% of FBS without treatment (untreated cells) and another portion was treated for 48 h with three different concentrations of selexipag (30 μM, 3 μM, or 0.3 μM); a further portion of cells was treated for 48 h with three different concentrations of ACT-333679 (10 μM, 1 μM or 0.1 μM) (Actelion Pharmaceutics, Switzerland), in accordance with recent studies [ 15 , 24 – 26 ].…”

“…Another possible explanation for the absence of a dose-response effect might depend on the expression level and activity of the IP receptor on the cell surface of cultured SSc skin fibroblasts/myofibroblasts. In a recent study it has been observed that several cell types, including pulmonary arterial smooth muscle cells (PASMCs), displayed a moderate degree of constitutive IP receptor internalization and treatment with prostacyclin analogs (such as beraprost, treprostinil) induced strong depletion of this receptor from the cell surface, whereas the non-prostanoid agonists selexipag and ACT-333679 did not induce these effects [ 15 ]. Moreover, ACT-333679 1 μM and 10 μM induced the same increase in the expression of the IP receptor on the cell surface, which was higher than that induced by ACT-333679 0.1 μM [ 15 ].…”

Diverse Pharmacology of Prostacyclin Mimetics: Implications for Pulmonary Hypertension

“…Mechanistic and functional differences between prostanoid and non-prostanoid IP agonists are now beginning to emerge that need to be carefully considered in a clinical context. Both ralinepag and MRE-269 behave as partial agonists in cAMP assays in CHO cells stably expressing the human IP receptor and in human PASMCs from PAH patients [35,37,38]. Partial agonism may in part explain why MRE-269 was about 65-fold less potent than iloprost at inhibiting contractions in human pulmonary arteries [19] and why selexipag has little overall effect on platelet aggregation in vivo [39].…”

“…Because this did not happen, it might suggest either a large IP receptor reserve (redundancy) in the system and/or that IP function recovers sufficiently well in culture before agonist treatment. Other studies have suggested that partial agonism leads to limited β-arrestin recruitment and less IP receptor internalisation, and hence a lower likelihood of receptor desensitisation with MRE-269 compared to other analogues [38]. Against this notion, MRE269 still remains the weakest anti-proliferative agent when compared to other prostacyclin analogues in the same assay [37].…”