The mechanistic basis for prostacyclin action in pulmonary hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease of the small pulmonary arteries in which patients suffer from elevated pulmonary arterial pressure, right ventricular failure and a reduction in gas exchange. Left untreated the median survival from diagnosis is ~2.8 years, though outcome is significantly worse if patients have underlying pulmonary fibrosis or scleroderma. Injury to the endothelium probably initiates the disease process, with increased production of vasoconstrictors (endothelin and thromboxane) and growth factors accompanying the loss of vasodilator and anti-platelet agents, prostacyclin and nitric oxide, which results in vascular remodelling. To date prostacyclin therapy still remains the most efficacious treatment for PAH, although its short half-life and cumbersome delivery (continuous infusion) meant analogues with improved stability and alternative routes of delivery were developed. Classically, prostacyclin agents are thought to produce haemodynamic and anti-proliferative effects through prostacyclin (IP) receptors coupled to cyclic AMP generation, though other prostanoid receptors may contribute (EP2, EP4) or counterbalance (EP1, EP3) these responses. Increasing evidence suggests peroxisome proliferator-activated receptors (PPARs) are also cellular targets for prostacyclin agonists, regulating cell growth, inflammation and apoptosis through these transcription factors. Activation involves ligand binding and/or membrane receptors but probably not cyclic AMP. Here we discuss recent advances in our understanding of PPARs and how they may represent an important therapeutic target in PA

The mechanistic basis of prostacyclin and its stable analogues in pulmonary arterial hypertension: Role of membrane versus nuclear receptors

Pulmonary arterial hypertension (PAH) is a progressive disease of distal pulmonary arteries in which patients suffer from elevated pulmonary arterial pressure, extensive vascular remodelling and right ventricular failure. To date prostacyclin (PGI2) therapy remains the most efficacious treatment for PAH and is the only approved monotherapy to have a positive impact on long-term survival. A key thing to note is that improvement exceeds that predicted from vasodilator testing strongly suggesting that additional mechanisms contribute to the therapeutic benefit of prostacyclins in PAH. Given these agents have potent antiproliferative, anti-inflammatory and endothelial regenerating properties suggests therapeutic benefit might result from a slowing, stabilization or even some reversal of vascular remodelling in vivo. This review discusses evidence that the pharmacology of each prostacyclin (IP) receptor agonist so far developed is distinct, with non-IP receptor targets clearly contributing to the therapeutic and side effect profile of PGI2 (EP3), iloprost (EP1), treprostinil (EP2, DP1) along with a family of nuclear receptors known as peroxisome proliferator-activated receptors (PPARs), to which PGI2 and some analogues directly bind. These targets are functionally expressed to varying degrees in arteries, veins, platelets, fibroblasts and inflammatory cells and are likely to be involved in the biological actions of prostacylins. Recently, a highly selective IP agonist, selexipag has been developed for PAH. This agent should prove useful in distinguishing IP from other prostanoid receptors or PPAR binding effects in human tissue. It remains to be determined whether selectivity for the IP receptor gives rise to a superior or inferior clinical benefit in PAH

Do anionic phospholipids serve as cofactors or second messengers for the regulation of activity of cloned ATP-sensitive K+ channels?

The regulation of ion channels by anionic phospholipids is currently very topical. An outstanding issue is whether phosphatidylinositol 4,5-diphosphate and related species act as true second messengers in signaling or behave in a manner analogous to an enzymatic cofactor. This question is especially pertinent regarding ATP-sensitive K+ channels in smooth muscle, for which there is substantial literature supporting inhibitory regulation by hormones. In this study, we have examined regulation of the potential cloned equivalents of the smooth muscle ATP-sensitive K+ channel (SUR2B/Kir6.1 and SUR2B/Kir6.2). We find that both can be inhibited via the G(q/11)-coupled muscarinic M3 receptor but that the pathways by which this occurs are different. Our data show that SUR2B/Kir6.1 is inhibited by protein kinase C and binds anionic phospholipids with high affinity, such that potential physiological fluctuations in their levels do not influence channel activity. In contrast, Kir6.2 is not regulated by protein kinase C but binds anionic phospholipids with low affinity. In this case, phosphatidylinositol 4,5-diphosphate and related species have the potential to act as second messengers in signaling. Thus, Kir6.1 and Kir6.2 are regulated by distinct inhibitory mechanisms

Selexipag in the management of pulmonary arterial hypertension: an update

Selexipag is a compound that was designed to overcome the issues associated with oral administration of prostanoid compounds, beraprost and treprostinil in the treatment of pulmonary hypertension (PAH). As a selective IP agonist, it was designed to avoid the off-target prostanoid effects especially in the gastrointestinal system. To place this compound in context, this paper briefly reviews the efficacy, tolerability, and safety of subcutaneous, inhaled, and oral prostanoid preparations and comparesthemto selexipag. Selexipag is the first agent targeting a prostanoid receptor where a reduction in the primary efficacy morbidity/mortality composite end-point has been demonstrated. While safety outcomes favor selexipag over placebo, tolerability issues remain. Efficacy in terms of improvement in effort tolerance, hemodynamic and mortality benefit is less than seen with IV therapy. This is the first prostanoid demonstrated in a clinical trial to have added benefit in those on background double combination therapy and the first non IV prostanoid to demonstrate outcome benefit in the connective tissue disease (CTD) population in a randomized controlled trial

Binding and activity of the prostacyclin receptor (IP) agonists, treprostinil and iloprost, at human prostanoid receptors: Treprostinil is a potent DP1 and EP2 agonist

The prostacyclin analogues, iloprost and treprostinil are extensively used in treating pulmonary hypertension. Their binding profile and corresponding biochemical cellular responses on human prostanoid receptors expressed in cell lines, have now been compared. Iloprost had high binding affinity for EP1 and IP receptors (Ki 1.1 and 3.9 nM, respectively), low affinity for FP, EP3 or EP4 receptors, and very low affinity for EP2, DP1 or TP receptors. By contrast, treprostinil had high affinity for the DP1, EP2 and IP receptors (Ki 4.4, 3.6 and 32 nM, respectively), low affinity for EP1 and EP4 receptors and even lower affinity for EP3, FP and TP receptors. In functional assays, iloprost had similar high activity in elevating cyclic AMP levels in cells expressing the human IP receptor and stimulating calcium influx in cells expressing EP1 receptors (EC50 0.37 and 0.3 nM, respectively) with the rank order of activity on the other receptors comparable to the binding assays. As with binding studies, treprostinil elevated cyclic AMP with a similar high potency in cells expressing DP1, IP and EP2 receptors (EC50 0.6, 1.9 and 6.2 nM, respectively), but had low activity at the other receptors. Activation of IP, DP1 and EP2 receptors, as with treprostinil, can all result in vasodilatation of human pulmonary arteries. However, activation of EP1 receptors can provoke vasoconstriction, and hence may offset the IP-receptor mediated vasodilator effects of iloprost. Treprostinil may therefore differ from iloprost in its overall beneficial pulmonary vasorelaxant profile and other pharmacological actions, especially in diseases where the IP receptor is down-regulated

Synthetic routes to treprostinil N-acyl methylsulfonamide

The synthesis of the prodrug candidate, treprostinil N-acyl methylsulfonamide 5 was accomplished from treprostinil 2 utilising protecting group strategies. A more direct synthesis for the prodrug was also achieved using a treprostinil triol precursor 12 and bromoacetyl acylmethylsulfonamide 14. The overall yield of treprostinil N-acyl sulfonamide 5 directly from the triol precursor 12 is similar to the protecting group strategies because deprotonation of the acidic proton in the bromoacetyl acylmethylsulfonamide 14 reduces electrophilicity. However, the more direct route using the treprostinil triol precursor holds greater promise as a strategy to prepare a wide range of treprostinil prodrug candidates. Treprostinil N-acyl methylsulfonamide prodrug 5 exhibited a 30-fold decrease in the potency at the human prostacyclin (IP) receptor compared to treprostinil 2 in an in vitro cyclic AMP assay

Phosphodiesterase-type 3 inhibitor potentiates cAMP generation and antiproliferative effects of treprostinil in pulmonary arterial smooth muscle cells from patients with pulmonary hypertension

The prostacyclin class of drugs are used in pulmonary arterial hypertension (PAH), a vascular proliferative disease. While evidence suggests agents improve survival, they eventually stop working. Thus ways are being sort to improve their clinical efficacy. We hypothesised that prostacyclin action could be enhanced by inhibition of phosphodiesterase type 3 (PDE3), a major regulator of cyclic AMP levels in the lung, whose activity appears increased in PAH [1]. Cell proliferation (assessed by cell number) and cyclic AMP assays were performed in pulmonary arterial smooth muscle cells derived from patients with idiopathic PAH (n=6). Treprostinil (1μM) significantly (p<0.001) increased cAMP by 7.0±0.6 fold (n=6) at 30 mins but not thereafter. In the presence of the relatively selective PDE3 inhibitor, cilostamide (1μM), treprostinil produced twice as much cAMP (13.1±0.8 compared to 25.0±2.7 pmol/mg protein, n=3), and levels remained significantly (p<0.05) elevated at 24 hr compared to analogue alone (10.8±1.8 versus 3.22±0.8, pmol/mg protein). Cilostamide (1 & 10μM) also suppressed growth induced by 10% FBS by 20%, as did treprostinil (1μM). The combination however, produced further growth suppression of 35% (p<0.05) and 47% (P<0.001) for low and high PDE inhibitor dose, respectively. In conclusion, PDE3 inhibition may help improve prostacyclin analogue action in IPAH by prolonging elevation in cAMP and enhancing the antiproliferative effects of these agents. Reference: 1. Murray F, et al. Am J Physiol 2007; 292: L294-L303. Supported by an unrestricted educational grant from United Therapeutics

Pulmonary vasoconstrictor action of KCNQ potassium channel blockers

KCNQ channels have been widely studied in the nervous system, heart and inner ear, where they have important physiological functions. Recent reports indicate that KCNQ channels may also be expressed in portal vein where they are suggested to influence spontaneous contractile activity. The biophysical properties of K+ currents mediated by KCNQ channels resemble a current underlying the resting K+ conductance and resting potential of pulmonary artery smooth muscle cells. We therefore investigated a possible role of KCNQ channels in regulating the function of pulmonary arteries by determining the ability of the selective KCNQ channel blockers, linopirdine and XE991, to promote pulmonary vasoconstriction. Linopirdine and XE991 both contracted rat and mouse pulmonary arteries but had little effect on mesenteric arteries. In each case the maximum contraction was almost as large as the response to 50 mM K+. Linopirdine had an EC50 of around 1 μM and XE991 was almost 10-fold more potent. Neither removal of the endothelium nor exposure to phentolamine or α,β-methylene ATP, to block α1-adrenoceptors or P2X receptors, respectively, affected the contraction. Contraction was abolished in Ca2+-free solution and in the presence of 1 μM nifedipine or 10 μM levcromakalim