Biochemical Pharmacology 84 (2012) 68–75 Contents lists available at SciVerse ScienceDirect Biochemical Pharmacology jo urnal homepage: www.elsevier.com/locate/biochempharm Binding and activity of the prostacyclin receptor (IP) agonists, treprostinil and iloprost, at human prostanoid receptors: Treprostinil is a potent DP1 and EP2 agonist a b b c, Brendan J. Whittle , Adam M. Silverstein , David M. Mottola , Lucie H. Clapp * a William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK b United Therapeutics Corporation, 55 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA c Centre for Clinical Pharmacology, Division of Medicine, University College London, Rayne Building, London WC1E 6JF, UK A R T I C L E I N F O A B S T R A C T Article history: The prostacyclin analogues, iloprost and treprostinil are extensively used in treating pulmonary Received 24 January 2012 hypertension. Their binding profile and corresponding biochemical cellular responses on human prostanoid Accepted 19 March 2012 receptors expressed in cell lines, have now been compared. Iloprost had high binding affinity for EP1 and IP Available online 27 March 2012 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 Keywords: and 32 nM, respectively), low affinity for EP1 and EP4 receptors and even lower affinity for EP3, FP and TP Prostacyclin analogues receptors. In functional assays, iloprost had similar high activity in elevating cyclic AMP levels in cells Radioligand binding expressing the human IP receptor and stimulating calcium influx in cells expressing EP1 receptors (EC50 0.37 Cyclic AMP and 0.3 nM, respectively) with the rank order of activity on the other receptors comparable to the binding Calcium influx Prostanoid receptors 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. ß 2012 Elsevier Inc. Open access under CC BY-NC-ND license. 1. Introduction pulmonary hypertension, peripheral vascular disease as well as Raynaud’s phenomenon and digital ulcers associated with scleroder- The endogenous prostanoid, prostacyclin, is of substantial ma [7–13]. In particular, iloprost and treprostinil are currently used therapeutic benefit in the treatment of the highly debilitating extensively in Europe and the US for the treatment of pulmonary disease, pulmonary hypertension [1–4]. Prostacyclin itself is however arterial hypertension [14–18]. chemically unstable at physiological temperatures and pH, and As with most other mediators, prostaglandins such as prostacy- rapidly decomposes to a relatively inactive breakdown product as clin elicit their molecular, pharmacological and biochemical effects reviewed by Whittle and colleagues [5,6]. Therefore, the early clinical through binding and activation of specific receptor sites [19]. It was use of prostacyclin, as the chemically synthesised material epopros- initially established by pharmacological techniques that there was a tenol, necessitated the use of a high pH formulation and ice packs for range of specific receptors for the naturally occurring prostanoids its prolonged intravenous use. The development of chemically stable (see [20]) and these receptors have been subsequently cloned and prostacyclin analogues such as iloprost, treprostinil and beraprost expressed [19,21]. The original classification of the different obviated the requirement for such a formulation [6]. These agents prostanoid receptors [20,22,23] has remained essentially intact have been used clinically for different indications, including since the early proposals [24]. Thus, the receptors are identified as the IP, EP1, EP2, EP3, EP4, DP (now DP1, see below), FP and TP receptor [23–25]. The IP, EP2, EP4 and DP1 receptors are classically known to * Corresponding author at: Centre for Clinical Pharmacology, Division of be Gs-coupled receptors linked to cyclic AMP (cAMP) generation, Medicine, Floor 3, Rayne Building, University College, 5 University Street, London while EP1, FP and TP receptors couple to calcium mobilisation WC1E 6JF, UK. Tel.: +44 020 7679 6180; fax: +44 20 7679 6212. pathways through Gq, Gi and as yet unidentified G proteins [19,25]. E-mail addresses: [email protected] (B.J. Whittle), [email protected] There are several splice variants of EP3 which can couple negatively com (A.M. Silverstein), [email protected] (D.M. Mottola), [email protected] (L.H. Clapp). or positively to Gi or Gs, respectively [19]. 0006-2952 ß 2012 Elsevier Inc.Open access under CC BY-NC-ND license. http://dx.doi.org/10.1016/j.bcp.2012.03.012 B.J. Whittle et al. / Biochemical Pharmacology 84 (2012) 68–75 69 The natural ligand for the IP receptor is prostacyclin (PGI2), with previously described [21,34,35]. Briefly, cells from each cell line prostaglandin E2 (PGE2) for the EP receptors, PGF2a for the FP stably expressing the recombinant human prostanoid receptor receptors and thromboxane A2 for the TP receptor [24]. A recent were spun down at 4 8C and the cell pellet suspended in a 50 mM pharmacological study has suggested evidence for a second IP Tris/HCl (pH 7.4) buffer containing 5 mM EDTA, 20 mm NaCl, 5 mM receptor on human airway epithelial cells that mediates the KCl, 5 mM MgCl2, 1.5 mM CaCl2, 10 mg/ml trypsin inhibitor, 1 mg/ inhibition of cytokine release [26]. This is not thought to be a splice ml leupeptin and 75 mg/ml phenylmethylsulphonyl fluoride. variant although its occurrence elsewhere has not been described. Cell lysis was performed by ultra sonication (3 min at 4 8C) The original classification of the DP receptor with prostaglandin D2 using a Vibro cell 72405, followed by centrifugation (Beckman (PGD2) as the natural ligand has now been designated as DP1 [24]. Avanti J30I) of the resulting homogenate at 4 8C (50,000 Â g for This takes into account the more recently identified DP2 receptor or 15 min). The membrane pellet was resuspended in fresh Tris buffer CRTh2 receptor, that while recognising PGD2, is more closely containing 10% glycerol and stored as aliquots at À70 8C until used associated with chemo-attractant molecules and has no significant in the binding studies. Proteins levels were determined using the homology with the other prostanoid receptors [24]. Bradford method and the optimised quantity of protein used in the Despite their extensive clinical use over the past decade, there is binding studies was 16 mg for the TP receptor, 20 mg for the EP2, relatively little direct comparative pharmacology of iloprost and EP3, EP4 and FP receptors, 40 mg for the IP receptor and 60 mg per treprostinil in experimental systems and models. It is generally sample for the EP1 and DP1 receptors. Incubations were carried out 3 assumed that both are potent agonists at the prostacyclin IP receptor using nanomolar concentrations of the appropriate [ H] radioli- and that such agonist activity predominantly underlies their gand (Table 1) in the absence or presence of various concentrations respective responses, including their potent vasodilator effects in of the prostacyclin analogue (final solvent concentration was kept the pulmonary vasculature, at least under physiological conditions constant). Total binding was determined in the presence of vehicle. [27–29]. Indeed, based on this premise, novel agents that are highly Non-specific binding was determined in the presence of 650– selective agonists at the IP receptor such as the non-prostanoid 5000-fold excess of the corresponding non-labelled ligand. moiety, selexipag, are being developed for clinical utilities including Following a 60–120 min incubation of ligands at room tempera- pulmonary hypertension [30,31]. However, the situation is more ture (Table 1), samples were filtered rapidly under vacuum complex, since the prostacyclins appear to have functionally through glass fibre filters, dried, and then counted for radioactivity relevant effects at other prostanoid receptors as reviewed by Clapp in a scintillation counter. and Patel [32]. The specific ligand binding was calculated as the difference Although the receptor binding profile of iloprost, including its between total binding measured in the presence of radioligand high affinity for the IP as well as the EP1, and EP3 receptor, has been alone and nonspecific binding determined in the presence of an reported for both murine and human prostanoid receptors [21,33], excess of unlabelled ligand, as performed in the laboratory at Cerep there has been no reported comparable evaluation of treprostinil. (Le bois l’Eveˆque, France). Specific binding for ligands reached Because of the multiple pathophysiological processes involved in equilibrium after 30–40 min of incubation at room temperature, pulmonary hypertension, there is a need to understand more about was stable for greater than 2 h and was determined to be saturable. the respective pharmacology of these two extensively used Results are expressed as a percent of the control specific binding prostacyclins. Thus, the current study investigates the binding obtained. profile of treprostinil on human prostanoid receptors, individually Competition curves for each data-set were generated by non- expressed in separate cell lines, and has directly compared this linear regression analysis of the data (Prism 4.03; GraphPad, San profile to that of iloprost in the same studies.