Selective Prostacyclin Receptor Agonist Selexipag, in Contrast to Prostacyclin Analogs, Does Not Evoke Paradoxical Vasoconstriction of the Rat Femoral Artery

Selexipag [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide] is a selective nonprostanoid prostacyclin (PGI2) receptor (IP receptor) agonist that is approved for the treatment of pulmonary arterial hypertension (PAH). In contrast to selexipag, PGI2 analogs used in the clinic are nonselective agonists at prostanoid receptors and can also activate contractile prostaglandin E receptor 3 (EP3) receptors. Leg pain is a common side effect in patients receiving treatment with PGI2 analogs and peripheral vasoconstriction can be responsible for side effects related to muscular ischemia. This study tested the hypothesis that PGI2 analogs could cause paradoxical vasoconstriction of the femoral artery via EP3 receptor activation but that only vasorelaxation would be observed in response to selexipag and its active metabolite ACT-333679 [{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}acetic acid]. Selexipag and ACT-333679 relaxed rings of the isolated rat femoral artery contracted with either prostaglandin F2α (PGF2α) or the α1 adrenoceptor (α1AR) agonist phenylephrine. ACT-333679 also inhibited contraction of the femoral artery to sympathetic nerve stimulation. In contrast, PGI2 analogs (iloprost, beraprost, and treprostinil) caused additional contraction of arterial rings precontracted with phenylephrine, which was reverted to relaxation by antagonism of EP3 receptors. Treprostinil augmented contraction of the femoral artery to sympathetic nerve stimulation in an EP3 receptor–dependent manner. Mechanistically, concomitant EP3 and α1AR receptor activation synergistically constricted femoral arteries. It is concluded that selexipag and ACT-333679 are vasorelaxants of the rat femoral artery and, unlike PGI2 analogs, do not cause paradoxical vasoconstriction via activation of EP3 receptors. EP3 receptor–mediated vasoconstriction may contribute to the well documented peripheral muscle pain reported in patients with PAH receiving PGI2 analogs. Leg pain may be less in patients treated with selexipag.


Introduction
Selexipag [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide] is a selective and orally bioavailable prostacyclin (PGI 2 ) receptor (IP receptor) agonist (Kuwano et al., 2007) that is approved for the treatment of pulmonary arterial hypertension (PAH). Selexipag lowered the risk of the primary composite end point of death or a complication related to PAH in newly treated patients or in patients already treated with one or two other classes of PAH therapies compared with patients who received placebo in the GRIPHON phase 3 clinical trial (Sitbon et al., 2015). Restoration of IP receptor signaling compensates for the reduced production of PGI 2 in PAH (Christman et al., 1992;Tuder et al., 1999) through mechanisms that include vasodilatation and inhibition of exaggerated vascular smooth muscle cell proliferation (Fetalvero et al., 2007;Smyth et al., 2009). Selexipag and its active metabolite (isopropyl)amino]butoxy}acetic acid; previously known as  have nonprostanoid structures and possess higher selectivity than PGI 2 analogs for the IP receptor over other prostanoid receptor subtypes in binding and functional cellular assays (Kuwano et al., 2007;Gatfield et al., 2016). In contrast, PGI 2 analogs used in the clinical management of PAH are not selective for the IP receptor and can activate other prostanoid receptor subtypes (Abramovitz et al., 2000;Kuwano et al., 2008;Whittle et al., 2012) as the vasorelaxant efficacy of treprostinil and beraprost, but not selexipag, is reduced via activation of contractile prostaglandin E receptor 3 (EP 3 ) receptors in the pulmonary artery under conditions associated with PAH (Kuwano et al., 2008;Morrison et al., 2012).
Leg pain is a common side effect in patients receiving treatment with PGI 2 analogs and although the effect may be neuropathic in origin (Pagani-Estévez et al., 2017), an additional vascular component can also be considered. Adrenergic https://doi.org/10.1124/jpet.117.246058.
ABBREVIATIONS: a 1 AR, a 1 adrenergic receptor; [4][5] activity is increased in the legs of patients with PAH (Velez-Roa et al., 2004) and potent contractile synergy has been reported between a 1 -adrenoceptors and EP 3 receptors in preclinical studies (Hung et al., 2006), a phenomenon that could contribute to the peripheral pain reported with PGI 2 analogs.
This study tested the hypothesis that selexipag and ACT-333679, unlike nonselective PGI 2 analogs (iloprost, beraprost, and treprostinil), cause only relaxation of the femoral artery without paradoxical EP 3 receptor-mediated vasoconstriction.

Animals
Original studies in animals were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health and were approved by the local Basel-Landschaft cantonal veterinary office (Switzerland). Twelve-week-old male Wistar rats were obtained from the Biotechnology and Animal Breeding Division (Harlan, Füllinsdorf, Switzerland). All rats were housed in climate-controlled conditions with a 12-hour light/dark cycle and had free access to normal pelleted rat chow and drinking water. In certain experiments, pulmonary hypertension (PH) was induced in rats by a single injection of monocrotaline (MCT; 60 mg/kg, i.p.). Vehicle control rats were treated in parallel. Endothelial function was tested 30 days after injection of MCT (Iglarz et al., 2008).

Rat Isolated Femoral Artery
After euthanasia, rings of the rat femoral artery were prepared using a standard technique. Briefly, the right and left femoral arteries were isolated. Two arterial rings (1.5 mm) were prepared from each artery, and vessels were suspended between 40-mm stainless steel wires in a Mulvany-Halpern myograph system (10 ml) containing modified Krebs-Henseleit buffer with the following composition: 115 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO 4 , 1.5 mM KH 2 PO 4 2.5 mM CaCl 2 , 25 mM NaHCO 3 , and 10 mM glucose. Care was taken to avoid damage to the endothelium. Bathing solution was maintained at 37°C and aerated with 95% O 2 /5% CO 2 (pH 7.4). An initial resting force of 3.9 mN was applied to the vessel (Duckles et al., 1985), and changes in force generation were measured using an isometric force recorder (Multi Wire Myograph System model 610 M, version 2.2; DMT A/S, Aarhus, Denmark) coupled to a EMKA data acquisition system (EMKA Technologies Inc., Paris, France). Viability of the femoral artery was tested by measuring contraction to KCl (60 mM) and the presence of a functional endothelium was confirmed by measuring the ability of acetylcholine (10 mM) to relax arterial rings contracted with U46619 (9,11-dideoxy-9a,11a-metha-noepoxy prostaglandin F 2a ; 1 mM). Mean relaxation to acetylcholine was 81.6% 6 1.2% for all rings tested. In certain experiments, contraction of the femoral artery to electrical field stimulation (EFS) was measured. Arterial rings were placed between platinum electrodes and stimulated every 5 minutes (17 V, 0.5-millisecond pulse width, 10 seconds, 4-24 Hz) using an electrical stimulator (EMKA Technologies Inc.). Two frequency-contraction curves were obtained in each vessel: an initial control response, followed (after a period of recovery) by a second curve in the presence of drug vehicle or test compound(s). Contraction in the presence of test compound was expressed as a percentage of the maximal contraction in the first control response.

Rat Isolated Pulmonary Artery
Rings of the extralobar pulmonary artery were prepared from rats using standard techniques. Vessels were suspended between stainless steel wires in a 10-ml tissue bath set-up and processed in a similar manner to that described for the femoral artery. An initial resting force of 4.9 mN was applied to vessels.

Experimental Protocols
Relaxation of the Pulmonary and Femoral Artery from Control and MCT-PH Rats. Rings of the pulmonary and femoral artery were contracted with phenylephrine (1 mM). When the developed force had stabilized, relaxation to acetylcholine (10 mM) was measured.
Relaxation of the Femoral Artery. Rings of the femoral artery were contracted with either prostaglandin F 2a or phenylephrine (3.5 6 0.9 and 3.0 6 0.5 mM, respectively) to give matched submaximal contraction relative to KCl (60 mM) (50.8% 6 2.5% and 50.4% 6 2.7%, respectively). Cumulative concentration-relaxation curves to selexipag, ACT-333679, iloprost, beraprost, or treprostinil were obtained when the developed force had stabilized. The interval between additions of higher concentrations of compounds to the baths was determined by the time required for the response to reach plateau. In experiments that sought to characterize the identity of the receptor mediating responses to test compounds, rings of the femoral artery were exposed to either vehicle or receptor antagonists for 30 minutes prior to obtaining cumulative concentration-response curves to agonists. The choice and concentrations of the following receptor antagonists were based on published data: DBTSA [(2E)-3-(39,49dichlorobiphenyl-2-yl)-N-(2-thienylsulfonyl)acrylamide; EP 3 receptor] (Gallant et al., 2002;Kuwano et al., 2008) (Lumley et al., 1989).
Contraction of the Femoral Artery to EFS. Frequencycontraction curves (4-24 Hz) were first obtained in the absence or presence of tetrodotoxin (0.1 mM; 10 minutes) and prazosin (0.1 mM; 10 minutes) to establish that the smooth muscle contraction was neuronal in origin and mediated via activation of a 1 ARs (Zacharia et al., 2004). Contraction of the femoral artery to EFS was abolished by tetrodotoxin (0.1 mM) and prazosin (0.1 mM) (n 5 3, data not shown).
In separate experiments, the influence of ACT-333679 or treprostinil (both at 10 mM, 20-minute incubation) on EFS-induced contraction was measured. DBTSA (1 mM) and GR32191B (1 mM) were added to the bath 20 minutes prior to the addition of treprostinil.
Contraction of the Femoral Artery to Agonists. Cumulative concentration-contraction curves to the EP 1/3 receptor agonist sulprostone were obtained in rings of the femoral artery. The ability of a subthreshold concentration of sulprostone to contract the femoral artery was measured after exposure of the artery to phenylephrine (0.1 mM; 10 minutes), and the role of a 1 -adrenoceptors and EP 3 receptors in this response was investigated by prior incubation with either prazosin (0.1 mM; 20 minutes) or DBTSA (1 mM; 20 minutes).

Statistical Analyses
Relaxation of the rat femoral artery to test compounds is expressed as a percentage of the contraction, and contractile responses are expressed as a percentage of the reference contraction to KCl (60 mM). Results are presented as the mean 6 S.E.M. In some experiments, the S.E.M. values are smaller than the data symbol. n values refer to the number of animals. Best-fit analyses of graphs were performed using GraphPad Prism software (version 7.02 for Windows, www.graphpad.com; GraphPad Software Inc., La Jolla, CA). pEC 50 values are defined as the negative logarithm of the concentration of agonist that evokes a half maximal response. The effects of receptor antagonists on responses of the femoral artery to analogs of PGI 2 were quantified by comparing calculated areas under the agonist concentration-response curves in the absence and presence of antagonists. Calculation of the area under the curve is an integrated analytical method for quantifying the response to an agonist over the whole range of concentrations tested (Hermann et al., 2003;Liang et al., 2010;Morrison et al., 2012). Statistical comparisons between control and treated groups were performed using the paired t test (two-tailed). Differences were considered significant at P , 0.05.

Endothelial Function of the Pulmonary and Femoral Artery in MCT-Induced PH Rats
Relaxation of the extralobar pulmonary and femoral artery to acetylcholine was measured using rings precontracted with the selective a 1 adrenoceptor (a 1 AR) agonist phenylephrine. Acetylcholine (10 mM) relaxed rings of the femoral artery from both control and MCT-PH rats, whereas relaxation of the pulmonary artery to acetylcholine was significantly less in arterial rings from MCT-PH rats (Table 1).

Selexipag and ACT-333679 Relax the Femoral Artery
As femoral arteries from MCT-PH rats displayed a normal endothelial function, the remaining experiments were conducted in femoral arteries from healthy Wistar rats. The effects of selexipag and its metabolite ACT-333679 on isometric force development in the rat femoral artery were measured using rings precontracted with equi-effective concentrations of either prostaglandin F 2a (PGF 2a ) or the selective a 1 AR agonist phenylephrine. Both selexipag (Fig. 1A) and ACT-333679 (Fig. 1B) relaxed the femoral artery. No statistically significant difference in relaxation (area under the curve) to either selexipag or ACT-333679 was observed in the femoral artery contracted with PGF 2a or phenylephrine ( Fig.  1; Table 2). EFS (4-24 Hz) contracted the femoral artery via endogenously released norepinephrine (Fig. 2). The maximum contraction under control conditions was 91.1% 6 7.6% relative to KCl (60 mM). ACT-333679 (10 mM) significantly inhibited contraction of the femoral artery by EFS (4-24 Hz; area under the curve: 1234 6 135.9 and 580.8 6 69.3 for the control and ACT-333679, respectively; P , 0.05, n 5 6; Fig. 2).

PGI 2 Analogs Constrict the Femoral Artery
The effects of PGI 2 analogs on the rat femoral artery were compared with those of selexipag and ACT-333679 in rings precontracted with equi-effective concentrations of either PGF 2a or phenylephrine. Although iloprost, beraprost, and treprostinil evoked concentration-dependent relaxation of the femoral artery contracted with PGF 2a (Fig. 3), these PGI 2 analogs did not cause vasorelaxation but rather induced further contraction in femoral arterial rings precontracted with phenylephrine (Fig. 3). The maximum contraction to   Fig. 2. Effect of ACT-333679 (10 mM) on contraction of the rat femoral artery to EFS. *P , 0.05; **P , 0.01 (n = 6/group).

a 1 ARs and EP 3 Receptors Act Synergistically in the Femoral Artery
Since reactivity of the femoral artery to PGI 2 analogs was only modulated during a 1 AR stimulation, the potential pharmacological interaction between contractile EP 3 receptors and a 1 ARs was investigated. The EP 1/3 receptor agonist sulprostone caused concentration-dependent contraction of the rat femoral artery (Fig. 6A; pEC 50 5 6.4 6 0.3, E max 5 140.6% 6 15.6%). Sulprostone at a concentration that did not cause contraction by itself (subthreshold concentration of 1 nM) was able to contract the femoral artery in the presence of phenylephrine (0.1 mM) (Fig. 6B). Next, the identity of the receptor subtype involved in the exaggerated contraction to sulprostone in the presence of phenylephrine was determined. EP 3 receptor antagonist DBTSA (1 mM) and prazosin (0.1 mM; selective a 1 AR antagonist) significantly reduced sulprostoneevoked contraction (Fig. 6B). Selective EP 1 receptor antagonist SC51322 (1 mM) did not inhibit contraction to sulprostone (47.4% 6 10.5% and 38.5% 6 6.9% for the control and treated groups, respectively; P . 0.05, n 5 4).

Discussion
The results of this study demonstrate the functional impact of the selectivity of selexipag and its metabolite for the IP receptor over other prostanoid receptors. Relaxation of the femoral artery to selexipag and ACT-333679 is not modulated by coactivation of contractile EP 3 receptors, nor is it dependent on the nature of the contractile agent used to raise vascular tone. In contrast, PGI 2 analogs activate EP 3 receptors to contract the femoral artery in the presence of phenylephrine, and treprostinil augments contraction to nerve-released norepinephrine.
We established that endothelial function was preserved in the femoral, but not pulmonary, artery from MCT-PH rats, demonstrating the vascular selectivity of this PH model. Further experiments using the femoral artery were therefore performed in the presence of a functional vascular endothelium. The femoral artery was chosen for investigation since its occlusion contributes to leg pain in patients with peripheral artery disease (Beard 2000). This artery is also predominantly used in preclinical models of leg ischemia (Krishna et al., 2016;Queme et al., 2017).
Vasorelaxation of the femoral artery to selexipag and ACT-333679 was similar in rings precontracted with either PGF 2a or the a 1 AR agonist phenylephrine. These data are in good agreement with previous findings in the pulmonary artery (Kuwano et al., 2008;Morrison et al., 2012). Reactivity to analogs of PGI 2 was markedly different from that measured in response to selexipag and ACT-333679. PGI 2 analogs relaxed the femoral artery precontracted with PGF 2a but caused further contraction of the femoral artery precontracted with phenylephrine. This augmented contraction to PGI 2 analogs might be caused by activation of contractile EP 3 receptors, since antagonism of EP 3 receptors revealed modest relaxation to all PGI 2 analogs tested. Contraction of the femoral artery to PGI 2 analogs measured during a 1 AR activation contrasted with the weak relaxation observed under the same conditions in pulmonary artery (Morrison et al., 2012). These data suggest an important synergy between EP 3 receptors and the adrenergic system in the femoral artery.
Differential effects of ACT-333679 and analogs of PGI 2 were also observed after transmural sympathetic nerve stimulation. ACT-333679, at a concentration that evoked maximal relaxation of the femoral artery, inhibited arterial contraction to EFS. This inhibitory effect of ACT-333679 is considered to be mediated via postsynaptic IP receptors in a manner similar to that observed for PGI 2 in the rabbit mesenteric artery (Armstrong et al., 1979). The same concentration of treprostinil, however, significantly augmented contraction to EFS in an EP 3 receptor-dependent manner. Sensitivity of EFSinduced contraction to tetrodotoxin and prazosin confirmed the nerve origin and critical involvement of a 1 ARs in this response (Zacharia et al., 2004). Thus, the ability of treprostinil to augment contraction of the femoral artery to endogenously released norepinephrine is consistent with postsynaptic interplay between a 1 ARs and EP 3 receptors.
Marked contractile synergy between EP 3 receptors and a 1 ARs has been described in the rat femoral artery (Hung et al., 2006). This artery receives dense sympathetic innervation and possesses high norepinephrine content (Todd 1980;  Selexipag and the Femoral Artery Duckles et al., 1985;Stassen et al., 1998). Thus, the femoral artery is suitable for study of the potential pharmacological interplay between EP 3 receptors and a 1 ARs and its effect on vascular responsiveness to selexipag and analogs of PGI 2 . Synergy between a 1 ARs and EP 3 receptors in the femoral artery was further supported by the observations that a subthreshold concentration of the EP 1/3 receptor agonist sulprostone evoked significant contraction of the femoral artery only in the presence of phenylephrine. Activation of both EP 3 and a 1 ARs receptors was required, since contraction to sulprostone was abolished by either DBTSA or prazosin. The contractile synergy between femoral EP 3 receptors and a 1 ARs described here and by others (Hung et al., 2006) may contribute to the well documented peripheral muscle pain (myalgia) reported in patients with PAH receiving treatment with PGI 2 analogs (Tapson et al., 2012(Tapson et al., , 2013Pagani-Estévez et al., 2017). Involvement of other lower limb arteries that are under adrenergic control (e.g., the popliteal artery) (Sada et al., 1985) cannot be excluded. Although the development of pain is considered to arise from IP and EP 3 receptor-dependent sensitization of sensory afferent neurons (Nakae et al., 2005;Southall and Vasko, 2001), our data support an additional vascular mechanism. Leg ischemia is commonly associated with pain in the calf and thigh muscles while walking, due to restriction of blood flow through the femoral artery (Beard 2000). In addition, reduced blood flow and tissue oxygenation, as occur after exaggerated vasoconstriction, promote the production of metabolism-derived pain mediators (Queme et al., 2017). The high selectivity of selexipag and its metabolite for the prostacyclin IP receptor precludes EP 3 receptor-mediated vasoconstriction and sensitization of afferent neurons, which might translate into improved tolerability over PGI 2 analogs in patients with PAH. In conclusion, this study described differences in the pharmacology of the selective prostacyclin IP receptor agonists selexipag and ACT-333679 and nonselective analogs of PGI 2 in the rat femoral artery. Selexipag and ACT-333679 relaxed the femoral artery, whereas EP 3 receptor-mediated contraction to PGI 2 analogs was exacerbated during a 1 AR stimulation. DBTSA (1 mM) and prazosin (0.1 mM) inhibit contraction to sulprostone in the presence of phenylephrine. *P , 0.05; **P , 0.01 (n = 6/group).