Pulmonary arterial hypertension (PAH) is a life-threatening disorder characterized by a progressive increase in pulmonary arterial pressure and pulmonary vascular resistance, leading ultimately to right ventricular failure and death (Rich et al., 1987; Farber and Loscalzo, 2004). The pathogenesis of PAH includes at least three processes occurring in small pulmonary arteries (SPA): vasoconstriction, vascular remodeling, and thrombosis (Rubin, 1997; Runo and Loyd, 2003).

Prostacyclin (PGI₂) is a potent endogenous vasodilator and inhibitor of platelet aggregation that acts at the PGI₂ receptor (IP receptor) (Namba et al., 1994). PGI₂ contributes to the maintenance of homeostasis and has various other physiological effects in many organ systems (Narumiya et al., 1999). A decrease in PGI₂ production and an imbalance between PGI₂ and thromboxane A₂ has been reported in PAH (Christman et al., 1992; Tuder et al., 1999), and IP receptor knockout results in a worsening of pulmonary hypertension (PH) (Hoshikawa et al., 2001). These findings indicate that the PGI₂-IP system is involved in the progression of PAH. Treatment with IP receptor agonists or transfer of the PGI₂ synthase gene ameliorates PH in rats treated with monocrotaline (MCT) (Miyata et al., 1996; Nagaya et al., 2000).

When epoprostenol (PGI₂) therapy was developed in the 1990s, it improved the outcome of PAH treatment. Long-term treatment with PGI₂ markedly improves exercise tolerance and survival (Barst et al., 1996). However, PGI₂ therapy requires continuous infusion through a central venous catheter because of the very short half-life of PGI₂ (3–5 min). Despite its success in the treatment of PAH in some patients, PGI₂ infusion is still far from an ideal therapy because of its high cost, the discomfort associated with its administration, and the high risk of complications from infection (Humbert et al., 1999; Sitbon et al., 2002). Many kinds of PGI₂ analog have been synthesized to improve on the chemical stability of PGI₂, and some of them, such as beraprost, iloprost, and treprostinil, have been applied to the clinical treatment of PAH (Olschewski et al., 2002; Simonneau et al., 2002). Nevertheless, their duration of action is so short that continuous infusion or frequent administration is still needed.

To overcome the drawbacks of PGI₂ and its analogs related to their short half-lives, we synthesized a novel diphenylpyrazine derivative, NS-304. NS-304 is an orally available and long-acting IP receptor agonist prodrug, and its active form, MRE-269, is a highly selective IP receptor agonist (Kuwano et al., 2007). In addition to IP receptor agonists, endothelin receptor antagonists and phosphodiesterase type 5 inhibitors are also effective treatments for PAH as orally available drugs (Rubin et al., 2002; Galiè et al., 2005). Clinical studies have shown beneficial and significant effects of combination therapy with these three classes of drugs (McLaughlin et al., 2006; Mathai et al., 2007). However, an ideal combination therapy cannot be provided at present because there is no orally available and long-lasting drug in the IP receptor agonist class. It is hoped that NS-304 will satisfy this unmet need and allow the development of an ideal combination therapy that includes members of all three classes of orally available drug.

The binding affinity of MRE-269 for the human IP receptor is over 130 times its affinity for other human prostanoid receptors. Most PGI₂ analogs, such as beraprost and iloprost, show poor selectivity for the IP receptor because they also have high affinity for EP receptor subtypes, especially the EP₃ receptor (Kiriyama et al., 1997; Abramovitz et al., 2000; Kuwano et al., 2007). Because EP₃ receptor agonists cause arterial contraction (Qian et al., 1994; Jones et al., 1998), EP₃-receptor-mediated agonism interferes with IP-receptor-mediated vasorelaxation (Jones et al., 1997; Chan and Jones, 2004). In fact, we have reported that the effect of beraprost on the increase in femoral skin blood flow in rats is inferior to that of the more selective IP receptor agonist prodrug NS-304 (Kuwano et al., 2007).

In the present study, we show that NS-304 ameliorated various pathological processes associated with PH in MCT-treated rats, and that the pharmacological characteristics of its active form, MRE-269, in the vasodilation of pulmonary artery preparations were superior to those of beraprost and iloprost.

Materials and Methods

Materials

NS-304, MRE-269, and the EP₃ receptor antagonist (2E)-3-(3′,4′-dichlorobiphenyl-2-yl)-N-(2-thienylsulfonyl)acrylamide (hereinafter referred to as DBTSA) were synthesized in our laboratory as described previously (Gallant et al., 2002; Kuwano et al., 2007). Beraprost was purchased from Chinoin (Budapest, Hungary); iloprost, PGF_2α, and sulprostone were obtained from Cayman Chemical (Ann Arbor, MI); and MCT was obtained from Sigma-Aldrich (St. Louis, MO).

Animals

Male Sprague-Dawley rats (Japan SLC, Hamamatsu, Japan) were housed in cages under a 12-h light/dark cycle and allowed free access to pellet chow and tap water. All animal procedures were approved by the Committee for the Institutional Care and Use of Animals of Nippon Shinyaku Co., Ltd.

Measurement of Intracellular cAMP

Chinese hamster ovary (CHO) cells expressing the human EP₃ receptor were produced as described previously (Kuwano et al., 2007), cultured in 24-well plates (1 × 10⁵ cells/well), washed with Hanks' balanced salt solution buffered with 10 mM HEPES, pH 7.4, and preincubated at 37°C for 60 min in the same solution containing the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (500 μM). Then, the test agent and 1 μM forskolin were added to each well and, after incubation for an additional 15 min at 37°C, the reaction was terminated by the addition of perchloric acid solution, and the quenched reaction mixture was frozen for 2 h at -80°C. The thawed reaction mixture was centrifuged, a sample of supernatant was removed, and the cAMP in the supernatant was measured with an enzyme-linked immunosorbent assay kit (GE Healthcare, Chalfont St. Giles, UK) according to the manufacturer's instructions.

Rat Model of Monocrotaline-Induced Pulmonary Hypertension

Pulmonary Artery Hypertrophy and Relaxation Response, Hemodynamic Measurements, and Assessment of Right Ventricular Hypertrophy. Rats weighing 180 to 210 g were divided into groups and given a single s.c. injection of 40 mg/kg MCT, after which they were orally administered NS-304 at 1 mg/kg, beraprost at 0.1 mg/kg, or vehicle twice daily for 19 days.

Survival. Rats weighing 250 to 300 g were divided into groups and given a single s.c. injection of 60 mg/kg MCT, after which they were orally administered NS-304 at 1 mg/kg or vehicle twice daily for 45 days.

Preparation of Pulmonary Arteries for Measurement of Relaxant Response to Acetylcholine

The day after the final administration of drug to MCT-treated rats, the right main pulmonary arteries (2 mm in external diameter) were isolated and cut into rings 3 to 4 mm in length. The arterial rings were precontracted with 10 μM PGF_2α, and the relaxant responses induced by various concentrations of acetylcholine were measured as described below and expressed relative to the papaverine-induced relaxant response.

Assessment of Pulmonary Arterial Wall Hypertrophy

Histological analysis of the medial wall thickness of the peripheral pulmonary arteries was performed as described by Ono and Voelkel (1991), with some modifications. In brief, the lungs were immersed in 10% neutral buffered formalin and embedded in paraffin blocks. Four-micrometer sections cut from the middle region of the left lung were stained with elastin van Gieson stain and examined under a microscope at 400× magnification. For each lung section, 10 muscular arteries showing closed, circular sections 25 to 100 μm in external diameter were selected at random, and their external diameter and medial wall thickness were measured. The percentage medial wall thickness was calculated as [(medial wall thickness × 2)/external diameter] × 100.

Hemodynamic Measurements and Assessment of Right Ventricular Hypertrophy

A polyethylene tube was inserted through the right jugular vein into the right ventricle under urethane anesthesia and connected to a pressure transducer (model TP-300T; Nihon Kohden, Tokyo, Japan). The right ventricular systolic pressure (RVSP) was measured with the pressure transducer connected to a carrier amplifier (model AP-641G; Nihon Kohden). Heart rate (HR) was measured with a tachometer (model AT-601G; Nihon Kohden). After the hemodynamic measurements had been made, the heart was removed and the right ventricular free wall and the left ventricle plus septum were isolated and weighed. The ratio of the weight of the right ventricle to that of the left ventricle plus septum [RV/(LV+S)] was calculated as an index of right ventricular hypertrophy (RVH).

Measurement of Relaxant and Contractile Responses in Pulmonary Arteries

Large pulmonary arteries (LPA) (main arteries; external diameter ≈ 2 mm) and SPA (second or third branches; external diameter ≈ 0.3 mm) were isolated. The arteries were cut into rings 3 to 4 mm and 1 mm in length, respectively. LPA without endothelium [LPA(-)] was prepared from LPA with endothelium [LPA(+)] by gently rubbing the intimal surface of LPA(+) with cotton thread and briefly washing the surface with buffer. Because SPA was too small to keep the endothelium intact, only SPA without endothelium [SPA(-)] was prepared. Arterial rings were mounted between two wires in a 37°C organ bath containing modified Krebs-Ringer bicarbonate solution, pH 7.4. One wire was fixed, and the other was connected to an isometric force-displacement transducer (model T7-30-240; Orientec, Tokyo, Japan) and a carrier amplifier (model AP-621G; Nihon Kohden). At the end of each experiment, 100 μM papaverine was applied to produce maximal relaxation. For observation of relaxant responses, arterial rings were precontracted with 10 μM PGF_2α before the induction of relaxation by test drugs. The drugs were applied directly to the arterial preparations in cumulative concentrations. Contraction was induced with test drugs. For observation of the effect of an EP₃ receptor antagonist, DBTSA, on the relaxant and contractile responses, arterial rings were treated with the antagonist at a concentration of 3 μM before measurement of the response. For determination of the effect of MCT on contractile and relaxant responses in LPA(+), LPA was isolated from rats 19 or 20 days after injection of 40 mg/kg MCT, and the responses were measured as described above.

Statistical Analysis

First, the homogeneity of variance between pairs of groups was analyzed by the F-test. When the variance was homogenous, statistical differences between groups were analyzed by Student's t test. When the variance was not homogenous, statistical differences were analyzed by the Welch test. The statistical significance of differences among three or more groups was evaluated by Dunnett's test. Survival curves for both groups were calculated by the Kaplan-Meier method and compared by the log-rank test. A P value of less than 0.05 was considered statistically significant. All analyses were performed with SAS version 8.2 (SAS Institute Inc., Cary, NC).

Results

Relaxant Response to Acetylcholine in the Pulmonary Artery. Acetylcholine induced vasodilation of the pulmonary artery in a concentration-dependent manner in normal rats (Fig. 1A). This relaxant response was markedly attenuated in MCT-treated rats (MCT group). The maximal relaxant responses in normal and MCT-treated rats were 81 and 25%, respectively, of that induced by papaverine. Repeated treatment of MCT-treated rats with NS-304 significantly suppressed this deterioration of the relaxant response, producing a recovery of the maximal response to approximately 50% of that induced by papaverine.

Hypertrophy of the Pulmonary Arterial Wall. MCT treatment of rats induced marked and significant hypertrophy of the pulmonary arterial wall, the percentage wall thickness being 32.2 ± 2.5 in the MCT group versus 16.2 ± 1.0 in the normal group (Fig. 1B), whereas the percentage wall thickness in the NS-304 group, at 23.5 ± 1.5, was significantly lower than in the MCT group. Representative photomicrographs of a pulmonary arterial cross-section for each group also show that the hypertrophy of the arterial wall was markedly reduced by treatment with NS-304 (Fig. 1C).

Right Ventricular Systolic Pressure and Right Ventricular Hypertrophy. RVSP in the MCT group, at 68 ± 4 mm Hg, was significantly higher than the value of 36 ± 1 mm Hg observed in the normal group (Fig. 2A). Both the NS-304 and the beraprost group demonstrated a significantly lower RVSP than the MCT group, with values of 51 ± 2 and 57 ± 3 mm Hg, respectively. The RV/(LV+S) ratio in the MCT group, 0.500 ± 0.027, was also significantly higher than the value of 0.279 ± 0.006 observed in normal group (Fig. 2B). Treatment with NS-304 significantly reduced the ratio to 0.380 ± 0.019, whereas treatment with beraprost produced no significant decrease (0.441 ± 0.026). There were no significant differences in HR among the groups (Fig. 2C).

Effect of NS-304 on Survival. The survival rate of MCT-treated rats at 45 days was 33%, and NS-304 significantly increased this survival rate to 73% (Fig. 2D; P = 0.0307, log-rank test).

Relaxant Response of Pulmonary Artery Preparations to IP Receptor Agonists. MRE-269 induced concentration-dependent vasodilation in LPA(+), LPA(-), and SPA(-) (Fig. 3A). There were no significant differences in the concentration-response curves for MRE-269 between LPA(-) and SPA(-), but MRE-269-induced vasodilation in LPA was slightly attenuated by endothelium removal at higher concentrations. Although beraprost induced vasodilation in LPA(+), LPA(-), and SPA(-), the relaxant response was attenuated at higher concentrations in all three artery preparations (Fig. 3B). Beraprost-induced vasodilation in LPA was markedly attenuated by endothelium removal, the maximal relaxation at 3 μM being significantly reduced from 81.3 ± 3.5 to 55.3 ± 5.1%. Beraprost-induced vasodilation in SPA(-) was less than in LPA, the maximal relaxation at 3 μM being only 21.5 ± 3.7%. The rank order of the strength of beraprost-induced vasodilation for the three pulmonary artery preparations was LPA(+) > LPA(-) > SPA(-). Iloprost also induced concentration-dependent vasodilation in all three artery preparations (Fig. 3C), but endothelium removal had no significant effect on iloprost-induced vasodilation in LPA. The concentration-response curve for iloprost in SPA(-) was shifted to the right compared with the curve for LPA(-). The rank order of the strength of iloprost-induced vasodilation was LPA(+)≈LPA(-) > SPA(-).

EP₃-Receptor-Agonist-Mediated Contraction in Pulmonary Artery Preparations. The EP₃ receptor agonist sulprostone induced concentration-dependent vasoconstriction in LPA(+), LPA(-), and SPA(-) (Fig. 4A), and the vasoconstriction in SPA(-) was significantly greater than in LPA(-). The EP₃ receptor antagonist DBTSA attenuated sulprostone-induced vasoconstriction in LPA(-) (Fig. 4B). Beraprost and sulprostone decreased cAMP production evoked by forskolin in CHO cells expressing the human EP₃ receptor (Fig. 5A). This finding indicates that beraprost has EP₃ receptor agonist activity. Iloprost also showed EP₃ receptor agonism (data not shown). Beraprost induced vasoconstriction in LPA(-) (Fig. 5B) and SPA(-) (Fig. 5C), and DBTSA suppressed the vasoconstriction.

Effect of an EP₃ Antagonist on the Relaxation of Pulmonary Artery Preparations Induced by IP Receptor Agonists. The EP₃ receptor antagonist DBTSA had no significant effect on the concentration-response curve for MRE-269-induced vasodilation in SPA(-) (Fig. 6A). In contrast, beraprost-induced vasodilation in SPA was enhanced by treatment with DBTSA, and the maximal relaxation induced by beraprost, observed at 3 μM, was significantly increased from 27.4 ± 7.7 to 65.1 ± 6.0% by DBTSA treatment (Fig. 6B). Beraprost-induced vasoconstriction occurred at beraprost concentrations above 3 μM in spite of DBTSA treatment, as evidenced by the observed attenuation of the relaxant response. Although the vasodilation induced by iloprost at lower concentrations was enhanced by DBTSA treatment, iloprost at concentrations greater than 1 μM induced marked vasoconstriction (Fig. 6C).

Effects of MRE-269, Beraprost, and Iloprost on LPA from MCT-Treated Rats. Vasoconstriction induced by sulprostone, beraprost, or iloprost in rat LPA(+) was increased by treatment of rats with MCT, but MRE-269 induced no vasoconstriction in either normal or MCT-treated rats (Fig. 7). The vasodilation induced by beraprost or iloprost, but not that induced by MRE-269, was more strongly attenuated in LPA from MCT-treated rats than from normal rats (Fig. 8).

Discussion

NS-304 is an orally available and long-acting IP receptor agonist prodrug, and its active form, MRE-269, is a highly selective IP receptor agonist (Kuwano et al., 2007). In the present study we show that, in rats with MCT-induced PH, twice-daily treatment with NS-304 ameliorated the impaired relaxant response to acetylcholine in pulmonary artery preparations and reduced the hypertrophy of the pulmonary arterial wall induced by MCT. These results suggest that NS-304 ameliorates endothelial dysfunction and inhibits the pathological proliferation of intimal smooth muscle cells in the injured arteries. NS-304 also significantly reduced the RVH and RVSP without affecting the HR and significantly increased the survival rate of MCT-treated rats. The positive effect of NS-304 on survival was similar to that observed in this rat PH model after transfer of the PGI₂ synthase gene (Nagaya et al., 2000) or administration of beraprost (Itoh et al., 2004). Our results show that NS-304 suppressed various pathological processes associated with PH in rats.

Although 0.1 mg/kg is probably the optimal dose of beraprost for the amelioration of MCT-induced PH in rats (Miyata et al., 1996; Ueno et al., 2002), in the present study, beraprost at this dosage significantly reduced the RVSP but not the RVH. A pharmacokinetic study of NS-304 in humans showed a long plasma elimination half-life for MRE-269 (Kuwano et al., 2007), approximately eight times that of beraprost (Toda, 1988; Demolis et al., 1993). In contrast, in rats the elimination half-life of beraprost was slightly longer than that of MRE-269 after oral administration of NS-304 (Matsumoto et al., 1989; Kuwano et al., 2007). Nevertheless, MRE-269 was no less effective than beraprost in MCT-treated rats. Because the binding affinity of MRE-269 for the rat IP receptor (K_i = 220 nM) is approximately one-tenth that of beraprost (K_i = 19 nM) (Kuwano et al., 2007), the dose of NS-304 used was 10 times the dose of beraprost used. The binding affinity of MRE-269 (K_i = 20 nM) for the human IP receptor is about as high as that of beraprost (K_i = 39 nM) (Kuwano et al., 2007). Differences in the potency of MRE-269 are probably due to species differences in the binding affinity of MRE-269 for the IP receptor. Similar species differences have been reported for a nonprostanoid IP receptor agonist, octimibate (Merritt et al., 1991).

We have previously found that the efficacy of beraprost in increasing femoral skin blood flow in rats is lower than that of NS-304, and we suggested that the EP₃ receptor agonist activity of beraprost attenuates its efficacy at the IP receptor (Kuwano et al., 2007). To investigate the effect on vasodilation of differences among the receptor selectivity profiles of MRE-269, beraprost, and iloprost, we assessed the relaxant response to these compounds in pulmonary artery preparations. Although the receptor selectivity profiles of MRE-269 and PGI₂ analogs among rat prostanoid receptors are unknown, we have previously shown that MRE-269 is highly selective for the human IP receptor (K_i for IP = 20 nM; K_i for EP₃ >10 μM), whereas beraprost (K_i for IP = 39 nM; K_i for EP₃ = 680 nM) and iloprost (K_i for IP = 11 nM; K_i for EP₁ = 11 nM; K_i for EP₃ = 56 nM) show poor selectivity for the human IP receptor because of their relatively high affinities for human EP subtypes (Abramovitz et al., 2000; Kuwano et al., 2007). In the present study, the relaxant responses of the pulmonary artery preparations to the compounds were clearly different. MRE-269 induced vasodilation equally in LPA(-) and SPA(-), whereas beraprost and iloprost induced less vasodilation in SPA(-) than in LPA(-). The efficacy of MRE-269 in inducing vasodilation in pulmonary arteries was greater than that of beraprost, especially in SPA(-).

EP₃ receptor agonists cause pulmonary arterial contraction (Qian et al., 1994; Norel et al., 2004), and EP₃-receptor-mediated agonism interferes with IP-receptor-mediated vasorelaxation (Jones et al., 1997; Chan and Jones, 2004). In the present study, an EP₃ receptor agonist, sulprostone, induced vasoconstriction in both LPA(-) and SPA(-), and the vasoconstriction observed in SPA(-) was more potent than that observed in LPA(-). Sulprostone-induced vasoconstriction in LPA(-) was suppressed by an EP₃ receptor antagonist, DBTSA, which has a high affinity for the human EP₃ receptor (K_i = 25 nM) (Gallant et al., 2002). These results suggest that EP₃-receptor-mediated vasoconstriction in SPA is greater than in LPA, and therefore that the EP₃ receptor density in SPA is probably higher than in LPA. In the present study, beraprost and iloprost showed apparent EP₃ receptor agonism, as judged by their suppression of cAMP production in cells expressing the EP₃ receptor. Beraprost induced vasoconstriction in both LPA(-) and SPA(-), and DBTSA completely inhibited this vasoconstriction. Moreover, treatment with DBTSA enhanced the vasodilation induced by beraprost or iloprost in SPA, but not the vasodilation induced by MRE-269. These findings indicate that the vasodilation in SPA induced by beraprost or iloprost, but not that induced by MRE-269, is counteracted by EP₃ receptor agonism. Beraprost induced vasoconstriction in LPA and SPA at concentrations of more than 10 μM; in contrast, significant attenuation of beraprost-induced vasodilation started at concentrations as low as 1 nM. In the contractile response induced by beraprost, because IP-receptor-mediated vasodilation is expected to occur concomitantly with the vasoconstriction, it was possible that vasodilation canceled out the vasoconstriction at lower concentrations of beraprost. Consistent with this possibility, beraprost-induced vasoconstriction was observed only at higher concentrations. Because the progression of PAH mainly affects the small pulmonary arteries, MRE-269, which can equally relax both LPA and SPA, has an advantage over beraprost and iloprost, which preferentially relax LPA, as a medication for PAH.

Iloprost induced equal vasodilation in LPA irrespective of the presence or absence of endothelium, and MRE-269-induced vasodilation in LPA was slightly attenuated by endothelium removal only at higher concentrations. However, there was no significant difference in the maximal relaxation induced by either MRE-269 or iloprost in the presence and in the absence of endothelium. In contrast, the vasodilation induced by beraprost was markedly attenuated by endothelium removal. Because PAH is usually accompanied by endothelial dysfunction, the ability to induce an endothelium-independent relaxant response is a desirable pharmacological characteristic in a drug intended for the treatment of PAH. Treatment of rats with MCT led to endothelial dysfunction in the pulmonary arteries, as well as to an increase in EP₃-receptor-mediated vasoconstriction. MRE-269 induced vasodilation equally in LPA from normal and MCT-treated rats. In contrast, vasodilation induced by beraprost or iloprost was markedly attenuated in LPA from MCT-treated rats compared with normal rats. Vasoconstriction induced by beraprost or iloprost was intensified by treatment with MCT. This vasoconstriction seems to contribute to the attenuation of vasodilation induced by these compounds. Furthermore, repeated treatment with NS-304 ameliorated endothelial dysfunction in the pulmonary arteries of MCT-treated rats. We have therefore shown that NS-304 could both protect the endothelium and relax pulmonary arteries with impaired endothelium. Although the PGI₂ analogs iloprost and beraprost have similar chemical structures, they unexpectedly showed different dependencies of their vasodilation activity on the presence or absence of endothelium. The reason for this is unknown, but it may be related to the fact that iloprost has agonist activity at the EP₁ receptor in addition to the IP and EP₃ receptors (Abramovitz et al., 2000). In the treatment of PAH with either PGI₂ receptor agonist, dose escalation is commonly required (Barst et al., 1996), and this is likely to cause significant EP₃ agonism, thereby attenuating IP-receptor-mediated vasorelaxation.

In conclusion, NS-304 was found to ameliorate various pathological processes associated with PH in MCT-treated rats, and its active form, MRE-269, showed suitable pharmacological characteristics in the vasodilation of pulmonary artery preparations for a drug intended to treat PAH. Its favorable pharmacological profile is based in part on its high selectivity for the IP receptor. NS-304 is therefore a promising alternative medication for PAH with prospects for good patient compliance.