title page differential effects of selexipag and prostacyclin analogs in rat pulmonary...

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TITLE PAGE

Differential effects of selexipag and prostacyclin analogs in rat pulmonary artery

Keith Morrison, Rolf Studer, Roland Ernst, Franck Haag, Katalin Kauser, Martine Clozel

Drug Discovery Department, Actelion Pharmaceuticals, Ltd

Allschwil, Switzerland

JPET Fast Forward. Published on August 23, 2012 as DOI:10.1124/jpet.112.197152

Copyright 2012 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on August 23, 2012 as DOI: 10.1124/jpet.112.197152

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RUNNING TITLE PAGE

Running Title: Selexipag and rat pulmonary artery

Corresponding Author: Keith Morrison Ph.D.,

Drug Discovery Department,

Actelion Pharmaceuticals Ltd.,

Gewerbestrasse 16,

Allschwil CH4123

Switzerland

E-mail: [email protected]

Phone: +41 61 5656565

Text Pages: 29

Number of tables: 3

Number of Figures: 8

Number of References: 50

Abstract word count: 247

Introduction word count: 750

Discussion word count: 1457

Abbreviations: PGI2, prostacyclin; IP receptor, PGI2 receptor; EP1 receptor,

prostaglandin E receptor 1; EP3 receptor, prostaglandin E receptor 3; DP1 receptor,

prostaglandin D2 receptor; TP receptor, thromboxane A2 receptor; EPA, extralobar

pulmonary artery; IPA, intralobar pulmonary artery; MCT, monocrotaline; PAH,

pulmonary arterial hypertension; PASMC, pulmonary arterial smooth muscle cells;

PGF2α, prostaglandin F2α; QPCR, quantitative polymerase chain reaction; RVSP, right

ventricular systolic pressure; ACT-333679, {4-[(5,6-diphenylpyrazin-2-

yl)(isopropyl)amino]butoxy}acetic acid; selexipag, 2-{4-[(5,6-diphenylpyrazin-2-


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yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide; DBTSA, (2E)-3-(3',4'-

dichlorobiphenyl-2-yl)-N-(2-thienylsulfonyl)acrylamide; SC51322, 8-chloro-2-[3-[(2-

furanylmethyl)thio]-1-oxopropyl]hydrazide, dibenz[b,f][1,4]oxazepine-10(11H)-

carboxylic acid hydrazide; GR32191B, (4Z)-7-[(1R,2R,3S,5S)-5-([1,1’-biphenyl]-4-

ylmethoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-hetenoic acid.

Section: Drug Discovery and Translational Medicine


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ABSTRACT

ACT-333679 is the main metabolite of the selective prostacyclin (PGI2) receptor (IP

receptor) agonist selexipag. The goal of this study was to determine the influence of IP

receptor selectivity on vasorelaxant efficacy of ACT-333679 and the PGI2 analog

treprostinil in pulmonary artery under conditions associated with PAH. Selexipag and

ACT-333679 evoked full relaxation of pulmonary artery from control and MCT-PAH

rats, and ACT-333679 relaxed normal pulmonary artery contracted with either ET-1 or

phenylephrine. In contrast, treprostinil evoked weaker relaxation than ACT-333679 of

control pulmonary artery and failed to induce relaxation of pulmonary artery from MCT-

PAH rats. Treprostinil did not evoke relaxation of normal pulmonary artery contracted

with either ET-1 or phenylephrine. Expression of EP3 receptor mRNA was increased in

pulmonary artery from MCT-PAH rats. In contraction experiments, the selective EP3

receptor agonist sulprostone evoked significantly greater contraction of pulmonary artery

from MCT-PAH compared to control rats. The presence of a threshold concentration of

ET-1 significantly augmented the contractile response to sulprostone in normal

pulmonary artery. ACT-333679 did not evoke direct contraction of rat pulmonary artery,

whereas treprostinil evoked concentration-dependent contraction that was inhibited by the

EP3 receptor antagonist DBTSA. Antagonism of EP3 receptors also revealed a relaxant


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response to treprostinil in normal pulmonary artery contracted with ET-1. These data

demonstrate that the relaxant efficacy of the selective IP receptor agonist selexipag and

its metabolite ACT-333679 is not modified under conditions associated with PAH,

whereas relaxation to treprostinil may be limited in the presence of mediators of disease.


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INTRODUCTION

Selexipag is an orally available IP receptor agonist in clinical development for the

treatment of PAH (Simonneau et al, 2012; GRIPHON Phase 3 clinical trial). Both

selexipag and its active metabolite ACT-333679 (previously known as MRE-269)

possess high selectivity for the IP receptor over other prostanoid receptors, and as such

can be distinguished from PGI2 analogs currently used in the management of PAH

(Kuwano et al, 2007). Progression of PAH is associated with reduced PGI2 production

(Christman et al, 1992; Tuder et al, 1999), and restoration of IP receptor signaling using

analogs of PGI2 is an effective strategy in the treatment of the disease (Gomberg-

Maitland and Olschewski, 2008). Clinical efficacy and tolerability of PGI2 analogs,

however, may be compromised by concomitant activation of other prostanoid receptors.

The selectivity of PGI2 replacement therapies for the IP receptor, therefore, is emerging

as an important consideration in the management of PAH. For example, vasorelaxation

to iloprost and beraprost is attenuated in pulmonary artery from MCT-PAH rats (Kuwano

et al, 2008) via a mechanism that involves co-activation of the contractile EP3 receptor.

In contrast, ACT-333679 has at least 130-fold selectivity for the human IP receptor over

other prostanoid receptors (Kuwano et al, 2007), and vasorelaxation of rat pulmonary

artery evoked by ACT-333679 is not modulated by antagonism of EP3 receptors


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(Kuwano et al, 2008). Moreover, gastric side-effects including cramping, nausea and

vomiting may develop via EP3 receptor-dependent mechanisms. Analogs of PGI2

contract gastric smooth muscle via stimulation of EP3 receptors (Morrison et al, 2010).

Activation of the EP3 receptor subtype mediates disruption of gastric contractility (Pal et

al, 2007, Forrest et al, 2009) and underlies the development of emesis (Kan et al, 2002).

Selexipag does not evoke gastric contraction, nor disrupts gastric function in the rat

stomach (Morrison et al, 2010). A role for EP3 receptors has also been invoked in the

development of peripheral pain reported in patients receiving treatment with PGI2 analogs

(Minami et al, 2001; Southall and Vasko, 2001). Thus, the development of a selective IP

receptor agonist that is devoid of off-target effects may provide improved efficacy and

tolerability in patients with PAH.

Infusion of treprostinil via the subcutaneous or intravenous routes is effective in

the clinical management of PAH (Simonneau et al, 2002; Tapson et al, 2006). Also,

treprostinil reduces pulmonary arterial pressure, as measured by right ventricular systolic

pressure (RVSP), and total pulmonary vascular resistance (TPVR) in the rat MCT-PAH

model (Yang et al, 2010), but the contribution of dilatation of the pulmonary artery to this

effect is not clear. Little information on the direct vasorelaxant efficacy of treprostinil in

pulmonary artery ex vivo is available.


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The present study sought to determine the influence of IP receptor selectivity of

ACT-333679 and treprostinil on vasorelaxation of the rat pulmonary artery under

pathological conditions associated with PAH. Experiments were performed using

pulmonary artery isolated from the rat MCT-PAH model. Although development of

neointimal and plexiform lesions, characteristic of the human disease (Tuder et al, 2007),

is not a feature of this rat model, pulmonary hypertension in MCT-PAH rats is associated

with sustained pulmonary arterial vasoconstriction (Oka et al, 2008). The pulmonary

artery isolated from this model provides a suitable functional system in which to evaluate

vasorelaxant properties of test compounds. Dysfunction of both the ET-1 and

adrenergic systems underlie the development of PAH (Rubens et al, 2001; Salvi, 1999),

and circulating levels of ET-1 and catecholamines are significantly increased in the MCT

rat model of PAH (Clozel et al, 2006). Thus, the current study measured the ability of

ACT-333679 and treprostinil to relax pulmonary artery from MCT-PAH rats and control

pulmonary artery precontracted with ET-1 or the α-adrenoceptor agonist phenylephrine.

The expression levels of mRNA encoding prostanoid receptors in the MCT rat model of

PAH were also analyzed to provide a molecular mechanistic explanation for potential

differences in arterial reactivity to the test compounds.


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The data presented here highlight the importance of IP receptor selectivity for full

vasorelaxant efficacy of IP receptor agonists under pathological conditions associated

with PAH. Selexipag and its active metabolite ACT-333679 exert full relaxant efficacy,

whereas the ability of treprostinil to evoke pulmonary arterial vasorelaxation is

significantly diminished in disease. The limited relaxant efficacy of treprostinil observed

in this study may result from co-activation of contractile EP3 receptors, which are up-

regulated in the MCT rat model of PAH. Potentiation of EP3 receptor-mediated

contraction by subthreshold concentrations of pathological mediators (ET-1 and

catecholamines) in control pulmonary arteries substantiates the impact of this contractile

mechanism in disease development.


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METHODS

Animals. Male Wistar rats (12 wks) were obtained from Harlan Laboratories B.V.

(Venray, Netherlands). All rats were housed in climate-controlled conditions with a 12 h

light/dark cycle and had free access to normal pelleted rat chow and drinking water in

accordance with local guidelines (Basel-Landschaft cantonal veterinary office). In

certain experiments, PAH was induced in rats by a single injection of monocrotaline

(MCT; 60 mg/kg, i.p.). Vehicle control rats were treated in parallel. Disease was

allowed to progress for thirty days before experimentation (Iglarz et al, 2008).

Development of disease was confirmed by an increase in the weight of right ventricle

(RV/LV+S ratio: control versus MCT-PAH, 0.33 ± 0.03 g versus 0.81 ± 0.1 g, P < 0.05)

and endothelial dysfunction as measured by attenuated relaxation to acetylcholine (pEC50

values: control versus MCT-PAH, 7.23 ± 0.04 versus 6.06 ± 0.13, P < 0.0001; Emax

values: control versus MCT-PAH, 82.33 ± 1.73 % versus 39.79 ± 5.26 %, P < 0.0001).

Rat isolated pulmonary artery. Following euthanasia by CO2 asphyxiation, rings of

extralobar (EPA) and intralobar (IPA) pulmonary artery were prepared from rats using

standard techniques. Two or four arterial rings, EPA (1.5 mm) and IPA (1.0 mm),


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respectively, were prepared from each animal. Vessels were suspended between stainless

wires in either a standard tissue bath set-up (EPA) or a Mulvany-Halpern myograph

system (IPA) containing modified Krebs-Henseleit buffer (10 ml) of the following

composition (mM): NaCl 115; KCl 4.7; MgSO4 1.2; KH2PO4 1.5; CaCl2 2.5; NaHCO3

25; glucose 10. Care was taken to avoid damage to the endothelium during preparation

of arterial rings. In certain experiments, the vascular endothelium was removed by

abrasion with the tip of a watchmaker’s forceps. Bathing solution was maintained at

37oC and aerated with 95%O2/ 5%CO2/ (pH7.4). An initial resting force of 4.9 mN

(EPA) or 3.9 mN (IPA) was applied to vessels, 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 pulmonary artery was tested

by measuring contraction to KCl (60 mM) and the presence of a functional endothelium

confirmed by measuring the ability of acetylcholine 10-5 M to relax arterial rings

contracted with phenylephrine (10-6 M; EPA) or U46619 (10-6 M; IPA).


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Experimental protocols

Relaxation of pulmonary artery from control and MCT-PAH rats: rings of pulmonary

artery were contracted with prostaglandin F2α (10-5 M). When the developed force had

stabilized, cumulative concentration-relaxation curves to selexipag, ACT-333679,

beraprost or treprostinil were obtained. The interval between additions of higher

concentrations of compounds to the baths was determined by the time required for the

response to reach plateau.

Relaxation of pulmonary artery contracted with ET-1 or phenylephrine: rings of EPA and

IPA, in the presence or absence of endothelium, were contracted with either ET-1 (3 x 10-

10 M) or phenylephrine (1-3 x 10-6 M) before obtaining cumulative concentration-

relaxation curves to ACT-333679 and treprostinil.

Contraction of pulmonary artery: cumulative concentration-contraction curves to the

EP3/EP1 receptor agonist sulprostone were obtained in rings of EPA from control and

MCT-PAH rats. Separate experiments measured the contractile responses of normal EPA

and IPA to ACT-333679 and treprostinil. In experiments that sought to characterize the

identity of the receptor mediating contraction to test compounds, rings of normal IPA

were exposed to either drug vehicle or receptor antagonists for 20 minutes prior to

obtaining cumulative concentration-response curves to agonists. The choice and


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concentrations of receptor antagonists were based on published data. The following

receptor antagonists were used: DBTSA (EP3 receptor; Gallant et al, 2002, Kuwano et al,

2008); SC51322 (EP1 receptor; McCormick et al, 2010) and GR32191 (TP receptor;

Lumley et al 1989).

Measurement of receptor mRNA expression.

Prostanoid receptor mRNA expression was measured by QPCR in arteries from normal

and MCT-PAH Wistar rats. Analyses were performed using the main, left and right

branches of the pulmonary artery, thoracic aorta and the 2nd branch of the mesenteric

artery. Total RNA was isolated from arteries (30 mg samples) using the RNeasy micro

fibrous tissue kit according to the manufacturer’s protocol (QIAGEN, Germany).

Remaining genomic DNA was digested using the DNAse free kit (Ambion, USA). The

quantity of RNA was analyzed using a Nanodrop spectrophotometer (Thermo Fisher,

USA) and RNA quality was assessed using a Bioanalyzer 2100 (Agilent, USA). Total

RNA was reverse transcribed with the high capacity cDNA archive kit (Applied

Biosystems, USA). QPCR was performed on an ABI 7500 machine using TaqMan

probes (Applied Biosystems, USA). The following TaqMan assays from Applied


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Biosystems were used for mRNA detection: IP-Rn01764022_m1, DP-Rn04223963_g1,

EP1-Rn01432713_s1, EP3-Rn0121735_m1, TP-Rn00690601_m1 and for normalization

18s-4319413E, B2M-Rn00560865_m1 and GUS-Rn00566655_m1. Results were

calculated using the delta delta cT method, which allows comparison between gene

expression values for different genes based on an identical linear scale. A value of 1 is

defined as no expression. Final results are presented as the PAH:control rat ratio.

Materials. Selexipag, ACT-333679 and DBTSA ((2E)-3-(3',4'-dichlorobiphenyl-2-yl)-

N-(2-thienylsulfonyl)acrylamide) were synthesized by Nippon Shinyaku Co. Ltd (Kyoto,

Japan). Beraprost, treprostinil, SC51322 and sulprostone were obtained from Cayman

Chemical. Acetylcholine, GR32191B, phenylephrine, and prostaglandin F2α were

purchased from Sigma (St Louis, MO, USA).

Statistical evaluation of results. Responses of rat pulmonary artery to test compounds

are expressed either as a percentage of the precontraction, or of the reference contraction

to KCl (60 mM). Contraction to phenylephrine and ET-1 is measured in mN. Results are

presented as mean ± S.E.M. In some experiments, the SEM values are smaller than the

data symbol. n values refer to the number of animals. pEC50 values are defined as the


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negative logarithm of the concentration of agonist that evokes half maximal response.

The effects of receptor antagonists on responses of pulmonary artery to analogs of PGI2

were quantified by comparing calculated areas under the agonist concentration-response

curves in the absence and presence of antagonists. Statistical comparisons between

control and treated groups were performed using either student's paired t test or student’s

unpaired t test (two-tailed). Significance was accepted at P < 0.05.


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RESULTS

Selexipag/ACT-333679, but not prostacyclin analogs, relax pulmonary artery from

MCT-PAH rats

The abilities of selexipag, metabolite ACT-333679, and the analogs of prostacyclin,

beraprost and treprostinil, to relax rat pulmonary artery were measured. Arterial rings

from control and MCT-PAH rats were contracted to equal degrees with PGF2α (10-5 M)

(control versus MCT-PAH, P > 0.05). Selexipag and ACT-333679 evoked

concentration-dependent relaxation of EPA rings contracted with PGF2α (10-5 M).

Relaxation was similar in EPA from control and MCT-PAH rats (table 1 and figure 1)

(areas under curves, P > 0.05). Beraprost and treprostinil evoked concentration-

dependent relaxation of control rat EPA, but Emax values were significantly less than that

measured in response to selexipag and ACT-333679 (table 1 and figure 1). Similarly,

relaxation of EPA from MCT-PAH rats to beraprost and treprostinil was significantly

reduced compared to control EPA (table 1 and figure 1) (areas under curves: beraprost,

P < 0.05, treprostinil, P < 0.0001). In particular, relaxation to treprostinil was abolished

in EPA from MCT-PAH rats.


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ACT-333679, but not treprostinil, relaxes pulmonary artery contracted with ET-1

ET-1 is a key mediator in the development of PAH (Rubens et al, 2001; Clozel et al,

2006). Thus, the ability of ACT-333679 and treprostinil to evoke vasorelaxation of

pulmonary artery contracted with ET-1 was measured both in the presence and absence of

functional endothelium. Rings of EPA and IPA from normal rats were contracted with

ET-1 (3 x 10-10 M) and when tone had stabilized, cumulative concentration-response

curves to ACT-333679 and treprostinil were obtained. Contraction to ET-1 was similar

in the presence or absence of endothelium within each pulmonary artery group (table 2).

ACT-333679 evoked concentration-dependent relaxation of normal EPA and IPA rings

(table 3 and figure 2). In contrast, treprostinil did not evoke relaxation of either EPA or

IPA rings, even at the highest concentration tested (table 3 and figure 2). Removal of

the endothelium did not influence the responsiveness of EPA and IPA rings to treprostinil.

Removal of endothelial cells in IPA, however, caused a small but statistically significant

decrease in the pEC50 value for ACT-333679 (table 3).


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ACT-333679, but not treprostinil, relaxes pulmonary artery contracted with

phenylephrine

Hyperactivation of the sympathetic nervous system is reported in PAH (Haneda et al,

1983; Salvi, 1999; Nagaya et al, 2000; Velez-Roa et al, 2004), and circulating levels of

catecholamines are increased in the MCT model of PAH (Clozel et al, 2006).

Vasorelaxation of EPA and IPA rings to ACT-333679 and treprostinil, in the presence

and absence of endothelium, was therefore measured following contraction of rings with

the α1-adrenoceptor agonist phenylephrine. ACT-333679 evoked concentration-

dependent relaxation of normal EPA and IPA rings contracted with phenylephrine,

whereas treprostinil did not evoke relaxation (table 3 and figure 3). Removal of the

endothelium did not influence the responsiveness of EPA and IPA rings to treprostinil. A

small but statistically significant decrease in the pEC50 value for ACT-333679 was

measured upon removal of the endothelium in IPA (table 3), but no significant

differences in responses of IPA, with or without endothelium, were observed over the full

range of concentrations tested (areas under curve: with and without endothelium, P >

0.05).


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Sulprostone contracts pulmonary artery from control and MCT-PAH rats

The potential role of EP3 receptors in modulating vasoreactivity of the pulmonary artery

from MCT-induced PAH rats was investigated. First, the ability of the selective EP3

receptor agonist sulprostone to contract rat EPA from control and MCT-PAH rats was

measured. Sulprostone evoked concentration-dependent contraction of EPA which was

significantly more pronounced in rings from MCT-PAH compared to control rats (figure

4) (Emax values control versus MCT-PAH: 62.7 ± 7.8 % versus 168.5 ± 20.6 %, P <

0.001).

Differential expression of prostanoid receptors from control and MCT-PAH rats

The expression profile of prostanoid receptors in pulmonary artery from control and

MCT-PAH rats was measured (figure 5). TP and EP1 showed the highest basal

expression, IP and EP3 a moderate expression and DP1 a low expression (data not

shown). mRNA which encoded the IP receptor was significantly increased in all

segments of pulmonary artery analyzed from MCT-PAH rats (P < 0.01). Similarly, EP3

was significantly upregulated in the main branch of MCT-PAH pulmonary artery (P <

0.05). In contrast, DP1 (P < 0.05), EP1 (P < 0.001) and TP (P < 0.01) were significantly

downregulated in arteries from MCT-PAH rats.


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Effect of threshold concentration of ET-1 on contraction of pulmonary artery to

sulprostone

Since the function of the EP3 receptor subtype appears to be increased in pulmonary

artery from MCT-PAH rats, the influence of ET-1 on contraction of control pulmonary

artery to sulprostone was measured. Sulprostone and ET-1 each evoked concentration-

dependent contraction of rat EPA (figure 6; sulprostone: pEC50 5.0 ± 0.4, Emax 37.9 ±

8.3 %; ET-1: pEC50 9.6 ± 0.09, Emax 131.9 ± 4.3 %). Next, the effect of the threshold

concentration of ET-1 on contraction of rat EPA to a threshold concentration of

sulprostone was tested. Sulprostone (3 x 10-6 M) alone evoked a small contraction of rat

EPA (3.9 ± 0.9 %) (figure 6). Addition of sulprostone after an initial exposure of EPA to

the threshold concentration of ET-1 (10-10 M, contraction 2.5 ± 0.6 %), evoked a

significantly greater contraction than that observed in the absence of ET-1 (66.0 ±

11.2 %; P < 0.05) (figure 6). Similar data were obtained when sulprostone was

combined with a threshold concentration of phenylephrine (10-8 M) in rat EPA (data not

shown).


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EP1, EP3 and TP receptors and contraction of pulmonary artery from control rats

Contraction of normal rat EPA and IPA to ACT-333679 and treprostinil was measured.

ACT-333679 did not contract rings of pulmonary artery, whereas treprostinil evoked

concentration-dependent contraction of both EPA and IPA. Contraction to treprostinil

was more pronounced in IPA than in EPA rings (EPA: Emax 22.5 ± 5.8 %, IPA: Emax 62.8

± 11.9 %) (figure 7). The EP3 receptor antagonist DBTSA (3 x 10-6 M) abolished

contraction of rat IPA to treprostinil (figure 7). The EP1 receptor antagonist SC51322

(10-6 M) had no effect on contraction of IPA to treprostinil (control versus treated: pEC50,

4.8 ± 0.15 versus 4.8 ± 0.15, P > 0.05; Emax, 52.5 ± 5.5 % versus 50.8 ± 5.6 %, P > 0.05).

Similarly, antagonism of TP receptors with GR32191B (10-6 M) did not significantly

inhibit contraction to treprostinil (control versus treated: pEC50, 4.5 ± 0.4 versus 4.5 ± 0.3,

P > 0.05; Emax, 34.8 ± 11.3 % versus 22.0 ± 5.2 %, P > 0.05).

EP3 receptors and relaxation of IPA from normal rats

The influence of DBTSA on the relaxant response of rat IPA to treprostinil was next

measured. Rings of IPA, in the absence or presence of DBTSA, were contracted to the

same level with ET-1 (control versus DBTSA groups, 3.08 ± 0.5 mN versus 2.8 ± 0.6 mN,

P > 0.05). In the absence of DBTSA, treprostinil evoked minimal relaxation of IPA


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(figure 8). Antagonism of EP3 receptors with DBTSA, however, revealed a significant

concentration-dependent relaxation of rat IPA to treprostinil (figure 8) (control versus

treated: areas under curves, P < 0.05).


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DISCUSSION

Selexipag and the active metabolite ACT-333679 can be distinguished from analogs of

PGI2 based on vasorelaxant efficacy in the rat pulmonary artery. Specifically, both

selexipag and ACT-333679, but not the PGI2 analog treprostinil, evoke concentration-

dependent relaxation of pulmonary artery under pathological conditions associated with

PAH. Increased expression of mRNA which encodes EP3 receptors and enhanced

pharmacological activity of this receptor subtype are measured in pulmonary artery from

the MCT rat model of PAH, but do not influence the relaxant efficacy of the selective IP

receptor agonist ACT-333679. The limited vasorelaxant efficacy of treprostinil was

partially restored following EP3 receptor blockade, indicating that co-activation of

contractile EP3 receptors attenuates the relaxant action of treprostinil.

ACT-333679 is highly selective for the human IP receptor (Kuwano et al, 2007).

The Ki value for ACT-333679 at the IP receptor is 130-fold lower than that at other

prostanoid receptors. The affinity values for ACT-333679 at the rat IP receptor is 2.2 x

10-7 M, and would be expected to activate IP receptors at the concentrations used in the

current study. Treprostinil, however, is a non-selective agonist at prostanoid receptors

(Whittle et al, 2012). In particular, treprostinil binds and activates DP1 and EP2 receptors

in addition to the IP receptor. Treprostinil regulates macrophage function and promotes


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survival of bacteria via an EP2 receptor-dependent mechanism (Aronoff et al, 2007).

Also, treprostinil activates contractile EP3 receptors in gastric (Morrison et al, 2010) and

vascular smooth muscle (Orie and Clapp, 2011).

The relaxant response to ACT-333679 was similar in pulmonary artery from

control and MCT-PAH rats. These data confirm previous findings (Kuwano et al, 2008).

In contrast, treprostinil evoked weaker relaxation than ACT-333679 in pulmonary artery

from control rats, and failed to relax pulmonary artery from MCT-PAH rats. Diminished

relaxation of pulmonary artery from MCT-PAH rats to iloprost and beraprost has been

reported (Kuwano et al, 2008), but treprostinil appears to be particularly sensitive to the

pathological conditions associated with PAH. This possibility was further examined in

experiments that measured the relaxant efficacy of ACT-333679 and treprostinil in

pulmonary artery contracted with ET-1 or the α1-adrenoceptor agonist phenylephrine.

Dysfunction of both the ET-1 and adrenergic systems underlie the progression of PAH

(Rubens et al, 2001; Salvi, 1999), and circulating levels of ET-1 and catecholamines are

significantly increased in the rat MCT-PAH model (Clozel et al, 2006). The relaxant

profile of ACT-333679 and treprostinil was remarkably similar to that observed in MCT-

PAH rats: ACT-333679 evoked concentration-dependent relaxation of all arterial rings


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tested, whereas treprostinil was unable to relax pulmonary artery contracted with ET-1 or

phenylephrine.

Treprostinil can activate contractile EP3 receptors that counter the vasorelaxant

properties of the drug (Orie and Clapp, 2011). Contraction evoked by the selective EP3

receptor agonist sulprostone (e.g. Dong et al, 1986) is significantly greater in pulmonary

artery from MCT-PAH rats than in control rats, and is consistent with previous findings

(Kuwano et al, 2008). Hyper-responsiveness of the MCT-PAH pulmonary artery to

sulprostone may reflect an increase in the number of EP3 receptors. Receptor density is a

key determinant of both potency and efficacy of an agonist (Neubig et al, 2003).

Molecular analyses of pulmonary artery from control and MCT-PAH rats revealed a

modest but statistically significant up-regulation of the expression of mRNA encoding

EP3 receptors in the disease condition. Thus, both expression and pharmacological

activity of contractile EP3 receptors are enhanced in pulmonary artery from MCT-PAH

rats. A differential regulation of IP and TP receptor mRNA expression was also noted:

an up-regulation of IP receptor mRNA whereas down-regulation of mRNA encoding TP

receptors was recorded. These data are suggestive of a pharmacological compensatory

mechanism to combat disease progression through improved PGI2 signaling (IP receptor)

and diminished deleterious effects of TXA2 (TP receptor) (Christman et al, 1992; Tuder


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et al, 1999). Published information on the regulation of prostanoid receptors in PAH is,

however, contradictory. Down-regulation in expression of IP receptors is reported in

pulmonary arterial smooth muscle cells (PASMCs) cultured from MCT-PAH rats (Lai et

al, 2008), and in PASMCs and non-defined lung tissue from human PAH patients (Lai et

al, 2008; Falcetti et al, 2010). In contrast, a genomewide RNA expression study did not

observe significant regulation of IP or EP3 receptor mRNA in lung tissue samples from

PAH patients (Rajkumar et al., 2010). Differences in observations between studies may

reflect the experimental approaches adopted. For example, in the present study receptor

mRNA was measured in arterial blood vessels that were directly isolated from MCT-

PAH rats. An additional cell culture stage was included in previous work and may have

incurred changes in receptor expression (e.g. Boess et al, 2003). Also, non-vascular

components in snap-frozen lung tissue may have altered the receptor expression profile.

Despite the variability of results, any potential changes in expression of IP receptors

should affect the interaction with selexipag and treprostinil equally, since both drugs bind

the human IP receptor with similar affinity (Ki values, 2 x 10-8 M and 3.2 x 10-8 M,

respectively) (Kuwano et al, 2007; Whittle et al, 2012). Vascular responsiveness to a

non-selective prostanoid receptor agonist, such as treprostinil, however, may be more


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susceptible to changes in the overall expression pattern and pharmacological activity of

prostanoid receptors.

Increased pharmacological activity of the EP3 receptor was also evident in

combination experiments that measured contraction to sulprostone and ET-1 or

phenylephrine in pulmonary arteries isolated from control rats. Contraction of pulmonary

artery to sulprostone was significantly augmented in the presence of a threshold

concentration of ET-1 or phenylephrine, and raises the possibility of a synergistic

interaction between the EP3 and endothelin and adrenergic receptor systems. Contractile

synergism between ET-1 and serotonin is reported in the human mammary artery (Yang

et al, 1990), and significant synergy exists between the α-adrenoceptor and EP3 receptors

in rat femoral artery (Hung et al, 2006). Co-activation of EP3 receptors under

pathological conditions of PAH may limit the therapeutic efficacy of treprostinil. The

current study and other work (Orie and Clapp, 2011) demonstrate a clear role for the EP3

receptor subtype in vasoconstriction to treprostinil. The beneficial effects of IP receptor

signaling on vascular wall remodeling in PAH can potentially be tempered by excessive

vasoconstriction. Indeed, the EP3 receptor mediates contraction of human pulmonary

artery (Qian et al, 1994; Walch et al, 1999; Maddox et al, 1985; Haye-Legrand et al,

1987; Norel et al, 1991), and may modulate relaxant IP receptor function (Jones et al,


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1997; Chan and Jones, 2004). Also, EP3 receptor signaling is pro-fibrotic (White et al,

2008; Bozyk and Moore, 2011; Harding and LaPointe, 2011) and may limit the beneficial

effects of treprostinil in PAH.

Failure of treprostinil to evoke relaxation of the pulmonary artery from MCT-

PAH rats may not be fully explained by the small down-regulation of mRNA encoding

relaxant DP1 receptors in this model. A role for EP2 receptors can similarly be

discounted since no change in expression of mRNA encoding this receptor subtype is

measured in MCT-PAH rats (Lai et al, 2008). Indeed, agonism at DP1 and EP2 receptors

does not evoke relaxation of human pulmonary artery (Walch et al, 1999).

The vasorelaxant efficacy of ACT-333679 measured here in rat pulmonary artery

ex vivo reflects the high selectivity of this compound for the relaxant IP receptor

(Kuwano et al, 2007). Further, vasorelaxation evoked by ACT-333679 under

pathological conditions associated with PAH (absence of endothelium, ET-1 and α-

adrenoceptor stimulation) supports the therapeutic profile of selexipag for the treatment

of PAH. The combined vasodilator and anti-proliferative properties of ACT-333679

(Kuwano et al, 2007, 2008) likely lead to the prolonged survival of MCT-PAH rats

(Kuwano et al, 2008) and contribute to the significant decrease in pulmonary vascular

resistance in patients with PAH (Simonneau et al, 2012). In contrast, the present data


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suggest that the therapeutic benefits of treprostinil in the management of PAH may not be

attributed solely to direct vasodilatation. Indeed, only a small, but statistically significant,

reduction in RVSP is reported in the MCT-PAH rat (Yang et al, 2010). Infusion of

treprostinil in a therapeutic regimen effectively prevents smooth muscle proliferation and

progression of PAH in the rat MCT-PAH model (Yang et al, 2010), although the impact

on survival is not reported. Also, treprostinil exerts anti-proliferative effects via an IP-

receptor dependent mechanism in a recombinant cell system (Falcetti et al, 2007).

In conclusion, these data demonstrate that selexipag/ACT-333679 and treprostinil

differ in their vasorelaxant effects in pulmonary artery. Selexipag/ACT-333679 relax

pulmonary artery under conditions associated with PAH whereas treprostinil fails to

evoke relaxation. The relaxant efficacy of treprostinil is dependent on the nature of the

agonist used to contract the pulmonary artery, and is also attenuated by off-target

agonism at EP3 receptors. Thus, a high degree of selectivity for the target IP receptor

may help maintain therapeutic efficacy and minimize unwanted side-effects of IP

receptor agonists.


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ACKNOWLEDGMENTS

The authors thank Marie Schnoebelen for technical assistance.


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AUTHORSHIP CONTRIBUTIONS

Participated in research design: Morrison, Studer, Kauser and Clozel

Conducted experiments: Studer, Ernst and Haag

Performed data analysis: Morrison, Studer, Ernst and Haag

Wrote or contributed to the writing of the manuscript: Morrison, Studer, Kauser and

Clozel


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LEGENDS FOR FIGURES

Figure 1) Relaxation of pulmonary artery (EPA) from control and MCT-PAH rats to

selexipag (A), ACT-333679 (B), beraprost (C) and treprostinil (D). Rings of EPA were

contracted with PGF2α (10-5 M). Data are shown as mean ± S.E.M.; n = 4.

Figure 2) Relaxation of EPA (A and B) and IPA (C and D) in the presence and

absence of endothelium to ACT-333679 and treprostinil. EPA and IPA were contracted

with ET-1 (3 x 10-10 M). Data are shown as mean ± S.E.M.; n = 4.

Figure 3) Relaxation of EPA (A and B) and IPA (C and D) in the presence and

absence of endothelium to ACT-333679 and treprostinil. EPA and IPA were contracted

with phenylephrine (1-3 x 10-6 M). Data are shown as mean ± S.E.M.; n = 4.

Figure 4) Contraction of pulmonary artery (EPA) from control and MCT-PAH rats

to sulprostone. Data are shown as mean ± S.E.M.; n = 6.


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Figure 5) Expression of prostanoid receptors in pulmonary artery from control and

MCT-PAH rats. Data are expressed as the PAH:control rat ratio. S.E.M. values are

omitted for clarity; * P < 0.05, ** P < 0.01, *** P < 0.001; n = 6.

Figure 6) Contraction of rat pulmonary artery (EPA) to sulprostone and ET-1.

Concentration-dependent contraction of EPA to sulprostone (A) and ET-1 (B).

Representative traces of contractile responses of EPA to sulprostone (3 x 10-6 M) alone

(C) or in combination with ET-1 (10-10 M) (D). Quantification of contractile data shown

in traces (E). Data are shown as mean ± S.E.M.; * P < 0.05; n = 4.

Figure 7) Contraction of rat pulmonary artery to ACT-333679 and treprostinil.

Concentration-dependent contraction of EPA and IPA to ACT-333679 (A) and

treprostinil (B). DBTSA (3 x 10-6 M) inhibits contraction of IPA to treprostinil (C). Data

are expressed as mean ± S.E.M.; * P < 0.05; n = 4.

Figure 8) Effect of DBTSA (3 x 10-6 M) on relaxation of rat pulmonary artery (IPA,

with endothelium) to treprostinil. IPA was contracted with ET-1 (3 x 10-10 M). Data are

expressed as mean ± S.E.M.; n = 4.


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TABLES

Table 1. Summary of relaxation data for selexipag, ACT-333679, beraprost and

treprostinil in pulmonary artery from control and MCT-PAH rats.

Selexipag

ACT-333679

beraprost

treprostinil

Control

pEC50

Emax

5.2 ± 0.1

98.4 ± 2.1

5.9 ± 0.1

102.3 ± 0.7

6.18 ± 0.2

81.1 ± 6.9*

6.88 ± 0.1

72.8 ± 3.7**

MCT-PAH

pEC50

Emax

4.9 ± 0.1

104.6 ± 2.4

5.5 ± 0.1

104.5 ± 1.0

4.7 ± 0.2

57.5 ± 7.8**

nd

4.3 ± 2.3***

Emax, % of reference contraction to KCl 60mM

*, P < 0.05; **, P < 0.001; ***, P < 0.0001 comparison with Emax value for ACT-333679


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Table 2. Contraction of rat pulmonary artery to ET-1 and phenylephrine.

Contractile agent/

relaxant

EPA

IPA

With

endothelium

Without

endothelium

With

endothelium

Without

endothelium

ET-1/

ACT-333679

treprostinil

8.9 ± 1.3

8.1 ± 0.9

10.3 ± 1.1

10.5 ± 1.0

3.5 ± 0.4

2.7 ± 0.3

3.2 ± 0.3

2.8 ± 0.3

phenylephrine/

ACT-333679

treprostinil

5.5 ± 0.8

6.5 ± 0.9

7.6 ± 1.2

6.7 ± 0.5

3.1 ± 0.2

3.1 ± 0.1

2.8 ± 0.3

3.4 ± 0.2

Values, mN


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Table 3. Relaxant potency of ACT-333679 in rat pulmonary artery.

Contractile agent

EPA

IPA

With

endothelium

Without

endothelium

With

endothelium

Without

endothelium

ET-1

5.0 ± 0.3

4.8 ± 0.3

5.4 ± 0.2

4.5 ± 0.2*

phenylephrine

4.9 ± 0.1

4.7 ± 0.1

5.1 ± 0.2

4.5 ± 0.1*

Values, pEC50

* , P < 0.05, comparison between IPA with and without endothelium


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