The selexipag active metabolite ACT-333679 displays strong anti-contractile and anti

55 Pages·2017·2.66 MB·English

by  John Gatfield

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The selexipag active metabolite ACT-333679 displays strong anti-contractile and anti-
remodeling effects, but low -arrestin recruitment and desensitization potential

John Gatfield ≠, Katalin Menyhart, Daniel Wanner, Carmela Gnerre, Lucile Monnier, Keith
Morrison, Patrick Hess, Marc Iglarz, Martine Clozel and Oliver Nayler

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Running title: Efficacy and desensitization profile of selexipag
Corresponding author: John Gatfield
Actelion Pharmaceuticals Ltd
Gewerbestrasse 16
4123 Allschwil
Switzerland
[phone] (phone)
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[email protected] d
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Text pages: 44 m
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Number of tables: 4 t.a
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Number of figures: 11 u
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Number of references: 43 .o
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Abstract: 235 words
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Introduction: 605 words J
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Discussion: 982 words ls
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Recommended section assignment: Cardiovascular a
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Abbreviations:
ACT-333679 or MRE-269, {4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}acetic acid;
AT , angiotensin receptor type 1; BSA. bovine serum albumin; CHO, Chinese hamster
1
ovary; DTT, dithiothreitol; EC , concentation leading to half-maximal stimulation; ECM,
50
extracellular matrix; E , maximal efficacy; EP receptor, prostaglandin E2 receptor type 1;
max 1
ET-1, endothelin-1; FBS, fetal bovine serum; GPCR, G protein-coupld receptor; h, human;
HBSS, Hank´s balanced salt solution; HEK, human embryonic kidney; HEPES, 4-(2-
2
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hydroxyethyl)-1-piperazineethanesulfonic acid; IBMX, 3-Isobutyl-1-methylxanthine; IP
receptor, prostaglindin I receptor; LC/MS, high-performance liquid chromatography coupled
2
to mass spectrometry; MAP, mean arterial blood pressure; MCT, monocrotaline; MLC,
myosine light chain; MLCK, myosin light chain kinase; MPAP, mean pulmonary arterial blood
pressure; PAH, pulmonary arterial hyptersion; PASMC, pulmonary arterial smooth muscle
cells; PDGF, platelet-derived growth factor; PGI , prostaglandin I or prostacyclin; PH,
2 2
pulmonary hypertension; PMSF, phenylmethylsulfonylfluoride; selexipag, 2-{4-[(5,6-
diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide; SD, standard
deviation; SEM, standard error of the mean; SHR, spontaneously hypertensive rats; S1P1, D
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sphingosine-1-phosphate receptor type 1 lo
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ABSTRACT
Prostacyclin (PGI ) receptor (IP receptor) agonists, which are indicated for the treatment of
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pulmonary arterial hypertension (PAH), increase cytosolic cAMP levels and thereby inhibit
pulmonary vasoconstriction, pulmonary arterial smooth muscle cell (PASMC) proliferation
and extracellular matrix synthesis. Selexipag (Uptravi®) is the first non-prostanoid IP
receptor agonist, it is available orally and was recently approved for the treatment of PAH. In
this study we show that the active metabolite of selexipag and the main contributor to clinical
efficacy, ACT-333679 (previously known as MRE-269), , behaved as full agonist in multiple
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PAH-relevant receptor-distal - or downstream cellular assays with a maximal efficacy w
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comparable to that of the prototypic PGI2 analog iloprost: In PASMC, ACT-333679 potently ded
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induced cellular relaxation (EC =4.3 nM), inhibited cell proliferation (IC =4.0 nM) as well as m
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extracellular matrix synthesis (IC =8.3 nM). In contrast, ACT-333679 displayed partial t.as
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agonism in receptor-proximal - or upstream - cAMP accumulation assays (Emax=56%) when urn
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compared to iloprost and the PGI2 analogs beraprost and treprostinil (Emax~100%). Partial rg
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agonism of ACT-333679 also resulted in limited -arrestin recruitment (E =40%) and lack S
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of sustained IP receptor internalization, whereas all tested PGI analogs behaved as full Jo
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agonists in these desensitization-related assays. In line with these in vitro findings, selexipag, ls o
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but not treprostinil, displayed sustained efficacy in rat models of pulmonary and systemic arc
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hypertension. Thus, the partial agonism of ACT-333679 allows for full efficacy in amplified , 2
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receptor-distal PAH-relevant readouts while causing limited activity in desensitization-related
receptor-proximal readouts.
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INTRODUCTION
Prostacyclin (PGI ) is an arachidonic acid metabolite synthesized mainly through endothelial
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cells by cyclooxygenase enzymes in conjunction with PGI synthase. PGI serves as an
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autocrine/paracrine mediator, modulating various physiological processes by binding and
activating the prostacyclin receptor (IP receptor), a G-protein coupled receptor (GPCR) that
stimulates adenylate cyclase to synthesize cyclic AMP (cAMP) (Boie et al., 1994). The IP
receptor is highly expressed in cell types such as vascular smooth muscle cells, fibroblasts,
platelets and leucocytes. Insufficient PGI synthesis and IP receptor signaling have been
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linked to cardiovascular pathologies, especially pulmonary arterial hypertension (PAH) D
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(Christman et al., 1992; Tuder et al., 1999; Arehart et al., 2008). PAH is a rare disease lo
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characterized by increased pressure in the pulmonary circulation caused by constriction and fro
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progressive remodeling of the pulmonary vasculature and thus increased pulmonary vascular et.a
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resistance (Galie et al., 2009; Morrell et al., 2009). Exogenous supplementation of synthetic tjo
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PGI (epoprostenol) by continuous intravenous (i.v.) application was the first efficacious ls
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therapy in PAH (Barst et al., 1996). t A
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Currently, three PGI analogs have been marketed for treatment of PAH: iloprost, treprostinil T
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and beraprost (Olschewski et al., 2004). Several limitations of PGI analog therapy are rn
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known: they are not selective, i.e. they activate other prostanoid receptors, and they display M
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chemical and metabolic instability limiting their potential for oral administration (Kuwano et 1
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al., 2007; Whittle et al., 2012). In addition, being GPCR agonists, it has been suggested that 2
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PGI analogs might have the potential to cause tachyphylaxis (Rubin et al., 1990; Archer et
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al., 1996; Shapiro et al., 1997; Barst et al., 2003) making it necessary to escalate drug
dosing over time. Tachyphylaxis can be caused by different mechanisms. In the case of PGI
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and its analogs, one of the main mechanisms is thought to be classical receptor
internalization into the endocytic compartment (Hasse et al., 2003; Smyth et al., 2003; Smyth
et al., 2000; Reid et al., 2010).
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In order to overcome the aforementioned pharmacological limitations associated with PGI
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structures, Actelion/Nippon Shinyaku have developed a selective, oral non-prostanoid IP
receptor agonist, selexipag (2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-
(methylsulfonyl)acetamide, previously known as NS-304) (Asaki et al., 2015), which has
been recently approved in the US, the EU and Japan for the treatment of PAH (Sitbon et al.,
2015). After systemic absorption, selexipag, the parent drug, is hydrolyzed to the highly
potent metabolite ACT-333679 ({4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}acetic
acid, previously known as MRE-269), the major driver of clinical efficacy (Figure 1) (Kuwano
et al., 2007; Kuwano et al., 2008). ACT-333679 differentiates favorably from PGI2 analogs in D
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terms of its higher metabolic stability and its high IP receptor selectivity. To date, the in vitro lo
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activities of selexipag and in particular its active metabolite, ACT-333679, using PAH- fro
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relevant assays in PASMC have not been reported. Furthermore, it is currently not known e
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how ACT-333679 compares with PGI2 analogs regarding its IP receptor internalizing and tjo
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desensitizing properties. ls
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In the present study we compared the activity of ACT-333679 with that of the prototypic PGI2 at A
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analog iloprost by measuring diverse PAH-relevant parameters in PASMC such as cellular E
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relaxation, proliferation and extracellular matrix synthesis and found both compounds to be rn
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full agonists. We then uncovered unique partial agonism of ACT-333679 in receptor proximal n M
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readouts such as induction of cAMP synthesis, recruitment of -arrestin and IP receptor 1
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internalization. This partial agonism is in contrast to the full agonism displayed by iloprost 2
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and other PGI analogs (beraprost, treprostinil). Finally, the propensity of IP receptor
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agonists to induce tachyphylaxis was tested in vivo comparing selexipag with the PGI
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analog treprostinil. Our data suggest that the unique partial agonism exhibited by ACT-
333679 on receptor-proximal parameters reduces its potential for desensitization, while still
reaching maximal effects on downstream parameters relevant to PAH.
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MATERIALS AND METHODS
Compounds. Selexipag and ACT-333679 were synthesized by Nippon Shinyaku Co. Ltd
(Kyoto, Japan). Beraprost, iloprost and treprostinil were purchased from Cayman Chemical
(Ann Arbor, MI, USA).
Cell culture. For use in immunofluorescence microscopy, CHO-K1 cells expressing the
human IP receptor (CHO-hIP) were cultured in Ham’s F12 containing 10 % FBS heat-
inactivated (Brunschwig, Basel, Switzerland), 100 U/ml penicillin, 100 µg/ml streptomycin,
and 600 µg/ml geneticin (Life Technologies, Zug, Switzerland). CHO-hIP PathHunter® cells
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for beta-arrestin assays (DiscoveRx, Birmingham, UK) were cultured in Ham’s F12 nlo
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containing 10 % FBS heat-inactivated, 100 U/ml penicillin, 100 µg/ml streptomycin, 300 µg/ml fro
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hygromycin (Life Technologies, Zug, Switzerland) and 800 µg/ml geneticin. jp
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The human IP receptor-expressing cell pool T-RExTM-HEK-293-CMV.TO.FLAG-humanIP-C9 tjo
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(abbreviated T-REx-HEK-hIP) was generated by integrase-mediated homologous .o
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recombination of the IP receptor sequence into the T-RExTM HEK293 background. This t A
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expression system allows the tetracycline-inducible expression of genes of interest from one J
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defined insertion site and the generated cells were used (i) in cAMP experiments to evaluate als
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cAMP responses to IP receptor agonists at high and low expression levels and (ii) in flow M
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cytometry to observe agonist-induced IP receptor internalization. Parental T-RExTM-HEK293 11
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cells (Life Technologies, Zug, Switzerland) were cultivated in growth medium (DMEM + 3
GlutaMAXTM-I (Life Technologies, Zug, Switzerland), 10 % dialyzed FBS (Life
Technologies, Zug, Switzerland), 100 U/mL penicillin, 100 µg/mL streptomycin, 0.1 mg/ml
hygromycin B (Life Technologies, Zug, Switzerland), 5 g/ml blasticidin (Life
Technologies, Zug, Switzerland) and 1 % non-essential amino acid solution (Life
Technologies, Zug, Switzerland)). Recombinant T-REx-HEK-hIP cells were cultivated in
selection medium (growth medium containing 1 mg/ml Geneticin (Life Technologies, Zug,
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Switzerland) instead of hygromycin. Every second day the medium was exchanged with
fresh selection medium.
Human proximal pulmonary arterial smooth muscle cells (PASMC) (Cat.No.CC-2581
LotNo.0000200208, male, Lonza, Basel, Switzerland) were cultivated and propagated in
complete cell growth medium (SmBM, supplemented with SmGM-2 SingleQuots (0.2 %
hEGF, 0.1 % insulin, 0.2 % hFGF-B, 5 % FBS and 0.1 % gentamicin/amphotericin-B (Lonza,
Basel, Switzerland)) up to passage 4. For measurements related to proliferation and
extracellular matrix synthesis ([3H]-thymidine incorporation, p27(Kip1) and cyclin D1
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immunoblotting, [3H]-proline incorporation) cells were kept in this proliferation medium. For o
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measurements related to shape change (impedance, tomographic microscopy, cAMP and ad
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MLCK phosphorylation), cells were treated for 48 h with differentiation medium (SmBM with 4 from
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% FBS heat-inactivated (Brunschwig, Basel, Switzerland), 100 g/mL heparin sodium salt t.a
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(Tocris, Zug, Switzerland), 100 g/mL penicillin/streptomycin (Life Technologies, Zug, u
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Switzerland)). Heparin induces differentiation of vascular smooth muscle cells to the .org
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contractile phenotype characterized by a mature contractile apparatus (Beamish et al., S
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2010). Prior to stimulation with IP receptor agonists, differentiation or growth factors, cells Jo
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were brought into the quiescent state by cultivation in starvation medium (SmBM, 20 mM ls
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HEPES 100 g/mL penicillin/streptomycin (Life Technologies, Zug, Switzerland), 0.1 % a
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BSA, fatty acid-free (Calbiochem, San Diego, CA, USA)). Cells were used up to passage 4 , 2
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and harvested for seeding for the various assays at approximately 90 % cell density.
Impedance assays. PASMC were seeded into E-plates (ACEA Biosciences, San Diego,
CA, USA) at 5,000 cells/well and placed into the xCELLigance MP device (ACEA
Biosciences, San Diego, CA, USA). The next day, medium was exchanged for differentiation
medium. After 48 hours, cells starved for 6 hours in starvation medium and then subjected to
IP receptor agonists diluted in starvation medium and further cultivated for several hours.
Impedance measurements were performed during the whole experimental period. For
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analysis, impedance traces were aligned at the last time point before IP receptor agonist
addition, the vehicle baseline was subtracted and impedance minima within the first 3 hours
after agonist addition were used to generate concentration-response curves. EC and E
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values were calculated with the proprietary IC witch software using the compound intrinsic
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curve maximum and minimum as plateau values. Compound efficacies were compared to
the maximal efficacy of iloprost (100 %). For the ET-1/IP receptor agonist combination
experiments, cells were prepared as above and after the starvation period were stimulated
with 10 nM ET-1 (diluted in starvation medium), observed for 60 min until a response plateau
was reached and then stimulated with a dilution series of ACT-333679 or iloprost in D
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starvation medium, again followed by an observation period. For analysis, impedance traces lo
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were aligned at the last time point before ET-1 addition, the vehicle-vehicle baseline was fro
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subtracted and impedance minimum 60 min after IP receptor agonist addition were used to e
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generate concentration-response curves. tjo
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Tomographic microscopy. Human PASMC were seeded into 35 mm tissue culture dishes
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(FluoroDish, World Precision Instruments Inc., Sarasota, FL, USA) at 15,000 cells/dish, J
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grown overnight and then treated for 24 hours in differentiation medium. Then cells were ls
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starved for 3 hours by exchange for starvation medium with 20 mM HEPES. Dishes were a
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then placed on the tomographic microscope (3D Explorer, NanoLive, Lausanne, Switzerland) 1
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and 3D tomographic images (z-stacks) were taken at regular intervals (2 min). Within the
observation period, cells were treated with ET-1 (100 nM) for ~90 min followed by ACT-
333679 (1 M) or vehicle. The plane with the best focus was picked from every z-stack and
used to generate time lapse sequences. In order to get a smooth motion movie, the number
of frames were increased by a combination of motion interpolation and frame blending. In
brief, a similarity distance between pixels in different frames was defined by making use of
the intensities of surrounding pixels. A motion vector was defined from the position of each
pixel in frame "n" to the closest pixel in frame "n+1", based on the similarity distance. This
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vector allowed a smooth interpolation which was used to increase the frame-rate from 0.2 to
60 frames per second. Further post-processing steps like noise reduction, gamma and
contrast enhancement were applied to the final rendering of the video.
Immunoblotting. Isogenic T-REx-HEK-hIP cells were seeded at 15,000 cells/well into 96-
well plates, and supplemented after attachment with different concentrations of tetracycline
between 0 and 10 ng/mL to induce increasing IP receptor expression levels. After a 24 hour
induction period, cells were washed with phosphate buffered saline (PBS) and then lysed on
ice in 15 L/well of RIPA buffer (Sigma Aldrich, Buchs, Switzerland) containing 100 mM NaF,
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4 mM Na-orthovanadate, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM dithiothreitol w
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(DTT) and 100 U/mL benzonase (Sigma Aldrich, Buchs, Switzerland). The contents of one de
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well (15 L volume containing 10 g protein) were analyzed by SDS-PAGE and Western m
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blotting using mouse-anti-human IP receptor monoclonal antibody 13-Q (1 g/ml, Santa Cruz s
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Biotechnology Inc., Dallas, TX, USA) and HRP-coupled anti-mouse IgG secondary rn
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antibodies (GE Life Sciences, Glattbrugg, Switzerland). Membranes were treated with rg
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Western Lightning®-Enhanced Chemiluminescence Substrate (Perkin Elmer, S
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Schwerzenbach, Switzerland), and the chemiluminescence signal was recorded and o
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quantified using a chemiluminescence reader (LAS-4000) (Fujifilm, Tokyo, Japan) and the ls o
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corresponding software (Multigauge V3.0, Fujifilm). rc
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Human PASMC were seeded at 10,000 cells per well into 96-well plates that were pre- 02
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coated with fibronectin. The next morning the growth medium was exchanged for
differentiation medium and after 48 hours cells were switched for 6 hours to starvation
medium. The cells were then stimulated for 90 min at 37 oC with dilution series of IP receptor
agonists (in starvation medium; +/- 100 nM ET-1; for MLCK and MLC phosphorylation) or
with dilution series of IP receptor agonists for 24 hours at 37 oC (in starvation medium; +/- 50
ng/ml PDGF-BB; for cyclinD1 and p27/Kip1). Cells were lysed in RIPA buffer containing 100
mM NaF, 4 mM Na-orthovanadate, 1 mM PMSF, 1 mM DTT and 100 U/mL benzonase and
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