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CELLULAR AND MOLECULAR

In Vitro and in Vivo Pharmacological Characterization of Ethyl-4-{trans-4-[((2S)-2-hydroxy-3-{4-hydroxy-3[(methylsulfonyl)amino]-phenoxy}propyl) Amino]cyclohexyl}benzoate Hydrochloride (SAR150640), a New Potent and Selective Human ₃-Adrenoceptor Agonist for the Treatment of Preterm Labor

Exploratory Research Department, Sanofi-Midy Research Center, sanofi-aventis S.p.A., Milan, Italy (T.C., R.C., P.M., C.R., N.V., F.G.); Exploratory Research Department, sanofi-aventis Recherche & Development, Toulouse, France (M.P.); Drug Safety Evaluation, Vitry Sur Sein, Paris, France (Y.F.); DMPK, Montpellier, France (L.D.); Laboratoire de Physiopathologie et Pharmacologie Cardiovasculaires, Facultéde Médecine, Dijon, France (M.B.); Institut National de la Recherche Agronomique, Biologie du Développement et Reproduction, Centre de Recherches de Jouy, Jouy-en-Josas, France (G.G.); Institut National de la Santé et de la Recherche Médicale, Unité 767, Paris, France (M.B.-F., M.-J.L.); and Département de Pharmacologie, UPRES EA220, UFR des Saints Pères, Paris, France (C.A.)

Received December 27, 2006; accepted March 7, 2007.

	Abstract

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Ethyl-4-{trans-4-[((2S)-2-hydroxy-3-{4-hydroxy-3[(methylsulfonyl)amino] phenoxy}propyl) amino]cyclohexyl}benzoate hydrochloride (SAR150640) was characterized as a new potent and selective ₃-adrenoceptor agonist for the treatment of preterm labor. SAR150640 and its major metabolite, the corresponding acid 4-{trans-4-[((2S)-2-hydroxy-3-{4-hydroxy-3[(methylsulfonyl) amino] phenoxy}propyl)amino]cyclohexyl}benzoic acid (SSR500400), showed high affinity for ₃-adrenoceptors (K_i = 73 and 358 nM) and greater potency than (-)-isoproterenol in increasing cAMP production in membrane preparations from human neuroblastoma cells (SKNMC), which express native ₃-adrenoceptors (pEC₅₀ = 6.5, 6.2, and 5.1, respectively). SAR150640 and SSR500400 also increased cAMP production in membrane preparations from human uterine smooth muscle cells (UtSMC), which also express native ₃-adrenoceptors (pEC₅₀ = 7.7 and 7.7, respectively). In these cells, SAR150640 dose-dependently inhibited oxytocin-induced intracellular Ca²⁺ mobilization and extracellular signal-regulated kinase 1/2 phosphorylation. SAR150640 and SSR500400 had no ₁- or ₂-agonist or antagonist activity in guinea pig atrium and trachea, or in human isolated atrium and bronchus preparations. Both compounds concentration-dependently inhibited spontaneous contractions in human near-term myometrial strips, with greater potency than salbutamol and 4-[3-[(1,1-dimethylethyl)-amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (CGP12177) (pIC₅₀ = 6.4, 6.8, 5.9, and 5.8, respectively), but with similar potency to (-)-isoproterenol and atosiban (oxytocin/vasopressin V₁a receptor antagonist). SAR150640 also inhibited the contractions induced by oxytocin and prostaglandin F₂. In vivo, after intravenous administration, SAR150640 (1 and 6 mg/kg), but not atosiban (6 mg/kg), dose-dependently inhibited myometrial contractions in conscious unrestrained female cynomolgus monkeys, with no significant effects on heart rate or blood pressure. In contrast, salbutamol (50 and 250 µg/kg) had no inhibitory effect on uterine contractions, but it dose-dependently increased heart rate. These findings indicate a potential for the therapeutic use of SAR150640 in mammals during preterm labor.

The discovery of the ₃-adrenoceptor has stimulated the search for potent and selective ₃-adrenoceptor agonists for the treatment of various metabolic and nonmetabolic diseases (Hollenga et al., 1991; Bloom et al., 1992; Cecchi et al., 1994). However, most of these "first generation" agonists, although effective in rodents as antiobesity, antidiabetic, and smooth muscle relaxing agents, failed to achieve similar effects in humans. Although a number of new generation human selective ₃-adrenoceptor agonists have been developed in the past 10 years, and some are currently under clinical evaluation (Fisher et al., 1998; Sennitt et al., 1998; Konkar et al., 1999; Mathvink et al., 2000; Tanaka et al., 2001), none is yet available for the treatment of obesity, diabetes, or diseases of the genito-urinary or gastrointestinal system. We and others have reported the existence of functional ₃-adrenoceptors in the human myometrium (Bardou et al., 2000; Dennedy et al., 2001), where the ₃-adrenoceptor is predominant over the ₂-adrenoceptor, and, moreover, ₃-adrenoceptor expression increases at the end of pregnancy (Rouget et al., 2005). The ₃-adrenoceptor agonist SR59119A was more efficient than salbutamol in vitro in inhibiting human near-term myometrial spontaneous contractions (Bardou et al., 2000). In addition, the myometrial ₃-adrenoceptor seems to be resistant to agonist-induced desensitization (Rouget et al., 2004). By contrast, ₂-adrenoceptor expression is either decreased (Breuiller et al., 1987; Chanrachakul et al., 2003) or unchanged (Gsell et al., 2000) at the end of pregnancy, and there is a loss of response to ₂-mimetics after chronic exposure to these drugs (Berg et al., 1983). This might partly explain the lack of efficacy of ₂-mimetics at the end of pregnancy. In a series of newly synthesized molecules, we found, as a general rule, that compounds with an aryloxypropanolamine chemical structure were more potent agonists at human ₃-adrenoceptors than the corresponding derivatives based on a phenylethanolamine structure, typically present in the earlier agonists.

In the present study, we investigated the in vitro and in vivo biochemical and pharmacological profile of SAR150640 and its major metabolite SSR500400 (Fig. 1). SSR500400 is metabolically stable, and it is formed by rapid and extensive de-esterification of SAR150640 by human plasma esterases. The in vivo uterine and cardiovascular effects of SAR150640 were also studied in conscious unrestrained monkeys, using a telemetric recording system (Germain et al., 1986; Carbonne et al., 1998).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Chemical structures of SAR150640 and SSR500400.

	Materials and Methods

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Cells Cultures. SKNMC (ATTC HTB-10; American Type Culture Collection, Manassas, VA) were grown in 5% CO₂ atmosphere (37°C) in Eagle's minimal essential medium supplemented with 5% fetal calf serum, 450 µM sodium pyruvate, 40 µg/ml proline, and 10 µg/ml gentamicin. Human uterine smooth muscle cells (UtSMC) from Cambrex (East Rutherford, NJ) were grown in 5% CO₂ atmosphere (37°C) in SmGM-2 medium supplemented with 0.5 µg/ml human epidermal growth factor, 1 µg/ml human fibroblast growth factor, 5 mg/ml insulin, 5% fetal calf serum, and 10 µg/ml gentamicin. Culture medium was removed every other day, and cells were subcultured by treatment with 0.05% trypsin and 0.02% EDTA.

Membrane Preparations from Cell Lines. Cells were suspended in 2 mM Tris-HCl/2 mM EGTA. The cell suspension was placed in an ice bath and homogenized with a Potter Elvejhem homogenizer. The homogenates were centrifuged at 1500g for 5 min at 4°C. The resulting supernatants were centrifuged at 48,000g for 20 min at 4°C. The final pellet was resuspended in 80 mM Tris-HCl, and aliquots of membranes were used immediately. The protein content of the membrane fraction was determined by the Bradford method (Bradford, 1976).

-Adrenoreceptor Binding Studies. SAR150640 and SSR500400 were tested at different concentrations (10 nM–10 µM). Binding of 0.03 nM [¹²⁵I]cyanopindolol to membranes of CHO K-1 cells expressing human ₁-adrenoceptor was studied in an incubation buffer containing 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 1.5 mM CaCl₂, 120 mM NaCl, 1.4 mM ascorbic acid, and 10 mg/ml bovine serum albumin (Tris buffer). Nonspecific binding was determined in the presence of 100 µM(-)-propranolol; the reaction was started by addition of the membranes and followed by incubation for 2 h at 25°C. Binding of 0.2 nM [³H]CGP12177 to membranes of CHO expressing human ₂-adrenoceptor in Tris buffer was also investigated. Nonspecific binding was determined in the presence of 10 µM ICI 118551; the reaction was started by addition of the membranes, followed by incubation for 1 h at 25°C. Binding of 0.5 nM [¹²⁵I]cyanopindolol to membranes of HEK-293 cells expressing human ₃-adrenoceptors (isoform C of 408 amino acids) was measured in Tris buffer. Nonspecific binding was determined in the presence of 1 mM alprenolol; the reaction was started by addition of the membranes and followed by incubation for 90 min at 25°C. Specific binding was 95, 95, and 80% for ₁-, ₂-, and ₃-adrenoceptors, respectively. Binding studies on -adrenoceptors were done by MDS Pharma Services (Pharmacology Laboratories, Taipei, Taiwan, Republic of China)

cAMP Production in Membranes from Human SKNMC and UtSMC. cAMP production was assessed with membrane preparations (20 µg of proteins) incubated for 20 min at 37°C in the incubation buffer (120 mM Tris-HCl, 20 mM phosphocreatine, 5 U of creatine phosphokinase, 3.5 mM MgCl₂, 0.5 mM 3-isobutyl-1-methyl, 1 mM ATP, 0.1 mM GTP, and 1 mM dithiothreitol, pH 7.4) in a final volume of 100 µl. Different concentrations of (-)-isoproterenol, CGP12177 (only in SKNMC study), SAR150640, and SSR500400 or their respective solvents were also added to the incubation medium in presence or absence of the selective ₁- and ₂-adrenoceptor antagonists CGP20712 and ICI 118551 (both at 1 µM). The production of cAMP was assessed by a commercial kit (RPN225; GE Healthcare, Little Chalfont, Buckinghamshire, UK) and expressed as (-)-isoproterenol maximal effect. Forskolin (1 µM) was used as internal reference.

[³⁵S]GTPS Binding in Membrane Preparations from Human UtSMC. The binding of [³⁵S]GTPS (37 TBq/mmol, SJ 1320; GE Healthcare) was determined with human UtSMC membranes preparations (20 µg protein/well) using the SPA G protein-coupled receptor assay kit (RPNQ0210; GE Healthcare). [³⁵S]GTPS (4 nM) and 5 µM GDP were incubated in assay buffer (2 mM HEPES, 10 mM NaCl, 1 mM MgCl₂, 0.1 mM EDTA, pH 7.4, and 58 µM dithiothreitol) for 30 min at room temperature in the presence of different concentrations of SAR150640 or SSR500400. The incubation was performed in 96-well OptiPlate: MicroScint-20 (PerkinElmer Life and Analytical Sciences, Boston, MA) was added to each well (final volume 200 µl), and the plate was analyzed by a TopCount (PerkinElmer Life and Analytical Sciences, Boston, MA). Nonspecific binding was defined as binding in the presence of 50 µM GTPS.

ERK1/2 Phosphorylation. Human UtSMC (Cambrex) were incubated with different concentrations of SAR150640 for 10 min. In another set of experiments, to induce ERK activation, human Ut-SMC were incubated with 50 nM oxytocin for 10 min, before addition of 1 µM SAR150640 and incubation for another 10 to 20 min. At the end of the incubation, the culture medium was removed, and the cells were scraped into phosphate-buffered saline buffer and centrifuged. The cells pellet was extracted using the Nuclear Extract kit (40010; Active Motif Inc., Carlsbad, CA). Cells were lysed in complete lysis buffer [20 mM HEPES, 350 mM NaCl, 20% glycerol, 1% Triton X-100, 1 mM MgCl₂, 0.5 mM EDTA, 0.1 mM EGTA, and protease inhibitor cocktail (P-2714; Sigma-Aldrich, St. Louis, MO)] for 10 min on ice, under gentle agitation. The lysate was then centrifuged 20 min at 14,000g at 4°C. The supernatant was processed for SDS-polyacrylamide gel electrophoresis and Western blotting. Phospho-ERK1/2 and total ERK1/2 were detected with a rabbit anti-phospho-ERK 1/2 (9101; Cell Signaling Technology Inc., Danvers, MA) and a rabbit anti-total-ERK1/2 (9102; Cell Signaling Technology Inc.). Immunobands were detected by chemiluminescence and analyzed with the imaging system from Total Lab (Nonlinear Dynamics, Newcastle upon Tyne, UK).

Intracellular Ca²⁺. Human UtSMC were plated onto 96-well plates at 20,000 cells/well in SmGM-2 medium and grown for 2 to 3 days. The cells were incubated with 450 µM Fluo-4 AM (Invitrogen, Carlsbad, CA), 1% Pluronic acid (Sigma-Aldrich), and 25 mM probenecid (Sigma-Aldrich) at 37°C in 5% CO₂ for 1 h in the dark. Cells were then washed three times with Hanks' buffer containing 20 mM HEPES, 1 mM CaCl₂, 1 mM MgSO₄, and 2.5 mM probenecid. Fluo-4 calcium transient fluxes were measured with a spectrofluorometer (VictorV; PerkinElmer Life and Analytical Sciences) at 30°C (excitation 485 nm, emission 535 nm) over a period of 120 s after injection of 50 nM oxytocin (oxytocin concentration-dependently mobilized Ca²⁺ from the internal stores with an IC₅₀ of 34 nM). SAR150640 was administered 30 min before the oxytocin. Each concentration of SAR150640 was investigated in quadruplet. The response to oxytocin was evaluated as the area under the curve (AUC).

Functional Studies with Human Myometrial Strips. Myometrial biopsies were obtained from pregnant women delivered by elective caesarean section in the third trimester of pregnancy. All the patients had received an intravenous perfusion of oxytocin after delivery, but before excision of the sample. Myometrial strips were excised from an immediately subserosal site where the majority of the fibers are oriented longitudinally, at an antiplacental site. This study was approved by the "Comité Consultatif de Protection des Personnes pour la Recherche Biomédicale" (Dijon Hospital and Cochin Port-Royal Hospital-Paris, France), and all donors provided informed consent. Segments of myometrium (8–12 mm in length, 2–3 mm in thickness) were suspended isometrically under a resting tension of 2 g in a 10-ml organ bath containing a Krebs' solution (composition 118 NaCl mM, 5.4 mM KCl, 2.5 mM CaCl₂, 0.6 mM KH₂PO₄, 1.2 mM MgSO₄, 25 mM NaHCO₃, 11.7 mM glucose, and 0.1 mM ascorbic acid) maintained at 37°C, and they were continuously exposed to a mixture of 95% oxygen and 5% carbon dioxide, pH 7.4. One end of each strip was connected to a force displacement transducer, and tension changes were measured and recorded using IOX software (EMKA, Paris, France). After 1 h, during which the myometrial strips were washed every 15 min and the resting tension was readjusted to 2 g, the strips were allowed to equilibrate for another hour until they showed regular spontaneous contractile activity. Once contractions became regular in amplitude and frequency, cumulative concentration-response curves (CRC) (0.01–30 µM) were plotted, to characterize the effects of SAR150640 and SSR500400, salbutamol, (-)-isoproterenol, CGP12177, or atosiban. The exposure time for each additional concentration was around 20 to 40 min, the time necessary to reach a plateau of contractions. The isoproterenol maximal effect (E_max) was determined as the percentage of inhibition of the initial amplitude of spontaneous contractions. The drugs intrinsic effect (IA) was calculated versus isoproterenol maximal response taken as 100%. In a second set of experiments, once contractions had become regular, 1 µM prostaglandin PGF₂ or 1 nM oxytocin submaximal concentration was added to the bath, before plotting the CRC for SAR150640. To study -adrenoceptor desensitization, myometrial strips were incubated for 15 h, at room temperature, in preoxygenated Krebs' solution (composition as described above) containing either 10 µM SAR150640 or 10 µM salbutamol, or their solvents.

In Vivo Experiments in Cynomolgus Monkeys. Mature cyclic cynomolgus females (Macaca fascicularis, at least 3.5 years old, weighing 3.8–6.5 kg) were used for the in vivo studies. The experiments were performed in accordance with internationally accepted principles for care of laboratory animals (EEC Council Directive 86/609, OJ L 358, 1, December 12, 1987) and with the approval of a local ethical committee. The animals were housed under controlled environmental conditions (22 ± 1°C, 70 ± 5% relative humidity, and 12-h light from 6:30 AM to 6:30 PM) with water and food ad libitum. They received a daily supply of pellets for primates (SDS, Vigny, France) supplemented with varied fruits. The menstrual cycles were assessed by examining a vaginal smear with a cotton bud from day 20 of the cycle until the appearance of menstrual bleeding, which lasts on average 3 to 4 days. The mean duration of menstrual cycles is 28 days with ovulation usually occurring on days 11 to 12 of the cycle. After detection of their cycles, three animals were implanted at the beginning of the follicular phase (first part of the cycle) with a telemetric sensor for chronic recording of the electrocardiogram (ECG) and intrauterine pressure (IUP) (Chellman et al., 2004). Under general anesthesia, a telemetric device (TL11M3-D70-CCP; Data Sciences International, St. Paul, MN) was placed in the flank of the animal. The distal tip of the pressure catheter was inserted into the uterine cavity through the fundus after puncturing the uterine wall with a 14-gauge needle. A couple of electrodes fixed, through a subcutaneous tunnel, on the chest, were used for ECG recording. After surgery the animals received analgesics, and antibiotics to prevent infection. After 7 days of recovery, the monkeys were placed in a cage equipped with a receiving antenna (RLA3000; Data Sciences International) for IUP and ECG recordings. Radiosignals were transcoded to an analogical signal by the receiving system (R11CPA; Data Sciences International), and then they were digitized in real time (200 Hz) and stored on a computer. Data acquisition and replay was processed by NI-DAQ hardware and BioBench software 1.2 (National Instruments, Austin, TX). Physiological parameters were recorded daily (9:00 AM–5:00 PM). Because the control group showed clear circadian changes in uterine motility in this period, the effect of treatment was also compared with the uterine motility recordings obtained in controls, on the even hours of each day. Uterine contractions digitized at the final sampling rate of 3 Hz were exported and the AUC of each hour of IUP recording was calculated after subtraction of the basal tone. Heart rate was measured directly with the BioBench 1.2 software every 5 min (12 times/h) and averaged for every hour.

Drugs were injected as an i.v. bolus in the external saphenous vein. The volume injected did not exceed 1.2 ml. Two studies were performed. In the first study, two doses of SAR150640 (0.3 and 1 mg/kg) and salbutamol (50 and 250 µg/kg) were tested. In the second study, the effect of an i.v. bolus of 6 mg/kg atosiban (Tractocile) was tested and compared with the effect of an equivalent i.v. dose of SAR150640. SAR150640 was dissolved immediately before injection in 0.1% ascorbic acid at pH 5.5. Control monkeys received no drug treatment ("sham injection").

Data Analysis. Data are expressed as the mean ± S.E.M., and they were analyzed using analysis of variance followed by the Dunnett or Newman-Keuls test, as indicated in the figure legends. A probability level less than 0.05 was accepted as significant. Potency was expressed as pIC₅₀, or pEC₅₀, which are the -log IC₅₀ or -log EC₅₀, where IC₅₀ or EC₅₀ is the concentration of the agonist producing half its maximal response. In binding studies, IC₅₀ was calculated by nonlinear, least-squares regression analysis using an internal software Biost@t-SPEED v1.3 using the four-parameter logistic model according to Ratkowsky and Reedy (1986). The adjustment was obtained by nonlinear regression using the Marquardt algorithm in SAS version 8.2 (SAS Institute, Cary, NC) software under UNIX. The K_i values were calculated using the equation of Cheng and Prusoff (1973).

Chemicals. SAR150640 and SSR500400 were synthesized at the sanofi-aventis Research Center (Milan, Italy). ICI 118551, CGP20712, CGP12177, pentobarbital sodium, buffers, fetal calf serum, penicillin/streptomycin, gentamicin, oxytocin, salbutamol, isoproterenol, and PGF₂ were purchased from Sigma-Aldrich. [³⁵S]GTPS was from GE Healthcare. Tractocyle (commercial preparation of atosiban, 7.5 mg/ml) was from Ferring Pharmaceuticals Ltd. (Langley, UK). Ventolin (commercial preparation of salbutamol, 5 mg/5 ml, for in vivo studies) was from GlaxoSmithKline (Brentford, UK). The other chemicals were purchased from standard commercial sources. For in vitro studies, SAR150640 and SSR500400 were dissolved in dimethyl sulfoxide to obtain a 1 mM stock solution, and then they were diluted in water containing 0.1% ascorbic acid.

	Results

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Binding and in Vitro Selectivity. SAR150640 showed high affinity (K_i = 73 nM) for ₃-adrenoceptor binding sites (displacement of [¹²⁵I]cyanopindolol in membrane preparations from HEK-293 cells transfected with the ₃-adrenoceptor) and 20 to 50 times lower affinity for ₁- and ₂-adrenoceptor binding sites (Table 1). The metabolite SSR500400 also displaced [¹²⁵I]cyanopindolol from the ₃-adrenoceptor with approximately 5 times less affinity (K_i = 358) than the parent compound. SSR500400 up to 10 µM had no affinity for either ₁-or ₂-receptors.

View this table: [in this window] [in a new window]   TABLE 1 Comparative binding affinity (Ki) of SAR150640 and SSR500400 for human 3-, 2-, and 1-adrenoceptors Binding assay was performed in CHO cells expressing 2, 1-adrenoceptors or HEK-293 cells expressing 3-adrenoceptor. [125I]Cyanopindolol (0.03 nM for 1-adrenoceptor, 0.5 nM for 3-adrenoceptor) and [3H]CGP12177 (0.2 nM for 2-adrenoceptor) were used as radiolabeled ligands (see Materials and Methods for details). Values are mean ± S.E.M. of three assays.

The absence of affinity of both compounds for ₁- and ₂-adrenoceptors was confirmed by in vitro functional studies. At concentration up to 10 µM, they a) had no chronotropic or inotropic effects on isolated guinea pig and human atrium (₁-adrenoceptor response), b) did not relax guinea pig trachea or human bronchi (₂-adrenoceptor response), and c) did not prevent the action of -adrenoceptor agonists (isoproterenol or salbutamol) in these isolated preparations (data not shown).

To further evaluate their selectivity, SAR150640 and SSR500400 were tested in vitro at 1 and 10 µM on 100 targets (60 of them human) using receptor binding, ion channel binding, and cellular and enzymatic assays, including target known to interfere with human myometrium contractility. SAR150640 had no substantial activity in these assays, except for dopamine and noradrenaline transporter binding (IC₅₀ = 0.2–0.5 µM). However, no functional consequence could be detected in vivo and in vitro using known standard assays (i.e., no changes in noradrenaline response in vitro on rat and guinea pig isolated heart, and no behavioral or cardiovascular effects in monkeys or rats). SSR500400 had no effect on any in vitro assay up to 10 µM (data not shown).

In Vitro Functional Studies with Cells. The agonist properties of SAR150640 and SSR500400 were studied by measuring their ability to stimulate cAMP production in membrane preparations from human SKNMC, which express native human ₃-adrenoceptor (Table 2). In presence of the selective ₁- and ₂-adrenoceptor antagonists CGP20712 and ICI 118551 (both at 1 µM), SAR150640 and SSR500400 were more potent than (-)-isoproterenol (pEC₅₀ = 6.5 and 6.0 versus 5.1) with comparable efficacy. Unlike (-)-isoproterenol, SAR150640 and SSR500400 showed a similar potency and efficacy also in absence of the ₁- and ₂-adrenoceptor antagonists (Table 2). SAR150640 and SSR500400 showed similar potency but higher efficacy than the ₃-agonist, ₁- and ₂-antagonist CGP12177.

View this table: [in this window] [in a new window]   TABLE 2 SAR150640 and SSR500400-induced cAMP production in membrane preparations from SKNMC in absence or in presence of 1- and 2-antagonists CGP20712 + ICI 118551 (both at 1 µM) The IA is relative to the maximal effect of (–)-isoproterenol. Values are mean ± S.E.M. of four to five assays.

The stimulation of cAMP production by SAR150640 and SSR500400 was assessed on membrane preparations from isolated human myometrial cells that express native ₃-adrenoceptors (Fig. 2A). The two compounds showed similar potency (mean ± S.E.M.; pEC₅₀ = 7.7 ± 0.2 and 7.7 ± 0.1, respectively), but different efficacy (30 and 54% of 1 µM forskolin effect). The agonist activity of SAR150640 and SSR500400 was also assessed on [³⁵S]GTPS binding, pEC₅₀ (mean ± S.E.M.; 6.6 ± 0.1 and 6.6 ± 0.4, respectively) (Fig. 2B).

View larger version (24K): [in this window] [in a new window]   Fig. 2. Effects of SAR150640 and SSR500400 on cAMP production versus 1 µM forskolin effect (A) and GTPS binding (B) in human UtSMC membrane preparations. Mean ± S.E.M. of three or four experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Effect of SAR150640 on Ca2+ mobilization induced by 50 nM oxytocin in human UtSMC. Mean ± S.E.M. of seven experiments.

View larger version (27K): [in this window] [in a new window]   Fig. 4. Effect of SAR150640 on basal (A) or oxytocin-induced (B) ERK1/2 activation in human UtSMC. Mean ± S.E.M. of three or four experiments. For A, *, p < 0.05; **, p < 0.01 versus basal; °°, p < 0.01 versus SAR150640 10-7 M. For B, *, p < 0.05 versus oxytocin (Dunnett's test).

View larger version (17K): [in this window] [in a new window]   Fig. 5. Inhibition of spontaneous contractions in a preterm uterus strips: a representative tracing of SAR150640.

In near-term myometrial strips, SAR150640 inhibited the contractions evoked by 1 nM oxytocin and 1 µM PGF₂, with pIC₅₀ of 6.30 and 5.63, respectively (Fig. 6). The potential tolerance and cross-tolerance to the in vitro tocolytic action of SAR150640 was evaluated by preincubating human near-term myometrial strips for 15 h with 10 µM SAR150640 or 10 µM salbutamol (Fig. 7). The curves for inhibition of spontaneous contractions by SAR150640 were similar for nonincubated and SAR150640- or salbutamol-preincubated strips, indicating the absence of self-tolerance or salbutamol cross-tolerance for the myorelaxant action of SAR150640.

View larger version (25K): [in this window] [in a new window]   Fig. 6. Effect of SAR150640 on in vitro inhibition of spontaneous, oxytocin-, and PGF2-induced contractions in human near-term myometrium. Mean ± S.E.M. of three or four experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Effect of pretreatment (15 h) with 10 µM SAR150640 or salbutamol on the SAR150640-induced in vitro inhibition of human near-term spontaneous contractions. Mean ± S.E.M. of 10 specimens from five patients.

View larger version (30K): [in this window] [in a new window]   Fig. 8. Effect of an intravenous bolus of SAR150640 on uterine contractility (AUC of IUP activity) (A) and heart rate (B) in conscious unrestrained female primates. The percentage of resting state represents the mean value changes after the treatment relative to 1-h basal activity. A two-way analysis of variance with repeated measures is performed. Results are mean ± S.E.M. of four to nine experiments on three animals. *, p < 0.05; **, p < 0.01 versus basal (Newman-Keuls test).

A representative recording of uterine contractions from two female monkeys in the estrogenic or menstrual phase, treated with either SAR150640 or atosiban, is shown in Fig. 9. SAR150640 (6 mg/kg i.v.) substantially reduced the amplitude and frequency of uterine contractions for at least 3 h after injection. The same dose of atosiban only slightly inhibited the frequency of myometrial contractions. Prostration was only observed in one animal treated with atosiban. SAR150640 (6 mg/kg i.v.) had no effect on heart rate, blood pressure, or QT interval in these female monkeys (data not shown).

View larger version (68K): [in this window] [in a new window]   Fig. 9. Representative recordings of spontaneous uterine contractions after an intravenous bolus of 6 mg/kg SAR150640 or atosiban in conscious unrestrained female primates. A, top recording shows the effect of SAR150640 on the uterine contractions in estrogenic and menstrual phases. B, bottom recording shows the effect of atosiban on the uterine contraction in estrogenic and menstrual phases. The arrows correspond to the time of injection. These recordings are representative of two animals. One unit = 2.5 mm Hg.

	Discussion

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2

2

Should clinical trials confirm the results of the present study, this new ₃-adrenoceptor agonist will be the lead compound of a new class of therapeutic agents for the pharmacological management of premature delivery, which still constitutes an unmet medical need.

	Acknowledgements

 
We are indebted to the surgical teams of the Departments of Obstetrics at the university hospitals of CHU du Bocage (Prof. PaulSagot and co-workers, Dijon, France), Antoine Béclère (Prof. René Frydman and co-workers, Clamart, France), and Cochin Port-Royal (Prof. Dominique Cabrol and co-workers, Paris, France) for making myometrial tissues available to us and to Alberto Bianchetti for assistance in manuscript preparation. Special thanks go to Catherine Loustalot-Bardou who suggested assessing the presence of ₃-adrenoceptors in human myometrium.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.119123.

ABBREVIATIONS: SAR150640, ethyl-4-{trans-4-[((2S)-2-hydroxy-3-{4-hydroxy-3[(methylsulfonyl)amino]phenoxy}propyl) amino]cyclohexyl}benzoate hydrochloride; SSR500400, 4-{trans-4-[((2S)-2-hydroxy-3-{4-hydroxy-3[(methylsulfonyl) amino] phenoxy}propyl)amino]cyclohexyl}benzoic acid; SKNMC, human neuroblastoma cells; UtSMC, human uterine smooth muscle cell(s); CHO, Chinese hamster ovary; CGP12177, 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride; ICI 118551, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol; HEK, human embryonic kidney; GTPS, guanosine 5'-O-(3-thio)triphosphate; ERK, extracellular signal-regulated kinase; AUC, area under the curve; IA, intrinsic activity; CRC, concentration-response curve(s); ECG, electrocardiogram; IUP, intrauterine pressure; SR59119A, (1R)-1-(3-chlorophenyl)-2({[(2R)-7-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl]methyl}amino)ethanol hydrochloride; CGP20712, (±)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate salt.

Address correspondence to: Dr. Tiziano Croci, Sanofi-Midy Research Center, sanofi-aventis, S.p.A., Via G. B. Piranesi, 38, 20137 Milan, Italy. E-mail: tiziano.croci{at}sanofi-aventis.com

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