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CARDIOVASCULAR

In Vitro and in Vivo Pharmacological Characterization of BM-613 [N-n-Pentyl-N'-[2-(4'-methylphenylamino)-5-nitrobenzenesulfonyl]urea], a Novel Dual Thromboxane Synthase Inhibitor and Thromboxane Receptor Antagonist

Natural and Synthetic Drugs Research Centre, Department of Pharmacy, Laboratory of Medicinal Chemistry, University of Liège, Liège, Belgium (J.H., J.-M.D., B.P.); Department of Pharmacy, University of Namur, Namur, Belgium (S.R., B.M.); Programme in Integrative Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (D.R., N.Q., C.P.-A.); Department of Biochemistry, Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Ireland (L.P.K., H.M.R., B.T.K.); and Department of Biological Sciences, Imperial College, London, United Kingdom (F.V., J.T.)

Received October 15, 2004; accepted November 18, 2004.

	Abstract

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Thromboxane A₂ (TXA₂) is a key mediator of platelet aggregation and smooth muscle contraction. Its action is mediated by its G protein-coupled receptor of which two isoforms, termed TP and TP, occur in humans. TXA₂ has been implicated in pathologies such as cardiovascular diseases, pulmonary embolism, atherosclerosis, and asthma. This study describes the pharmacological characterization of BM-613 [N-n-pentyl-N'-[2-(4'-methylphenylamino)-5-nitrobenzenesulfonyl]urea], a new combined TXA₂ receptor antagonist and TXA₂ synthase inhibitor. It exhibits a strong affinity for human platelet TP receptors (IC₅₀ = 1.4 nM), TP and TP expressed in COS-7 cells (IC₅₀ = 2.1 and 3.1 nM, respectively), and TPs expressed in human coronary artery smooth muscle cells (IC₅₀ = 29 µM). BM-613 shows a weak ability to prevent contraction of isolated rat aorta (ED₅₀ = 1.52 µM) and guinea pig trachea (ED₅₀ = 2.5 µM) induced by TXA₂ agonist U-46619 (9.11-dideoxy-9.11-methanoepoxy-prostaglandin F₂). Besides, BM-613 antagonizes TP (IC₅₀ = 0.11 µM) and TP (IC₅₀ = 0.17 µM) calcium mobilization induced by U-46619 and inhibits human platelet aggregation induced by U-46619 (ED₅₀ = 0.278 µM), arachidonic acid (ED₅₀ = 0.375 µM), and the second wave of ADP. BM-613 also dose dependently prevents TXA₂ production by human platelets (IC₅₀ = 0.15 µM). In a rat model of ferric chloride-induced thrombosis, BM-613 significantly reduces weight of formed thrombus by 79, 49, and 28% at 5, 2, and 1 mg/kg i.v., respectively. In conclusion, BM-613 is a dual and potent TP receptor antagonist and TXA₂ synthase inhibitor characterized by a strong antiplatelet and antithrombotic potency. These results suggest that BM-613 could be a potential therapeutic drug for thrombotic disorders.

TXA₂ is a metabolite of arachidonic acid (AA), a 20-carbon essential fatty acid stored in membrane phospholipids. Free AA can be released mainly by phospholipase A₂ upon stimulation. TXA₂ is synthesized in two steps: first the action of cyclooxygenase (COX) on free AA leads to the formation of prostaglandin H₂ (PGH₂) which can be subsequently metabolized by thromboxane synthase (TXS) into TXA₂.

COX exists in two main isoforms, COX-1 and COX-2, which are encoded by two separate genes. COX-1 is expressed constitutively in most tissues mediating "housekeeping" functions, whereas COX-2 expression is mainly induced at sites of inflammation by various stimuli (Dogné et al., 2004a). The second is also expressed in a constitutive manner in endothelium (Cheng et al., 2002). TXA₂ synthesis mainly occurs in platelets where COX-1 and thromboxane synthase are highly expressed.

TXA₂ actions are mediated by its G protein-coupled receptor, referred to as the TP receptor (Coleman et al., 1994). In 1991, Hirata et al. reported that human TP receptor was coded by one gene (Hirata et al., 1991), and in 1994, Raychowdhury et al. highlighted the existence of an isoform generated by alternative splicing (Raychowdhury et al., 1994). The two isoforms, named TP and TP, share the same first 328 amino acids but differ by the length of their carboxy-terminal tails (15 amino acids of the isoform being replaced by 79 amino acids in the isoform).

Given the wide implications of TXA₂ in several pathologies, studies have been undertaken to find therapeutic agents able to counteract the negative effects of TXA₂. Indeed, therapeutic strategies include blocking the synthesis of TXA₂ by the inhibition of TXS, antagonizing its action at the receptor level, or both. Thus, several TP receptor antagonists (TXRAs), such as sulotroban (Stegmeier et al., 1984), thromboxane synthase inhibitors (TXSIs), such as furegrelate (Gorman et al., 1983), and drugs combining both properties, such as ridogrel and terbogrel, have been developed. Dual compounds proved to be more interesting in therapeutics and promising antithrombotic agents (Dogné et al., 2004a).

In addition, acetylsalicylic acid (ASA, aspirin) is a nonsteroidal, anti-inflammatory drug exerting its effects by irreversibly acetylating the active site of COX. In platelets, the inhibition is effective throughout the platelets' life time because they lack a nucleus. Since TXA₂ is mainly produced by platelets, low-dose ASA has become the most common anti-platelet therapy used for secondary prevention of cardiovascular disease. Nevertheless, ASA is far from being a panacea since it has been associated with Reye's syndrome and allergic reaction (asthma), and almost one-third of patients receiving low-dose ASA do not respond, demonstrating a link between platelet function and the persistence of TXA₂ production (Patrono, 2003). These latter developments have strengthened the interest for development of other anti-thrombotic drugs such as TXA₂ modulators.

Herein, we report the in vitro and in vivo pharmacological profile of BM-613 (Fig. 1), a new TXA₂ modulator synthesized in our laboratory. BM-613 is a chemical derivative of BM-573, another thromboxane modulator combining TXRA and TXSI activities that has been widely described in literature (Rolin et al., 2001; Dogné et al., 2004b; Ghuysen et al., 2004; Lambermont et al., 2004). BM-613 has been developed as a dual TXRA and TXSI characterized by high activity on platelets compared with smooth muscle. In this study, we present several in vitro tests performed to characterize BM-613 effects on TXS, platelets, and smooth muscle. We have also carried out specific binding and antagonism of intracellular calcium concentration ([Ca²⁺]_i) mobilization tests to compare the affinity and activity of our two compounds on the distinct TP receptors isoforms. Finally, an in vivo test is presented to evaluate the potential of BM-613 as an antithrombotic drug.

View larger version (7K): [in this window] [in a new window]   Fig. 1. Chemical structure of BM-613.

	Materials and Methods

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Materials
BM-613 and BM-573 were synthesized in our laboratory. The sodium salts were dissolved in propyleneglycol and diluted with physiological saline. Furegrelate sodium salt [5-(3-pyridinylmethyl)-2-benzofurancarboxylate], SQ-29548, [³H]SQ-29548, and U-46619 were purchased from Cayman Chemical (Ann Arbor, MI). They were dissolved in ethanol and diluted with physiological saline. Phosphate-buffered saline (PBS) was purchased from Cambrex Bio Science (Petit-Rechain, Belgium). dRhodamine terminator cycle sequencing kit was from Applied Biosystems (Foster City, CA). TP and TP cDNA were as described previously (Allan et al., 1996; Becker et al., 1998). Human coronary artery smooth muscle cells (HCASMCs) were obtained from CellWorks (Buckingham, UK). Dulbecco's modified Eagle's medium supplemented with 4.5 g/l glucose, fetal bovine serum, and antibiotic/antimycotic solution were purchased from Invitrogen (Carlsbad, CA).

Animals
Male Sprague-Dawley rats, weighing 250 to 300 g, were housed in a temperature-controlled room before being used in the present experiments. All experimental procedures and protocols used in this investigation have been carried out in accordance with the Declaration of Helsinki (Publication 85-23. revised 1985) and were reviewed and approved by the Ethics Committee of the Medical Faculty of the University of Liège.

Cell Culture and Transfection
COS-7 cells were maintained in Dulbecco's modified Eagle's medium (Hyclone Laboratories, Logan, UT) supplemented with 10% fetal bovine serum and 1% of solution containing 10,000 units/ml penicillin G, 10,000 µg/ml streptomycin, and 25 µg/ml amphotericin (Cellgro; Mediatech, Herndon, VA). Cells were grown at 37°C in a humidified atmosphere of 95% O₂ and 5% CO₂. cDNAs for the TP and TP were subcloned into pcDNA3; the resultant plasmids, pcDNA3:TP and pcDNA3:TP, were introduced into COS-7 cells by the DEAE-dextran/chloroquine method. After 48 h post-transfection, cells were harvested by centrifugation at 500g for 5 min and washed three times in phosphate-buffered saline. Protein concentrations were determined using the Bradford assay (Bradford, 1976). Cells were resuspended in buffer containing 25 mM HEPES, 125 mM NaCl, and 10 µM indomethacin, pH 7.4, and kept on ice for the binding study. TP expression was determined according to Miggin and Kinsella (1998) using 10 nM [³H]SQ-29548 and a single saturating amount of SQ-29548 (10 µM).

Sequence Analysis
TP, TP, and TPtailless sequences were confirmed on both strands using the ABI Big Dye Terminator Cycle Sequencing Ready Reaction kit, and the products were resolved on an ABI Prism 310 genetic analyzer (Applied Biosystems). The sequences were assembled and analyzed using the ClustalW Sequence analysis.

Radioligand Binding Assay
The binding experiments were performed on whole cells.

Human Platelet. The TXA₂ receptor binding study was carried out on human washed platelets suspended in a calcium- and magnesium-free Tyrode-HEPES buffer (137 mM NaCl, 2.7 mM KCl, 0.4 mM NaH₂PO₄, 12 mM NaHCO₃, 5 mM D-glucose, and HEPES; pH 7.4) to a concentration 2 x 10⁸ cells/ml (Dogné et al., 2000, 2001). Freshly prepared samples of this suspension (0.5 ml) were incubated with [³H]SQ-29548 (5 nM final concentration, 0.1 ml) for 60 min at 25°C. The displacement was initiated by addition of the studied ligand dissolved in the same buffer (0.4 ml). After incubation (30 min, 25°C), ice-cold Tris-HCl buffer (10 mM, pH 7.4; 4 ml) was added, the sample was rapidly filtered through a glass fiber filter (Whatman GF/C; Whatman, Maidstone, UK), and the tube rinsed twice with ice-cold buffer (4 ml). The filters were then placed in plastic scintillation vials containing an emulsion-type scintillation mixture (4 ml), and the radioactivity was counted. The amount of [³H]SQ-29548 specifically bound to human platelet TXA₂ receptors (Bs, %) was calculated from the following equation: Bs = 100 x (B - NSB)/Bt, where total binding (Bt) and nonspecific binding (NSB) are the radioactivity of 5 nM [³H]SQ-29548 bound to the platelets incubated in the absence of any competing ligand and in the presence of unlabeled 50 µM SQ-29548, respectively. B is the radioactivity of the filtered platelets incubated with 5 nM [³H]SQ-29548 and the studied compound at a concentration ranging from 10^-5 to 10^-10 M. In each experiment, NSB varied between 5 and 7% of Bt. For each drug, three concentration-response curves were performed in triplicate. The drug concentration which reduced the amount of specifically bound [³H]SQ-29548 by 50% (IC₅₀) was determined by nonlinear regression analysis (GraphPad Prism software; GraphPad Software, Inc., San Diego, CA).

TP and TP. Competition binding curves were carried out on COS-7 cells expressing TP receptor isoforms. Binding reactions were carried out on 5 x 10⁵ cells in a total volume of 0.2 ml in the above-mentioned buffer with 10 nM [³H]SQ-29548 added to all tubes in triplicate, containing various concentration of BM-613 and BM-573 (10^-6-10^-11 M) in 1 µl of ethanol. Additional tubes containing excess unlabeled 10 µM SQ-29548 were included to assess the extent of nonspecific binding. Binding was allowed to take place for 30 min at 37°C; free radioligand was removed by rapid vacuum filtration through Whatman GF/B glass fiber filters prewashed with the cell suspension buffer. The tubes and the filters were rapidly washed with ice-cold 10 mM Tris buffer, pH 7.4 (three times with 3 ml). The radioactivity on the filters containing the ligand-receptor complexes was counted in 10 ml of Ecolite scintillation fluid (MP Biomedicals, St. Laurent, QC, Canada) in a Beckman (model LS 3800) liquid scintillation counter.

Human Coronary Artery Smooth Muscle Cells. HCASMCs were maintained in HCASMC basal medium supplemented with HCASMC growth supplement (CellWorks) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. HCASMCs were resuspended in the binding buffer (25 mM Tris-HCl, pH 7.4, 5 mM CaCl₂, 10 µM indomethacin, 50 µg/ml phenylmethylsulfonyl fluoride). For the binding assay, 50 µg of protein was incubated with [³H]SQ-29548 (30 Ci/mol, 1 µM; PerkinElmer Life and Analytical Sciences, Boston, MA) in the presence of the drug at the indicated concentrations (0-100 µM) in a 0.1-ml reaction volume with vigorous shaking at room temperature for 60 min. The reaction was then terminated by adding 1 ml of ice-cold washing buffer (25 mM Tris-HCl, pH 7.4). The unbound ligand was filtered under vacuum through a Whatman GF/C glass filter (Whatman, Clifton, NJ) presoaked with the ice-cold washing buffer. The radioactivity of the TP receptor-bound [³H]SQ-29548 remaining on the glass filter was counted in 8 ml of scintillation mixture (PerkinElmer Life and Analytical Sciences) using a Beckman counter (Beckman Coulter, Fullerton, CA).

Human ex Vivo Platelet Aggregation
The antiaggregant potency has been determined according to the turbidimetric Born's method (Born and Cross, 1963). The blood was drawn from 10 healthy donors of both genders, aged 20 to 30 years. The subjects were free from medication for at least 14 days. No significant differences in the results were observed between the donors in our experiments. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared as described previously (Dogné et al., 2000, 2001). Platelet concentration of PRP was adjusted to 3 x 10⁸ cells/ml by dilution with PPP. Platelet aggregation of PRP was studied using a double channel aggregometer (Chronolog Corporation, Chicago, IL) connected to a linear recorder as described previously (Harris et al., 1979). PRP (294 µl) was added in a silanized cuvette and stirred (1000 rpm). Each drug was diluted (1 mM) in dimethyl sulfoxide (DMSO)/PBS (30:70) and preincubated in PRP for 3 min at 37°C before the aggregating agent was added. Platelet aggregation was initiated by addition of a fresh solution of arachidonic acid (600 µM final) or U-46619 (1 µM final). To evaluate platelet aggregation, the maximum increase in light transmission was determined from the aggregation curve 6 min after addition of the inducer. The drug concentration preventing 50% of platelet aggregation (ED₅₀) induced by arachidonic acid and U-46619 was calculated by nonlinear regression analysis (GraphPad Prism software) from at least three dose-response curves.

Rat Aorta Relaxation
Endothelium-denuded thoracic aorta rings, obtained from rats (Wistar, 250-300 g) anesthetized with sodium pentobarbital (80 mg/kg i.p.), were suspended under a tension of 1 g in a Krebs' solution (118 mM NaCl, 5.4 mM KCl, 2.5 mM CaCl₂, 1.5 mM MgCl₂·6H₂O, 25 mM NaHCO₃, 1.2 mM NaH₂PO₄, and 10 mM glucose, pH 7.4), which was bubbled with O₂, CO₂ (95:5%) at 37°C in a 20-ml tissue bath (EMKA Technologies, Paris, France). The muscle tension of the aortic rings was isometrically recorded with a force displacement transducer IT1 (EMKA Technologies). The buffer was renewed every 15 min during the equilibration period (1 h) before exposing the rings to 20 nM U-46619. When a stable tension was obtained (15 min), cumulative increasing concentrations of potential antagonist were added to the bath until tension returned to the baseline value. The ED₅₀ value of each drug was assessed for at least four concentration-response curves obtained from separate preparations and corresponded to the concentration which reduced to 50% the tension induced by 20 nM U-46619. The ED₅₀ values were calculated by nonlinear regression analysis (GraphPad Prism software).

Guinea Pig Trachea Relaxation
Tracheae were removed from guinea pigs (Hartley, 250-300 g) anesthetized with sodium pentobarbital (80 mg/kg i.p.) and carefully cleaned of connective tissue. Tracheal strips were suspended in the organ bath (20 ml), and the experiment progressed in the same conditions as those described above for the rat aorta, except for the concentration of 10 nM U-46619, the contraction inducer.

Calcium Measurements
HEK.TP and HEK.TP cell lines, stably overexpressing hemagglutinin-tagged forms of TP and TP in human embryonic kidney (HEK) 293 cells have been described previously (Walsh et al., 2000). HEK 293 cells or their stable cell line equivalents were routinely grown in minimal Eagle's medium containing 10% fetal bovine serum. For measurements of [Ca²⁺]_i mobilization, approximately 48 h before transfection, HEK.TP and HEK.TP stable cell lines were plated in 10-cm culture dishes at a density of 2 x 10⁶ cells/dish in 8 ml of media; thereafter, cells were transiently transfected with 10 µg of pADVA and 25 µg of pCMV:Gq, coding for Gq, using the calcium phosphate/DNA coprecipitation procedure essentially as described previously (Kinsella et al., 1997; Hayes et al., 1999). Measurement of [Ca²⁺]_i mobilization either in Gq-transfected HEK.TP and HEK.TP cells was carried out in Fura-2/acetoxymethyl ester-pre-loaded cell lines cells, essentially as described previously (Kinsella et al., 1997). Briefly, cells were stimulated at 50 s with either 1 µM U-46619, or for competition studies, BM-573 or BM-613; cells were prestimulated with 10^-9 to 10^-3 M BM-573 or BM-613 for 5 min before stimulation with 1 µM U-46619. In all cases, the stock solution (100 mM BM-573 or BM-613 in DMSO) was diluted with PBS to achieve the desired working solution and 20 µl of the vehicle or drug in vehicle was added to 2 ml of cells; the vehicle had no effect on [Ca²⁺]_i mobilization by either TP isoform and had no effect on experimental data. The ratio of the fluorescence at 340 nm to that at 380 nm is a measure of [Ca²⁺]_i (Grynkiewicz et al., 1985), assuming a K_d value of 225 nM Ca²⁺ for Fura-2/acetoxymethyl ester. The results presented in the Figs. 3 and 4 are either representative or mean data from three or four independent experiments. Alternatively, mean percentage reductions in U-46619-mediated [Ca²⁺]_i mobilization in the presence of BM-573 (10^-9-10^-3 M) are expressed as a percentage of U-46619-mediated [Ca²⁺]_i mobilization in the absence of BM-573.

View larger version (19K): [in this window] [in a new window]   Fig. 3. A and B, HEK.TP cells, transiently cotransfected with pCMV:Gq, were either stimulated with 1 µM U-46619 alone (A) or preincubated with BM-613 (10-7) for 5 min before stimulation with 1 µM U-46619 (B). Data presented in A and B are representative profiles from three independent experiments. C and D, HEK.TP cells, transiently cotransfected with pCMV:Gq, were either stimulated with 1 µM U-46619 alone (C) or preincubated with BM-613 (10-7) for 5 min before stimulation with 1 µM U-46619 (D). Data presented in C and D are representative profiles from three independent experiments.

View larger version (16K): [in this window] [in a new window]   Fig. 4. A and C, HEK.TP cells, transiently cotransfected with pCMV:Gq, were incubated with either BM-573 (A, 10-9-10-3 M) or BM-613 (C, 10-9-10-3 M) for 5 min followed by stimulation with 1 µM U-46619. B and D, HEK.TP cells, transiently cotransfected with pCMV:Gq, were incubated with either BM-573 (B, 10-9-10-3 M) or BM-613 (D, 10-9-10-3 M) for 5 min followed by stimulation with 1 µM U-46619. Mean changes in U-46619-mediated [Ca2+]i mobilization in the presence of the drug were expressed as a percentage of U-46619-mediated [Ca2+]i mobilization in the absence of the drug.

Thromboxane Synthase Activity
PRP preparation is identical to that described for the platelet aggregation experiments. Each drug was dissolved in DMSO and diluted with a Tyrode-HEPES buffer (137 mM NaCl, 2.7 mM KCl, 0.4 mM NaH₂PO₄·H₂O, 5 mM D-glucose, 12 mM NaHCO₃, and HEPES, pH 7.4). To 900 µl of PRP, 50 µl of 0.9% NaCl and 10 µl of drug solution were added. After 6-min incubation at 37°C under stirring (600 rpm), aggregation was induced by 40 µl of sodium arachidonate (0.6 mM final). After 4 min, the reaction was stopped by adding 50 µl of indomethacin (0.02 M in ethanol). The sample was immediately centrifuged (17,500g for 10 s), and the supernatant was removed and frozen (-80°C) until assayed for TXB₂. Basal and maximal production of TXB₂ was estimated in the absence and in the presence of arachidonic acid, respectively. Evaluation was done in triplicate on concentrations ranging from 10 to 0.1 µM. Thromboxane synthase activity was expressed as the TXB₂ production, which was measured by using a competitive enzyme immunoassay (TXB₂ enzyme immunoassay kit; Cayman Chemical).

Ferric Chloride-Induced Rat Arterial Thrombosis
The experiments were carried out according to the modification of the method described by Kurz et al. (1990). Rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.). After an abdominal midline incision, the abdominal aorta was exposed carefully. A filter paper disk (8 mm in diameter) saturated with 50% (w/v) ferric chloride solution was placed on the surface of the artery for 10 min. The artery was isolated 10 min after removing the disk and then opened lengthwise. The thrombus was scraped out and placed on a filter paper to remove any water, and its wet weight was measured immediately. Results are expressed in milligrams of thrombus weight per kilogram of rat weight. BM-613 (5, 2, 1, and 0.5 mg/kg) and placebo were injected intravenously 5 min before application of ferric chloride solution. The results are expressed as the mean of results from four separate experiments.

Statistical Analysis
Results are expressed as the mean ± S.E.M., and statistical significance was determined by a Mann-Whitney U test. Probability values of less than 0.05 were considered to be significant.

	Results

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In Vitro Functional Assays
Inhibition of Human Platelet Aggregation. The ability of BM-613 to prevent human platelet aggregation has been evaluated in vitro with three different agonists of platelet aggregation. BM-613 was able to inhibit (ED₅₀ = 0.278 ± 0.186 µM) platelet aggregation induced by the stable TXA₂ agonist U-46619 (1 µM). This effect is in the same order of potency as results obtained with BM-573 (ED₅₀ = 0.240 ± 0.013 µM). In these experiments, BM-613 produced a shift to the right of the concentration-response curve of U-46619 (data not shown). BM-613 also inhibited platelet aggregation induced by 600 µM arachidonic acid (ED₅₀ = 0.375 ± 0.050 µM) in the same concentration range as BM-573 (ED₅₀ = 0.125 ± 0.015 µM). ADP is a weak inducer of platelet aggregation by itself, and it induces secretion of TXA₂ among other proaggregating substances. BM-613 (1 and 10 µM) inhibited the second wave of platelet aggregation induced by ADP, which is due to the secretion of TXA₂ (Table 1).

Thromboxane Synthase Inhibitory Potency. Thromboxane synthase is the enzyme converting PGH₂ into TXA₂. BM-613 was evaluated in a thromboxane activity test and showed an inhibitory activity (IC₅₀ = 0.15 ± 0.13 µM) comparable with that obtained with BM-573 (IC₅₀ = 0.053 ± 0.028 µM). It was 100-fold more potent than the thromboxane synthase inhibitor furegrelate (IC₅₀ = 10.2 ± 6 µM), chosen as reference drug.

Inhibition of Rat Aorta and Guinea Pig Trachea Contraction Induced by U-46619. The TXA₂ receptor antagonism properties of BM-613 were studied on isolated rat thoracic aorta precontracted by the TXA₂ agonist U-46619 (20 nM) (Table 2). The concentration that caused 50% of relaxation (ED₅₀) was calculated from concentration-response curves (Table 2). On rat aorta, BM-613 (ED₅₀ = 2.5 ± 0.18 µM) was 100-fold less active than BM-573 (ED₅₀ = 28.4 ± 4.5 nM) and 1000-fold less active than SQ-29548 (ED₅₀ = 2.3 ± 0.07 nM), which was the most potent compound in these experiments. To evaluate the potential therapeutic interest of BM-613 in the treatment of asthma, the relaxing activity of BM-613 was measured on the guinea pig trachea contracted by 10 nM U-46619 (Table 2), which is considered as a potent constrictor of the bronchopulmonary tract (Coleman et al., 1981). Cumulative increasing concentrations of BM-613 caused a concentration-dependent relaxation of the contracted isolated trachea. The drug concentration required to decrease by 50% the muscular tonus (ED₅₀) induced by U-46619 was calculated from these curves (Table 2). BM-613 (ED₅₀ = 1.52 ± 0.25 µM) was much less active than BM-573 (ED₅₀ = 17.7 ± 3.9 nM) and than SQ-29548 (ED₅₀ = 3.8 ± 0.5 nM), which remains the most potent compound.

Radioligand Binding Assay. Affinity of BM-613 for TP receptors has been evaluated by measuring its ability to displace radiolabeled SQ-29548, a strong TXRA, using different cells types (Table 3). Binding experiments have been conducted on human washed platelets, human coronary artery smooth muscle cells, and COS-7 cells where TP and TP were expressed. BM-613 showed strong affinity (IC₅₀ = 1.4 ± 0.2 nM) for human platelet TP receptor. It was as active as BM-573 (IC₅₀ = 1.3 ± 0.1 nM) and 10-fold more potent than SQ-29548 (IC₅₀ = 21 ± 2 nM). Affinities of BM-613 for either TP (IC₅₀ = 2.1 ± 0.5 nM) or TP (IC₅₀ = 3.1 ± 0.7 nM) expressed alone in COS-7 cells are not significantly different (p = 0.28). BM-573 was slightly more potent in this test, but again, there was little difference in affinity for the two isoforms. In binding studies performed on HCASMCs, BM-613 (IC₅₀ = 29 ± 7 µM) showed an affinity comparable with the reference drug, BM-573 (IC₅₀ = 37 ± 1 µM).

Intracellular Signaling by Assessment of Intracellular Calcium Mobilization. The effect of BM-613 on intracellular signaling by the TP isoforms was investigated by comparing its effect to BM-573 in intracellular calcium mobilization mediated by TP and TP stably overexpressed in HEK 293 cells, in response to the selective TXA₂ mimetic U-46619. It has been reported that for efficient TP and TP coupling to phospholipase C activation in HEK 293 cells, it is necessary to cotransfect cells with a member of the G_q family of heterotrimeric G proteins (Kinsella et al., 1997; Walsh et al., 1998). Hence, throughout these studies, HEK.TP and HEK.TP cells were routinely cotransfected with pCMV:G_q, encoding G_q. In HEK.TP cells, both BM-573 (IC₅₀ = 0.17 µM) and BM-613 (IC₅₀ = 0.11 µM) exhibited concentration-dependent antagonism of U-46619-mediated [Ca²⁺]_i mobilization (Figs. 3 and 4). Similarly, in HEK.TP cells, both BM-573 (IC₅₀ = 0.38 µM) and BM-613 (IC₅₀ = 0.30 µM) antagonized U-46619-mediated [Ca²⁺]_i mobilization to levels that were not significantly different from those observed in HEK.TP cell lines (BM-573, p = 0.6; BM-613, p = 0.8; Table 4).

View this table: [in this window] [in a new window]   TABLE 4 Summary of IC50 determinations of effects of BM-573 and BM-613 on U-46619-mediated [Ca2+]i mobilization by TP and TP

In Vivo Antithrombotic Activity of BM-613
To further test the hypothesis that BM-613 was a potential antithrombotic agent acting on TP receptors and TXS, we evaluated its effect in a rat model of arterial thrombosis. It has been described previously that application of ferric chloride solution on vascular vessels induced thrombus formation (Kurz et al., 1990). In this model, topical application of ferric chloride [50% (w/v)] for 10 min to the abdominal aorta induced marked thrombi in vehicle-treated rats (8.95 ± 0.92 mg/kg). Intravenously injected at 5, 2, and 1 mg/kg, BM-613 significantly reduced the thrombus weight by 79, 49, and 28%, respectively (Fig. 5). At 0.5 mg/kg, BM-613 did not significantly reduce the thrombus weight. Intraperitoneally injected, BM-573 tested in the same model showed similar activity (Dogné et al., 2004b).

View larger version (12K): [in this window] [in a new window]   Fig. 5. Dose-dependent effects of BM-613 on thrombus weight (milligrams per kilogram) induced by application of ferric chloride solution [50% (w/v)] on rat abdominal aorta. Results are expressed as the mean ± S.E.M. *, p < 0.05. Data presented are representative of four independent experiments.

	Discussion

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TXA₂ is a key mediator involved in platelet aggregation and smooth muscle contraction. It is synthesized by the action of TXS on PGH₂, an unstable endoperoxide formed by action of COX on free AA. TXA₂ mediates its action by a specific G protein-coupled receptor named TP receptor. In humans but not in nonprimates, this receptor is expressed as two isoforms, termed TP and TP, that arise by alternative splicing. Since TXA₂ has been implicated in several pathologies such as myocardial infarction, thrombosis, and thrombotic disorders, unstable angina, pulmonary embolism, shock, atherosclerosis, preeclampsia, and asthma (Dogné et al., 2004a), efforts have been made to develop agents able to antagonize TXA₂ actions.

Aspirin is the antiplatelet drug commonly used for secondary prevention of cardiovascular disease. Nevertheless, ASA has been associated with Reye's syndrome and allergic reaction (asthma), and almost one-third of patients receiving low-dose ASA do not respond, demonstrating a link between platelet function and the persistence of TXA₂ production (Patrono, 2003). Although the etiology of this "aspirin resistance" is still not fully explained, some authors have pointed out the possible role of oxidative products such as the non-enzymatic isoprostane derivatives of AA acting on TP receptor (Cipollone et al., 2000) or COX-2-dependent TXA₂ formation (Patrono, 2003). Besides the aspirin resistance, there is growing evidence that TP receptor is deeply involved in growing of atherosclerotic plaque. Recently, Cayatte et al. (2000) have shown that the TP receptor antagonist S18886 reduces atherogenesis, whereas ASA has no effect, thus demonstrating the superiority of TP receptor antagonist in these tests (Cayatte et al., 2000). These results were explained by the TP receptor activation by other mediators that can still promote growth of the atherosclerotic plaque.

These recent developments in our knowledge on TXA₂ biology have highlighted the limitations of ASA and the growing interest of TXA₂ modulators in several pathologies. Here, we describe in vitro and in vivo pharmacological characterization of BM-613, a novel dual TXRA and TXSI developed in our laboratory. It is a close derivative of BM-573, another TXRA and TXSI described in the literature (Rolin et al., 2001; Dogné et al., 2004b; Ghuysen et al., 2004; Lambermont et al., 2004).

Our first aim was to determine the in vitro TP receptor antagonistic potency of BM-613 in a test of inhibition of human platelet aggregation. BM-613 was able to inhibit platelet aggregation induced by AA in the same concentration range as BM-573 (Table 1). Because COX inhibitors are also able to inhibit the AA-induced aggregation by blocking TXA₂ production, BM-613 activity was evaluated with U-46619, which is a stable TP agonist. We have shown in this experiment that BM-613 completely inhibited platelet aggregation induced by U-46619, thus demonstrating its action as TXRA (Table 1). Moreover, BM-613 produced a shift to the right of the concentration-response curve of U-46619, suggesting a competitive type antagonism of platelet TP receptors (data not shown). ADP provokes platelet aggregation by acting on specific purine receptors. Platelet aggregation induced by ADP is characterized by two waves. The first wave is due to a weak and reversible aggregation, which is the consequence of ADP action on its specific receptors. The second wave is due to TXA₂ synthesis and release, which provokes irreversible and complete aggregation. Our compound only inhibited the second wave (Table 1) of ADP-induced platelet aggregation, like other TXRAs (Reynaud et al., 2002). This result highlights the lack of action of BM-613 on ADP receptors and confirms specific activity on TP receptors.

Compounds combining both actions on TP receptors and TXS have proved to be more therapeutically interesting and promising as antithrombotic agents (Gresele et al., 1987). Indeed, stopping exclusively the production of TXA₂ provokes the accumulation of PGH₂, another TP receptor agonist (Coleman et al., 1981). In contrast, with pure TP receptor antagonists, the benefit of redirecting production of other prostanoids (such as prostacyclin) by accumulation of PGH₂ is lost. Thus, we also evaluated the potency of BM-613 as TXSI by investigating the production of the stable TXA₂ metabolite, thromboxane B₂ (TXB₂), by human platelets activated by arachidonic acid. The IC₅₀ value determined for BM-613 was 100-fold lower than that of furegrelate, a clinically evaluated TXSI used as reference drug. These results demonstrated the efficacy of BM-613 as a TXSI (Mohrland et al., 1990). Combined with the effect on platelet aggregation, BM-613 can be considered as a well balanced TXRA and TXSI. This is of great importance since the lack of efficacy of ridogrel was explained by its strong effect on TXS compared with TP receptors (Soyka et al., 1999).

Further functional experiments were conducted on isolated rat aorta and guinea pig trachea precontracted by U-46619 (Fig. 2). In these experiments, BM-573 has already shown good potency compared with SQ-29548 (Rolin et al., 2001). BM-613 showed a 100-fold decreased activity in these experiments compared with BM-573 (Table 2). This discrepancy between potency of BM-613 to inhibit platelet aggregation and to inhibit smooth muscle cell contraction could find several explanations. First, the TP receptor may present conformational differences between species used in the test (human, rat, and guinea pig). Second, several authors initially stated in the 1980s that at least two pharmacological subtypes of TP receptor existed (Mais et al., 1985; Saussy et al., 1985). The first subtype was thought to be present at the platelet surface (Takahara et al., 1990), responsible for platelet aggregation. The second subtype, detected in smooth muscle cells was thought to be responsible for smooth muscle contraction (Mais et al., 1988). However, it is still controversial whether the two isoforms are responsible for pharmacological differences described in some studies because none of the initial tissue-selective antagonists of TP receptors were able to discriminate between the two isoforms. Moreover, the exact physiological significance of the existence of two isoforms is still unclear, although it has been shown that TP was the only isoform expressed in platelets (Habib et al., 1999) and that both TP and TP were expressed in smooth muscle cells (Miggin and Kinsella, 1998). Recently, Qiao et al. (2003) showed for the first time that the hepoxilin-stable analog PBT-3 could selectively bind the TP isoform in transfected COS-7 cells. Thus, we postulated that BM-613 could have a greater affinity for TP than TP and that this could be coupled with higher activity as an antiplatelet agent.

View larger version (7K): [in this window] [in a new window]   Fig. 2. Dose-dependent antagonistic effect of BM-613 and BM-573 on U-46619 induced smooth muscle cell contraction. Experiments have been carried out on rat aorta and guinea pig trachea. Data presented are representative of four independent experiments.

To test this second hypothesis, we have performed binding experiments on several cell types. BM-613 and BM-573 affinity was evaluated on washed platelets, COS-7 cells expressing either TP or TP, and HCASMCs. In these experiments, BM-613 showed high affinity for human platelet TP receptor. BM-613 was also characterized by great affinity for both TP and TP expressed in COS-7 cells. This result confirmed the potent affinity of BM-613 for human platelet TP receptor of which TP is predominant (Habib et al., 1999). Since TP and TP are both expressed in smooth muscle cells (Miggin and Kinsella, 1998), lower affinity for TP should occur when performing tests on smooth muscle cells. BM-613 and BM-573 exhibited very similar affinities for HCASMC TP receptors, which is in accordance with their TP binding properties.

Since the binding affinity of a compound for different receptors cannot reflect its antagonistic or agonistic properties, we performed studies on intracellular signaling by TP and TP. BM-613 dose dependently inhibits [Ca²⁺]_i mobilization upon stimulation by U-46619 in HEK 293 cells cotransfected with Gq and either TP or TP (Figs. 3 and 4). Nevertheless, BM-613 exhibited no significant differences in antagonistic potency against the two isoforms in these experiments (Table 4). We conclude from these results that the weak activity of BM-613 in inhibiting smooth muscle cell contraction was due to interspecies differences in TP receptor configuration.

We further used a rat model of ferric chloride-induced thrombus formation to test the antithrombotic properties of BM-613. The drug, injected intravenously, was able to significantly reduce the weight of thrombus formed in the lumen of the aorta (Fig. 5). These results are in accordance with in vitro antiplatelet activity of BM-613 and with results obtained with BM-573 in similar conditions (Dogné et al., 2004b).

In conclusion, we have presented herein a new compound, BM-613, that is characterized by high affinity for human platelets and HCASMC TP receptors and TP and TP receptors. These binding affinities are comparable with those of BM-573, our compound used as reference. Moreover, our compound is an antagonist of human platelet TP receptors TP and TP and an inhibitor of TXS. Moreover, it is characterized by antagonist properties for platelet aggregation and calcium mobilization in the same range of concentration than the potent BM-573. Nevertheless, BM-613 is less powerful than reference BM-573 in counteracting rat and guinea pig smooth muscle contraction. It seems that this difference is due to interspecies variations. However, given the therapeutic interest of tissue-selective compounds, these results need to be confirmed in further experiments. BM-613 is also an in vivo antithrombotic agent, suggesting that it could be a potential therapeutic agent for cardiovascular diseases and thrombotic disorders.

	Acknowledgements

 
We are grateful to Dr. Perry V. Halushka (Medical University of South Carolina, Charleston, SC) for providing cDNA for the TP and TP receptor isoforms. We also thank Philippe Neven for excellent technical assistance.

	Footnotes

 
This work was supported by the Fonds National de la Recherche Scientifique and Wellcome Trust. J.H. is funded by the Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture.

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

doi:10.1124/jpet.104.079301.

ABBREVIATIONS: TXA₂, thromboxane A₂; AA, amino acid(s); COX, cyclooxygenase; PGH₂, prostaglandin endoperoxide H₂; TXS, thromboxane synthase; TXRA, thromboxane receptor antagonist; TXSI, thromboxane synthase inhibitor; ASA, acetylsalicylic acid; BM-613, N-n-pentyl-N'-[2-(4'-methylphenylamino)-5-nitrobenzenesulfonyl]urea; BM-573, N-tert-butyl-N'-[2-(4'-methylphenylamino)-5-nitrobenzenesulfonyl]urea; SQ-29548, [1S-[1,2(Z),3,4]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid; U-46619, 9.11-dideoxy-9.11-methanoepoxy-prostaglandinF₂; PBS, phosphate-buffered saline; HCASMC, human coronary artery smooth muscle cell; DMSO, dimethyl sulfoxide; PRP, platelet-rich plasma; PPP, platelet-poor plasma; HEK, human embryonic kidney; TXB₂, thromboxane B₂; S18886, 3-((6R)-6-{[(4-chlorophenyl)sulfonyl]amino}-2-methyl-5,6,7,8 tetrahydro-1-naphthalenyl) propanoic acid, sodium salt.

¹ These authors contributed equally to this work.

Address correspondence to: Julien Hanson, Natural and Synthetic Drugs Research Centre, Department of Pharmacy, Laboratory of Medicinal Chemistry, University of Liège, 1, Av de L'hôpital, 4000 Liège, Belgium. E-mail: j.hanson{at}ulg.ac.be

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