Kinins are 9 to 11 amino acid peptides known to be important mediators of pain, inflammation, and cardiovascular homeostasis. They are released in injured tissues from kininogen by activation of plasma or tissue kallikreins (Bhoola et al., 1992). Kinins exert their biological activities via the activation of two subtypes of G-protein coupled receptors, denoted B₁ and B₂ receptors (Regoli and Barabe, 1980; Regoli et al., 1998). Bradykinin (BK) and Lys⁰-BK are natural endogenous agonists of bradykinin B₂ receptors, whereas their kininase I-hydrolyzed metabolites des-Arg⁹-BK and Lys⁰-des-Arg⁹-BK are specific agonists of bradykinin B₁ receptors. B₁ peptide antagonists were obtained by replacing the C-terminal Phe residue of agonists by Leu. Human and rabbit B₁ receptors have a higher affinity for Lys⁰-des-Arg⁹,leu⁸-BK than for des-Arg⁹,Leu⁸-BK, whereas rat and mice receptors have equivalent affinities for these two antagonists (Regoli et al., 1998; Jones et al., 1999). Although the B₂ receptors are constitutively present in normal tissues, the B₁ receptors are poorly present in healthy tissues but highly inducible by tissue injury and treatment by bacterial endotoxin or inflammatory mediators such as cytokines (Marceau et al., 1998).

B₁ receptors are important mediators of chronic inflammation, especially with an immune component (Couture et al., 2001). The contribution of B₁ receptor activation in inflammation and pain process is supported by the demonstration that B₁ receptor knockout mice have a decreased response to nociceptive and pro-inflammatory stimuli. Mice lacking B₁ receptors show hypoalgesia and a decrease in neutrophil migration in pleurisy and peritonitis models (Pesquero et al., 2000; Araujo et al., 2001). The hyperalgesic response to Freund's adjuvant-induced inflammation is blunted (Ferreira et al., 2001). The therapeutic interest of B₁ receptor blockage is supported further by the pharmacological properties of B₁ peptide antagonists. Edema and hyperalgesia associated to burn injuries are attenuated by des-Arg⁹,Leu⁸-BK (Perkins et al., 1993; Rawlingson et al., 2001). The peptide inhibitor R-954 was shown to reduce eosinophil and neutrophil infiltration and airway hyperreactivity induced by lung inflammation (Gama Landgraf et al., 2003). DesArg⁹,Leu⁸-BK also inhibits carrageenan-induced hyperalgesia and the late phase of nociceptive response to formalin (Rupniak et al., 1997). In neuropathic pain models, B₁ peptide antagonists were also shown to inhibit hyperalgesia induced by peripheral nerve injury (Levy and Zochodne, 2000) and diabetes (Gabra and Sirois, 2002).

Bradykinin B₁ antagonists may have a therapeutic interest against chronic inflammatory diseases and inflammatory and neuropathic pain. Non-peptide antagonists with long-lasting efficacy and good bioavailability would be important tools to confirm the biological effects of B₁ inhibition and possibly to develop new anti-inflammatory and analgesic drugs.

Here, we describe some general biochemical and pharmacological activities of a novel non-peptide B₁ antagonist, SSR240612 (Fig. 1).

Materials and Methods

Binding Assays

Assay of [³H]Lys⁰-des-Arg⁹-BK Binding to B₁ Receptors. MRC5 human fibroblasts (American Type Culture Collection, Manassas, VA) and transfected HEK-293 cells expressing human B₁ receptors were routinely grown in Dulbecco's modified Eagle's medium (DMEM) with Glutamax-I (Life Technologies, Cergy Pontoise, France) supplemented with 10% fetal calf serum (Life Technologies) and antibiotics. MRC5 were incubated for 4 h in DMEM containing 0.5 μg/ml interleukin-1β (IL-1β) to induce B₁ receptor synthesis. Cells were scraped and homogenized for 1 min using a Polytron (setting 8) in 25 mM TES-HCl containing 1 mM 1-10 phenantrolin. Homogenates were centrifuged at 40,000g for 15 min at 4°C, and pellets were resuspended in the same buffer using the Polytron (setting 8) for 1 min. Membranes were pelleted at 40,000g for 10 min at 4°C, resuspended in the same buffer, and conserved at -80°C.

[³H]Lys⁰-des-Arg⁹-BK binding to cell membranes was performed in binding buffer of the following composition: 137 mM NaCl, 5.4 mM KCl, 1.05 mM MgCl₂, 1.8 mM CaCl₂, 1.2 mM NaH₂PO₄, 15.5 mM NaHCO₃, 10 mM HEPES, 1 g/l bovine serum albumin (BSA), 140 mg/l bacitracin, and 1 μM captopril, pH 7.4. Membranes were incubated for 30 min at 25°C in 500 μl of binding buffer containing 1 nM [³H]Lys⁰-des-Arg⁹-BK for competition curves or 0.1 to 10 nM for saturation isotherms. The reaction was terminated by filtration using a Brandel Harvester onto GF/B Whatman filters previously soaked for 2 h in 0.1% polyethyleneimine. Filters were washed three times with 5 ml of binding buffer, and radioactivity was determined by liquid scintillation spectrometry. Nonspecific binding was determined by the addition of 1 μM of unlabeled Lys⁰-des-Arg⁹BK.

Assay of [³H]BK Binding to Guinea Pig Ileum Homogenates (B₂ Receptors). The binding assay was performed according the method described by Manning et al. (1986). Male Hartley guinea pigs (Charles River Laboratories, Inc., Wilmington, MA) were killed by exsanguination under anesthesia.

A 3-cm section of ileum was removed and homogenized in ice-cold buffer (25 mM TES and 1 mM 1-10 phenantrolin, pH 6.8) for 30 s using a Polytron (setting 6). Homogenates were filtered through three layers of gauze and centrifuged at 40,000g for 10 min. Pellets were resuspended in the same buffer (Polytron for 30 s at setting 6), and the membranes were pelleted at 40,000g for 10 min and stored at -80°C. [³H]BK binding to ileum membranes was performed at 25°C for 90 min in 2 ml of binding buffer (25 mM TES, 1 mM 1-10 phenantrolin, 1 g/l BSA, 140 mg/l bacitracin, and 1 μM captopril, pH 6.8). Radioligand (0.1 nM) was incubated with membranes and varying concentrations of SSR240612 or BK. The incubation was terminated by filtration through GF/C Whatman filters previously soaked for 4 h in 0.1% polyethyleneimine. Filters were washed three times with 5 ml of binding buffer, and the radioactivity was measured by liquid scintillation spectrometry. Nonspecific binding was determined in the presence of 1 μM unlabeled BK.

Assay of [³H]BK Binding to Human Recombinant B₂ Receptor Membrane Preparations. Membrane preparations from a stable CHO-K1 cell line expressing the human recombinant B₂ receptor were purchased from Euroscreen (Brussels, Belgium). Binding assays were performed at 4°C for 45 min in 250 μl of 25 mM potassium phosphate buffer, pH 6.6; 0.2% BSA containing radioligand (0.46 nM), membranes (2 μg of protein), and varying concentrations of SSR240612. The incubation was terminated by filtration through GF/C Whatman filters previously soaked for 4 h in 0.1% polyethyleneimine. Filters were washed three times with 5 ml of binding buffer, and radioactivity was measured by liquid scintillation spectrometry. Nonspecific binding was determined in the presence of 1 μM unlabeled BK.

Calculation and Statistical Analysis. Equilibrium constant (K_d) and concentration of receptor sites (B_max) values were determined from Scatchard plots. Data from equilibrium binding (K_d and B_max) and competition experiments (IC₅₀) were analyzed using a nonlinear least-squares method (RS1). Results are expressed as the mean ± S.E.M. of three independent experiments performed in triplicate. Inhibition constant (K_i) values were calculated from the IC₅₀ values using the Cheng and Prusoff equation (Cheng and Prusoff, 1973).

Specificity Studies. In addition, the affinity of SSR240612 for various receptors and channels and its effects on various enzyme activities were tested by CEREP (Celles l'Evescault, France).

In Vitro Functional Assays

Measurement of Inositol Phosphate Accumulation in MRC5 Fibroblasts. [³H]Inositol phosphate1 accumulation was measured in MRC5 fibroblasts labeled with [³H]myoinositol according to the method described by Oury-Donat et al. (1994). Cells cultured in 6-well plates were labeled for 48 h with 5 μCi/ml [³H]myoinositol added to the culture medium (DMEM). Cells were then incubated for 4 h in DMEM containing 0.5 μg/ml IL-1β to induce B₁ receptor synthesis. Agonist stimulation of inositol phosphate 1 accumulation was performed in DMEM containing 50 mM LiCl and test compounds. Antagonists were added 15 min before 10 nM Lys⁰-des-Arg¹⁰BK. After 30 min of incubation at 37°C, the medium was discarded, and the reaction was stopped by rapid addition of 1 ml of cold methanol and 1 N HCl (v/v). The cells were scraped, and the suspension was transferred to a glass tube with 1 ml of chloroform and 20 μl of 12 N HCl. The aqueous phase was neutralized by 350 μl of 1 M NaHCO₃ and applied to 1 ml of Dowex AG1 × eight columns. [³H]inositol phosphate 1 was eluted with 0.2 M ammonium formate and 0.1 M formic acid. Radioactivity was measured by liquid scintillation spectrometry.

Isolated Organ Experiments. B₁ antagonistic properties of SSR240612 were tested on des-Arg⁹-BK-induced contractions of rabbit aorta and myenteric plexus of rat ileum. All experiments were performed in isolated organ baths containing an oxygenated (95% O₂/5% CO₂) and thermostated (37°C) Krebs-modified physiological salt solution, pH 7. 4.

Strips of rabbit aorta and myenteric plexus of rat ileum were prepared for tension recording. Tissues were rapidly excised from male albino rabbits (3–3.5 kg) and male Sprague-Dawley rats (100–150 g). The rat ileum was threaded into glass rods, and the mesenteric attachment was stroked tangentially with cotton to separate the longitudinal muscle and adherent myenteric plexus from the circular muscle.

Tissues were allowed to equilibrate under a resting tension (2 g for spirally cut strips from rabbit thoracic aorta, 0.5 g for rat myenteric plexus) and pre-incubated 20 to 22 (rabbit) or 4 (rat ileum) h in Krebs-modified physiological salt solution. Tension equivalent to the maximum response was obtained with 100 mM KCl for rabbit aorta and 40 mM for rat ileum.

Changes in tension were recorded isotonically with a length-displacement transducer (Ugo Basile, Comerio, Italy). Cumulative concentration-response curves were obtained in the absence and then, after repeated washings, in the presence of a fixed concentration of SSR240612 added 45 min (rabbit aorta) or 1 h (rat ileum) before the agonist. Each preparation was exposed to one concentration of antagonist.

The agonist concentration producing 50% of the maximal effect (EC₅₀) was calculated using a four-parameter logistic model according to Ratkowsky and Reedy (1986), with adjustment by nonlinear regression using the Levenberg-Marquardt algorithm in RS/1 software. The pA₂ for antagonists, as defined by Arunlakshana and Schild (1959), was obtained from linear regression of mean values of the log (dr - 1) versus the negative log of the antagonist concentration. Computer analysis was done as described by Tallarida and Murray (1987).

In Vivo Assays

All procedures have been approved by the Animal Care and Use Committee of Sanofi-Synthelabo Recherche (Montpellier, France) and were carried out in accordance with French legislation, the European Community Guidelines (86/609/EEC), and the declaration of Helsinki.

Des Arg⁹-BK-Induced Mice Paw Edema. Groups of eight male albino mice under isoflurane anesthesia received a 20-μl intraplantar injection into the right hind paw of 5 μg of IL-1β in phosphate-buffered saline/0.1% BSA. Forty minutes later (T = 0), mice received, under anesthesia, a 20-μl intraplantar injection in the same paw of des-Arg⁹-BK (10 μg/paw) in water.

SSR240612 or vehicle [5% (v/v) ethanol and 5% (v/v) Tween 80 in water] was administered by oral route at the doses of 1, 3, and 10 mg/kg 1 h before des-Arg⁹-BK injection and by intraperitoneal route at the doses of 0.1, 0.3, and 1 mg/kg 40 min before des-Arg⁹-BK injection. Paw volume was measured with a plethysmometer at T = -2 h (initial measurement) and at several times after edema induction (T = 20, 40, 60, and 120 min). Paw edema volume was expressed in milliliters as the difference between the paw volume at each time after edema induction and the initial paw volume. Results for each group are expressed as mean ± S.E.M. of individual paw edema volumes. Statistical analysis was performed after verification of normality and homogeneity of variances using repeated ANOVA, then Duncan's test, treated groups versus des-Arg⁹-BK control group.

Capsaicin Ear Edema. Groups of eight male albino mice, 28 to 33 g (Iffa Credo, L'Arbresele, France), were anesthetized with isoflurane. Capsaicin ear edema was induced by applying a 25 mg/ml capsaicin solution in absolute ethanol using a micropipette to the right ear (10 μl to both the dorsal and ventral surfaces) of each animal. Thirty minutes later, animals were again anesthetized using isoflurane and killed by cervical dislocation. Right (treated) and left (untreated) ears of each animal were removed by cutting horizontally across the indentation at the base of the ear. Ear weights were determined to the nearest one-tenth of a milligram, and the difference between the weights of the right and left ears (ear edema) was calculated for each animal.

SSR240612 at 0.3, 3, or 30 mg/kg or its vehicle [5% (v/v) Tween 80 and 5% (v/v) ethanol in water] were administered by the oral route under a 20 ml/kg volume 1 h before capsaicin edema induction. Dexamethasone-21-acetate, used as the reference compound, was administered at 1 mg/kg (as the salified compound) using the same route and volume of administration. Results for each group are expressed as means ± S.E.M. of individual ear edema. Statistical analysis was performed after verification of normality and homogeneity of variances using ANOVA, then Duncan's test, treated group versus capsaicin control group.

Splanchnic Artery Occlusion/Reperfusion. Male Sprague-Dawley rats (160–180 g) were fasted overnight before surgery. Animals were anesthetized with ketamine (0.3 ml i.p. of twice-diluted SBH-Ketamin injection 100 mg/ml) - xylazine (0.3 ml i.m. of twice diluted 2% Rompun) (Bayer Pharma, Puteaux, France). After mid-line laparatomy, the superior mesenteric artery was isolated near its aortic origin. The artery was ligated with surgical thread for 30 min. During this period, the intestinal tract was maintained at 37°C by placing it between gauze pads soaked with warmed saline solution. After 30 min, the ligature was removed, and a 45-min reperfusion period was applied. Sham-operated animals were subjected to the same surgical procedure except that the artery was not occluded. SSR240612 [1% (v/v) Tween 80 in water] was injected in a volume of 1 ml/kg into the jugular vein 5 min before removal of the ligature. Sham and control animals received the vehicle.

After the 45-min reperfusion period, the animals were killed by an overdose of urethane. The small bowel was isolated between pylorus and sacculus rotundus. The degree of the tissue damage was estimated by the reduction of the weight of intestine. The intestine was gently cleaned from all adherent soft and adipose tissues. The bowel was cut up longitudinally along the axis, and all components were removed from it by rinsing in saline. Wet weight of the tissue was measured using analytical scales.

Neutrophil accumulation in intestinal tissue was evaluated by measuring tissular activity of myeloperoxidase (MPO). Tissues were frozen in liquid nitrogen immediately after dissection and stored at -20°C before activity determination. Tissue samples were thawed and homogenized for 1 min in 15 ml of 50 mM potassium-phosphate buffer (pH 6.0) containing 0.5% (w/v) hexa-decyl-trimethyl-ammonium-bromid (SigmaAldrich, St. Louis, MO). The samples were frozen and thawed, then sonicated for 1 min. This was repeated once more. After the second sonication, samples were centrifuged for 30 min at 20,000g at 4°C. An aliquot (0.01 ml of four times diluted sample) of the supernatant was then allowed to react with the solution containing 0.44 mM tetramethylbenzidine (Fluka, Buchs, Switzerland) and 0.0033% H₂O₂. Absorbance change was measured at 650 nm by spectrophotometry.

Values are given as mean ± S.E.M. The significance of differences compared with the control group was obtained after performing the normality test and investigating homogeneity of variances (Bartlett test) using Dunnett's test in the case of intestinal weights and Kruskal-Wallis test (followed by Mann-Whitney test) for MPO determinations. P < 0.05 was considered as significant. All statistical analyses were performed using RS/1 software.

Ultraviolet Irradiation-Induced Hyperalgesia in Rat Paw. The protocol used is according to Perkins and Kelly (1993). In brief, under light anesthesia (12 mg of chloral hydrate + 1.9 mg pentobarbitone/ml), groups of 9 to 20 male Oncins France souche A rats (100–150 g, Iffa Credo) were exposed to ultraviolet light (maximal intensity = 6210 mJ/cm²) on the plantar surface of the left hind paw (UV-exposed paw) for 20 min (the right hind paw being protected = non-exposed paw). Forty-eight hours later, the time taken to withdraw (withdrawal latency) each hind paw was measured, following a heat stimulus (a focused radiant heat beam, Ugo Basile unit) applied to the underside of the paw. The hind paws were exposed to the heat stimulus in a random manner so that there was no consistent order of testing between exposed and non-exposed paws, and no rats had both paws exposed to heat stimulus immediately after each other. A cutoff time at 22 s was used to prevent blistering.

SSR240612 (suspended with 0.1% Tween 80 in saline) was administered by the oral route in a volume of 20 ml/kg, 2 h before the thermal hyperalgesia measurement. In the time course study, the compound was administered at 3 mg/kg p.o. 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h before measuring the withdrawal latencies in rats. Results are expressed in seconds as mean withdrawal latencies (s) ± S.E.M., and statistical analyses were performed using a two-way ANOVA followed by Dunnett's test.

Nociceptive Behavior Elicited by Intraplantar Injection of Formalin in Mice. The protocol used was identical to the one described by Rupniak et al. (1997). Groups of 10 to 12 male Oncins France souche 1 mice (30–35 g, Iffa Credo) received an intraplantar injection of formalin (20 μl of 2.5% saline solution) into one hind paw. Then, mice were placed in a cylindrical glass (15-cm height × 10-cm diameter), and the duration of licking directed at the injected paw was recorded during the late-phase (30–40 min) period after injection of formalin.

SSR240612 was suspended with 0.1% Tween 80 in saline and was administered by oral route in a volume of 5 ml/kg, 2 h before formalin administration. Results are expressed in seconds as mean licking time (s) ± S.E.M., and statistical analyses were performed using ANOVA followed by Dunnett's test.

Neurogenic Pain Induced by Sciatic Nerve Constriction in the Rat. Male Sprague-Dawley rats (Iffa Credo) weighing 175 to 200 g on arrival were used. They were housed five to a cage, had free access to standard laboratory food and tap water, and were kept on a 12-h light/dark cycle with light onset at 6 AM. All animals were allowed to acclimatize to the housing facilities for at least 1 week before experiments.

The unilateral mononeuropathy was produced on the right hind paw according to the method of Bennett and Xie (1998) and Attal et al. (1990). In brief, the animals were anesthetized with isoflurane (3%) followed by sodium pentobarbitone (50 mg/kg, i.p.). The common sciatic nerve was exposed by blunt dissection at the level of the mid-thigh, and four ligatures (5-0 chromic catgut, about 1-mm spacing) were placed around the sciatic nerve. After the surgery, the rats were housed in large cages with sawdust bedding to minimize possible painful mechanical stimulation. Nonoperated rats were used as control.

Rats were used 2 weeks after surgery, a time period required to obtain a stable abnormal pain behavior (Attal et al., 1990; Bennett and Xie, 1998). Thermal nociception was tested by measuring the struggle latency elicited by immersion of the hind paw into a 46°C water bath as extensively described (Attal et al., 1990; Lee et al., 1994; Idanpaan-Heikkila et al., 1997). For each rat, a control latency (mean of three consecutive trials, 45 min apart) was determined before administering the drug. The struggle latency was measured before (time 0) and 60, 120, and 180 min following either vehicle or SSR240612 oral administration at the dose of 10, 20, or 30 mg/kg. Data are expressed as mean ± S.E.M. (n = 9–21 rats per group) of struggle latencies (s). Control latency data from nerve injured and uninjured rats were analyzed by one-way ANOVA. Data from drug-treated animals were submitted to a two-way ANOVA.

Chemicals

SSR240612 (Fig. 1) was synthesized at Sanofi-Synthelabo Recherche and was used as its hydrochloride salt. Radioactive ligands were purchased from PerkinElmer Life and Analytical Sciences (Paris, France). All peptides were obtained from Bachem (Bubendorf, Switzerland).

Results

Receptor Binding. As shown in Table 1, SSR240612 displaced the binding of [³H]Lys⁰-des-Arg⁹-BK to membrane preparations of MRC5 human lung fibroblasts and of HEK cells expressing human B₁ receptors with K_i values of 0.48 and 0.73 nM, respectively. The affinity of SSR240612 for B₂ receptors labeled with [³H]BK was much lower with a K_i of 481 nM for guinea pig ileum membranes and a K_i of 358 nM for membrane preparations of CHO cells expressing human B₂ receptors. The affinity of SSR240612 for human fibroblast MRC5 B₁ receptors was higher than the affinity of agonist and antagonist peptides, Lys-des-Arg9-BK (1.63 nM), Lys-des-Arg9,Leu8-BK (2.44 nM), des-Arg9-BK (542 nM), and des-Arg9,Leu8-BK (152.5 nM) (Table 2). Saturation binding experiments were performed for the binding of [³H]Lys⁰-des-Arg⁹-BK to MRC5 membranes in the absence and in the presence of SSR240612 (0.3 and 1 nM). Scatchard analysis of the data showed that increasing concentrations of SSR240612 produced an increase in dissociation constant (K_d) without significant modification in receptor density (B_max) (Fig. 2), which is consistent with competitive antagonism.

In Vitro Functional Studies. Antagonistic activity of SSR240612 at human B₁ receptors was studied by measuring inhibition of inositol phosphate-1 formation induced by Lys⁰-des-Arg⁹-BK (10 nM) activation of the B₁ receptor in MRC5 human fibroblast cells. Inositol phosphate 1 formation was concentration dependently inhibited by SSR240612 with an IC₅₀ of 1.9 nM. The drug itself did not modify the level of inositol phosphate 1, showing a total absence of agonistic effect. In contrast, SSR240612 was unable to antagonize inositol phosphate-1 formation induced by BK (3 nM) activation of B₂ receptor in human fibroblast MRC5 (IC₅₀ > 1 μM).

In rat ileum and rabbit aorta, SSR240612 caused a concentration-dependent rightward shift of the concentration-response curves for des-Arg⁹-BK (Figs. 3 and 4). Schild plot analysis produced a pA₂ of 9.4 ± 0.1 and a slope of 1.5 ± 0.15 in the rat ileum and a pA₂ of 8.9 ± 0.1 and a slope of 1.06 ± 0.11 in the rabbit aorta.

In Vivo Studies. The in vivo antagonistic effects of SSR240612 were first investigated on B₁ agonist-induced paw edema in mice after induction of B₁ receptors by intraplantar injection of IL-1β. SSR240612 significantly inhibited the des-Arg⁹-BK-induced paw edema in the mice at the doses of 3 and 10 mg/kg p.o. and 0.3 and 1 mg/kg i.p. (Fig. 5).

The effects of SSR240612 were then studied on capsaicin-induced ear inflammation in mice. The drug administered per os 1 h before capsaicin significantly reduced the ear edema but in a non-concentration-dependent manner because the three doses tested, 0.3, 3, and 30 mg/kg, produced an inhibition of 43%, 44%, and 28% of the increase of ear weight (Fig. 6).

With regard to the postischemic intestinal reperfusion, the B₁ receptors, up-regulated by ischemia, are supposed to contribute to systemic vasodilatation and intestinal injury consequent to neurogenic reflex induced by reperfusion (Madeddu et al., 2001). SSR240612 administered intravenously at 0.03 and 0.3 mg/kg dose dependently inhibited the tissue destruction and neutrophil accumulation in the rat intestine, after splanchnic artery occlusion/reperfusion. Although the lower dose (0.03 mg/kg) was inactive, the 0.3 mg/kg dose (i.v.) caused marked and significant reduction of mucosal damage (57% inhibition of tissue weight decrease, P < 0.01) and neutrophil accumulation determined as MPO tissue activity (79% inhibition, P < 0.01) (Table 3).

In the thermal hyperalgesia induced by UV irradiation in rats, SSR240612 dose dependently and significantly increased the withdrawal latencies by 66% and 85% at 1 and 3 mg/kg p.o., respectively, with F(3,70) = 6.82, P < 0.001, the comparison between UV-exposed and non-exposed hind paw showing a highly significant effect: F(1,70) = 10.15, P < 0.002 (Fig. 7).

The time course study of SSR240612 at 3 mg/kg p.o. upon the ultraviolet-exposed hind paw showed a maximal and significant effect at 1 h postinjection, which was long-lasting (>6 h) (Fig. 8).

In the formalin model of inflammation in mice, the duration of the late phase of paw licking (30–40 min post-formalin injection) was dose dependently attenuated by SSR240612 at 10 and 30 mg/kg (27% and 69%, respectively), the effect being statistically significant at 30 mg/kg with F(1,38) = 11.37, P < 0.001, and an ID₅₀ of 18.7 mg/kg (16.2–22.2, 95% confidence limits) (Fig. 9).

As previously reported (Lee et al., 1994; Idanpaan-Heikkila et al., 1997), sciatic nerve injury produced a significant decrease [-45%, F(1,96) = 138.33, P < 0.01] in the struggle latency relative to control nonoperated rats (Fig. 10). In nerve-injured rats, oral SSR240612 administration significantly increased struggle latencies as compared with values before drug treatment. Two-way ANOVA (treatment × time) revealed a significant effect of drug treatment [F(3,211) = 5.21, P < 0.01] and time period [F(3,211) = 3.38, P < 0.05]. The antinociceptive effect of SSR240612 was observed at 120 and 180 min after drug administration at the dose of 20 (+44%, P < 0.01; and +38%, P < 0.05, at 120 and 180 min, respectively) and 30 mg/kg p.o. (+ 74%, P < 0.01; and + 57%, P < 0.05, at 120 and 180 min, respectively). In contrast, SSR240612 had no significant effect on struggle latencies in uninjured rats [treatment, F(2,171) = 1.87, P > 0.05; time, F(3,171) = 1.77, P > 0.05].

Discussion

The present study demonstrates that SSR240612 is a high-affinity, subtype-selective bradykinin B₁ antagonist. In binding experiments, SSR240612 inhibited binding of [³H]Lys⁰-des-Arg⁹-BK to human B₁ receptors with an inhibition constant (K_i) in the nanomolar range showing an affinity of the same order as human receptor-specific peptides (Lys-des-Arg⁹-BK and Lys-des-Arg⁹,Leu⁸-BK) and much higher than rat receptor-specific peptides (des-Arg9-BK and des-Arg9,Leu8-BK). The selectivity for B₁ versus B₂ receptors was in the range of 500- to 1000-fold. Finally, SSR240612 was tested over 100 (mainly human) receptor-binding, ion channel-binding, and enzyme assays including the following: receptors to adenosine (A1, A2A, and A3), adrenergic (α1, α2, β1, and β2), dopamine (D1 and D2), nicotinic and muscarinic (M1, M2, M3, M4, and M5), opiate (μ, κ, δ, and ORL1), serotonin (5-HT1A, 5-HT2A, 5-HT2C, 5-HT3, 5-HT5A, 5-HT6, and 5-HT7), angiotensin (AT1 and AT2), neurokinin (NK1, NK2, and NK3), calcitonin gene-related peptide (CGRP), cholecystokinin (CCK1 and CCK2), corticotropin releasing factor (CRF1 and CRF2), galanin (GAL1 and GAL2), neurotensin (NT1), vasopressin (V1A), hormones (glucocorticoid, estrogen, progesterone, and testosterone), ion channels (sodium, calcium, potassium, and chloride), cyclooxygenases (COX1 and COX2), phosphodiesterases (III and IV), and acetylcholinesterase. SSR240612, at concentrations up to 1 μM, was inactive (inhibition less than 50%), except in muscarinic M₂ receptor assay (IC50 = 0.48 μM) (data not shown).

The potent antagonism by SSR240612 of B₁ receptors has been demonstrated in vitro for several species in different functional assays. On the human receptors, SSR240612, potently inhibited B₁ receptor-mediated inositolphosphate-1 formation in the human fibroblast, MRC5. On animal receptors, SSR240612 inhibited des-Arg⁹-BK-induced contractions of rabbit aorta and rat ileum with similar potencies. These results show that SSR240612 is a potent antagonist of human, rabbit, and rat B₁ receptors.

In vivo, SSR240612 was shown to exert a potent antagonism of edema induced by des-Arg⁹-BK in the mouse hind paw after induction of B₁ receptor by intraplantar injection of IL-1β. SSR240612 was active by the oral route at the doses of 3 and 10 mg/kg.

SSR240612 was then studied in animal models where an enhanced endogenous B₁-specific tone is suspected to play a major role. B₁ receptor activation is known to induce proinflammatory effects including leukocyte accumulation, edema, and pain. In addition, B₁ receptors are mainly implicated in neurogenic inflammation processes. The B₁ antagonist des-Arg⁹,leu⁸-BK has been reported to inhibit the capsaicin-induced mouse ear edema by modulation of neurogenic inflammation (Mantione and Rodriguez, 1990). B₁ antagonists also block inflammation-like hypotension and plasma protein infiltration of the intestinal wall, observed during mesenteric postischemic reperfusion in the anesthetized rat, presumably regulated by a neurogenic reflex response (Madeddu et al., 2001). Likewise, SSR240612 caused a significant though not dose-dependent reduction of capsaicin-induced mice ear edema at the oral doses of 0.3 to 30 mg/kg and of mucosal damages and neutrophil accumulation produced by postischemic reperfusion of mesenteric artery in the rat at the dose of 0.3 mg/kg (intravenous route).

Because B₁ receptors are known to be important mediators of inflammatory pain, SSR240612 was tested on two pharmacological models, UV-induced hyperalgesia in rat paw, and the late phase of formalin test in mice, where pain or hyperalgesia were induced by local tissue injury. SSR240612, given orally, inhibited UV-induced hyperalgesia at the dose of 1 to 3 mg/kg. It significantly antagonized the second phase of formalin-induced pain at the dose of 30 mg/kg (oral administration).

B₁ receptors also have been suspected to modulate hyperalgesia induced by peripheral nerve injury. At 14 days after chronic constriction injury of the rat sciatic nerve, an increased expression of B₁ receptor mRNA has been reported in the lumbar dorsal root ganglia ipsilateral to the injured nerve site, coinciding with an analgesic effect of B₁ antagonist (Levy and Zochodne, 2000). In the same model of neuropathic pain, after 10 days of sciatic nerve constriction in the rat, SSR240612 antagonized hyperalgesia in the ipsilateral paw at the doses of 20 to 30 mg/kg.

In conclusion, SSR240612 is a highly potent, selective nonpeptide antagonist of the bradykinin B₁ receptor. SSR240612 displayed high potency for the B₁ receptor in all species tested (rat, mouse, rabbit, and human). SSR240612 was found orally active in several models of neurogenic inflammation and inflammatory pain. These results suggest that SSR240612 may have potential therapeutic interest for the treatment of chronic inflammation and pain and is a useful tool for further exploring the pathophysiological role of B₁ receptors.