The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N2-[(2,2-diphenylethoxy)acetyl]-l-arginine (SB 290157), functioned as a competitive antagonist of 125I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC50 of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca2+ mobilization in RBL-C3aR cells and human neutrophils with IC50s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca2+ mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRs. It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.

The anaphylatoxins C3a and C5a are proinflammatory 74- to 77-aa bioactive peptide fragments cleaved from the serum proteins C3 and C5 during complement activation. They elicit mast cell and basophil degranulation with release of histamine, cytokines, and other inflammatory mediators and induce smooth muscle contractions (reviewed in Ref. 1). C3a, originally thought not to be chemotactic, has recently been shown to be a potent chemotaxin for eosinophils, macrophages, and mast cells (2, 3, 4, 5, 6). Consistent with its actions on eosinophils, C3a, when administered to guinea pigs via i.v. injection or administered via intrabronchial instillation, has been implicated in allergic bronchospasm and pulmonary inflammation (7, 8, 9). C5a also causes increased vascular permeability and induces chemotaxis of neutrophils, monocytes, and astrocytes.

The primary structure of human C3a was first determined in 1975 (10) and the site responsible for binding to its receptor, the carboxyl-terminal residues, was defined shortly thereafter (11). The C3aR was suspected to be a G protein-coupled receptor (GPCR)3 as C3a-induced increases in intracellular Ca2+ and C3-mediated release of reactive oxygen species in human polymorphonuclear leukocytes were blocked by pretreatment with pertussis toxin (12, 13, 14). This was confirmed when the C3aR was cloned in 1996 and definitively demonstrated to be a GPCR (15, 16, 17). The primary structure of the C3aR is unique among the superfamily of GPCRs. The receptor has been cloned from four different species, human, rat, mouse, and guinea pig, and all possess an unusually large second extracellular domain in excess of 170 aa long. Another striking and unanticipated feature of the C3aR is the tissue distribution pattern, where it is widely expressed throughout the periphery. There is also abundant expression of the C3aR transcript and protein in the brain (15, 18, 19).

Insight into the pathophysiological roles of C3a and the C3aR is emerging from gene targeting studies; two groups have independently produced C3aR−/− mice by this approach (20, 21). C3aR−/− mice are more susceptible than wild-type mice to an i.v. challenge with LPS, implicating complement activation and the C3aR in events leading to endotoxin-induced septic shock (21). Consistent with the effects of C3a on mast cells, eosinophils, and smooth muscle, allergen-challenged OVA-sensitized C3aR−/− mice do not exhibit airway hyperresponsiveness to methacholine challenge when compared with OVA-sensitized wild-type mice (20). Similarly, in a C3aR-deficient inbred strain of guinea pigs (C2BB/R), which have a mutation encoding a stop codon within the coding sequence of the C3aR, we have noted significantly decreased bronchial reactivity in an OVA-induced asthma model (22). Allergen-challenged OVA-sensitized C2BB/R guinea pigs exhibited a 30% decrease in bronchial reactivity when compared with allergen-challenged OVA-sensitized C2BB/R+ guinea pigs which express an intact wild-type C3aR (22). These data obtained with Ag-sensitized C3aR−/− mice and guinea pigs implicate the C3aR in the pathogenesis of allergic asthma and suggest that C3aR antagonists may be useful in the treatment of this disease.

The lack of potent and selective C3aR antagonists has made it difficult to fully assess the relative contribution of C3a to the inflammatory processes elicited by the anaphylatoxins and the terminal complement complexes. Knowledge of the receptor-binding site of C3a and the molecular identification of the C3aR has facilitated discovery efforts to identify small molecule C3aR antagonists. We have used membranes from rat basophilic leukemia (RBL-2H3) cells stably expressing the human recombinant C3aR and radiolabeled C3a to establish a high throughput binding assay to screen for low m.w. nonpeptide C3aR antagonists. In this report, we describe the in vitro and preliminary in vivo pharmacological characterization of N2-[(2,2-diphenylethoxy)acetyl]-l-arginine (SB 290157), a potent and selective C3aR antagonist. Our data indicate that the compound should be a useful tool compound to help define the potential physiological role(s) of C3a.

RBL-2H3 cells (23), stably expressing the human, guinea pig, and mouse C3aRs, have previously been described (24, 25). The cell lines were maintained in Eagle’s MEM with Earle’s salts, with l-glutamine and nonessential amino acids (Life Technologies, Gaithersburg, MD) supplemented with 10% FBS (HyClone, Logan, UT) and 400 μg/ml G418 (Life Technologies)

High throughput scintillation proximity assay (SPA) for C3a antagonists.

A primary high throughput radioligand binding assay was established using membranes of RBL-C3aR cells. 125I-C3a binds to RBL-C3aR cells with high affinity (Kd = 8 pM) and is saturable.

All binding assays were performed in a 96-well microtiter plate (Wallac, Turku, Finland) format. Bolton-Hunter custom iodination was performed by NEN Research Products (Boston, MA) with a sp. act. of 2200 Ci/mmol. The binding buffer consists of 20 mM bis-Trispropane (pH 8.0) with 25 mM NaCl, 1 mM MgSO4, and 0.1 mM EDTA. Each well contains: 125I-C3a (16 pM), 70 μg wheat germ agglutinin SPA (Amersham, Arlington Heights, IL) beads, 0.20 μ g RBL-C3aR membranes, 23 μg/ml BSA, and 0.03% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in binding buffer. In addition, control wells for nonspecific binding included an excess of 15 nM unlabeled C3a.

Membranes were prebound to SPA beads for 30 min on ice while shaking. The mixture of membranes and beads was centrifuged for 3 min at 2000 rpm. The supernatant was removed, and the pellet was resuspended to original volume in binding buffer containing 50 μg/ml BSA before dispensing into microtiter plates. Antagonists were dissolved in neat DMSO to yield a 20× solution followed by a 1:1 mixture with H2O to yield a 10×, 50% DMSO working solution. The order of addition was 10 μl sample, 45 μl membrane-bound SPA beads followed by 45 μl radiolabeled ligand in binding buffer containing 0.06% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The plates were covered with plate sealers from Dynex Technologies (Chantilly, VA), shaken for 20 min, and incubated for an additional 40 min at room temperature. The plates were then centrifuged for 3 min at 2000 rpm followed by counting on the Wallac 1450 Micro β Plus Liquid Scintillation counter (Wallac, Gaithersburg, MD).

Binding for follow-up studies.

The binding assay was performed essentially as previously described (26). Briefly, 2–5 × 105 RBL-C3aR cells were incubated with 100 pM 125I-C3a (NEN, Boston, MA) and varying concentrations of antagonist at room temperature in 20 mM HEPES (pH 7.4), 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 0.25% BSA and 0.5 mM glucose (HAG-CM) for 45 min at room temperature. The unbound ligand was removed by vacuum filtration using the HV Millipore MultiScreen assay plate with a Durapore 0.45-μm pore size membrane (Millipore, Bedford, MA) equilibrated with HAG-CM. Filters were washed twice with 100 μl/well HAG-CM and dried. Plates were counted on a Beckman gamma counter 5500B (Beckman, Fullerton, CA). Data analysis was performed using KaleidaGraph v3.09 (Synergy Software, Reading, PA).

The flow cytometric internalization assay using C3aR-specific rabbit polyclonal antiserum (27) was performed as described (28). Concentrations of SB 290157, 1-naphthyloxyacetylarginine (SKF 63649), and N-[(3,5-dichlorophenyl)methyl]-N-(3-pyridinylcarbonyl)glycl-l-arginine] (SB 280936) ranging from 100 nM to 10 μM were tested for the ability to inhibit the internalization of the C3aR after stimulation of human neutrophils with 10 nM C3a, a concentration that induces an almost complete disappearance of the C3aR from the cell surface (>90% receptor internalization). Neutrophils were coincubated for 3 min at 37°C with compound and 10 nM C3a (Advanced Research Technologies, San Diego, CA) and then evaluated for receptor internalization.

C3aR Ca2+ mobilization studies were conducted using Fluo 3-loaded RBL-C3aR cells and a microtiter plate-based assay using a FLIPR (Molecular Devices, Sunnyvale, CA). Briefly, cells (∼80% confluent) were harvested and plated in 96-well black wall clear-bottom plates (Packard view plate) at ∼40,000 cells/well and grown in an incubator for 18–24 h. On the day of assay, the medium was aspirated and replaced with 100 μl Eagle’s MEM with Earle’s salts containing l-glutamine, 0.1% BSA, 4 μM fluo-3 acetoxymethyl ester; Molecular Probes, Eugene, OR) and 1.5 mM sulfinpyrazone. Plates were incubated for 60 min at 37°C; medium was aspirated, replaced with the same medium without fluo-3 acetoxymethyl ester, and incubated for 10 min at 37°C. Cells were washed three times and incubated at 37°C in 100 μl assay buffer (120 mM NaCl, 4.6 mM KCl, 1.03 mM KH2 PO4, 25 mM NaHCO3, 1.0 mM CaCl2, 1.1 mM MgCl2, 11 mM glucose, 20 mM HEPES (pH 7.4) with 1.5 mM sulfinpyrazone). Plates were placed into FLIPR for analysis as described previously (29). The maximal change in fluorescence after agonist addition was quantitated. The percent of maximal C3a-induced Ca2+ mobilization was determined for each concentration of antagonist. The IC50, defined as the concentration of test compound that inhibits 50% of the maximal response induced by 1 nM C3a, was obtained from concentration-response curves. For agonist potency the EC50 is defined as the concentration that produces 50% of the maximal C3a-induced response.

fura-2-acetoxymethyl ester-loaded human neutrophils or RBL-C3aR cells were assayed for a Ca2+ mobilization response, as described (30).

A chemiluminescent assay was used to quantitate ATP release from C3a-stimulated guinea pig platelets, as described (31). Varying concentrations of antagonist were mixed with 1 nM C3a, a concentration equivalent to an EC80 in this assay, and the reaction was initiated by the addition of platelets.

C3a-mediated chemotaxis of HMC-1 cells was assessed using Neuro Probe (Gaithersburg, MD) 96-well disposable chemotaxis plates (5 μm pore size). The top surface of the membrane was precoated with 100 ng laminin or fibronectin (Sigma, St. Louis, MO). Varying concentrations of C3a with and without antagonist were added in 28 μl RPMI 1640 to the lower wells. The filter was assembled, and 2 to 5 × 105 cells were added in 25 μl to the top well. Plates were incubated at 37°C and 5% CO2 for 60 min. Filters were removed, and the top surfaces of the membranes were rinsed with PBS; then the cells were stained with Diff-Quik (Baxter, Dade Division, Miami, FL). The number of cells migrated was quantitated microscopically by counting the cells in three successive high power fields.

Male Sprague Dawley normotensive rats weighing between 400 and 600 g were euthanized, and the tail was removed and placed in physiologic buffer. The tail was secured to a dissection board, the caudal artery was exposed, and a 30- to 40-mm-long section of the artery was dissected from the tail and placed into buffer. The artery section was cut into two segments of equal length, each segment was cannulated at both ends with PE50 tubing, and the tubing was secured with ties of 4-0 surgical silk. The cannulated arterial segments were mounted in a tubular glass chamber and were simultaneously perfused intraluminally and superfused extraluminally with oxygenated Krebs buffer at 38°C. The rate of intraluminal perfusion was 1 ml/min, and that of extraluminal superfusion was 2 ml/min. Under these conditions, the baseline perfusion pressure equilibrated to between 25 and 50 mm Hg. After a 20- to 30-min stabilization period, the periarterial sympathetic nerves were stimulated electrically every 30 s via platinum electrodes located at both ends of the chamber to obtain a brief, spike-like increase in perfusion pressure. The stimulation consisted of a 1-s train of square wave pulses at 70 V of 0.7 ms duration and a frequency of 15 Hz. These stimulation parameters resulted in a 50- to 100-mm Hg increase in perfusion pressure above baseline. When the response stabilized, one of the arterial segments was exposed to SB 290157 delivered in the superfusion flow, and the other artery was left untreated. After a 15-min exposure to SB 290157 (10 nM, 100 nM, and 1 μM), C3a (100 nM) was introduced in the superfusion flow to both arterial segments, and the effect on perfusion pressure was monitored. Typically, C3a enhanced the perfusion pressure. The C3a-mediated increase in perfusion pressure was rapidly desensitized (1–2 min).

Male Hartley guinea pigs were obtained from Charles River Breeding Laboratories (Raleigh, NC) and maintained in a barrier facility. Guinea pigs were placed four at a time into a plastic box (20 liters) that had been modified with an intake and exhaust port; a small fan in the lid increased aerosol circulation. An LPS aerosol (Sigma) dissolved in normal saline (30 μg/ml) was generated by a modified DeVilbiss Pulmosonic nebulizer (DeVilbiss, Somerset, PA) and delivered for 15 min into the box via the intake port at a rate of 250 ml/min. SB 290157 (30 mg/kg) or vehicle (20% polyethylene glycol 400 (PEG) in saline) was administered i.p. 1 h before and 4 h after LPS challenge and administered twice a day (b.i.d.) 6 h apart on the next day. A third group of animals were left unexposed to LPS and received vehicle alone. Bronchoalveolar lavages (BAL) were performed 48 h after LPS exposure. Guinea pigs were euthanized by pentobarbital overdose, and the lungs were lavaged with 50 ml Dulbecco’s PBS (5 × 10 ml), which was aspirated after a gentle chest massage. The BAL fluid was centrifuged, and the pellet was resuspended in 0.25% NaCl to lyse residual erythrocytes; after centrifugation, the pellet was resuspended again in 1 ml 0.9% NaCl. After total cells were counted, slides were prepared, stained, and differentiated as eosinophils, neutrophils, and mononuclear cells by counting a minimum of 200 cells and expressing the results as percentage of total cells as well as actual numbers of each type. This measurement and expression technique has been previously validated, by histological methods, as accurately reflecting endothelial and subendothelial airway leukocytosis (32). Cell number and percentages were statistically compared by ANOVA followed by Fisher’s protected least square difference test.

Male inbred Lewis rats were obtained from Charles River Breeding Laboratories (Raleigh, NC). Within a given experiment, only animals of the same age were used. Adjuvant-induced arthritis (AIA) was induced as described previously (33). Briefly, 0.75 mg of Mycobacterium butyricum (Difco, Detroit, MI) suspended in paraffin oil was injected into the base of the tail of male Lewis rats 6–8 wk old (160–180 g). Hind paw volumes were measured by a water displacement method on day 20 (34). SB 290157 was suspended in a vehicle consisting of 5% ethanol, 10% Cremaphor-El, and 85% saline and administered b.i.d. at 30, 10, and 3 mg/kg i.p. in a final volume of 0.5 ml starting on the day of adjuvant injection. Cages were modified to allow the compromised animals free access to food and water. Control animals were given vehicle alone. Change in paw volume is presented as mean and SEM of 10–12 animals/group, and the percentage inhibition of hind paw edema was calculated as described (34). For statistical analysis, paw volumes of rats treated with SB 290157 were compared with the untreated controls by Student’s t test.

A pharmacokinetic study was conducted using three male Hartley guinea pigs. Under aseptic conditions, each guinea pig received surgically implanted femoral and arterial vein catheters at least 5 days before the study day. On the study day, fed animals received SB 290157 (30 mg/kg) as a single i.p. bolus injection (3 ml/kg total volume). The dose solution was prepared in normal saline with 20% PEG. Blood samples were obtained from a arterial catheter at various time intervals after administration of SB 290157; plasma was isolated by centrifugation. Plasma concentrations of SB 290157 were quantified by liquid chromatography/mass spectroscopy (MS)/MS (lower limit of quantitation was 10 ng/ml). Noncompartmental methods were used for analysis of plasma concentration vs time data (35).

All animal experimental procedures were in accordance with protocols approved by the SmithKline Beecham Institutional Animal Care and Use Committee, and met or exceeded the standards of the American Association for the Accreditation of Laboratory Animal Care), the U.S. Department of Health and Human Services, and all local and federal animal welfare laws.

9-Fluorenylmethoxycarbonyl (F-moc)-arginine(Boc)2 Wang.

To F-moc-arginine(Boc)2 (1.8 g, 3 mmol) and Wang resin (2 g, 2 mmol) in CH2Cl2 (40 ml) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (573 mg, 3 mmol) and 4-dimethylaminopyridine (244 mg, 2 mmol). The mixture was shaken overnight and washed twice with dimethylformamide (DMF) and six times with CH2Cl2.

(2,2-Diphenylethoxy)acetic acid.

Into a 0°C solution of 2,2-diphenylethanol (1 g) in DMF (10 ml) under argon was added 60% sodium hydride (350 mg). The solution was stirred for 10 min, tert-butyl bromoacetate (888 μl) was added, and the solution was warmed to ambient temperature. After stirring for 30 min, the reaction was quenched with water (20 ml), and the aqueous solution was extracted with ether (25 ml). The organic layer was washed with water (20 ml) and brine (20 ml). The organic solution was dried (MgSO4), and silica gel flash chromatography (3% ethyl acetate-hexanes) yielded tert-butyl(2,2-diphenylethoxy)acetate. The intermediate was treated with 25% trifluoroacetic acid (TFA)-CH2Cl2 for 1 h. The solvent was removed, and the residual TFA was removed by azeotrope with toluene to yield the title compound. 1H nuclear magnetic resonance (CDCl3) δ 7.1–7.4 (multiplet (m), 10H), 4.32 (triplet (t), J = 8.4 Hz, 1H), 4.0–4.1 (m, 4H).

N2-[(2,2-Diphenylethoxy)acetyl]-l-arginine(Boc)2Wang.

F-moc-2-carginine(Boc)2 Wang (200 mg) was treated with 20% piperidine in CH2Cl2 (5 ml) for 30 min. The solvent was drained, and the resin was washed with CH2Cl2 (six times). To the resin in DMF (3.5 ml) was added (2,2-diphenylethoxy)acetic acid (92 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (69 mg), and 1-hydroxybenzotriazole (49 mg); and the mixture was shaken overnight. The solution was drained, and the resin was washed twice with DMF and six times with CH2Cl2.

The resin was treated with a solution of 2.5% triisopropylsilane in 1:1 TFA-CH2Cl2 for 90 min. The cleavage solution was collected, the solvent was removed under reduced pressure, and residual TFA was removed by azeotrope with toluene. The residue was washed twice with hexanes, and the title compound was purified by reversed-phase HPLC (acetonitrile/water/0.1% trifluoroacetic acid). MS (electrospray) m/e = 413.3 (M+H)+.

To identify a nonpeptide C3aR antagonist, a high thoughput radioligand binding assay was configured using membranes prepared from RBL-C3aR cells and 125I-C3a. Approximately 240,000 compounds from the SmithKline Beecham compound collection were tested in a high throughput screen, affording 64 confirmed active compounds. One of these compounds, SKF 63649, was further progressed as a selective C3aR antagonist (Fig. 1,A). Subsequent chemical optimization of this compound led to the discovery of SB 290157 (Fig. 1,B). The affinity of the two compounds for the C3aR was evaluated in 125I-C3a competitive binding experiments. SB 290157 was an order of magnitude higher affinity than SKF 63649 for the C3aR in this assay; the IC50 values were 200 and 3000 nM, respectively (Fig. 2,A). A related structure, SB 280936 (Fig. 1 C), showed no affinity for this receptor in competitive binding assays at concentrations up to 10 μM and was used as a negative control.

FIGURE 1.

Structures of SKF 63649 and analogs. A, SKF 63649; B, SB 290157; C, SB 280936 from this same chemical series but inactive and used as negative control.

FIGURE 1.

Structures of SKF 63649 and analogs. A, SKF 63649; B, SB 290157; C, SB 280936 from this same chemical series but inactive and used as negative control.

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FIGURE 2.

Binding and cellular functional assays. A, Competition binding by SKF 63649 and SB 290157 of 125I-C3a binding to RBL C3aR cells. Affinities of SKF 63649 and SB 290157 were evaluated in competitive binding experiments with 125I-C3a binding to RBL-C3aR cells as described in Materials and Methods. Values are the mean ± SD for n = 3. B, Inhibition of C3a-induced Ca2+ mobilization in human neutrophils by SKF 63649, SB 280936, and SB 290157). Values are the mean ± SEM for n = 2–3. C, Inhibition of C3a-mediated chemotaxis of HMC-1 cells by SB 290157. C3a-mediated chemotaxis was tested in the absence or presence of 5 μM compound.

FIGURE 2.

Binding and cellular functional assays. A, Competition binding by SKF 63649 and SB 290157 of 125I-C3a binding to RBL C3aR cells. Affinities of SKF 63649 and SB 290157 were evaluated in competitive binding experiments with 125I-C3a binding to RBL-C3aR cells as described in Materials and Methods. Values are the mean ± SD for n = 3. B, Inhibition of C3a-induced Ca2+ mobilization in human neutrophils by SKF 63649, SB 280936, and SB 290157). Values are the mean ± SEM for n = 2–3. C, Inhibition of C3a-mediated chemotaxis of HMC-1 cells by SB 290157. C3a-mediated chemotaxis was tested in the absence or presence of 5 μM compound.

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To determine whether the compounds were functional antagonists, a FLIPR-based C3a-induced Ca2+ mobilization assay in RBL-C3aR cells was used. SKF 63649 and SB 290157 demonstrated concentration-dependent inhibition of 1 nM C3a-induced Ca2+ mobilization with IC50s of 350 nM (n = 2) and 27.7 ± 2.9 nM, (n = 3), respectively (Fig. 2 B). At concentrations up to 20 μM, SB 280936 had no effect on C3a-induced Ca2+ mobilization in RBL-C3aR cells. Testing activity with cells that naturally express the C3aR, we looked at the ability of the antagonists to inhibit C3a-induced Ca2+ mobilization in freshly isolated peripheral blood neutrophils. Both compounds were antagonists with IC50s of 388 and 30 nM for SKF 63649 and SB 290157, respectively. SB 290157 was selective for the C3aR in that it did not antagonize C5a-induced Ca2+ mobilization in human neutrophils or in RBL-C5aR cells, nor did it inhibit Ca2+ mobilization responses for five other GPCRs on neutrophils, i.e., leukotriene B4, fMLP, platelet-activating factor, CXCR1, and CXCR2.

SB 290157 was evaluated for its ability to inhibit C3a-induced chemotaxis of HMC-1 cells, a human mast cell line that naturally expresses the C3aR and for which C3a is chemotactic (5, 6, 36). A concentration of 5 μM SB 290157 markedly inhibited C3a-mediated chemotaxis of HMC-1 cells (Fig. 2 C). SB 290157 had no effect on C5a-mediated chemotaxis of HMC-1 cells (data not shown).

The antagonists were tested for inhibition of C3a-induced internalization of the C3aR. A 3-min incubation of neutrophils with 10 nM C3a is sufficient to stimulate internalization of ∼90% of the C3aR. Both SKF 63649 and SB 290157 inhibited C3aR internalization induced by 10 nM C3a in a concentration-dependent manner (Fig. 3). In the presence of >1 μM concentrations of the antagonists the internalization of the C3aR induced by C3a was reduced by ∼50% (Fig. 3). SB 280936 had no effect on C3aR internalization in this assay (Fig. 3).

FIGURE 3.

Effect of SB 290157 and SKF 63649 of C3a-induced C3aR internalization of human neutrophils. Human neutrophils were coincubated for 3 min at 37°C with varying concentrations of test compound and a 10 nM C3a. Data are mean ± SD from three experiments.

FIGURE 3.

Effect of SB 290157 and SKF 63649 of C3a-induced C3aR internalization of human neutrophils. Human neutrophils were coincubated for 3 min at 37°C with varying concentrations of test compound and a 10 nM C3a. Data are mean ± SD from three experiments.

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In addition to functional antagonism of the human C3aR, SB 290157 also was a potent inhibitor of C3a-induced Ca2+ mobilization of RBL 2H3 cells stably expressing the mouse and guinea pig C3aRs (Table I). The IC50s for SB 290157 inhibition of C3a-induced Ca2+ mobilization of the mouse and guinea pig C3aRs were 7 and 12.5 nM, respectively. SB 280936 was inactive at both the mouse and guinea pig C3aRs.

Table I.

SB 290157 potently inhibits C3a-induced calcium mobilization in RBL-2H3 cells expressing guinea pig, mouse, or the human C3aRa

RBL-2H3 Cells Expressing C3aRSB 290157 IC50 (nM)
Guinea pig 12.5 (n = 2) 
Mouse 7 (n = 2) 
Human 27.7± 2.9 (n = 3) 
RBL-2H3 Cells Expressing C3aRSB 290157 IC50 (nM)
Guinea pig 12.5 (n = 2) 
Mouse 7 (n = 2) 
Human 27.7± 2.9 (n = 3) 
a

The C3aR antagonist activity of SB 290157 was evaluated on the mouse and guinea pig C3aRs using the FLIPR-based Ca2+ assay in RBL-2H3 cells stably expressing these receptors. The IC50, defined as the concentration of SB 290157 that inhibits 50% of the maximal response induced by 1 nM C3a, was obtained from concentration-response curves.

To assess the functional activity of the antagonists for endogenous C3aRs of species other than human, they were evaluated for the inhibition of 1 nM (EC80 concentration in this assay) C3a-induced ATP release from guinea pig platelets, cells that naturally express the C3aR (37). Both SKF 63649 and SB 290157 inhibited in a concentration-dependent manner with IC50 values of 385 ± 185 and 30 ± 14 nM, respectively (Table II).

Table II.

Inhibition of C3a-mediated ATP release from guinea pig plateletsa

CompoundIC50 (nM)
SB 280936 Inactive 
SB 63649 385 ± 185 
SB 290157 30 ± 14 
CompoundIC50 (nM)
SB 280936 Inactive 
SB 63649 385 ± 185 
SB 290157 30 ± 14 
a

Varying concentrations of compounds were tested for the ability to inhibit the EC80 of C3a (∼1 nM C3a) in the guinea pig platelet ATP release assay. The IC50 is the concentration that inhibited 50% of the 1 nM C3a response in absence of antagonist. Values of one typical experiment of three are presented.

At the tissue level, C3a is a spasmogen causing contraction of smooth muscle. SB 290157 was evaluated for the inhibition of C3a-induced contractile response to field stimulation of the perfused rat caudal artery. SB 290157 at concentrations of 100 nM and 1 μM completely abolished the effect of C3a, whereas a concentration of 10 nM was inactive (Fig. 4).

FIGURE 4.

Effect of SB 290157 on field stimulation in the perfused rat caudal artery. Sprague Dawley rat tail caudal arterial segments were mounted in a chamber, perfused intraluminally, and superfused extraluminally as described in Materials and Methods. The periarterial sympathetic nerves were stimulated electrically, resulting in a 50- to 100-mm Hg increase in perfusion pressure. One segment was exposed to SB 290157 in the superfusion flow at the designated concentration, and the control segment was left untreated. After 15 min exposure to compound (10 nM, 100 nM and 1 μM), C3a was added to superfusion fluid of both segments. The effect of compound on the C3a-induced increase in perfusion pressure was quantitated as percent of the 100 nM C3a increase. Values are means ± SEM.

FIGURE 4.

Effect of SB 290157 on field stimulation in the perfused rat caudal artery. Sprague Dawley rat tail caudal arterial segments were mounted in a chamber, perfused intraluminally, and superfused extraluminally as described in Materials and Methods. The periarterial sympathetic nerves were stimulated electrically, resulting in a 50- to 100-mm Hg increase in perfusion pressure. One segment was exposed to SB 290157 in the superfusion flow at the designated concentration, and the control segment was left untreated. After 15 min exposure to compound (10 nM, 100 nM and 1 μM), C3a was added to superfusion fluid of both segments. The effect of compound on the C3a-induced increase in perfusion pressure was quantitated as percent of the 100 nM C3a increase. Values are means ± SEM.

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The pharmacokinetic profile of SB 290157 was assessed in guinea pigs and mice after i.p. administration. The results of the guinea pig study are summarized in Fig. 5. When administered i.p. at a dose of 30 mg/kg, high and sustained plasma concentrations (>100 ng/ml, 0.25 μM) of SB 290157 were detected out to 8 h (Fig. 5). The Cmax attained was 7000 ng/ml, and the apparent half-life (t1/2) was 0.89 ± 0.26 h. Similar pharmacokinetic data were obtained after i.p. administration of SB 290157 to mice (t1/2 = 1.47 ± 0.10 h; data not shown).

FIGURE 5.

Pharmacokinetic profile of i.p. administered SB 290157 in guinea pigs. Guinea pigs were given 30 mg/kg SB 290157 (20% PEG in saline) i.p. Blood samples were obtained at various times, and plasma levels of SB 290157 were quantified as described in Materials and Methods. Values are the mean ± SD for the three guinea pigs studied.

FIGURE 5.

Pharmacokinetic profile of i.p. administered SB 290157 in guinea pigs. Guinea pigs were given 30 mg/kg SB 290157 (20% PEG in saline) i.p. Blood samples were obtained at various times, and plasma levels of SB 290157 were quantified as described in Materials and Methods. Values are the mean ± SD for the three guinea pigs studied.

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SB 290157 was evaluated in a guinea pig LPS-induced airway neutrophilia model. As seen in Fig. 6, LPS (10 μg/ml) administered as an aerosol produced an infiltration of leukocytes (5-fold higher than with unexposed animals), especially neutrophils (>1000-fold) 48 h after LPS exposure. The resultant airway neutrophilia was reduced (39%) by administration of SB 290157, 30 mg/kg i.p. b.i.d. (LPS + vehicle = 33.2 ± 3.0 million neutrophils or 50.4% of total leukocytes recovered; LPS + SB 290157 = 20.3 ± 1.7 million neutrophils or 31.5% of total leukocytes; p = 0.02, Fisher’s protected least square difference). Total leukocyte numbers for treated animals were not significantly different (LPS + vehicle = 65.1 ± 11.3 million; LPS + SB 290157 = 62.0 ± 10.1 million) from vehicle-treated animals.

FIGURE 6.

Effect of SB 290157 in the guinea pig lung neutrophilia model. Guinea pigs were given SB 290157 i.p. at 30 mg/kg or vehicle (20% PEG in saline) 1 h before and 4 h after LPS aerosol challenge and 6 h apart the next day. The BAL was performed 48 h after LPS exposure as described in Materials and Methods. Total cells were counted, and slides were prepared for counting eosinophils, neutrophils, and mononuclear cells with results expressed as percent of total cells. Values are the mean ± SEM, ∗, p < 0.05 vs LPS and vehicle.

FIGURE 6.

Effect of SB 290157 in the guinea pig lung neutrophilia model. Guinea pigs were given SB 290157 i.p. at 30 mg/kg or vehicle (20% PEG in saline) 1 h before and 4 h after LPS aerosol challenge and 6 h apart the next day. The BAL was performed 48 h after LPS exposure as described in Materials and Methods. Total cells were counted, and slides were prepared for counting eosinophils, neutrophils, and mononuclear cells with results expressed as percent of total cells. Values are the mean ± SEM, ∗, p < 0.05 vs LPS and vehicle.

Close modal

SB 290157 was also evaluated in an adjuvant-induced arthritis model using a prophylactic dosing protocol. Compound was administered to male Lewis rats starting on the day of adjuvant injection. SB 290157 was administered i.p. b.i.d., and paw inflammation was measured on day 20. There was 41% inhibition of paw edema on day 20 (p < 0.001) in animals that received a dose of 30 mg/kg b.i.d for 20 days. There was no significant effect on paw edema in rats that received 3 or 10 mg/kg SB 290157 b.i.d. (Fig. 7).

FIGURE 7.

Prophylactic activity of SB 290157 on paw edema in AIA in Lewis rats. Rats were treated with SB 290157 in 5% ethanol, 10% Cremaphor-El, and 85% saline at 3, 10, and 30 mg/kg i.p. b.i.d. or vehicle (AIA cont) alone starting on the day of adjuvant injection, and paw edema was measured on day 20 as described in Materials and Methods. Values are the mean ± SEM of 10–12 rats/group (inhibition expressed as % AIA controls). ∗, p < 0.001 vs AIA controls.

FIGURE 7.

Prophylactic activity of SB 290157 on paw edema in AIA in Lewis rats. Rats were treated with SB 290157 in 5% ethanol, 10% Cremaphor-El, and 85% saline at 3, 10, and 30 mg/kg i.p. b.i.d. or vehicle (AIA cont) alone starting on the day of adjuvant injection, and paw edema was measured on day 20 as described in Materials and Methods. Values are the mean ± SEM of 10–12 rats/group (inhibition expressed as % AIA controls). ∗, p < 0.001 vs AIA controls.

Close modal

A small molecule nonpeptide C3aR antagonist, SKF 63649, identified from a high throughput screen inhibited the C3aR binding with low micromolar affinity. After chemical optimization, to afford SB 290157, the affinity for the C3aR was increased by an order of magnitude. SB 290157 was a functional antagonist demonstrating equipotent inhibition of the C3a-induced Ca2+ mobilization response at the native receptor expressed on freshly isolated neutrophils, as well as at the recombinant C3aR stably expressed on RBL-2H3 cells. SB 290157 was a functional antagonist not only of the human C3aR but also of the mouse, rat, and guinea pig C3aRs. The potencies of SB 290157 for inhibition of C3a-induced Ca2+ mobilization of the mouse, guinea pig, and human receptors were similar (IC50 = 7–30 nM). This was somewhat surprising in light of the relatively low level of sequence identity (60–65% overall identity) between the C3aR from these different species (1). SB 290157 was selective for the C3aR and did not antagonize the C5aR or 5 other chemotactic GPCRs on human neutrophils.

There was good correlation between the antagonist potency of SB 290157 in the human neutrophil Ca2+ mobilization assay and the guinea pig ATP release assay. This result supports the recombinant receptor antagonist data demonstrating similar potency at endogenous C3aRs from two species. However, the antagonist potencies determined in the functional assays were ∼7-fold higher than the affinity estimated in the whole cell binding assay. This is likely due to the inherent differences in the assay protocols, including: differences in times of incubation for the functional assays (seconds) vs the equilibrium conditions (30–60 min) in the binding assay; the temperatures at which the assays were run (room temperature for the binding assay vs 37°C for the functional assay): or possibly the effect of iodination on the affinity of C3a for its receptor. The effect of iodination of C3a on its interaction with the C3aR appears to be minimal, because the affinities determined for C3a with the C3aR in competition binding assays were in good agreement with the published Kd for the C3aR (0.1–1.0 nM). In both binding and functional assays, SB 290157 was consistently 10-fold more potent as a C3aR antagonist than with the initial high throughput screening hit, SKF 63649.

The C3aR antagonist compounds had a significant effect on C3a-induced C3aR internalization, inhibiting by almost 50% the number of receptors internalized in response to challenge with 10 nM C3a. At doses of <10 μM, SB 290157 appeared to be a more potent antagonist of C3a-induced receptor internalization than SKF 63649, consistent with the potency obtained with this compound in the binding and functional assays.

Marked inhibition of C3a-mediated chemotaxis of HMC-1 cells and of the C3a-induced contractile response to field stimulation in perfused rat caudal arteries was also noted with SB 290157. Concentration response studies were difficult to perform in these assays, but SB 290157 antagonized mouse, rat, and guinea pig C3a receptors with potencies equivalent to the potency vs the human C3aR. These data, combined with the determination that after i.p. administration to mice and guinea pigs plasma levels of SB 290157 were high and sustained, indicated that it was a suitable compound for study in animal models to help define the physiological and pathophysiological role of C3a and the C3aR.

The pathophysiological role of C3a has been difficult to assess because as a result of complement activation both C3a and C5a are released into the circulation. Although C5a is generally more potent in its actions than C3a, the plasma concentration of C3a is significantly higher (∼10-fold) than that of C5a (38). Another factor complicating studies of the in vivo actions of C3a is the rapid inactivation of this peptide via the cleavage of the amino-terminal arginine residue by carboxypeptidase N. The resultant peptide, C3a(desArg), is inactive at the C3aR (13, 39), therefore serum carboxypeptidase N inhibitors have been used for in vivo studies to increase the effectiveness of C3a (40). Several groups have assessed the pulmonary and cardiac effects of C3a in animals after direct administration. Intrabronchial instillation of C3a into guinea pigs induced acute pulmonary injury and bronchospasm (8, 9). The i.v. injection of C3a into guinea pigs caused a rapid neutropenia with resultant sequestration of neutrophils within lung tissue (7), whereas C3a administered by coronary bolus to guinea pigs induced cardiac dysfunction (41).

Recent reports with C3aR-deficient guinea pigs showed decreased bronchial reactivity in a OVA-induced asthma model compared with control animals (22). In addition, another recent study with C3aR−/− mice has demonstrated decreased airway hyperresponsivness to methacholine challenge compared with wild-type mice (20). These studies suggested a potential role for C3a in inflammatory pulmonary diseases such as asthma and acute respiratory distress syndrome.

We studied the C3aR antagonist, SB 290157, in two animal models of inflammation. In the first, SB 290157 inhibited neutrophil recruitment and accumulation in a guinea pig LPS-induced airway neutrophilia model. The inhibitory activity appeared to be specific for neutrophils as the number of neutrophils recovered in the challenged lungs was decreased, but there was no significant inhibition of the total number of cells recovered. This is somewhat surprising because C3a is not chemotactic for neutrophils, although they express the C3aR, demonstrate specific binding, and respond to C3a with a transient calcium response (27). The effect of SB 290157 may be a secondary rather than a direct effect on neutrophil recruitment.

SB 290157 was also tested in a disease-modifying rat model of AIA. Antiinflammatory activity was observed in Lewis rats that received SB 290157, 30 mg/kg i.p. b.i.d. There was a significant reduction (41%) in paw swelling as compared with the control untreated animals. This is significant activity for the C3aR antagonist in an aggressive arthritis model and potentially implicates C3a in the pathogenesis of this disease.

Our data indicate that SB 290157 is a high affinity, selective, and competitive C3aR antagonist. It is active in two in vivo models of inflammation; therefore, it shows promise as a tool compound for further studies to elucidate physiological and pathophysiological role(s) of C3aR activation.

3

Abbreviations used in this paper: GPCR, G protein-coupled receptor; SPA, scintillation proximity assay; FLIPR, fluorometric imaging plate reader; BAL, bronchoalveolar lavage; AIA, adjuvant-induced arthritis; DMF, dimethylformamide; TFA, trifluoroacetic acid; F-moc, 9-fluorenylmethoxycarbonyl; Boc,tert-butoxycarbonyl; RBL, rat basophilic leukemia; HAG-CM, 20 mM HEPES (pH 7.4), 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 0.25% BSA, and 0.5 mM glucose; SB 290157, N2-[(2,2-diphenylethoxy)acetyl]-l-arginine; SKF 63649, 1-naphthyloxyacetylarginine; PEG, polyethylene glycol 400; b.i.d., twice a day; m, multiplet; t, triplet.

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