C3a receptor (IC50 = 200 nM)

At an IC50 of 200 nM, SB 290157 acts as a competitive antagonist of 125I-C3a radioligand binding to rat basophilic leukemia-2H3 cells expressing human C3aR (RBL-C3aR). With IC50 values of 27.7 and 28 nM, respectively, SB 290157 inhibits C3a-induced Ca2+ mobilization and C3a-induced C3aR internalization in RBL-C3aR cells and human neutrophils. Because it does not agonistically interact with the C5aR or six other chemotactic G protein-coupled receptors, SB 290157 exhibits selectivity for the C3aR. Additionally, RBL-2H3 cells expressing the mouse and guinea pig C3aRs are inhibited by SB 290157 when it comes to C3a-induced Ca2+ mobilization. It potently inhibits both the C3a-induced potentiation of the contractile response to field stimulation of the perfused rat caudal artery and the C3a-mediated ATP release from guinea pig platelets[1].

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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, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s 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 Ca(2+) 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 [1].

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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 SB290157 (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.

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 SB290157, 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.

SB290157 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. 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).

In addition to functional antagonism of the human C3aR, SB290157 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 SB290157 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.

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. Both SKF 63649 and SB290157 inhibited in a concentration-dependent manner with IC50 values of 385 ± 185 and 30 ± 14 nM, respectively [1].

In models of guinea pig LPS-induced airway neutrophilia and rat adjuvant-induced arthritis, SB290157 reduces paw edema and inhibits neutrophil recruitment, respectively[1]. Only after three hours is the antagonist effective to diminish joint swelling; at 30 mg/kg, there is an observed 50% suppression of joint swelling. At three hours, the C3 level is much lower than in naive mice who exhibit complement consumption. Additionally, it is noted that the C3 activation increases in accordance with the anti-OVA pAb's graded concentration [2].

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SB290157 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 SB290157, 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.
SB290157 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. [1].

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It was investigated whether the C3a-receptor antagonist (C3aRA) SB290157 was involved in the suppression of anti-OVA pAb-induced arthritis because it is well known that anaphylatoxin C3a plays a crucial role in the development of an effective inflammatory response during complement activation. Anti-OVA pAb-induced arthritis was induced in DBA/1J mice by administration of anti-OVA pAb 0.5 h prior to intra-articular (i.a.) injection of OVA (0 h). Two peaks of joint swelling were observed at 0.5 and 3 h. The role of C3aRA in arthritis was investigated by injection of SB290157 at concentrations of 10 and 30 mg/kg at 0 and 2 h. The antagonist was able to reduce joint swelling only at 3 h, and about 50% inhibition of joint swelling was observed with the concentration of 30 mg/kg. The C3 level was significantly decreased at 3 h compared with naïve mice showing complement consumption. Furthermore, the C3 activation was observed and increased corresponding to the graded concentration of anti-OVA pAb. The results also revealed that the C3aRA was able to reduce the expression of IL-1beta in synovial tissue. Taken together, the results suggested that C3aRA may be effective in the inhibition of arthritis [2].

Receptor internalization assay [1]
The flow cytometric internalization assay using C3aR-specific rabbit polyclonal antiserum was performed as described. 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 and then evaluated for receptor internalization.

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Binding assays [1]
High throughput scintillation proximity assay (SPA) for C3a antagonists. [1]
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 format. Bolton-Hunter custom iodination was performed by NEN Research Products 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 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, 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.

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Binding for follow-up studies. [1]
The binding assay was performed essentially as previously described (26). Briefly, 2–5 × 105 RBL-C3aR cells were incubated with 100 pM 125I-C3a 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 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. Data analysis was performed using KaleidaGraph v3.09.

HMC-1 chemotaxis assay [1]
C3a-mediated chemotaxis of HMC-1 cells was assessed using Neuro Probe 96-well disposable chemotaxis plates (5 μm pore size). The top surface of the membrane was precoated with 100 ng laminin or fibronectin. 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.

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Fluorometric imaging plate reader (FLIPR) Ca2+ mobilization assay [1]
C3aR Ca2+ mobilization studies were conducted using Fluo 3-loaded RBL-C3aR cells and a microtiter plate-based assay using a FLIPR. 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) 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. 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.

Contraction of rat caudal artery [1]
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 SB290157 delivered in the superfusion flow, and the other artery was left untreated. After a 15-min exposure to SB290157 (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).

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Guinea pig airway neutrophilia model [1]
Male Hartley guinea pigs were obtained from Charles River Breeding Laboratories 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 dissolved in normal saline (30 μg/ml) was generated by a modified DeVilbiss Pulmosonic nebulizer and delivered for 15 min into the box via the intake port at a rate of 250 ml/min. SB290157 (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. Cell number and percentages were statistically compared by ANOVA followed by Fisher’s protected least square difference test.

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Adjuvant-induced arthritis [1]
Male inbred Lewis rats were obtained from Charles River Breeding Laboratories. Within a given experiment, only animals of the same age were used. Adjuvant-induced arthritis (AIA) was induced as described previously. Briefly, 0.75 mg of Mycobacterium butyricum 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. SB290157 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. For statistical analysis, paw volumes of rats treated with SB290157 were compared with the untreated controls by Student’s t test.

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Pharmacokinetic studies in guinea pigs [1]
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 SB290157 (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 SB290157; plasma was isolated by centrifugation. Plasma concentrations of SB290157 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. Induction of arthritis into DBA/1J mice [2]
Mice were given 0.2 ml of rat anti-OVA polyclonal antibody (10 mg/ml) by intravenous (i.v.) injection 0.5 h before OVA administration (−0.5 h). OVA (10 µ g) was dissolved in 25 µ l PBS and given by intra-articular (i.a.) injection (0 h). The OVA injection alone was used as the baseline. The net increase in joint thickness attributable to anti-OVA pAb injection was calculated by subtracting the joint thickness of OVA-injected nonimmunized mice from that of the anti-OVA pAb–injected mice. Administration of SB290157, a C3aR antagonist, (10 or 30 mg/kg) was injected i.p. two times, at 0 (right after OVA injection) and 2 h while 5% ethanol in PBS was used as a vehicle control. Joint swelling was measured using a dial thickness gauge before injection, at 0.5 h, and then every hour until 5 h after OVA injection.

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). [1]

[1]. Identification of a selective nonpeptide antagonist of the anaphylatoxin C3areceptor that demonstrates antiinflammatory activity in animal models. J Immunol. 2001 May 15;166(10):6341-8.

[2]. Effect of the C3a-receptor antagonist SB 290157 on anti-OVA polyclonalantibody-induced arthritis. J Pharmacol Sci. 2010;112(1):56-63.

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. 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.

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. 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. [1]

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In conclusion, the present data indicate that SB 290157 can inhibit the induction of arthritis by lowering the level of joint swelling, neutrophil migration, and IL-1 β production. The current findings suggested that C3a may be involved in the aggravation of arthritis by using SB 290157. Therefore, SB 290157 may be effective in the inhibition of arthritis. [2]

C24H29F3N4O6

526.505476713181

526.203

C, 54.75; H, 5.55; F, 10.83; N, 10.64; O, 18.23

1140525-25-2

1140525-25-2 (TFA);259218-28-5;

16760645

White to yellow solid powder

5

10

12

37

619

1

C1=CC=C(C=C1)C(COCC(=O)N[C@@H](CCCN=C(N)N)C(=O)O)C2=CC=CC=C2.C(=O)(C(F)(F)F)O

ZJRMPPVJAQWGEG-FYZYNONXSA-N

InChI=1S/C22H28N4O4.C2HF3O2/c23-22(24)25-13-7-12-19(21(28)29)26-20(27)15-30-14-18(16-8-3-1-4-9-16)17-10-5-2-6-11-17;3-2(4,5)1(6)7/h1-6,8-11,18-19H,7,12-15H2,(H,26,27)(H,28,29)(H4,23,24,25);(H,6,7)/t19-;/m0./s1

(2S)-5-(diaminomethylideneamino)-2-[[2-(2,2-diphenylethoxy)acetyl]amino]pentanoic acid;2,2,2-trifluoroacetic acid

1140525-25-2; SB290157 trifluoroacetate; SB 290157 trifluoroacetate salt; SB290157 (trifluoroacetate); (2S)-5-(diaminomethylideneamino)-2-[[2-(2,2-diphenylethoxy)acetyl]amino]pentanoic acid;2,2,2-trifluoroacetic acid; SB 290157 trifluoroacetate; N2-[2-(2,2-Diphenylethoxy)acetyl]-L-arginine 2,2,2-Trifluoroacetate;; SB290157 trifluoroacetate salt;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~189.93 mM)
Ethanol : ~100 mg/mL (~189.93 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (9.50 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear EtOH stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 5 mg/mL (9.50 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 5 mg/mL (9.50 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (3.95 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: ≥ 2.08 mg/mL (3.95 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 6: ≥ 2.08 mg/mL (3.95 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)