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Departments of
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Molecular Biology,
Medicinal Chemistry,
Pulmonary Biology,
Molecular Screening Technologies,
¶ Renal Biology,
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Drug Metabolism, and
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Bone and Cartilage Biology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA; and
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Institute of Medical Microbiology, Medical School Hannover, Hannover, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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)
Binding assays
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 20x solution followed by a 1:1 mixture with
H2O to yield a 10x, 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 
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 x 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).
Receptor internalization assay
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.
Fluorometric imaging plate reader (FLIPR) Ca2+ mobilization assay
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.
Suspended cell Ca2+ mobilization
fura-2-acetoxymethyl ester-loaded human neutrophils or RBL-C3aR cells were assayed for a Ca2+ mobilization response, as described (30).
Guinea pig platelet ATP release assay
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.
HMC-1 chemotaxis assay
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 x 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.
Contraction of rat caudal artery
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).
Guinea pig airway neutrophilia model
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 x 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.
Adjuvant-induced arthritis
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.
Pharmacokinetic studies in guinea pigs
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.
Synthesis of SB 290157
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) 
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.
N2-[(2,2-Diphenylethoxy)acetyl]-L-arginine.
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)+.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 SB 290157 (Fig. 1B). 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. 2A). A related structure, SB
280936 (Fig. 1C), showed no affinity for this receptor in
competitive binding assays at concentrations up to 10 µM and was used
as a negative control.
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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. 2C). 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).
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). 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.
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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).
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, 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Footnotes |
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2 Current address: Sunesis Pharmaceuticals, 3696 Haven Avenue, No. C, Redwood City, CA 94063
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.
Received for publication December 19, 2000. Accepted for publication March 9, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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K. Kambas, M. M. Markiewski, I. A. Pneumatikos, S. S. Rafail, V. Theodorou, D. Konstantonis, I. Kourtzelis, M. N. Doumas, P. Magotti, R. A. DeAngelis, et al. C5a and TNF-{alpha} Up-Regulate the Expression of Tissue Factor in Intra-Alveolar Neutrophils of Patients with the Acute Respiratory Distress Syndrome J. Immunol., June 1, 2008; 180(11): 7368 - 7375. [Abstract] [Full Text] [PDF]
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A. C. Jacobson, J. J. Weis, and J. H. Weis Complement Receptors 1 and 2 Influence the Immune Environment in a B Cell Receptor-Independent Manner J. Immunol., April 1, 2008; 180(7): 5057 - 5066. [Abstract] [Full Text] [PDF]
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 X. Zhang, Y. Kimura, C. Fang, L. Zhou, G. Sfyroera, J. D. Lambris, R. A. Wetsel, T. Miwa, and W.-C. Song Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo Blood, July 1, 2007; 110(1): 228 - 236. [Abstract] [Full Text] [PDF]
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 P. Dillard, R. A. Wetsel, and S. M. Drouin Complement C3a Regulates Muc5ac Expression by Airway Clara Cells Independently of Th2 Responses Am. J. Respir. Crit. Care Med., June 15, 2007; 175(12): 1250 - 1258. [Abstract] [Full Text] [PDF]
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J. M. Thurman, A. M. Lenderink, P. A. Royer, K. E. Coleman, J. Zhou, J. D. Lambris, R. A. Nemenoff, R. J. Quigg, and V. M. Holers C3a Is Required for the Production of CXC Chemokines by Tubular Epithelial Cells after Renal Ishemia/Reperfusion J. Immunol., February 1, 2007; 178(3): 1819 - 1828. [Abstract] [Full Text] [PDF]
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R. Purwar, M. Wittmann, J. Zwirner, M. Oppermann, M. Kracht, O. Dittrich-Breiholz, R. Gutzmer, and T. Werfel Induction of C3 and CCL2 by C3a in Keratinocytes: A Novel Autocrine Amplification Loop of Inflammatory Skin Reactions J. Immunol., October 1, 2006; 177(7): 4444 - 4450. [Abstract] [Full Text] [PDF]
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K. Ritis, M. Doumas, D. Mastellos, A. Micheli, S. Giaglis, P. Magotti, S. Rafail, G. Kartalis, P. Sideras, and J. D. Lambris A Novel C5a Receptor-Tissue Factor Cross-Talk in Neutrophils Links Innate Immunity to Coagulation Pathways J. Immunol., October 1, 2006; 177(7): 4794 - 4802. [Abstract] [Full Text] [PDF]
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 J Mocco, W. J. Mack, A. F. Ducruet, S. A. Sosunov, M. E. Sughrue, B. G. Hassid, M. N. Nair, I. Laufer, R. J. Komotar, M. Claire, et al. Complement Component C3 Mediates Inflammatory Injury Following Focal Cerebral Ischemia Circ. Res., July 21, 2006; 99(2): 209 - 217. [Abstract] [Full Text] [PDF]
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 M. Nozaki, B. J. Raisler, E. Sakurai, J. V. Sarma, S. R. Barnum, J. D. Lambris, Y. Chen, K. Zhang, B. K. Ambati, J. Z. Baffi, et al. Drusen complement components C3a and C5a promote choroidal neovascularization PNAS, February 14, 2006; 103(7): 2328 - 2333. [Abstract] [Full Text] [PDF]
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 M. Schaefer, S. Konrad, J. Thalmann, C. Rheinheimer, K. Johswich, B. Sohns, and A. Klos The Transcription Factors AP-1 and Ets Are Regulators of C3a Receptor Expression J. Biol. Chem., December 23, 2005; 280(51): 42113 - 42123. [Abstract] [Full Text] [PDF]
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M. Honczarenko, M. Z. Ratajczak, A. Nicholson-Weller, and L. E. Silberstein Complement C3a Enhances CXCL12 (SDF-1)-Mediated Chemotaxis of Bone Marrow Hematopoietic Cells Independently of C3a Receptor J. Immunol., September 15, 2005; 175(6): 3698 - 3706. [Abstract] [Full Text] [PDF]
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L. Bao, I. Osawe, M. Haas, and R. J. Quigg Signaling through Up-Regulated C3a Receptor Is Key to the Development of Experimental Lupus Nephritis J. Immunol., August 1, 2005; 175(3): 1947 - 1955. [Abstract] [Full Text] [PDF]
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A. G. Therien, R. Baelder, and J. Kohl Agonist Activity of the Small Molecule C3aR Ligand SB 290157 J. Immunol., June 15, 2005; 174(12): 7479 - 7480. [Full Text] [PDF]
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R. Baelder, B. Fuchs, W. Bautsch, J. Zwirner, J. Kohl, H. G Hoymann, T. Glaab, V. Erpenbeck, N. Krug, and A. Braun Pharmacological Targeting of Anaphylatoxin Receptors during the Effector Phase of Allergic Asthma Suppresses Airway Hyperresponsiveness and Airway Inflammation J. Immunol., January 15, 2005; 174(2): 783 - 789. [Abstract] [Full Text] [PDF]
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J. Godau, T. Heller, H. Hawlisch, M. Trappe, E. Howells, J. Best, J. Zwirner, J. S. Verbeek, P. M. Hogarth, C. Gerard, et al. C5a Initiates the Inflammatory Cascade in Immune Complex Peritonitis J. Immunol., September 1, 2004; 173(5): 3437 - 3445. [Abstract] [Full Text] [PDF]
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J. Ratajczak, R. Reca, M. Kucia, M. Majka, D. J. Allendorf, J. T. Baran, A. Janowska-Wieczorek, R. A. Wetsel, G. D. Ross, and M. Z. Ratajczak Mobilization studies in mice deficient in either C3 or C3a receptor (C3aR) reveal a novel role for complement in retention of hematopoietic stem/progenitor cells in bone marrow Blood, March 15, 2004; 103(6): 2071 - 2078. [Abstract] [Full Text] [PDF]
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 H. F. Ismail, J. Zhang, R. G. Lynch, Y. Wang, and D. J. Berg Role for Complement in Development of Helicobacter-Induced Gastritis in Interleukin-10-Deficient Mice Infect. Immun., December 1, 2003; 71(12): 7140 - 7148. [Abstract] [Full Text] [PDF]
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R. Reca, D. Mastellos, M. Majka, L. Marquez, J. Ratajczak, S. Franchini, A. Glodek, M. Honczarenko, L. A. Spruce, A. Janowska-Wieczorek, et al. Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1 Blood, May 15, 2003; 101(10): 3784 - 3793. [Abstract] [Full Text] [PDF]
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T. E. Mollnes, O.-L. Brekke, M. Fung, H. Fure, D. Christiansen, G. Bergseth, V. Videm, K. T. Lappegard, J. Kohl, and J. D. Lambris Essential role of the C5a receptor in E coli-induced oxidative burst and phagocytosis revealed by a novel lepirudin-based human whole blood model of inflammation Blood, August 13, 2002; 100(5): 1869 - 1877. [Abstract] [Full Text] [PDF]
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