^125I-IL-8-CXCR2 ( IC50 = 22 nM )

In vitro activity: SB225002 effectively suppresses both IL-8 and GROalpha-induced human and rabbit neutrophil chemotaxis, as well as GROα-stimulated calcium mobilization.[1] In WHCO1 cells, SB 225002 significantly lowers the amounts of phosphorylated ERK1/2 and suppresses cell division.[2] Moreover, SB225002 exhibits antitumor activity by inhibiting microtubules.[3]

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A chemical lead, which selectively inhibited CXCR2 was discovered by high throughput screening and chemically optimized. SB225002 (N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea) is the first reported potent and selective non-peptide inhibitor of a chemokine receptor. It is an antagonist of 125I-IL-8 binding to CXCR2 with an IC50 = 22 nM. SB 225002 showed >150-fold selectivity over CXCR1 and four other 7-TMRs tested. In vitro, SB 225002 potently inhibited human and rabbit neutrophil chemotaxis induced by both IL-8 and GROalpha.
To determine the feasibility of targeting individual IL-8 receptors with non-peptide, low molecular weight antagonists, a high throughput screen was configured using 125I-IL-8 binding to membranes of CHO-CXCR1 or CHO-CXCR2 cells. One compound identified from this screen was SK&F 83589 (Fig. 1 A), which selectively inhibited 125I-IL-8 binding to CHO-CXCR2 with an IC50 of 500 nm. Chemical modification of SK&F 83589 led to SB 225002,N-(2-hydroxy-4-nitrophenyl)-N′-(2-bromophenyl)urea (Fig. 1 B), which inhibited 125I-IL-8 binding to CHO-CXCR2 membranes with an IC50 = 22 nm (Fig. 2). SB 225002, at concentrations up to 3.3 μm (Fig. 2), failed to significantly inhibit the binding of 125I-IL-8 to CHO-CXCR1, or [3H]fMLP, [3H]LTB4, [3H]LTD4, or 125I-C5a to their cognate receptors. SB225002 was, therefore, at leased >150-fold selective for CXCR2 over the other 7-TMRs tested.
To determine if SB225002 was a functional CXCR2 antagonist, we monitored its effects on intracellular calcium mobilization stimulated by IL-8 or GROα. Cross-desensitization studies with Me2SO differentiated HL60 cells indicated that these cells predominantly express CXCR2 (∼80%) with a smaller number of CXCR1 (∼20%) receptors (Fig. 3 A). In these cells, SB 225002 produced a concentration-dependent inhibition of both IL-8- and GROα-mediated calcium mobilization with IC50 values of 8 and 10 nm, respectively (Fig. 3 B). Similarly, in 3ASubE cells stably transfected with CXCR2, SB 225002 dose-dependently inhibited calcium mobilization induced by both GROα and IL-8, with IC50values of 20 and 40 nm, respectively (Fig. 3 B). In contrast to HL60 cells, human neutrophils express equal numbers of CXCR1 and CXCR2 on their cell surface. In these cells, SB 225002 inhibited GROα-, but not IL-8-, stimulated calcium mobilization (IC50 values = 30 nm and >10 μm, respectively, Fig. 3 C). In addition, in RBL-2H3 cells, stably transfected with CXCR1, SB 225002 failed to inhibit calcium mobilization induced by either IL-8 or LTD4(IC50 > 10 μm, Fig. 3 C). The failure of SB 225002 to block IL-8-induced calcium mobilization in human neutrophils, presumably reflects the ability of IL-8 to circumvent the blockade of CXCR2 by activating CXCR1, which was not inhibited by this compound. SB 225002 also demonstrated functional selectivity, since it failed to inhibit calcium mobilization induced by optimal concentrations of LTB4 in PMNs (Fig. 3 C), or RANTES, MIP-1α, or MCP-1 in monocytes (data not shown). [1]

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RObeta enhances transcription of EGR-1, via the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, which can be blocked by a specific antagonist of CXCR2 (SB 225002) or specific antibody to GRObeta. WHCO1 cells treated with SB 225002 exhibited a 40% reduction in cell proliferation. [2]

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SB225002 (SB) is an IL-8 receptor B (IL-8RB) antagonist that has previously been shown to inhibit IL-8-based cancer cell invasion, and to possess in vivo anti-inflammatory and anti-nociceptive effects. The present study presented an evidence for the cell cycle-targeting activity of SB225002/SB in a panel of p53-mutant human cancer cell lines of different origin, and investigated the underlying molecular mechanisms. A combination of cell cycle analysis, immunocytometry, immunoblotting, and RNA interference revealed that SB induced a BubR1-dependent mitotic arrest. Mechanistically, SB was shown to possess a microtubule destabilizing activity evidenced by hyperphosphorylation of Bcl2 and BclxL, suppression of microtubule polymerization and induction of a prometaphase arrest. Molecular docking studies suggested that SB has a good affinity toward vinblastine-binding site on β-tubulin subunit. Of note, SB265610 which is a close structural analog of SB225002 with a potent IL-8RB antagonistic activity did not exhibit a similar antimitotic activity. Importantly, in P-glycoprotein overexpressing NCI/Adr-Res cells the antitumor activity of SB was unaffected by multidrug resistance. Interestingly, the mechanisms of SB-induced cell death were cell-line dependent, where in invasive hepatocellular carcinoma HLE cells the significant contribution of BAK-dependent mitochondrial apoptosis was demonstrated. Conversely, SB activated p38 MAPK signaling in colorectal adenocarcinoma cells SW480, and pharmacologic inhibition of p38 MAPK activity revealed its key role in mediating SB-induced caspase-independent cell death. In summary, the present study introduced SB as a promising antitumor agent which has the potential to exert its activity through dual mechanisms involving microtubules targeting and interference with IL-8-drivin cancer progression.[3]

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Effects of CXCR2 siRNA or an antagonist on ICC cell proliferation [4]
The effect of CXCR2 inhibition by siRNA or an antagonist (SB225002) on ICC cell proliferation was assessed in vitro. The cells were harvested 3 days after CXCR2 siRNA administration, and were counted by a crystal violet staining procedure. CXCR2 siRNA significantly inhibited ICC cell proliferation in both RBE (P = .009) and SSP25 cells (P = .01) compared with the control (Fig 2, A and B left). The inhibition of CXCR2 was also performed using a CXCR2 antagonist, SB225002. ICC cells were harvested 3 days after SB225002 administration, and were counted using the same method. SB225002 suppressed ICC cell proliferation in both RBE (P = .03) and SSP25 (P = .02) cells as well as the siRNA did, (Fig 2, A and B right). Effects of CXCR2 siRNA or antagonist on ICC cell migration [4]
In the wound assay, both ICC cell lines migrated away from the original site of adhesion to the plate over a 24-hour period in the control group. On the contrary, both CXCR2 siRNA and SB225002 suppressed cell migration compared with control (Fig 2, C, left photographs shows representative result of RBE cells). As a result of counted the number of migrated cells in 10 high-power fields (×200), both CXCR2 siRNA and SB225002 suppressed ICC cell migration compared with the control with significant difference (Fig 2, C, right graphs; siRNA in RBE, P = .04; SB225002 in RBE, P = .018; siRNA in SSP25, P = .004; and SB225002 in SSP25, P = .001).

SB225002 specifically inhibits the neutrophil margination induced by IL-8 in rabbits. [1] The growth of transplanted subcutaneous tumors is suppressed by SB225002 (1 mg/kg i.p.) in a mouse intrahepatic cholangiocellular carcinoma model. [4] Furthermore, SB225002 exhibits persistent antinociceptive properties and mitigates colitis induced by TNBS in mice models. [5] [6]

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In vivo, SB225002 selectively blocked IL-8-induced neutrophil margination in rabbits. [1]
In rabbits, an intravenous infusion of LTB4, or other chemotactic factors, rapidly promotes neutrophil shape change and margination of neutrophils to the microcapillary endothelial cells of the lung (27, 30, 31, 32). Thus, this is the basis of a useful model to study the initial stages of neutrophil activation and attachment to the endothelium. As seen in Fig. 5,A and B, administration of IL-8 or fMLP resulted in rapid margination of neutrophils (62 and 68%, respectively), which lasted throughout the infusion period (30 min). Co-administration of SB 225002 (Fig. 5, A and B) inhibited, in a dose-dependent manner, IL-8-, but not fMLP-, mediated PMN sequestration (Fig. 5 C).

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Growth suppression in subcutaneous SSP25 cell tumors [4]
Male BALB/c nude mice were given subcutaneous injections of 5 × 106 SSP25 cells in the flank. At 7 days after cancer cell injection, either 1 mg/kg of SB225002 (n = 4), or DMSO (0.001%) as a control (n = 4) was administered by intraperitoneal injection once every 2 days until 28 days after the cancer cell injection. The growth suppression of tumors by SB225002 is shown in Fig 2, E. Although complete tumor disappearance was not found in the SB225002 group, SB225002 injections significantly suppressed tumor growth. No suppression was observed in the control group (P = .02 at 21 days and P = .021 at 28 days).

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This study evaluated the antinociceptive effects of the selective and non-peptide CXCR2 antagonist SB225002 in mouse models of pain. As assessed in different tests of spontaneous nociception, intraperitoneal (i.p.) administration of SB225002 caused consistent and dose-related reduction of acetic acid-induced abdominal constrictions, whereas it did not significantly affect the nociception evoked by formalin, capsaicin, glutamate or phorbol ester acetate (PMA). Systemic treatment with SB225002 strikingly reduced the spontaneous nociception induced by 8-bromo-cAMP (8-Br-cAMP), or mechanical hypernociception induced by prostaglandin E(2) (PGE(2)), epinephrine, or the keratinocyte-derived chemokine (KC). In the carrageenan model, SB225002 markedly reduced mechanical hypernociception when administered by i.p., intrathecal (i.t.) or intracerebroventricular (i.c.v.) routes, or even when co-administered with carrageenan into the mouse paw, indicating peripheral and central sites of action for SB225002. In addition, i.p. treatment with SB225002 significantly attenuated the increase in MPO activity or the elevation of IL-1beta, TNFalpha or KC levels following carrageenan injection. In the persistent models of pain evoked by complete Freund's adjuvant (CFA) or by the partial ligation of the sciatic nerve (PLSN), the repeated administration of SB225002 displayed prominent and long-lasting antinociceptive effects. Notably, SB225002 did not evoke unspecific central effects, as evaluated in the open-field and rota-rod tests, or even in the latency responses for thermal stimuli. Our data confirm the previous notion on the critical role exerted by chemokines in pain, indicating that selective CXCR2 antagonists, such as SB225002, might well represent interesting and innovative alternatives for the management of both acute and chronic pain. [5]

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Although neutrophils are strongly implicated in eliminating pathogens, excessive recruitment may cause tissue damage. Therefore, reducing cell influx during an inflammatory process may be a potential target for treating inflammatory bowel diseases (IBD). As CXCR2 is involved in neutrophil migration, this study aimed to evaluate whether the systemic therapeutic treatment with selective CXCR2 antagonist SB225002 ameliorates experimental colitis, which was induced in mice by 2,4,6-trinitrobenzene sulfonic acid (TNBS). After colitis establishment (24 h), mice were treated with SB225002. At later time-points, up to 72 h, mice were monitored for body weight loss and overall mortality. At the time of sacrifice, colonic tissues were scored for macro- and microscopic damage, and cytokine levels, myeloperoxidase (MPO) activity, and protein expression were analyzed. TNBS administration induced macro- and microscopic damage in colon tissue, leading in most cases to animal death. Curative treatment with SB225002 significantly reduced all of the parameters analyzed, leading to an improvement of inflammatory signs. SB225002 reduced neutrophil influx, MPO activity, IL-1beta, MIP-2, and keratinocyte-derived chemokine (KC) levels and the expression of vascular endothelial growth factor, inducible NO synthase, and cyclooxygenase-2 proteins into the colon tissue. Levels of IL-4 and IL-10 were increased significantly in the colons of animals treated with SB225002. Additionally, curative treatment with mouse anti-KC significantly reduced MPO activity and colonic damage. These results taken together demonstrate that a selective blockade of CXCR2 consistently reduced TNBS-induced colitis, suggesting that the use of SB225002 is a potential therapeutic approach for the treatment of IBD and other related inflammatory disorders[6].

There are ready membranes for CHO-CXCR1 and CHO-CXCR2. The experiments are carried out in 96-well microtiter plates with 1.0 μg/mL membrane protein in 20 mM Bis-Tris-propane, pH 8.0, and 1.2 mM MgSO₄, 0.1 mM EDTA, 25 mM NaCl, and 0.03% CHAPS and SB225002 (10 mM stock in Me₂SO) added at the indicated concentrations. Under standard binding conditions, the final Me₂SO concentration is less than 1%. When 0.25 nM ¹²⁵I-IL-8 (2,200 Ci/mmol) is added, binding is started. The plate is harvested onto a glass fiber filtermat blocked with 1% polyethyleneimine and 0.5% BSA after a one-hour incubation period at room temperature. The plate is then cleaned three times using 25 mM NaCl, 10 mM Tris•HCl, 1 mM MgSO₄, 0.5 mMEDTA, 0.03% CHAPS, and pH 7.4. The filter is sealed in a sample bag with 10 mL of Wallac 205 Betaplate liquid scintillation fluid after it has dried, and a Wallac 1205 Betaplate liquid scintillation counter is used to count the results.[1]

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Tubulin polymerization assay [3]
Fluorescence-based tubulin polymerization assay kit was used according to the manufacturer instructions. A representative of 4 independent experiments was shown. Inhibition of CXCR2 by knock down with small interfering RNA and an antagonist [4]
Inhibition of CXCR2 was performed by 2 strategies. Silencing of CXCR2 gene expression was performed using small interfering RNA (siRNA) in vitro. The ICC cells were transfected with a mixture of the CXCR2 siRNA plasmid (20 mmol/L; and Lipofectamine 2000. At 24 hours after transfection, the medium was replaced by fresh medium with 5% fetal bovine serum for ≤72 hours post-transfection. As a control of CXCR2 siRNA, Stealth RNAi siRNA Negative Control Duplex was transfected by the same method. SB225002, a small molecule antagonist of CXCR2, was used to inhibit CXCR2 in vitro and in vivo in the mice model described below. 0.001% dimethylsulfoxide (DMSO) was used as solvent control.

Three esophageal squamous cell carcinoma cell lines—WHCO1, WHCO5, and WHCO6—are cultured in DMEM containing 10% FCS at 37°C in a humidified atmosphere with 5% CO₂. These cell lines were initially established from surgical biopsies of primary esophageal squamous cell carcinomas. To perform MTT assays, use the Cell Proliferation kit. In 96-well plates, 1.5×10³ cells are plated with a final volume of 180 μL DMEM per well. Cells are treated with 400 nM of SB 225002, and as a control, 0.001% DMSO (solvent) is added. Following the specified incubation time, each well receives 18 μL of the MTT labeling reagent (final concentration 0.5 mg/mL), which is then incubated for 4 hours in a humidified environment. The solubilization solution is poured into each well in a volume of 180,000 microliters, and the plates are then left at 37°C overnight. A microtiter plate reader is used to measure the samples' spectrophotometric absorbance at 595 nm.

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Inhibition of Ca2+ Mobilization [1]
Human neutrophils were separated from whole blood of healthy volunteers by the one-step Hypaque-Ficoll method. HL60 cells were differentiated, under incubation conditions, with Me2SO (0.5%) for 3 days. Cells (PMN, HL60, CXCR1-RBL-2H3, or 3ASubE) were loaded with Fura-2AM as described previously. For antagonist studies, SB225002 (final Me2SO < 0.35%) was added at the indicated concentrations, to 106 cells/ml in Krebs-Ringer-Henseleit buffer, followed 15 s later by agonist at the designated concentration. The maximal calcium concentration attained after agonist stimulation was quantitated as described previously.

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Inhibition of Neutrophil Chemotaxis [1]
Neutrophils were washed twice with phosphate-buffered saline (PBS) and resuspended in PBS containing 1 mm MgCl2 and 1 mmCaCl2. Cell motility was determined using a modified Boyden chamber procedure as described For measurement of chemotaxis, lower chambers were filled with 30 μl of IL-8 (1 nm) or GROα (10 nm), the empty upper chambers were lowered into place, and 50 μl of a PMN suspension (5 × 106 cells/ml), without (control) or with SB 225002, was added at the indicated concentrations. SB225002, dissolved in Me2SO (100%) at 10 mg/ml, was diluted in PBS to the desired concentration; the final Me2SO concentration was <0.1%. Neutrophil migration proceeded for 60 min at 37 °C in the cell incubator, after which the chamber was disassembled. Following fixation (75% methanol) and staining (Diff-Quick) migrated cells were counted in four successive high power fields (HPF).

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Cell proliferation, wound, and invasion assay [4]
The ICC cell line, RBE and SSP25 (3 × 104) were inhibited by CXCR2 siRNA (20 mmol/l) or an antagonist of CXCR2 (SB225002, 50 nm/l). The Cells were harvested at 3 days after administration of the inhibitor, and counted by a crystal violet staining procedure. Empty siRNA (20 mmol/L) or DMSO (0.001%) was used as a control. Invasion and wound assays have been described previously.23 For the wound assay, RBE and SSP25 cells had CXCR2 inhibited by CXCR2 siRNA or SB225002. After 24 hours of growth, the cells on one half of each plate were scraped away with a sterile razor blade. The remaining cells were washed 3 times with phosphate-buffered saline and incubated for 24 hours with serum-free RPMI. Finally, the cells were fixed on the plate and stained with hematoxylin and eosin. Ten high-power fields (×200) were randomly selected and 2 researchers counted the number of migrated cancer cells. The RBE and SSP25 cells with inhibited CXCR2 by CXCR2 siRNA were added to the inner cup of a Matrigel invasion chamber. Invasion was estimated after 22 hours of cultivation.

Mice: Male 7-8 week old wildtype (C57BL/6J, Harlan) and ApoE^−/− mice, raised on the same C57BL/6 background, are given a normal or cholesterol-rich diet for six weeks before having a left-sided middle cerebral artery occlusion (MCAO) for 20 minutes or having a sham operation performed. The treatment paradigms that animals receive are assigned at random, and experimenters are blinded throughout the entire intervention and data analysis process. Intraperitoneally (i.p.) injections of the vehicle (1% DMSO in PBS) or the selective CXCR2 antagonist SB225002 (2 mg/kg) are given at 0, 24, and 48 hours after ischemia. In additional studies, CXCR2 is blocked specifically by intraperitoneal injection (i.p.) of 300 μL of neutralizing rabbit anti-CXCR2 serum at 0 hours, 24 hours, and 48 hours after ischemia. NRS, or normal rabbit serum, was used as the control in the later investigations. In certain tests, an intraperitoneal injection of 200 μg anti-mouse Ly6G is used to deplete neutrophils 24 hours prior to and 24 hours following ischemia. 200 μg of an isotype control antibody are used as the control in these tests.\nRats: For the purposes of this study, each dam produces 10–12 Sprague-Dawley rat pups. Injections of SB225002(1 or 3 mg/kg) diluted in NS solution containing 0.33 % Tween 80, or vehicle (NS solution containing 0.33 % Tween 80) are given intraperitoneally to the pups 30 minutes prior to lipopolysaccharide (LPS) administration and right after hypoxic ischemia (HI). There are four groups of pups that are randomly assigned: vehicle (NS injections 30 minutes before LPS administration and immediately after HI, N=18), SB-1 (1 mg/kg, N=14) and SB-3 (3 mg/kg, N=18) (SB225002 injections 30 minutes before LPS administration and immediately after HI).

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\n\nInhibition of Neutrophil Sequestration in Vivo [1]
\nThe in vivo neutrophil sequestration model was performed in rabbits as reported previously. Using sterile techniques, rabbits were surgically fitted with an implanted cannula in the external jugular vein. IL-8 (150 ng/kg/min) or fMLP (5 ng/kg/min) was directly infused into the blood in the absence or presence of SB225002 (1.39–5.5 μg/kg/min) via the marginal ear vein. Blood samples were withdrawn at 2.5–5-min intervals via the vascular access port in the external jugular. White blood cell counts were determined with a Coulter counter, and differential counts were done using blood smears stained with Diff-Quick. Percent change of PMN count was determined relative to the base-line value.\n

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\n\nExperimental animal models [3]
\nMale Balb/c nude mice, 6–7 weeks old, were purchased from Japan CLEA and were used for a subcutaneous tumor model. SSP25 cells at a density of 5 × 106 were injected into one part of the flank of each mouse. At 7 days after the cancer cell injection, either 1 mg/kg of SB225002 as a CXCR2 antagonist (n = 4) or DMSO (0.001%) as a control (n = 4) was administered by intraperitoneal injection once every 2 days until 35 days after the cancer cell injection. The tumor size was measured once weekly using calipers. The tumor volumes were determined using the formula, volume = width2 × length × 0.52. All of the mice were maintained under conditions free of specified pathogens and all of the experimental animal research protocols in this study were submitted and approved by the experimental animal review committee of Hyogo College of Medicine.\n

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\n\nAcetic acid-induced abdominal constrictions [5]
\nAbdominal constrictions induced by the intraperitoneal (i.p.) injection of acetic acid (0.6%) were accomplished according to the procedures described previously (Vaz et al., 1996). Animals were treated with different doses of SB225002 (0.1–1 mg/kg, i.p.) or vehicle (10 ml/kg, 1% Tween 80 in 0.9% NaCl solution), 30 min prior to the injection of acetic acid. The animals were observed individually and the number of abdominal constrictions was cumulatively counted over a period of 20 min after acetic acid injection, and considered as indicative of nociception. Dipyrone (60 mg/kg, i.p., 30 min) was used as a positive control drug.\n

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\n\nFormalin-induced nociception [5]
\nThe procedure used was similar to that described previously (Mendes et al., 2000). Animals were treated with SB225002 (1 or 3 mg/kg, i.p.) or with vehicle (10 ml/kg, 1% Tween 80 in 0.9% NaCl solution), 30 min before formalin injection. Subsequently, mice received a 20-μl intraplantar (i.pl.) injection of 2.5% formalin solution into the right hindpaw. The animals were placed immediately in a glass cylinder (20 cm in diameter), and the time spent licking the injected paw over a period of 30 min was considered as indicative of nociception.\n

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\n\nOvert nociception models [5]
\nThe effects of SB225002 were further evaluated in other mouse models of spontaneous nociception. For that purpose, animals were pre-treated with SB225002 (1 or 3 mg/kg, i.p.) or vehicle (10 ml/kg, 1% Tween 80 in 0.9% NaCl solution), 30 min before the algogen injection. Mice received a 20-μl i.pl. injection of one of the following agents: capsaicin (5.2 nmol/paw; Sakurada et al., 1992), glutamate (30 μmol/paw; Beirith et al., 2002), phorbol myristate acetate (PMA) (30 ng/paw; Taniguchi et al., 1997), or 8-Br-cAMP (10 nmol/paw; Otuki et al., 2005), into the right hindpaw. The animals were observed individually in transparent glass cylinders (20 cm in diameter) during a period of 5 min after capsaicin, 15 min after glutamate, 45 min after PMA, or 10 min after 8-Br-cAMP. The amount of time spent licking the injected paw was registered with a chronometer and considered as indicative of nociception.\n

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\n\nMechanical hypernociception induced by KC, PGE2 or epinephrine [5]
\nTo evaluate mechanical hypernociception, mice were treated with SB225002 (1 mg/kg, i.p.) or vehicle (10 ml/kg, 1% Tween 80 in 0.9% NaCl solution), 30 min before the i.pl. injection of KC (10 ng/paw, Cunha et al., 2005), prostaglandin E2 (PGE2) (0.1 nmol/paw, Kassuya et al., 2007) or epinephrine (100 ng/paw, Khasar et al., 2005). The mechanical hypernociception was measured with Von Frey filaments (VFH), as described below, at different time-points after the i.pl. injection of the algogenic mediators.\n

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\n\nMechanical hypernociception induced by carrageenan [5]
\nFor the induction of inflammatory pain, mice received an i.pl. injection of 50 μl of carrageenan (300 μg/paw) under the surface of the right hindpaw (Quintão et al., 2005). To assess the systemic effect of drug treatment, mice received SB225002 (0.1–3 mg/kg, i.p.) or vehicle (10 ml/kg, 1% Tween 80 in 0.9% NaCl solution), 30 min before carrageenan injection. In order to evaluate the possible site of action of SB225002 (central or peripheral), a separate group of animals received an i.pl. injection of SB225002 (35–106 μg/paw), co-administered with carrageenan (300 μg/paw). Additional groups of animals received an intrathecal (i.t.) or intracerebroventricular (i.c.v.) injection of 5 μl of SB225002 (3.5–35 μg/site), 10 min before the application of carrageenan. The i.t injections were performed according to the method described by Hylden and Wilcox (1980) with some modifications. For the i.t. injections, the animals were conscious to avoid possible anesthetic interference. The needle connected to a microsyringe by a polyethylene tube was introduced through the skin, and a volume of 5 μl of vehicle solution (control) or SB225002 was injected into the L5–L6 vertebral space. For i.c.v. injections, the animals were lightly anesthetized with isoflurane and 5 μl of SB225002 solution was injected directly into the lateral ventricle (coordinates from bregma: 1 mm lateral; 1 mm rostral; 3 mm vertical) as described previously by Laursen and Belknap (1986). The mechanical hypernociception of all groups was assessed by means of VFH, for up to 24 h after carrageenan administration, as described below.\n

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\n\nMechanical hypernociception induced by complete Freund adjuvant (CFA) [5]
\nTo produce a persistent inflammatory response, mice received a 20-μl i.pl. injection of CFA (1 mg/ml heat-killed and dried Mycobacterium tuberculosis; each ml of vehicle contained 0.85 ml paraffin oil plus 0.15 ml mannide monooleate) into the right hindpaw (Quintão et al., 2005). To observe the effects of repeated treatment, SB225002 (1 mg/kg) was administered orally twice a day (12 × 12 h) for a period of 7 days. The evaluation of the mechanical hypernociception was assessed every day using VFH, 6 h after the first daily administration. Control animals received the vehicle (10 ml/kg, 1% Tween 80 in 0.9% NaCl solution), under the same schedule of treatment adopted for SB225002.\n

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\n\nSurgical procedures of partial ligation of sciatic nerve (PLSN) [5]
\nTo evaluate neuropathic pain-like behaviour, the procedure used was similar to that described by Quintão et al. (2005). Mice were anesthetized with 7% chloral hydrate (8 ml/kg; i.p.). PLSN was performed by tying 1/3–1/2 of the dorsal portion of the sciatic nerve with an 8.0 silk suture. In sham-operated control group, the sciatic nerve was exposed without ligation. Following a period of recovery (4 days after the surgical procedures), animals submitted to PLSN were treated systemically with SB225002 (1 mg/kg, i.p.) or vehicle (10 ml/kg, Tween 80 plus 0.9% NaCl solution), twice a day (12 × 12 h) for a period of 5 days after the surgery, and then evaluated with VFH for up to 6 h after the treatment.\n

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\n\nHindpaw withdrawal response induced by von Frey hairs [5]
\nFor the evaluation of mechanical allodynia, mice were placed individually in clear Plexiglas boxes (9 × 7 × 11 cm) on elevated wire mesh platforms to allow access to the ventral surface of the right hindpaw. The withdrawal response frequency was measured following 10 applications (duration of 1 s each) of von Frey hairs (VFH, Stoelting, Chicago, IL, USA). Stimuli were delivered from below, to the plantar surface of the right hindpaw. The animals were acclimatized for 30 min before behavioural testing and the mechanical hypernociception was evaluated at several time-points. The VFH of 0.6 g produces a mean withdrawal frequency of about 15%, which is considered to be an adequate value for the measurement of mechanical hypernociception (Quintão et al., 2005). Therefore, 0.6 g VFH was used throughout this study. In order to determine the basal mechanical thresholds, all the groups were evaluated before the test or surgical procedures.\n

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\n\nMyeloperoxidase (MPO) activity [5]
\nNeutrophil recruitment to the mouse paw was assessed indirectly by means of tissue myeloperoxidase (MPO) activity, according to the method described beforehand (Cunha et al., 2005). For this purpose, animals were treated with SB225002 (0.1–3 mg/kg, i.p.) and 30 min after, they received a 50-μl i.pl. injection of carrageenan (300 μg/paw) into the right paw. Saline-injected paws were used as control. Animals were sacrificed 6 h after the application of carrageenan. The subcutaneous tissue of the paws was removed, homogenized at 5% (w/v) in EDTA/NaCl buffer (pH 4.7) and centrifuged at 10,000 rpm for 15 min at 4 °C. The pellet was resuspended in 0.5% hexadecyltrimethyl ammonium bromide buffer (pH 5.4), and the samples were frozen and thawed three times in liquid nitrogen. Upon thawing, the samples were recentrifuged (10,000 rpm, 15 min, 4 °C), and 25 μl of the supernatant was used for the MPO assay. The enzymatic reaction was assessed with 1.6 mM tetramethylbenzidine, 80 mM NaPO4, and 0.3 mM hydrogen peroxide. The absorbance was measured at 650 nm, and the results are expressed as OD per milligram of tissue.\n

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\n\n Determination of IL-1β, TNFα or KC levels in the mouse paw [5]
\nTissue levels of the proinflammatory cytokines IL-1β, TNFα or KC were measured according to the protocol described by Cunha et al. (2005). The animals were treated with SB225002 (0.1–3 mg/kg, i.p.), and after 30 min they received a 50-μl i.pl. injection of carrageenan (300 μg/paw) into the right paw. The mice were sacrificed 6 h after the injection of carrageenan. Saline-injected paws were used as control. Tissues were placed in PBS (PBS; pH 7.4; NaCl 137 mM, KCl 2.7 mM, Na2HPO4 8.1 mM, KH2PO4 1.5 mM) containing NaCl 0.4 M, PMSF 0.1 M, EDTA 10 mM, 0.05% of Tween 20, 0.5% of BSA and 2 mg/ml of aprotinin, homogenized, centrifuged at 3000g for 10 min and stored at −70 °C until further analysis. Cytokine levels were evaluated using an ELISA kit according to the manufacturer's recommendations.\n

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\n\nMeasurement of non-specific effects [5]
\nTo exclude possible non-specific effects of SB225002 on motor coordination, locomotor activity, or the response latencies, mice were tested on the rota-rod, open-field and hot-plate paradigms, respectively (Quintão et al., 2005). Different groups of animals were pre-treated with SB225002 (1 and 3 mg/kg i.p.) or vehicle (10 ml/kg, i.p.) and they were submitted to all tests.\n

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\n\nAnimal treatment [6]
\nN-(2-Hydroxy-4-nitrophenyl)-N9-(2-bromophenyl) urea (SB225002) was synthesized as described before. To evaluate the potential therapeutic effect of SB225002 in the experimental colitis, animals received different doses of SB225002 twice per day (0.1, 0.3, and 1 mg/kg, i.p.), 24 h after the colitis induction. At 24 h or 72 h following colitis induction, the macroscopic score and myeloperoxidase (MPO) activity were analyzed. The most effective dose (0.3 mg/Kg) was used for the other experiments, such as cytokine contents, COX-2, VEGF, and iNOS protein expression. Similar protocol treatments were carried out using the positive control drug dexamethasone (1 mg/kg, s.c.) and anti-mouse KC (30 μg /kg, i.v.). SB225002 was dissolved in 0.9% NaCl solution containing 0.33% Tween 80 just before use, and control mice were treated with this vehicle.

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[3]. Biochem Pharmacol. 2013 Jun 15;85(12):1741-52.

[4]. Surgery. 2014 Apr;155(4):640-9.

[5]. Eur J Pain. 2010 Jan;14(1):23-31.

[6]. J Leukoc Biol. 2008 Oct;84(4):1213-21.

SB225002 belongs to the phenylurea class of compounds. Its structure is urea, with one amino group replaced by (2-bromophenyl)amino and the other by (2-hydroxy-4-nitrophenyl)amino. It is a potent CXCR2 chemokine receptor antagonist (IC50 = 22 nM). SB225002 possesses various pharmacological activities, including anti-inflammatory, antitumor, analgesic, apoptosis-inducing, and CXCR2 antagonistic activities. It is a nitrophenol, belonging to the bromobenzene and phenylurea classes of compounds. Interleukin-8 (IL-8) and its closely related glutamate-leucine-arginine (ELR) CXC chemokines, including growth-associated oncogenes (GRO) α, GROβ, GROγ, and epithelial cell-derived neutrophil-activating peptide-78 (ENA-78), are potent neutrophil chemotactic and activating peptides, considered major mediators of inflammation. IL-8 activates neutrophils by binding to two different seven-transmembrane (7-TMR) G protein-coupled receptors, CXCR1 (IL-8RA) and CXCR2 (IL-8RB), while GROα, GROβ, GROγ, and ENA-78 bind to and activate CXCR2 only. A lead compound selectively inhibiting CXCR2 was identified through high-throughput screening and chemically optimized. SB 225002 (N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea) is the first reported highly potent and selective non-peptide chemokine receptor inhibitor. It antagonizes the binding of 125I-IL-8 to CXCR2 with an IC50 of 22 nM. SB 225002 exhibits selectivity of over 150-fold against CXCR1 and four other tested 7-TMRs. In vitro experiments showed that SB 225002 could effectively inhibit IL-8 and GROα-induced neutrophil chemotaxis in humans and rabbits. In vivo experiments showed that SB 225002 could selectively block IL-8-induced rabbit neutrophil marginalization. The results of this study indicate that CXCR2 is responsible for IL-8-induced neutrophil chemotaxis and marginalization. This selective antagonist will become an effective tool compound for studying the role of CXCR2 in inflammatory diseases in which neutrophils play an important role. [1] Growth-associated oncogenes (GRO) are members of the CXC chemokine subfamily and play an important role in inflammation and wound healing. CXC chemokines have been found to be associated with tumorigenesis, angiogenesis and metastasis. Although GRO has been reported to be upregulated in a variety of human cancers, the expression and role of GRO and its receptor CXCR2 in esophageal cancer are still unclear. In this study, real-time reverse transcription PCR (RT-PCR) and immunohistochemistry were used to confirm that GROα, GROβ and CXCR2 were upregulated in esophageal tumor tissues. Furthermore, GROα, GROβ, and CXCR2 were constitutively expressed in the WHCO1 cell line, an esophageal cancer cell line used as a model system in this study. GROβ enhances EGR-1 transcription through the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, which can be blocked by a specific antagonist of CXCR2 (SB 225002) or a specific antibody against GROβ. SB 225002 treatment reduced WHCO1 cell proliferation by 40%. Stable WHCO1 GROα RNA interference (RNAi) clones showed a 43% reduction in GROα mRNA levels by real-time RT-PCR, and a 60% reduction in phosphorylated ERK1/2 levels under fluorescence microscopy. Stable clones expressing GROβ RNAi showed a reduction of over 95% in GROβ mRNA levels, and an 80% reduction in phosphorylated ERK1/2 levels under fluorescence microscopy. Furthermore, these clones expressing GROα RNAi and GROβ RNAi showed a 20% and 50% reduction in cell proliferation, respectively. Our results indicate that the GROα-CXCR2 and GROβ-CXCR2 signaling pathways significantly contribute to the proliferation of esophageal cancer cells, and that this autocrine signaling pathway may be involved in the development of esophageal tumors. [2]

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Background/Objective: Complete surgical resection is the only curative treatment for intrahepatic cholangiocarcinoma (ICC), but the prognosis of this disease is extremely poor. There is an urgent need for a new treatment method. CXC chemokine receptor 2 (CXCR2) is closely related to tumorigenesis and metastasis in human cancers. This study aimed to investigate the inhibitory effect of blocking CXCR2 on ICC growth. Materials and Methods: The human ICC cell lines RBE and SSP25 were used to evaluate the role of CXCR2. CXCR2 was blocked using CXCR2 small interfering RNA (siRNA) and an antagonist (SB225002). The inhibitory effect of blocking CXCR2 was verified by proliferation, migration and invasion assays. SSP25 tumor model was established subcutaneously in athymic nude mice and SB225002 was administered to the mice. Immunohistochemical staining was used to detect the expression of CXCR2 in 34 ICC specimens. We investigated the relationship between CXCR2 expression and ICC prognosis. Results: Patients with high CXCR2 expression in ICC had significantly worse prognoses (P = .004). CXCR2 siRNA treatment significantly inhibited CXCR2 expression in RBE and SSP25 cells. Compared with the control group, both CXCR2 siRNA and SB225002 significantly inhibited cell proliferation, migration and invasion. SB225002 also inhibited the growth of transplanted subcutaneous tumors (P = .02). Conclusion: Our results indicate that blocking CXCR2 can significantly inhibit the occurrence and development of ICC. Blocking CXCR2 may be a promising treatment for ICC. [4]

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In this study, we found that SB225002 showed significant analgesic effects in both acute and persistent pain mouse models. It is well known that various cell types, including neutrophils, monocytes, endothelial cells, and neurons, express CXCR2 ligands or receptors (Charo and Ransohoff, 2006). In the different experimental models evaluated in this study, chemokines produced by these cells may reach sensory nerve endings, leading to nociceptor sensitization. Furthermore, these chemokines may also activate other pain-related pathways (White and Wilson, 2008), such as PKA-cAMP pathway activation or the release of prostaglandins and sympathomimetic amines. Finally, peripheral inflammation and/or injury are associated with the upregulation of chemokines and their receptors in the central nervous system (Gosselin et al., 2008). It is reasonable to hypothesize that SB225002 may block pain generation by inhibiting CXCR2 activation at any of the aforementioned levels. In summary, our findings extend the view that chemokine receptors play a crucial role in pain variability and point to selective CXCR2 antagonists as a promising therapeutic option. [5] Finally, SB225002 treatment induced elevated levels of anti-inflammatory cytokines (IL-4 and IL-10), decreased levels of IL-1β, MIP-2 and KC, and reduced expression of iNOS, VEGF and COX-2 proteins, which greatly alleviated tissue damage and signs of inflammation. Most importantly, it significantly improved the survival rate of mice with TNBS-induced colitis. Notably, SB225002 has a therapeutic effect; that is, it can alleviate TNBS-induced colitis after inflammation has been established. Our data not only suggest that CXCR2 is an interesting and attractive target for IBD treatment (currently, there is still a lack of therapies for IBD), but also strongly suggest that the selective non-peptide CXCR2 antagonist SB225002 is a potential candidate for the treatment of IBD. [6]

C13H10BRN3O4

352.14

350.985

C, 44.34; H, 2.86; Br, 22.69; N, 11.93; O, 18.17

182498-32-4

3854666

White solid powder

1.8±0.1 g/cm3

401.6±45.0 °C at 760 mmHg

196.7±28.7 °C

0.0±1.0 mmHg at 25°C

1.765

4.36

3

4

2

21

390

0

O=C(NC1C(Br)=CC=CC=1)NC1C(O)=CC([N+](=O)[O-])=CC=1

MQBZVUNNWUIPMK-UHFFFAOYSA-N

InChI=1S/C13H10BrN3O4/c14-9-3-1-2-4-10(9)15-13(19)16-11-6-5-8(17(20)21)7-12(11)18/h1-7,18H,(H2,15,16,19)

1-(2-bromophenyl)-3-(2-hydroxy-4-nitrophenyl)urea

SB225002; 182498-32-4; sb 225,002; 1-(2-bromophenyl)-3-(2-hydroxy-4-nitrophenyl)urea; SB225,002; SB-225,002; N-(2-Bromophenyl)-N'-(2-hydroxy-4-nitrophenyl)urea; N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea; Urea, N-(2-bromophenyl)-N'-(2-hydroxy-4-nitrophenyl)-; SB 225002; SB225002

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 (e.g. under nitrogen), 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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.10 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.91 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 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 3: 2.08 mg/mL (5.91 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 4: 2% DMSO +Castor oil : 10mg/mL

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