RS 504393
(Synonyms: 6-甲基-1,1'-[2-(5-甲基-2-苯基-4-恶唑基)乙基]-螺[4H-3-1,1-苯并恶嗪-4,4'-哌啶]-2(1H)-酮,RS-504393; RS504393) 目录号 : GC15489
RS 504393是一种选择性的CCR2趋化因子受体拮抗剂,对人类重组CCR2受体和CCR1受体的IC50值分别为89nM和100μM。
Cas No.:300816-15-3
Sample solution is provided at 25 µL, 10mM.
RS 504393 is a selective CCR2 chemokine receptor antagonist, with IC50 values of 89nM and 100μM for human recombinant CCR2 and CCR1 receptors, respectively[1]. CCR2 is a key mediator for inflammatory monocyte activation and migration. RS 504393 can be applied for the treatment of autoimmune arthritis and cancer pain [2].
In vitro, when primary microglia were treated with RS 504393 (10μM) for 1 hour before MCP-1 stimulation, it inhibited microglial activation and reduced the expression of P2X4R in the cultured microglia [3]. Treatment of fibroblast-like synoviocytes (FLS) with RS 504393 (20μM) for 5 minutes inhibited the activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2 in CCL2 culture [4].
In vivo, RS 504393 (4mg/kg/day) was orally administered to mice with osteoarthritis (OA) for 4 weeks, which improved the changes in the cartilage and bones of the mice. RS 504393 could alleviate pain in the early stage of osteoarthritis development, but its therapeutic effect weakened in the later stage of the disease [5]. RS 504393 (4mg/kg/day, 8mg/kg/day) was administered subcutaneously once to mice with bleomycin-induced scleroderma, which reduced dermal fibrosis and dermal thickness in the mice, as well as the number of mast cells and myofibroblasts in the skin and lungs, and the amount of collagen in the skin and lungs. RS 504393 could inhibit pulmonary inflammation and fibrosis caused by bleomycin [6].
References:
[1] Mirzadegan T, et al. Identification of the binding site for a novel class of CCR2b chemokine receptor antagonists: binding to a common chemokine receptor motif within the helical bundle. J Biol Chem. 2000 Aug 18;275(33):25562-71.
[2] Carter P H. Progress in the discovery of CC chemokine receptor 2 antagonists, 2009–2012[J]. Expert opinion on therapeutic patents, 2013, 23(5): 549-568.
[3] Yan Y, Liang Y, Ding T, et al. PI3K/Akt signaling pathway may be involved in MCP-1-induced P2X4R expression in cultured microglia and cancer-induced bone pain rats[J]. Neuroscience letters, 2019, 701: 100-105.
[4] Nanki T, Nagasaka K, Hayashida K, et al. Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis[J]. The Journal of Immunology, 2001, 167(9): 5381-5385.
[5] Longobardi L, Temple J D, Tagliafierro L, et al. Role of the CC chemokine receptor-2 in a murine model of injury-induced osteoarthritis[J]. Osteoarthritis and cartilage, 2017, 25(6): 914-925.
[6] Ishikawa M, Yamamoto T. Antifibrogenic effects of C‐C chemokine receptor type 2 antagonist in a bleomycin‐induced scleroderma model[J]. Experimental Dermatology, 2021, 30(1): 179-184.
RS 504393是一种选择性的CCR2趋化因子受体拮抗剂,对人类重组CCR2受体和CCR1受体的IC50值分别为89nM和100μM[1]。CCR2是炎症性单核细胞激活及迁移的关键介质。RS 504393可以应用于治疗关节炎免疫发病以及癌症疼痛 [2]。
在体外,在MCP-1刺激前用RS 504393(10μM)处理小胶质细胞1小时,抑制了小胶质细胞活化并降低了培养的小胶质细胞中的P2X4R表达 [3]。用RS 504393(20μM)处理成纤维细胞样上皮细胞(FLS)5分钟,抑制了在CCL2培养下细胞外信号相关激酶1(ERK1)和ERK2的激活 [4]。
在体内,RS 504393(4mg/kg/day)通过口服治疗骨关节炎(OA)小鼠4周,改善了小鼠的骨关节软骨和骨骼的变化。RS 504393在骨关节炎发展的早期能够缓解疼痛,而在疾病后期其治疗效果减弱 [5]。RS 504393(4mg/kg/day,8mg/kg/day)通过皮下注射一次治疗博来霉素诱导的硬皮病小鼠,降低了小鼠真皮纤维化和真皮厚度,也降低了小鼠皮肤中肥大细胞和肌成纤维细胞的数量以及皮肤和肺中的胶原蛋白量。RS 504393可抑制博来霉素引发的肺部炎症和纤维化[6]。
Kinase experiment [1]: | |
Preparation Method | Isolated mast cells were sensitized by incubation with anti-DNP IgE (SPE7; Sigma) in RPMI1640 containing 10ng/mL of murine recombinant IL-3, 10ng/mL of recombinant SCF, and 5% murine serum. The cells were then washed with HBSS containing 10ng/mL of murine recombinant IL-3, 10ng/mL of recombinant SCF, 0.04% BSA, and 10mM HEPES. Transfer the cell suspension to three groups of wells on a 96-well U-shaped bottom plate at a concentration of 2 to 8×10^4 cells/100 microliters, and let it stand at 37°C for 10 minutes. Then, couple it with anti-DNP-IgE antibody first, and then stimulate with DNP-albumin. Add recombinant MCP-1 (1, 10, and 100 pg/mL) and/or RS 504393 (500 nM) simultaneously or at different times.After 45 minutes, the plate was centrifuged at 290g for 5 minutes at 4°C. The β-hexosaminidase activity of the culture supernatant was determined using a published protocol. 13 Fifty-microliter aliquots of the supernatant were placed in wells of another 96-well plate together with 100μL of 2.5mM p-nitrophenyl-N-acetyl β-d glucosaminide (Sigma-Aldrich) solubilized in 0.04M citrate buffer adjusted to pH 4.5 with disodium phosphate. After incubation at 37°C for 90 minutes, the reactions were terminated by addition of 50μL of 0.4M glycin adjusted to pH 10.7 with sodium hydroxide. The colored product was measured at 405nm with a reference filter of 570nm. The relative release of β-hexosaminidase was defined as the activity in the supernatant of the tested cells divided by the activity in the positive control cell supernatant, multiplied by 100. Compound 48/80 stimulus was used for assay control. |
Reaction Conditions | 500nM;90min |
Applications | RS 504393 inhibits MCP-1-induced chemotaxis with an IC50 of 330nM. Treatment with RS 504393 significantly inhibits allergen-induced release of β-hexosaminidase. Without the initiation of an allergen, MCP-1 induces degranulation of mast cells, which is completely inhibited by RS 504393. |
Cell experiment [2]: | |
Cell lines | Microglial cells |
Preparation Method | The spinal microglia cells were isolated from Sprague-Dawley neonatal pups. A mixed glial cell culture was prepared by resuspending the cell suspension in DMEM containing 10% heat-inactivated FBS, 100U/ml penicillin and 0.1mg/ml streptomycin to promote the isolation of microglia from the culture flask. After centrifugation at 800rpm for 8 minutes at 4°C, the cells were resuspended in fresh culture medium for all subsequent experiments. The cells were treated with exogenous recombinant MCP-1 (10, 30 or 100ng/ml) for 30 minutes. The CCR2 antagonist RS 504393 (10μM) and the PI3K inhibitor LY 294002 (10μM) were pre-incubated with the microglia for 20 minutes and 1 hour respectively before MCP-1 (100ng/ml) stimulation, and the Iba1 immunostaining was used for determination. |
Reaction Conditions | 10μM; 1h |
Applications | The selective CCR2 antagonist RS 504393 blocked the activation of microglia and reduced the expression of P2X4R in the cultured microglia, as well as inhibiting the expressions of MCP-1 and p-Akt. |
Animal experiment [3]: | |
Animal models | C57BL/6 |
Preparation Method | Injury-induced-osteoarthritis (OA) was induced in twelve-week-old male C57BL/6 mice (Jackson) by transecting the menisco-tibial ligament. These mice received a continuous 4-week treatment of CCR2 antagonist RS 504393 (4mg/kg/day) dissolved in 0.6% DMSO at different stages of disease progression. The mice underwent pain behavior assessment at 2, 4, 8, and 12 weeks after the DMM surgery and were euthanized. Dissected knees were fixed, μCT scanned, then prepared for histology. Evaluate the condition of cartilage and bone lesions and examine the degeneration of the joints. |
Dosage form | 4mg/kg/day for 4 weeks; oral |
Applications | In the early stage of osteoarthritis, the use of the CCR2 antagonist RS 504393 can improve the changes in joint cartilage and bone, and alleviate pain. However, its therapeutic effect is weaker in the later stage of the disease. |
References: |
Cas No. | 300816-15-3 | SDF | |
别名 | 6-甲基-1,1'-[2-(5-甲基-2-苯基-4-恶唑基)乙基]-螺[4H-3-1,1-苯并恶嗪-4,4'-哌啶]-2(1H)-酮,RS-504393; RS504393 | ||
化学名 | 6-methyl-1'-(2-(5-methyl-2-phenyloxazol-4-yl)ethyl)spiro[benzo[d][1,3]oxazine-4,4'-piperidin]-2(1H)-one | ||
Canonical SMILES | CC1=CC2=C(NC(OC23CCN(CCC4=C(C)OC(C5=CC=CC=C5)=N4)CC3)=O)C=C1 | ||
分子式 | C25H27N3O3 | 分子量 | 417.5 |
溶解度 | DMF: 10 mg/ml,DMF:PBS (pH 7.2) (1:9): 0.1 mg/ml,DMSO: 1 mg/ml,Ethanol: Slightly Soluble | 储存条件 | Store at -20°C |
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1 mg | 5 mg | 10 mg |
1 mM | 2.3952 mL | 11.976 mL | 23.9521 mL |
5 mM | 0.479 mL | 2.3952 mL | 4.7904 mL |
10 mM | 0.2395 mL | 1.1976 mL | 2.3952 mL |
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