AR-C118925XX   中文名称: AR-C 118925XX

P2Y2 Receptor

ATP和UTP增加了细胞内糖原含量，增强了肌动蛋白纤维应激反应，促进了GC细胞的增殖和迁移，而P2RY2竞争拮抗剂AR-C118925XX逆转了ATP诱导的变化。shRNA敲低P2RY2表达抑制GC细胞的增殖。激活P2RY2增加了GC细胞中Snail、Vimentin和β-catenin的表达，下调了E-cadherin的表达，而AR-C118925XX则降低了ATP诱导的这些基因的表达。P2RY2激活AKT/GSK-3beta/VEGF信号促进GC细胞增殖，P13/AKT信号通路LY294002逆转相应现象，但AR-C118925XX与LY294002未发现协同药理作用。［2］

AR-C118925XX (ip) 可减少博莱霉素引起的小鼠皮肤厚度的增加 [1]。
P2Y2受体拮抗剂AR-C118925XX可显著抑制博莱霉素诱导的小鼠的真皮纤维化[1]
注射博来霉素7天后，小鼠皮肤中ATP的数量显著增加（图6a），这表明博来霉素诱导的皮肤损伤和炎症可能会增强皮肤中各种细胞的ATP释放。最后，我们检测了P2Y2受体拮抗剂AR-C118925XX注射对博莱霉素诱导的小鼠真皮纤维化的影响。博莱霉素诱导的皮肤纤维化小鼠腹腔注射AR-C118925XX或磷酸盐缓冲盐水。AR-C118925XX注射可显著抑制博来霉素增强的真皮厚度（图6b和c）。我们证实，通过Masson-Trichrome染色，AR-C118925XX注射可显著减轻博来霉素诱导的真皮纤维化（图6d）。AR-C118925XX注射可显著抑制博来霉素诱导的病变皮肤中α-平滑肌肌动蛋白+肌成纤维细胞、CD68+巨噬细胞和CD3+ T细胞的数量（图6e）。AR-C118925XX注射剂有抑制病变皮肤博莱霉素诱导的T细胞数量的趋势，但差异无统计学意义。这些结果提示，博莱霉素诱导的皮肤ATP可能与皮肤纤维化有关，AR-C118925XX可能对体内皮肤纤维化有抑制作用。

Flu-3AM试验[2]
钙离子荧光探针测量游离钙浓度的变化。将AGS和HGC-27细胞接种于24孔板，分别用ATP（25、50、100 μM）、UTP （100 μM）、AR-C118925XX （10 μM）和ATP + AR-C118925XX （10 μM）处理细胞30 min。加入300µl 4µM Fluo-3AM工作液，37℃孵育1 h， PBS洗涤3次。平均Fluo-3荧光信号从三个独立的实验中得到。

实时PCR分析[1]
人真皮成纤维细胞在DMEM中按指定浓度（含或不含ATP或腺苷）孵育指定时间。用非选择性P2受体拮抗剂舒拉明（100 μmol/L）、P2X4受体拮抗剂5-BDBD （100 μmol/L）、P2X7受体拮抗剂AZ11645373 （1 μmol/L）、P2Y1受体拮抗剂MRS2179 （100 μmol/L）、P2Y2受体拮抗剂<强>AR-C118925XX<强> (10 μmol/L)和山奈酚（30 μmol/L）、P2Y11受体拮抗剂NF157 （50 μmol/L）、P2Y12受体拮抗剂氯吡格雷（30 μmol/L）、P2Y14受体拮抗剂PPTN盐酸（1 μmol/L）、p38抑制剂SB203580 （10 μmol/L）和BIRB796 （10 μmol/L）作用30分钟，然后用1 mmol/L ATP刺激1小时。

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Western blot检测[1]
根据先前描述的方案进行Western blot分析（Motegi et al., 2011a, 2011b）。为了研究舒拉明、SB203580、AR-C118925XX和山奈酚对ATP诱导的p38磷酸化或I型胶原生成的影响，用舒拉明（100 μmol/L）、SB203580 （10 μmol/L）、AR-C118925XX (10 μmol/L)、山奈酚（10 nmol/L）或DMSO（对照）预处理细胞30分钟，然后用ATP （1 mmol/L）刺激1小时，用ATP和/或可溶性IL-6受体（100 ng/ml）刺激48小时。

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PAS糖原含量检测[2]
将AGS和HGC-27细胞置于24孔板上，分别用或不加ATP （100 μM）、UTP （100 μM）、AR-C118925XX （10 μM）、ATP + AR-C118925XX （10 μM）、ATP + LY294002 （20 mM）、ATP + AR-C118925XX (10 μM) + LY294002 （20 mM）或shCon/shP2RY2处理24 h。加PAS固定液20 min， PBS洗涤3次。加入氧化剂15分钟，然后用PBS洗涤。用亚硫酸钠溶液洗涤细胞2次，每次2 min。加入梅耶苏木精染料溶液2 min， PBS洗涤。倒置显微镜下观察PAS染色阳性细胞数量，使用Image Pro Plus 6.0软件进行计数。

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细胞创面愈合试验[2]
将AGS和HGC-27细胞接种于6孔板。将细胞均匀地覆盖整个孔后。用无菌10µl枪头划痕。加入含10% FBS的1640培养基，培养24 h。细胞分别为ATP （100 μM）、UTP （100 μM）、AR-C118925XX （10 μM）、ATP + AR-C118925XX （10 μM）、ATP + LY294002 （20mM）、ATP + AR-C118925XX (10 μM) + LY294002 （20mM）或shCon/shP2RY2。在倒置显微镜下拍摄0和24 h的照片，并测量细胞划痕之间的伤口愈合百分比。

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细胞迁移能力评价[2]
采用24孔Transwell实验室分析GC细胞的侵袭能力。上插片滤液中加入200µl无血清细胞悬液（2 × 104个细胞），下插片中加入200µl含10% FBS的1640培养基。细胞分别用ATP （100 μM）、UTP （100 μM）、AR-C118925XX （10 μM）、ATP + AR-C118925XX （10 μM）、ATP + AR-C118925XX （20 μM）、ATP + AR-C118925XX （20mM）、ATP + AR-C118925XX (10 μM) + LY294002 （20mM）处理或未处理24 h， 4%多聚甲醛固定30 min，结晶紫染色，在显微镜下按5个随机场计数。

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肌动蛋白纤维标记法[2]
接种于24孔板的AGS和HGC-27细胞分别用ATP （100 μM）和ATP + AR-C118925XX （10 μM）处理24 h，然后用PBS洗涤。用4%多聚甲醛在冰上固定细胞15分钟，然后用PBS洗涤3次。加入400 μl 0.5% Trion X-100，作用10 min， PBS洗涤3次。加入200 μl PBS，用3 μl YF荧光标记的phalloidin溶液稀释，孵育30 min， PBS洗涤。然后在激光共聚焦显微镜（Lakar sp5）下随机选取5个视场并拍照。

Evaluation of tumor growth by in vivo experiment [2]
BALB/c nude mice were reared in an aseptic environment controlled by light and temperature in the laboratory. AGS cells were collected and reconstituted in PBS (100 μl), and approximately 4 × 106 cells. Cells were injected subcutaneously into the lateral thigh of 4-week-old male nude mice, and the xenografts were allowed to grow. When the tumor was grown to 1 week later, the mice were randomly divided into three groups with six mice in each group. The PBS (control), ATP (100 µM), or ATP + AR-C118925XX (10 µM) were injected into xenotransplant tissue at twice a week. The tumor size was measured with Vernier calipers, induced tumor volume = [length × width2]/2 for about 1 month.

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Bleomycin-induced skin fibrosis model [1]
Dermal fibrosis was induced in 8-week-old C57BL/6 mice with injections of bleomycin. Injections of 300 μl of bleomycin at a concentration of 1 mg/ml were given five times per week for 2 weeks as previously described (Yokoyama et al., 2018). To examine the effect of AR-C118925XX , mice intraperitoneally received AR-C118925XX (7 mg/kg/day) dissolved in 100 μl of DMSO or DMSO alone five times per week for 2 weeks.

[1]. The Regulation of Skin Fibrosis in Systemic Sclerosis by Extracellular ATP via P2Y2 Purinergic Receptor. J Invest Dermatol. 2019 Apr;139(4):890-899.

[2]. AKT/GSK-3beta/VEGF signaling is involved in P2RY2 activation-induced the proliferation and metastasis of gastric cancer. Carcinogenesis. 2022 Dec 5:bgac095.

Tissue injury/hypoxia and oxidative stress induced-extracellular adenosine triphosphate (ATP) can act as damage-associated molecular pattern molecules, which initiate inflammatory response. Our objective was to elucidate the role of extracellular ATP in skin fibrosis in systemic sclerosis (SSc). We identified that hypoxia enhanced ATP release and that extracellular ATP enhanced IL-6 production more significantly in SSc fibroblasts than in normal fibroblasts. There were no significant differences of P2X and P2Y receptor expression levels between normal and SSc fibroblasts. Nonselective P2 receptor antagonist and selective P2Y2 receptor antagonists, kaempferol and AR-C118925XX, significantly inhibited ATP-induced IL-6 production and phosphorylation of p38 in SSc fibroblasts. ATP-induced IL-6 production was significantly inhibited by p38 inhibitors, SB203580, and doramapimod. Collagen type I production in SSc fibroblasts by ATP-induced IL-6/IL-6 receptor trans-signaling was inhibited by kaempferol and SB203580. The amount of ATP in bleomycin-treated skin was increased, and administration of AR-C118925XX significantly inhibited bleomycin-induced dermal fibrosis in mice. These results suggest that vasculopathy-induced hypoxia and oxidative stress might enhance ATP release in the dermis in SSc and that extracellular ATP-induced phosphorylation of p38 via P2Y2 receptor might enhance IL-6 and collagen type I production in SSc fibroblasts. P2Y2 receptor antagonist therapy could be a treatment for skin sclerosis in patients with SSc. [1]

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Studies have revealed the contribution of ATP-G-protein-coupled P2Y2 receptor (P2RY2) in tumor progression, but the role of P2RY2 in regulating the progression of gastric cancer (GC) and related molecular mechanisms are relatively lacking. Therefore, this study investigates the effects of P2RY2 on the proliferation and migration of GC through in vivo and in vitro experiments. The results showed that P2RY2 was expressed in GC tissues and GC cell lines. Adenosine triphosphate (ATP) increased the calcium influx in AGS and HGC-27 cells, and was dose-dependent with ATP concentration. ATP and UTP increased the intracellular glycogen content, enhanced the actin fiber stress response, and promoted the proliferation and migration of GC cells, while P2RY2 competitive antagonist AR-C118925XX reversed the changes induced by ATP. Knockdown of P2RY2 expression by shRNA inhibited the proliferation of GC cells. Activation of P2RY2 increased the expression of Snail, Vimentin, and β-catenin in GC cells, and down-regulated the expression of E-cadherin, while AR-C118925XX decreased the expression of these genes induced by ATP. Activation of P2RY2 activated AKT/GSK-3beta/VEGF signal to promote the proliferation of GC cells, and the P13/AKT signaling pathway LY294002 reversed the corresponding phenomenon, but no synergistic pharmacological properties of AR-C118925XX and LY294002 have been found. In vivo experiments showed that ATP-induced tumor growth, while AR-C118925XX inhibited ATP-induced tumor growth. Our conclusion is that P2RY2 activated the AKT/GSK-3beta/VEGF signal to promote the proliferation and migration of GC, suggesting that P2RY2 may be a new potential target for the treatment of GC.[2]

C28H23N7O3S

537.592323541641

537.158

C, 62.56; H, 4.31; N, 18.24; O, 8.93; S, 5.96

216657-60-2

54210200

Light yellow to yellow solid powder

1.5±0.1 g/cm3

1.756

4.78

161

3

7

5

39

1010

0

PVKNPGQAFNALOI-UHFFFAOYSA-N

InChI=1S/C28H23N7O3S/c1-15-3-8-20-17(11-15)5-6-18-12-16(2)4-9-21(18)24(20)22-14-35(28(37)30-26(22)39)13-19-7-10-23(38-19)25(36)29-27-31-33-34-32-27/h3-12,14,24H,13H2,1-2H3,(H,30,37,39)(H2,29,31,32,33,34,36)

5-[[5-(6,13-dimethyl-2-tricyclo[9.4.0.03,8]pentadeca-1(11),3(8),4,6,9,12,14-heptaenyl)-2-oxo-4-sulfanylidenepyrimidin-1-yl]methyl]-N-(2H-tetrazol-5-yl)furan-2-carboxamide

AR-C118925XX; 216657-60-2; AR-C 118925XX; 5-[[5-(2,8-Dimethyl-5H-dibenzo[a,d]cyclohepten-5-yl)-3,4-dihydro-2-oxo-4-thioxo-1(2H)-pyrimidinyl]methyl]-N-2H-tetrazol-5-yl-2-furancarboxamide; CHEMBL4082045; 5-((5-(2,8-Dimethyl-5H-dibenzo[a,d][7]annulen-5-yl)-2-oxo-4-thioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-(1H-tetrazol-5-yl)furan-2-carboxamide; 5-[(5-{6,13-dimethyltricyclo[9.4.0.0^{3,8}]pentadeca-1(11),3,5,7,9,12,14-heptaen-2-yl}-2-oxo-4-sulfanylidene-1,2,3,4-tetrahydropyrimidin-1-yl)methyl]-N-(1H-1,2,3,4-tetrazol-5-yl)furan-2-carboxamide; 5-[[5-(6,13-dimethyl-2-tricyclo[9.4.0.03,8]pentadeca-1(11),3(8),4,6,9,12,14-heptaenyl)-2-oxo-4-sulfanylidenepyrimidin-1-yl]methyl]-N-(2H-tetrazol-5-yl)furan-2-carboxamide;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

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 (~186.02 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
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6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
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