LY 255283   中文名称: 1-[5-乙基-2-羟基-4-[[6-甲基-6-(1H-四唑-5-基)庚基]氧基]苯基]乙酮

LTB4 receptor/BLT2 (IC50 = 100 nM)

LY255283 竞争性降低肺实质对 LTB 的收缩反应 (pA2=7.2)[2]。 LY255283（10 μM，7 天）可显着抑制高胰岛素血症 253 J-BV 逆转胰岛素抵抗 [4]。

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白三烯B4（LTB4）诱导人中性粒细胞的许多功能变化，包括超氧化物释放和CD11b/CD18（Mo1）介导的对各种底物的粘附，如钥匙孔血蓝蛋白（KLH）。这些影响取决于时间和浓度。中性粒细胞粘附对LTB4的刺激作用的敏感性至少是超氧化物产生的10倍。评估了两种LTB4受体拮抗剂LY255283（1-（5-乙基-2-羟基-4-（6-甲基-6-（1H-四唑-5-基）庚氧基）-苯基）乙酮）和SC-41930钠盐（7-[3-（4-乙酰基-3-甲氧基-2-丙基苯氧基）丙氧基]-3,4-二氢-8-丙基-2H-1-苯并吡喃-2-羧酸）对人中性粒细胞超氧化物产生和粘附的影响。尽管中性粒细胞对LTB4诱导的刺激更敏感，但中性粒细胞粘附对LY255283和SC-41930抑制的敏感性至少比超氧化物产生低100倍。两种LTB4受体拮抗剂在这些模型中的表现相似。这些化合物不抑制粒细胞/巨噬细胞集落刺激因子（GM-CSF）诱导的中性粒细胞反应。[1]

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研究了白三烯（LT）B4受体拮抗剂LY255283对豚鼠肺的作用。LTB4和LY255283分别以9.9和7.0的pKi值从肺膜上的结合位点置换[3H]LTB4。在结合研究的功能相关性中，LY255283竞争性地降低了肺实质对LTB4的收缩反应（pA2=7.2）。[2]

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在这项研究中，免疫组织化学检查显示，白三烯B（4）受体BLT2在晚期恶性膀胱癌（人类移行细胞癌）中过表达，与进展阶段成正比，具有很高的预后意义（p<0.001）。用特异性拮抗剂LY255283阻断BLT2或siRNA敲低显著抑制高侵袭性253J-BV膀胱癌症细胞的侵袭性[4]。
此外，侵袭性253 J-BV细胞的数量和最大侵袭距离高于MCF-10A和SV-HUC-1细胞，并且通过用LY255283而不是U75302治疗显著降低（图3A，右），这表明BLT2信号的丧失降低了侵袭性膀胱癌症细胞的侵袭潜力。此外，当我们研究向LTB4和12（S）-HETE的迁移时，这两种已知是趋化因子的BLT2配体[20]，LTB4或12（S”-HETE显著增强了253个J-BV细胞的趋化迁移，并被LY255283或siBLT2治疗阻断，但程度远低于U75302（图3B）。有趣的是，单独用LY255283处理，即不使用LTB4或12（S）-HETE，显著抑制了253个J-BV细胞的基础活性；这暗示BLT2信号的自主基础激活[4]。

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NF-κB是253个J-BV细胞中BLT2-ROS级联反应的下游靶点[4]
NF-κB和AP-1是明确定义的重做调节转录因子，在癌症进展过程中驱动许多侵袭基因的转录。我们使用EMSA和免疫荧光分析来研究NF-κB或AP-1的激活是否位于BLT2-Nox-ROS连接信号的下游。LY255283和DPI处理253个J-BV细胞抑制了NF-κB DNA结合活性（图5A）。此外，我们发现高度侵袭性的253 J-BV细胞显示出强烈的核荧光，反映了NF-κB p65亚基的核转位。此外，用LY255283预处理，而不是U75302预处理，显著降低了p65 NF-κB的核水平（图5B）。相比之下，c-Jun不受影响，表明BLT2在AP-1信号传导中不起作用（数据未显示）。此外，DPI预处理（图5B）也显著降低了NF-κB的活化。总之，我们的结果表明，在高度侵袭性的253 J-BV膀胱癌症细胞中，NF-κB位于BLT2–Nox1/4–ROS级联的下游。此外，用四种不同的NF-κB抑制剂（SN-50、PDTC、Bay11-7082或Bay11-7085）治疗减弱了253个J-BV细胞的侵袭性（图5C）。

LY255283 (3, 30 mg/kg) 改善了猪的脂多糖传感器 ARDS，这可能是由于 PMN 募集以类似于舞蹈麻醉的方式将激活剂点燃到肺泡中的结果 [3]。 LY255283（2.5 mg/kg，腹腔注射）的结果表明膀胱癌主要是由 BLT2-Nox-ROS-NF-κB 级联引起的 [4]。

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LTB4产生气道阻塞，静脉注射（ED50=2.8 mg/kg）或口服（ED50=11.0 mg/kg）的LY255283可减轻气道阻塞。LY255283没有降低对组胺LTD4和血栓素模拟物U46619的收缩反应。该化合物也不抑制环氧化酶或5-脂氧合酶。我们得出结论，LY255283选择性拮抗肺组织对LTB4的药理学反应，似乎是研究LTB4在肺部疾病中作用的有用工具。[2]

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在对照组猪中，脂多糖诱导低氧血症、肺动脉高压和中性粒细胞活化（CORE/MORE比值增加）。这些变化被LY255283减弱，特别是当猪注射了更高剂量的化合物时。该药物还减弱了脂多糖诱导的肺气隙中性粒细胞的募集，这是通过在240分钟时进行的支气管肺泡灌洗来评估的，尽管脂多糖引起的肺白细胞隔离程度没有受到影响。 结论：LY255283以剂量依赖的方式改善了脂多糖诱导的猪ARDS，可能是通过阻断活化PMNs向肺泡的募集[3]。

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BLT2信号传导对于高度侵袭性膀胱癌症细胞的转移定植至关重要[4]
接下来，我们使用了一种名为实验性转移的检测方法来评估BLT2信号耗竭对转移的体内影响。我们将1×106个未经处理的253个J-BV细胞或用LY255283或U75302预处理的细胞注射到5周龄无胸腺小鼠的侧尾静脉中，然后确定肺部形成的转移结节的数量和大小。在注射253个J-BV细胞后3天和5天，腹腔内注射0.25 mg/kg U75302或2.5 mg/kg LY255283的剂量，与之前使用的剂量相似。注射后12周，在所有分析的小鼠中，未经治疗的肿瘤细胞在每个肺部形成了12-18个转移性结节，在用U75302治疗的小鼠中发现了类似数量的结节。相比之下，在用LY255283治疗的小鼠中，每个肺只形成0-3个结节（图6A，上图），组织学分析证实微转移病变的数量显著减少（图6A（下图））。

细胞活力测定[4]
细胞类型： 253 个 J-BV 细胞。
测试浓度：5 或 10 μM。
孵化持续时间：7天。
实验结果：抑制 BLT2 信号传导可减弱 253 个 J-BV 细胞的侵袭性迁移。

Animal/Disease Models: Mice (injected with 253 J-BV cells) [4].
Doses: 2.5 mg/kg.
Route of Administration: IP injection 3 and 5 days after cell injection.
Experimental Results: Twelve weeks after injection, mice treated with LY255283 developed only 0-3 nodules per lung, and histological analysis confirmed a significant reduction in the number of micrometastatic lesions.

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Experimental and spontaneous metastasis assays and morphological and histological analyses [4]
Male nude mice were inoculated between 5 and 8 weeks of age for experimental or spontaneous metastasis assays. Cultured 253 J-BV cells (1 × 106 cells) were pretreated with BLT antagonists for 24 h to ensure the inhibition of BLT signaling and then briefly treated with 0.025% trypsin and 0.1% EDTA in Hanks’ balanced salt solution (HBSS). The cells were then resuspended in HBSS and, within 1 h, were injected in a 0.1-ml volume into the lateral tail vein using a 30-gauge needle. For inhibitor experiments, dimethyl sulfoxide (DMSO), 0.25 mg/kg U75302, or 2.5 mg/kg LY255283 was intraperitoneally injected 3 and 5 days after injection of cells. The mice were maintained under aseptic barrier conditions until sacrifice 12 weeks after cell injection (n = 3 in each group). To identify experimental pulmonary metastases, the number of lung surface metastasis nodules larger than 0.2 mm in diameter was counted after euthanasia.
To assay spontaneous metastasis, mice were anesthetized with ketamine and xylazine, after which a lower midline incision was made, and viable tumor cells (2 × 106 cells in 0.05 ml) in HBSS were implanted in the bladder wall. The formation of a bulla indicated a satisfactory injection. The bladder was then returned to the abdominal cavity, and the abdominal wall was closed with a single layer of metal clips. For inhibitor experiments, 14 days after the surgery DMSO or the aforementioned dose of LY255283 or U75302 (n = 4 for each group) was injected intraperitoneally three times with intervals of 5 days between injections. The mice were killed and necropsied 9 weeks after tumor cell implantation. The primary tumors were removed and weighed, and the presence of metastases (liver) was determined grossly and microscopically. The livers and bladders were dissected and fixed in 4% formalin, processed, and embedded in paraffin.

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Eighteen hours before being studied, pigs were injected with lipopolysaccharide (20 micrograms/kg). From 0 to 60 minutes, pigs received either Ringer's lactate solution (n = 5) or lipopolysaccharide (250 micrograms/kg). Among the pigs that were infused with lipopolysaccharide, nine received no other treatment, six received a low dose of LY255283 (30 mg/kg loading dose; 3 mg/kg-hr infusion), and six received a high dose of LY255283 (30 mg/kg loading dose; 30 mg/kg-hr). In vivo PMN activation was assessed with an automated chemiluminescence assay wherein results are expressed as CORE/MORE (i.e., the ratio of complement-opsonized zymosan receptor expression on circulating cells [CORE] divided by the maximal complement-opsonized zymosan receptor expression induced by incubating the cells in vitro with LTB4 or platelet-activating factor [MORE]). [3]

[1]. Effects of two leukotriene B4 (LTB4) receptor antagonists (LY255283 and SC-41930) on LTB4-induced human neutrophil adhesion and superoxide production. Prostaglandins Leukot Essent Fatty Acids. 1991 Aug;43(4):267-71.

[2]. Pulmonary actions of LY255283, a leukotriene B4 receptor antagonist. Eur J Pharmacol. 1992 Nov 13;223(1):57-64.

[3]. LY255283, a novel leukotriene B4 receptor antagonist, limits activation of neutrophils and prevents acute lung injury induced by endotoxin in pigs. Surgery. 1993 Aug;114(2):191-8.

[4]. BLT2 promotes the invasion and metastasis of aggressive bladder cancer cells through a reactive oxygen species-linked pathway. Free Radic Biol Med. 2010 Sep 15;49(6):1072-81.

1-[5-ethyl-2-hydroxy-4-[6-methyl-6-(2H-tetrazol-5-yl)heptoxy]phenyl]ethanone is an aromatic ketone.

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Background: Polymorphonuclear neutrophils (PMNs) have been implicated in the pathogenesis of the adult respiratory distress syndrome (ARDS). Because leukotriene B4 (LTB4) is a potent activator of PMNs, we sought to determine whether LY255283, an LTB4 receptor antagonist, could block PMN activation and lung injury in a porcine model of lipopolysaccharide-induced ARDS. [3]

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Aggressive bladder cancer is a major cause of morbidity and mortality. Despite the fact that metastatic disease results in death in the majority of bladder cancer cases, the molecular events regulating the invasive phenotype of aggressive bladder cancer are not well understood. In this study, immunohistochemical examination showed that the leukotriene B(4) receptor BLT2 is overexpressed in advanced malignant bladder cancers (human transitional cell carcinomas) in proportion to advancing stages, with high prognostic significance (p<0.001). Blockade of BLT2 with the specific antagonist LY255283 or siRNA knockdown significantly suppressed the invasiveness of highly aggressive 253J-BV bladder cancer cells. Moreover, our results demonstrated that BLT2 mediates invasiveness through a signaling pathway dependent on NAD(P)H oxidase (Nox) 1- and Nox4-induced generation of reactive oxygen species (ROS) and subsequent NF-kappaB stimulation. Metastasis of 253J-BV cells in mice was also dramatically suppressed by inhibition of BLT2 or its signaling. These findings suggest that a BLT2-Nox-ROS-NF-kappaB cascade plays a critical role in bladder cancer invasion and metastasis. [4]

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We further confirmed the effect of BLT2 inhibition on the metastatic phenotype of aggressive bladder cancer cells by carrying out orthotopic metastasis assays. For these assays, we initially injected 253 J-BV cells into the bladder wall, as described under Materials and methods. Beginning 14 days later, three intraperitoneal injections of DMSO, LY255283, or U75302 (n = 4 per group) were administered with 5-day intervals between injections. We then analyzed tumor growth and the metastatic phenotype. As expected, all four mice treated with LY255283 showed a great reduction in metastasis (Fig. 6B). This is in contrast to findings in the untreated and U75302-treated mice, which developed large tumors in their bladders within 9 weeks as well as small metastatic nodules (< 0.2 mm diameter) in their livers (Fig. 6B). Taken together, these in vivo findings further confirm that BLT2 signaling plays a critical role in the metastasis of 253 J-BV bladder cancer cells.
TCC of the urinary bladder is responsive to conventional chemotherapeutic agents; however, the response is often short-lived, as chemoresistance can develop rapidly. Despite an initial chemotherapeutic response, most patients with advanced or metastatic TCC of the bladder die from progression of their disease (median survival, < 2 years). Thus, the development of new therapeutic agents that improve the outcome for patients with advanced bladder cancer is urgently needed. Here we show that a BLT2-linked cascade is critical for invasion and for the metastatic phenotype of aggressive bladder cancer. Notably, expression of BLT2, but not BLT1, was elevated in proportion to the tumor stage in bladder cancer specimens and metastatic bladder cancer cells. In addition, the level of BLT2 expression had high prognostic significance (p < 0.001). The fact that inhibition of BLT2 signaling by LY255283 or siBLT2 suppressed the invasive and metastatic potential in highly metastatic bladder cancer 253 J-BV cells (Fig. 2, Fig. 3, Fig. 6) suggests that the BLT2-linked cascade may be specifically required for invasion and metastasis in advanced bladder cancers in vivo [3].

C19H28N4O3

360.450624465942

360.216

C, 63.31; H, 7.83; N, 15.54; O, 13.32

117690-79-6

122023

Off-white to light yellow solid powder

1.2±0.1 g/cm3

573.4±60.0 °C at 760 mmHg

160-162 °C

300.6±32.9 °C

0.0±1.6 mmHg at 25°C

1.553

4.04

100.99

2

6

10

26

447

0

O(C1C=C(C(C(C)=O)=CC=1CC)O)CCCCCC(C1N=NNN=1)(C)C

WCGXJPFHTHQNJL-UHFFFAOYSA-N

InChI=1S/C19H28N4O3/c1-5-14-11-15(13(2)24)16(25)12-17(14)26-10-8-6-7-9-19(3,4)18-20-22-23-21-18/h11-12,25H,5-10H2,1-4H3,(H,20,21,22,23)

1-[5-ethyl-2-hydroxy-4-[6-methyl-6-(2H-tetrazol-5-yl)heptoxy]phenyl]ethanone

LY-255283; LY255283; LY255,283; LY-255,283; LY 255,283; UNII-H037W1I5AL; (1-(5-Ethyl-2-hydroxy-4-(6-methyl-6-(1H-tetrazol-5-yl)heptyloxy)phenyl)ethanone); DTXSID30151872; CGS 23356; ...; 117690-79-6; LY 255283

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

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 (~277.43 mM)

配方 1 中的溶解度: 2.5 mg/mL (6.94 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.94 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.94 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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；
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