YM-58483(BTP2)   中文名称: N-(4-(3,5-二(三氟甲基)-1H-吡唑-1-基)苯基)-4-甲基-1,2,3-噻二唑-5-甲酰胺

Ca(2+) release-activated Ca(2+) (CRAC) channels; store-operated Ca2+ entry (SOCE)

在不改变 CD11b 和 GFAP 表达的情况下，YM-58483 可以降低 P-ERK 和 P-CREB 的水平。此外，YM-58483 还可抑制肠道 IL-1β、TNF-α 和 PGE2 的产生 [1]。在单向混合溶液反应 (mLR) 中，YM-58483 和环孢菌素 A 均抑制 T 细胞生长，IC50 值分别为 330 和 12.7 nM [2]。 YM-58483 的 IC50 值为 460 和 310 nM，可抑制 RBL-2H3 细胞（梯度碱性稀释细胞系）中 IgE 刺激的白三烯 (LT) 和组胺的合成。 YM-58483 的 IC50 值分别为 125 和 148 nM，还抑制由植物血凝素-P (PHA) 触发的人外周血细胞中 IL-5 和 IL-13 的产生，该机制的效力比泼尼松龙[3]。在用植物血凝素刺激的人全血细胞中，YM-58483 减少用亮氨酸刺激的鼠 Th2 T 细胞克隆 (D10.G4) 中 IL-5 和 IL-4 的产生。 IC50值与已发表的CRAC通道抑制值相似（约100 nM）[4]。

鞘内注射 300 μM（1.5 nmol）和 1000 μM（10 nmol）剂量的 YM-58483 对 SNL 产生强烈的中枢镇痛作用[1]。 YM-58483（1-30 mg/kg，po）和环孢素A（1-30 mg/kg，po）抑制供体抗细胞毒性T（CTL）活性和IFN-γ产生，并减少供体T的量脾脏中的细胞，特别是供体 CD8+ T 细胞。 YM-58483（1-10 mg/kg，口服）和环孢素 A（2、10 mg/kg，口服）可降低绵羊红细胞 (SRBC) 引起的迟发型超敏反应 (DTH) [2]。 M-58483（30 mg/kg，肘部）显着降低卵清蛋白（OVA）致敏豚鼠中卵清蛋白（OVA）诱导的延迟型收缩，而 YM-58483（3-30 mg/kg，面部）和泼尼松龙（100 mg/ kg，面部）显着且完全抑制 OVA 产生的气道高反应性 (AHR) [3]。 YM-58483 缓冲因呼吸进入气道而引起的嗜酸性粒细胞，并降低由棕色挪威苏格兰威士忌中心主动致敏引起的活化气道中的 IL-4 和半胱氨酸泡沫白三烯水平。途径 YM-58483 可防止主动致敏的豚鼠强烈诱导晚期死亡和嗜酸性呼吸[4]。

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YM-58483对神经性疼痛有镇痛作用，但其机制尚不清楚。本实验在大鼠脊髓神经结扎（SNL）诱导的神经性疼痛模型上进行，试图探索YM-58483（BTP2）减弱神经性疼痛的机制。在雄性Sprague-Dawley大鼠中结扎左L5以产生SNL神经性疼痛模型。在鞘内注射YM-58483和赋形剂前后，采用上下法和Hargreaves法测量大鼠的戒断阈值。通过免疫荧光法定位脊髓背角中的SOCCs。Western blot检测脊髓水平磷酸化ERK和磷酸化CREB、CD11b和GFAP蛋白的表达，酶联免疫吸附试验（ELISA）检测促炎细胞因子（IL-1β、TNF-α、PGE2）的释放。与对照组+赋形剂相比，鞘内注射300μM（1.5 nmol）和1000μM（10 nmol）的YM-58483对SNL大鼠产生了显著的中枢镇痛作用（n=7，P<0.001）。然而，与生理盐水相比，两者都不能预防神经性疼痛的发展（P<0.001）。免疫荧光染色显示，Orai1和STIM1（SOCCs的两个关键成分）位于脊髓背角神经元中。Western blot显示，YM-58483可以降低P-ERK和P-CREB的水平（n=10，#P<0.05），而不影响CD11b和GFAP的表达（n=10）（#P>0.05）。与对照组+载体相比，YM-58483还抑制了脊髓IL-1β、TNF-α和PGE2的释放（n=5，#P<0.001）。YM-58483的镇痛机制可能是通过抑制神经元中的中枢ERK/CREB信号传导，减少中枢IL-1β、TNF-α和PGE2的释放，从而降低SNL大鼠脊髓背角的神经元兴奋性。[1]

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YM-58483/BTP2是储存操作性Ca2+进入（SOCE）的阻断剂，它调节淋巴细胞等非兴奋细胞的激活YM-58483（BTP2）已被报道可抑制T细胞中细胞因子的产生和增殖，并可作为治疗支气管哮喘的可能候选药物。本研究调查了YM-58483与细胞介导的免疫反应相关的药理学特征和治疗潜力。在小鼠移植物抗宿主病（GVHD）模型中，YM-58483（1-30mg/kg，口服）和环孢菌素A（1-30mmg/kg，口服）抑制了供体抗宿主细胞毒性T淋巴细胞（CTL）活性和IFN-γ产生，还减少了脾脏中供体T细胞的数量，特别是供体CD8+T细胞。YM-58483和环孢菌素A在单向混合淋巴细胞反应（MLR）中抑制T细胞增殖，IC50值分别为330和12.7 nM。此外，YM-58483（1-10mg/kg，口服）和环孢菌素A（2.10mg/kg，口服）抑制了绵羊红细胞（SRBC）诱导的迟发型超敏反应（DTH）。这些结果表明，抑制SOCE可以预防抗原诱导的T细胞反应，这些反应参与了自身免疫性疾病，如自身免疫性肝炎和类风湿性关节炎。[2]

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T细胞在各种免疫和过敏性疾病的发病机制中起着调节作用，包括人类哮喘。最近，有报道称吡唑衍生物YM-58483（BTP2）能有效抑制T细胞中钙（2+）释放激活的钙（CRAC）通道和白细胞介素（IL）-2的产生。我们研究了YM-58483（BTP2）对体外T辅助2型（Th2）细胞因子产生和体内抗原诱导的气道哮喘反应的影响。YM-58483抑制了锥蛋白刺激的小鼠Th2 T细胞克隆（D10.G4.1）中IL-4和IL-5的产生，以及植物血凝素刺激的人全血细胞中IL-5的生产，其IC（50）值与CRAC通道抑制的报告值相当（约100nM）。YM-58483抑制了抗原诱导的嗜酸性粒细胞浸润气道，并降低了主动致敏Brown Norway大鼠炎症气道中IL-4和半胱氨酰白三烯的含量。此外，口服YM-58483可预防主动致敏豚鼠抗原诱导的晚期哮喘支气管收缩和嗜酸性粒细胞浸润。这些数据表明，通过CRAC通道抑制Ca（2+）内流可以预防抗原诱导的气道炎症，可能是通过抑制Th2细胞因子的产生和炎症介质的释放。因此，YM-58483可用于治疗支气管哮喘的气道炎症。[4]

PHA诱导全人外周血产生IL-5和IL-13[3]
PHA被用作刺激剂，以模拟抗原在T淋巴细胞中引发的初始反应。人外周血取自健康志愿者。肝素化人外周全血在RPMI1640培养基中用青霉素-链霉素以1:3.5的比例稀释。稀释的血液（450μl）用不同浓度的YM-58483（BTP2）（10-1000 nM）或泼尼松龙（1-100 nM）预处理，然后在37°C下用PHA（10μg/ml）在24孔板（总体积：500μl）中刺激48小时。将平板离心（250g，在4°C下离心10分钟），然后收集上清液并在-20°C下冷冻直至使用。分别使用抗IL-5单克隆抗体和人IL-13 ELISA试剂盒DuoSet通过ELISA测量上清液中IL-5和IL-13的水平。

DNP诱导RBL-2H3细胞组胺释放和LTs产生[3]
RBL-2H3细胞是一种大鼠嗜碱性白血病细胞系，其表型与粘膜肥大细胞相似，购自美国典型培养物保藏中心，并在含有10%FBS、25 mM HEPES、2 g/l NaHCO3和青霉素（100 U/ml）/链霉素（100μg/ml）的RPMI 1640培养基中在5%CO2加湿气氛中保持单层培养。使用DNP和之前描述的方法，在稍作修改后，诱导RBL-2H3细胞释放介体。RBL-2H3细胞与单克隆抗DNP IgE（100 ng/ml）在1000 ml旋转培养瓶中以3×105个细胞/ml的密度培养24小时。洗涤后，在测定缓冲液（5 mM HEPES、140 mM NaCl、5 mM KCl、0.6 mM MgCl2、1 mM CaCl2、5 mM d（+）-葡萄糖，pH 7.4）中用IgE（8×10~5个细胞）预处理的细胞在37°C下用抗原DNP-BSA（0.1-100 ng/ml）刺激96孔板（总体积：200μl）20分钟。刺激后，细胞离心（250g，在4°C下10分钟），然后取出上清液并在-0°C下冷冻直至使用。分别使用组胺ELISA试剂盒和LTC4/D4/E4 EIA试剂盒测定上清液中组胺和LTC4/D4/E4（LT）的水平。为了测量测试化合物的效果，用不同浓度的YM-58483（BTP2）（30-3000nM）或泼尼松龙（10μM）预处理IgE引发的细胞，然后用DNP-BSA（30ng/ml）刺激。

Induction of graft-versus-host disease (GVHD)[3]
For the in vivo experiments, YM-58483(BTP2) was suspended in 0.5% methylcellulose solution for oral administration at a volume of 10 ml/kg.
Induction of GVHD was performed according to a previously described method [27]. Single-cell suspensions were prepared in HBSS from the spleen of female C57BL/6 mice (donor strain, H-2b) and female BDF1 mice (recipient strain, H-2b/d). The spleen cell suspensions were filtered through 70 μm-pore sterile nylon mesh, and then, to lyse the erythrocytes, resuspended in 3 ml of Tris ammonium chloride (0.26 M NH4Cl, 0.017 M Tris, pH 7.6), agitated for 2 min at room temperature, and then washed twice with HBSS. For the induction of GVHD, female BDF1 recipient mice were given a injection of spleen cells (5 × 10~7 cells) from sex- and age-matched C57BL/6 in the tail vein on day 0. As a negative control, female BDF1 mice were injected with sex- and age-matched syngeneic (BDF1) mice spleen cells (5 × 10~7 cells). As a normal control, female BDF1 mice were injected with 200 μl HBSS alone. YM-58483(BTP2) and cyclosporine A were administered orally once daily from day 0 to day 9 (10 consecutive days). Spleen cells from GVHD, negative control, and normal mice were used to examine anti-host CTL activity and the ability to produce IFN-γ on day 10. Cell subsets were also identified on day 10.

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YM-58483, has analgesic effects on neuropathic pain, but its mechanism is unclear. This experiment performed on spinal nerve ligation (SNL)-induced neuropathic pain model in rats tries to explore the mechanism, whereby YM-58483(BTP2) attenuates neuropathic pain. The left L5 was ligated to produce the SNL neuropathic pain model in male Sprague-Dawley rats. The withdrawal threshold of rats was measured by the up-down method and Hargreaves' method before and after intrathecal administration of YM-58483 and vehicle. The SOCCs in the spinal dorsal horn were located by immunofluorescence. The expression of phosphorylated ERK and phosphorylated CREB, CD11b, and GFAP proteins in spinal level was tested by Western blot, while the release of proinflammatory cytokines (IL-1β, TNF-α, PGE2) was measured by enzyme-linked immunosorbent assay (ELISA). Intrathecal YM-58483 at the concentration of 300 μM (1.5 nmol) and 1000 μM (10 nmol) produced a significant central analgesic effect on the SNL rats, compared with control + vehicle (n = 7, P < 0.001). However, both could not prevent the development of neuropathic pain, compared with normal + saline (P < 0.001). Immunofluorescent staining revealed that Orai1 and STIM1 (the two key components of SOCCs) were located in the spinal dorsal horn neurons. Western blot showed that YM-58483 could decrease the levels of P-ERK and P-CREB (n = 10, #P < 0.05), without affecting the expression of CD11b and GFAP (n = 10, #P > 0.05). YM-58483 also inhibited the release of spinal cord IL-1β, TNF-α, and PGE2, compared with control + vehicle (n = 5, #P < 0.001). The analgesic mechanism of YM-58483 may be via inhibiting central ERK/CREB signaling in the neurons and decreasing central IL-1β, TNF-α, and PGE2 release to reduce neuronal excitability in the spinal dorsal horn of the SNL rats.[1]

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YM-58483/BTP2 is a blocker of store-operated Ca2+ entry (SOCE), which regulates the activation of non-excitable cells such as lymphocytes. YM-58483(BTP2) has been reported to inhibit cytokine production and proliferation in T cells, and to be useful as a probable medicinal candidate for treatment of bronchial asthma. The present study investigated the pharmacological profile and therapeutic potential of YM-58483 in relation to cell-mediated immune responses. In the mouse graft-versus-host disease (GVHD) model, YM-58483 (1-30 mg/kg, p.o.) and cyclosporine A (1-30 mg/kg, p.o.) inhibited donor anti-host cytotoxic T lymphocyte (CTL) activity and IFN-gamma production, and also reduced the number of donor T cells, especially donor CD8+ T cells, in the spleen. YM-58483 and cyclosporine A inhibited T cell proliferation in a one-way mixed lymphocyte reaction (MLR) with IC50 values of 330 and 12.7 nM, respectively. Additionally, YM-58483 (1-10 mg/kg, p.o.) and cyclosporine A (2, 10 mg/kg, p.o.) inhibited the sheep red blood cell (SRBC)-induced delayed type hypersensitivity (DTH) response. These results suggest that the inhibition of SOCE leads to the prevention of antigen-induced T cell responses, which participate in autoimmune diseases such as autoimmune hepatitis and rheumatoid arthritis.[2]

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T cells play a regulatory role in the pathogenesis of various immune and allergic diseases, including human asthma. Recently, it was reported that a pyrazole derivative, YM-58483(BTP2), potently inhibits Ca(2+) release-activated Ca(2+) (CRAC) channels and interleukin (IL)-2 production in T cells. We investigated the effects of YM-58483(BTP2) on T helper type 2 (Th2) cytokine production in vitro and antigen-induced airway asthmatic responses in vivo. YM-58483 inhibited IL-4 and IL-5 production in a conalbumine-stimulated murine Th2 T cell clone (D10.G4.1), and IL-5 production in phytohemagglutinin-stimulated human whole blood cells with IC(50) values comparable to those reported for its CRAC channel inhibition (around 100 nM). YM-58483 inhibited antigen-induced eosinophil infiltration into airways, and decreased IL-4 and cysteinyl-leukotrienes content in inflammatory airways induced in actively sensitized Brown Norway rats. Furthermore, orally administered YM-58483 prevented antigen-induced late phase asthmatic bronchoconstriction and eosinophil infiltration in actively sensitized guinea pigs. These data suggest that the inhibition of Ca(2+) influx through CRAC channel leads to the prevention of antigen-induced airway inflammation, probably via the inhibition of Th2 cytokine production and inflammatory mediators release. YM-58483 may therefore be useful for treating airway inflammation in bronchial asthma.[4]

[1]. The Central Analgesic Mechanism of YM-58483 in Attenuating Neuropathic Pain in Rats. Cell Mol Neurobiol. 2016 Oct;36(7):1035-43.

[2]. Characterization of YM-58483/BTP2, a novel store-operated Ca2+ entry blocker, on T cell-mediated immune responses in vivo. Int Immunopharmacol. 2008 Dec 20;8(13-14):1787-9.

[3]. The suppressive effects of YM-58483/BTP-2, a store-operated Ca2+ entry blocker, on inflammatory mediator release in vitro and airway responses in vivo. Pulm Pharmacol Ther. 2008;21(2):360-9.

[4]. YM-58483, a selective CRAC channel inhibitor, prevents antigen-induced airway eosinophilia and late phase asthmatic responses via Th2 cytokine inhibition in animal models. Eur J Pharmacol. 2007 Apr 10;560(2-3):225-33.

YM-58483/BTP-2, 4-methyl-4'-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-1,2,3-thiadiazole-5-carboxanilide, blocks the store-operated Ca2+ entry (SOCE) that mediates the activation of non-excitable cells. This study investigated the pharmacological profile and therapeutic potential of YM-58483 as anti-asthma drug. YM-58483 inhibited DNP antigen-induced histamine release from and leukotrienes (LTs) production in IgE-primed RBL-2H3 cells, a rat basophilic leukemia cell line, with IC50 values of 460 and 310 nM, respectively. Prednisolone did not inhibit either of these responses. YM-58483 also inhibited phytohemagglutinin-P (PHA)-stimulated IL-5 and IL-13 production in human peripheral blood cells with IC50 values of 125 and 148 nM, respectively, which is approximately 5 times less potent than prednisolone. YM-58483 (30 mg/kg, p.o.) significantly suppressed ovalbumin (OVA)-induced bronchoconstriction in OVA-sensitized guinea pigs, whereas prednisolone did not. YM-58483 (3-30 mg/kg, p.o.) and prednisolone (100mg/kg, p.o.) both significantly and completely suppressed airway hyperresponsiveness (AHR) caused by OVA exposure. Since YM-58483 inhibits two major characteristic symptoms of bronchial asthma, namely bronchoconstriction and AHR via the suppression of inflammatory mediator and cytokine production, SOCE inhibition is a potential approach for treatment.[3]

C15H9F6N5OS

421.3214

421.043

C, 42.76; H, 2.15; F, 27.06; N, 16.62; O, 3.80; S, 7.61

223499-30-7

223499-30-7;

2455

White to off-white solid powder

1.6±0.1 g/cm3

1.608

3.77

100.94

1

11

3

28

568

0

XPRZIORDEVHURQ-UHFFFAOYSA-N

InChI=1S/C15H9F6N5OS/c1-7-12(28-25-23-7)13(27)22-8-2-4-9(5-3-8)26-11(15(19,20)21)6-10(24-26)14(16,17)18/h2-6H,1H3,(H,22,27)

N-[4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide

YM-58483; YM 58483; YM58483; BTP 2; 223499-30-7; YM-58483; btp2; CRAC Channel Inhibitor, BTP2; N-[4-[3,5-bis(trifluoromethyl)pyrazol-1-yl]phenyl]-4-methylthiadiazole-5-carboxamide; N-(4-(3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide; N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide; CHEMBL262766; BTP-2; BTP2.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~296.69 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.93 mM) (饱和度未知) 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), 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 (5.93 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：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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