sPLA2/secretory phospholipase A2 (IC50 = 9 nM)

在两个时间间隔的细胞裂解物中观察到的 RA 诱导的 MUC16 蛋白升高完全被维普拉地钠（10 μM；24 和 48 小时；HCjE 细胞）治疗所抑制 [2]。 vespladib钠（10μM）给药可强烈抑制HCjE细胞中RA诱导的MUC16表达，24小时抑制率为100%，48小时抑制率为99%[2]。

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Varespladib（LY-315920）是重组人IIA组非胰腺分泌型PLA2（sPLA2）的强效选择性抑制剂。在显色分离酶测定中，Varespladib（LY-315920）抑制sPLA2活性，IC50为9+/-1 nM或7.3 x 10（-6）摩尔分数，接近该测定的刺激测量极限。Varespladib (LY-315920)的真实效力是通过脱氧胆酸盐/磷脂酰胆碱测定法确定的，摩尔分数为1.5 x 10（-6）。LY315920对人、IB组、胰腺sPLA2的活性低40倍，对细胞质PLA2和环氧化酶的组成型和诱导型没有活性。LY315920抑制了人sPLA2诱导的豚鼠肺支气管肺泡灌洗液细胞释放血栓素A2（TXA2），IC50为0.79微M。N-甲酰基-甲磺酰基-亮酰基-苯丙氨酸或花生四烯酸对这些细胞释放TXA2没有抑制作用。[1]

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在使用广谱PLA2抑制剂ArA抑制RA诱导的MUC16上调后，我们试图确定IIA组sPLA2的特异性抑制剂Varespladib (LY-315920)是否会影响RA诱导的MUC16表达。我们通过实时PCR检测了HCjE培养物中MUC16 mRNA的表达水平，这些培养物分别用载体（DMSO）、100nM RA、100nM RA+10μmVarespladib（LY-315920）或单独使用10μm Varespladib (LY-315920)处理24和48小时。如图6所示，添加10μm LY315920显著抑制了RA诱导的MUC16表达，24小时时抑制率为100%，48小时时抑制了99%。如图7所示，添加sPLA2 IIA的特异性抑制剂LY315920可完全抑制RA诱导的MUC16蛋白在24（p<0.01）和48（p<0.0001）小时时在细胞裂解物中检测到的增加[2]。

给予伐瑞拉迪巴钠后，观察到对豚鼠肺支气管灌洗细胞中人 sPLA2 诱导的血栓素 A2 (TXA2) 释放的抑制作用的 IC50 为 0.79 μM。伐瑞拉迪钠的 ED50 为 16.1 mg/kg[1]。
在采集支气管肺泡灌洗液细胞前5分钟静脉注射Varespladib（LY-315920），导致sPLA2诱导的TXA2产生受到抑制，ED50为16.1mg/kg。用sPLA2攻击豚鼠肺胸膜条产生收缩反应，这些反应被Varespladib（LY-31592 0）以浓度依赖的方式抑制，表观KB为83+/-14nM。花生四烯酸诱导的收缩反应没有改变。向表达人sPLA2蛋白的转基因小鼠静脉或口服Varespladib（LY-315920），在4小时的时间过程中以剂量相关的方式抑制血清sPLA2活性Varespladib（LY-315920）是一种强效且选择性的sPLA2抑制剂，代表了一类新的抗炎药SPI。该药物目前正在进行临床评估，应有助于确定sPLA2在各种炎症性疾病状态中的作用[1]。

磷脂酶A2抑制剂治疗[2]
为了研究RA对MUC16的调节是否与sPLA2有关，在如上所述与100 nM RA加100μM ArA（抑制剂或载体（DMSO）单独培养24和48小时的HCjE细胞中，测定了广谱PLA2抑制剂马兜铃酸（ArA）对MUC16mRNA水平的影响。在这些实验之后，测试了IIA组分泌型磷脂酶A2特异性抑制剂Varespladib (LY-315920)的效果。HCjE细胞分别用100nM RA、100nM RA加10μmVarespladib（LY-315920）、抑制剂或载体（DMSO）单独处理24和48小时。分别通过实时PCR和Western blot分析测定MUC16 mRNA和蛋白质水平。两种抑制剂都进行了两次实验，每次实验重复两次。

蛋白质印迹分析 [2]
细胞类型： HCjE 细胞
测试浓度： 10 μM
孵育时间： 24 hrs（小时）、48 hrs（小时）
实验结果：RA 诱导的 MUC16 蛋白表达在两个时间点均受到显着抑制。

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RT-PCR[2]
细胞类型： HCjE 细胞
测试浓度： 10 μM
孵育时间：24小时、48小时
实验结果：显着抑制RA诱导的MUC16表达，24小时时达到100%（ hrs（小时））和 48 hrs（hrs（小时））时为 99%。

Animal/Disease Models: Male Hartley guinea pig (300-500 g) [1]
Doses: 3 mg/kg, 10 mg/kg and 30 mg/kg
Route of Administration: intravenous (iv) (iv)injection (pharmacokinetic/PK/PK study)
Experimental Results: sPLA2 activity in BAL Sustained inhibition of liquid was observed. diminished human sPLA2-induced TXA2 production on guinea pig BAL cells.

[1]. Pharmacology of LY315920/S-5920, [[3-(aminooxoacetyl)-2-ethyl-1- (phenylmethyl)-1H-indol-4-yl]oxy] acetate, a potent and selective secretory phospholipase A2 inhibitor: A new class of anti-inflammatory drugs, SPI. J Pharmacol Exp Ther. 1.

[2]. Effect of retinoic acid on gene expression in human conjunctival epithelium: secretory phospholipase A2 mediates retinoic acid induction of MUC16. Invest Ophthalmol Vis Sci. 2005 Nov;46(11):4050-61.

Varespladib Sodium is an intravenously administered inhibitor of secretory phospholipase A2 (sPLA2) with orphan drug designation. Varespladib is in trial for prevention of Acute Chest Syndrome in the treatment of sickle cell disease.

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Varespladib is a member of the class of indoles that is 1H-indole substituted by benzyl, ethyl, oxamoyl, and carboxymethoxy groups at positions 1, 2, 3, and 4, respectively. It is an oral secretory phospholipase A2 inhibitor and exhibits anti-inflammatory effects. It has a role as an EC 3.1.1.4 (phospholipase A2) inhibitor, an anti-inflammatory drug and an antidote. It is a member of indoles, a member of benzenes, an aromatic ether, a dicarboxylic acid monoamide, a monocarboxylic acid and a primary carboxamide. It is a conjugate acid of a varespladib(1-).
Varespladib has been investigated for the treatment and prevention of Sickle Cell Disease, Vaso-occlusive Crisis, and Acute Coronary Syndrome.

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LY315920 is a potent, selective inhibitor of recombinant human, group IIA, nonpancreatic secretory PLA2 (sPLA2). In a chromogenic isolated enzyme assay, LY315920 inhibited sPLA2 activity with an IC50 of 9 +/- 1 nM or 7.3 x 10(-6) mole fraction, which approached the stiochiometric limit of this assay. The true potency of LY315920 was defined using a deoxycholate/phosphatidylcholine assay with a mole fraction of 1.5 x 10(-6). LY315920 was 40-fold less active against human, group IB, pancreatic sPLA2 and was inactive against cytosolic PLA2 and the constitutive and inducible forms of cyclooxygenase. Human sPLA2-induced release of thromboxane A2 (TXA2) from isolated guinea pig lung bronchoalveolar lavage cells was inhibited by LY315920 with an IC50 of 0.79 microM. The release of TXA2 from these cells by N-formyl-methionyl-leucyl-phenylalanine or arachidonic acid was not inhibited. The i.v. administration of LY315920, 5 min before harvesting the bronchoalveolar lavage cells, resulted in the inhibition of sPLA2-induced production of TXA2 with an ED50 of 16.1 mg/kg. Challenge of guinea pig lung pleural strips with sPLA2 produced contractile responses that were suppressed in a concentration-dependent manner by LY315920 with an apparent KB of 83 +/- 14 nM. Contractile responses induced by arachidonic acid were not altered. Intravenous or oral administration of LY315920 to transgenic mice expressing the human sPLA2 protein inhibited serum sPLA2 activity in a dose-related manner over a 4-h time course. LY315920 is a potent and selective sPLA2 inhibitor and represents a new class of anti-inflammatory agent designated SPI. This agent is currently undergoing clinical evaluation and should help to define the role of sPLA2 in various inflammatory disease states. [1]

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Purpose: How vitamin A contributes to the maintenance of the wet-surfaced phenotype at the ocular surface is not well understood. This study sought to identify vitamin A-responsive genes in ocular surface epithelia using gene microarray analysis of cultures of a human conjunctival epithelial (HCjE) cell line grown with all-trans-retinoic acid (RA). The analysis showed that secretory phospholipase A(2) group IIA (sPLA(2)-IIA) was the gene most upregulated by RA, followed by the membrane-associated mucin MUC16 at a later time point. Since eicosanoids, the product of arachidonic acid generated by the PLA(2) family, have been shown to increase mucin production, this study sought to determine whether sPLA(2) mediates the RA induction of MUC16.

Methods: HCjE cells were cultured with or without RA for 3, 6, 24, and 48 hours. Complementary RNA prepared from RNA of the HCjE cells was hybridized to human gene chips and analyzed using commercial software. Microarray data on mucin expression were validated by real-time PCR. To investigate whether sPLA(2) is associated with RA-induced MUC16 upregulation, HCjE cells were incubated with RA and the broad-spectrum PLA(2) inhibitor aristolochic acid (ArA) or the specific sPLA(2)-IIA inhibitor LY315920, followed by analysis of MUC16 mRNA and protein by real-time PCR and Western blot analysis.

Results: After RA addition, 28 transcripts were upregulated and 6 downregulated by more than twofold (P < 0.01) at both 3 and 6 hours (early phase). Eighty gene transcripts were upregulated and 45 downregulated at both 24 and 48 hours (late phase). Group IIA sPLA(2), significantly upregulated by 24 hours, and MUC16 were the most upregulated RNAs by RA at 48 hours. sPLA(2) upregulation by RA was confirmed by Western blot analysis. When HCjE cells were incubated with RA plus ArA or specific inhibitor of sPLA(2)-IIA, LY315920, the RA-induced MUC16 mRNA was significantly reduced (P < 0.01).

Conclusions: The RA-associated upregulation of membrane-associated mucin MUC16 at late phase appears to be through sPLA(2)-IIA. Upregulation of this hydrophilic membrane-associated mucin may be one of the important mechanisms by which vitamin A facilitates maintenance of the wet-surfaced phenotype on the ocular surface. [2]

C21H19N2NAO5

402.375736474991

402.119

C, 62.68; H, 4.76; N, 6.96; Na, 5.71; O, 19.88

172733-42-5

Varespladib;172732-68-2

23674730

Off-white to light yellow solid powder

1.749

114.45

1

5

8

29

596

0

CCC1=C(C2=C(N1CC3=CC=CC=C3)C=CC=C2OCC(=O)[O-])C(=O)C(=O)N.[Na+]

XZZUHXILQXLTGV-UHFFFAOYSA-M

InChI=1S/C21H20N2O5.Na/c1-2-14-19(20(26)21(22)27)18-15(9-6-10-16(18)28-12-17(24)25)23(14)11-13-7-4-3-5-8-13;/h3-10H,2,11-12H2,1H3,(H2,22,27)(H,24,25);/q;+1/p-1

sodium;2-(1-benzyl-2-ethyl-3-oxamoylindol-4-yl)oxyacetate

Varespladib sodium; 172733-42-5; UNII-F6M52CDT0W; LY315920-Na; Varespladib Sodium [USAN]; LY315920-Sodium salt; F6M52CDT0W; Varespladib (sodium);

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，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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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));
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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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