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Varespladib (LY-315920)（10 μM；24 和 48 小时；HCjE 细胞）疗法所抑制 [2]。用 Varaspladib（10 μM；24 和 48 小时；HCjE 细胞）治疗可显着减少 RA 产生的 MUC16 量，24 小时时减少 100%，48 小时时减少 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]。

Varespladib (LY-315920)治疗的 IC50 为 0.79 μM，可防止人 sPLA2 诱导的豚鼠肺支气管肺泡灌洗细胞释放血栓素 A2 (TXA2)。 Varespladib (LY-315920) 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]。

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ApoE−/−标准模型[3]
对照组或A-002（varespladib methyl/LY333013/S-3013处理组）动物的体重在时间0时相似（赋形剂：27.2±2.6，30mg/kg A-002:26.7±2.3，90mg/kg A-002:37.3±2.5）。在西方饮食的16周内，Vehicle, 30 mg/kga-002（varespladib methyl/LY333013/S-3013和90 mg/kg a-002组）的体重分别增加了约155%、150%和141%（图2）。使用重复测量的双向方差分析，以时间和治疗为变量，两组之间的体重没有统计学上的显著差异。

研究开始时，各组的血浆胆固醇水平没有显著差异。然而，与对照组相比，每天两次服用a-002（varespladib methyl/LY333013/S-3013，剂量为30或90 mg/kg，治疗1个月后，总胆固醇显著降低（图3）。在整个4个月的治疗期间，这种效果保持一致。每次治疗4个月后，赋形剂、30mg/kg A-002和90mg/kg A-002组的血浆胆固醇分别变化了+15%、-10%和-12%（图3）。血浆胆固醇浓度没有明显的剂量反应效应。

A-002（varespladib methyl/LY333013/S-3013）治疗对斑块含量有显著影响，斑块含量以动脉粥样硬化斑块在主动脉管腔表面的占有率表示。载体治疗的小鼠斑块覆盖率约为12.6%±0.7%，而每天两次用30mg/kg A-002治疗的小鼠为6.3%±0.6%，每天两次服用90mg/kg A-002的小鼠为6.7%±0.8%。这表明与仅接受制剂载体的治疗组相比，A-002治疗组的斑块含量显著降低（P<0.05）（图4）。

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血管紧张素II ApoE−/−模型[3]
血管紧张素II在水中每天两次或在5%阿拉伯胶中每天两次配制，导致主动脉斑块覆盖率相似（分别为18%±3.3%和14.4%±4.8%，图5）A-002（varespladib methyl/LY333013/S-3013（30mg/kg）显著降低了主动脉的斑块覆盖率（8%±3%，而未经药物治疗输注血管紧张素II时为18%±3.3%，P<0.025）。在没有血管紧张素II的情况下，动脉粥样硬化的背景量（3.8%±0.6%，皮下盐水泵和每天两次水）明显低于输注血管紧张素Ⅱ的情况（18%±3.3%，P<0.025）。代表性的人脸图像如图6所示。

评估每组小鼠的主动脉瘤发生率。在未输注血管紧张素II的情况下，未观察到动脉瘤。输注水中配制的血管紧张素II导致25%的动脉瘤发生率，输注阿拉伯胶载体中配制的血管紧缩素II导致22.2%的动脉瘤发病率A-002（varespladib methyl/LY333013/S-3013治疗（30mg/kg，每天两次）在输注阿拉伯胶中配制的血管紧张素II的小鼠中完全预防了动脉瘤的形成（Prob>ChiSq=0.0096）（表1）。

sPLA2抑制[3]
根据其他地方描述的显色方法，测量了Varespladib（LY-315920）/A-001对sPLA2 V和X组酶抑制的内在活性。

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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）和48 hrs（小时）
实验结果：在两个时间点均显着抑制RA诱导的MUC16蛋白表达。

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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小时和48小时
实验结果：显着抑制RA诱导的MUC16表达，24小时时为100％，48小时时为99％ hrs（小时）。

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

Plasma Levels and Dose Selection [3]
Figure 1 shows plasma levels of Varespladib (LY-315920)/A-001 in serum after a single oral dose of A-002 (varespladib methyl/LY333013/S-3013. Levels of Varespladib (LY-315920)/A-001 were detectable in all samples from dosed mice at 10, 30, and 90 mg/kg. The 2 highest doses, 30 and 90 mg/kg A-002 (varespladib methyl/LY333013/S-3013, reached concentrations of Varespladib (LY-315920)/A-001 in plasma that were greater than the IC50 values for sPLA2 groups V and X, respectively, throughout the dosing period.

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Plasma Levels [3]
Plasma levels of A-001/Varespladib (LY-315920) (the active moiety of A-002 (varespladib methyl/LY333013/S-3013) were measured in a single-dose experiment in ApoE−/− mice. Animals were dosed by oral gavage as explained above with 10, 30, or 90 mg/kg ofA-002 (varespladib methyl/LY333013/S-3013. Blood was sampled at different time points using the tail clip method. Plasma was collected and stored at −70°C for analysis of the presence of A-001/Varespladib (LY-315920) by high-performance liquid chromatography-mass spectrometry. Plasma from dosed animals (25 μL) was combined with 5 μL of 20 μM LY329722 (internal standard), 25 μL of isopropanol, and 50 μL of acetonitrile in a polypropylene microfuge tube. The mixture was vortexed, incubated for 10 minutes at 37°C, and centrifuged at 12,000g at ambient temperature. Supernatant (65 μL) was decanted into 430 μL of water and 5 μL of formic acid in an 800-μL glass autosampler vial. The vial was capped, vortexed, and placed in a Surveyor autosampler. Injections (25 μL) were made onto a 2.1 × 100 mm, 5 μm, C8 Discovery reversed-phase column equipped with a 2.1 × 4 mm Phenomenex C8 guard column. Initial chromatography conditions were set to 95% mobile phase A (0.1% formic acid in 5% isopropanol) and 5% mobile phase B (0.1% formic acid in acetonitrile containing 5% isopropanol). At 1-minute postinjection, a gradient elution from 5% B to 60% B over 6 minutes gave retention times of 6.07 and 6.47 minutes for A-002 (varespladib methyl/LY333013/S-3013 and internal standard, respectively. Analytes were detected by electrospray, atmospheric pressure ionization, ion trap mass spectrometry. Capillary voltage was 4.5 kV and capillary temperature was 310°C. Nitrogen sheath flow was set at 75 arbitrary units. Collision-induced dissociation was optimized at 35% normalized collision energy. Transitions selective for the tandem mass spectrometric detection of A-001/Varespladib (LY-315920) and internal standard resulted from the loss of ammonia and carbon monoxide radical and were 381→336 m/z and 395→350 m/z, respectively. Peak areas for A-001 and internal standard were used to compute response factors and unknowns were interpolated against a linear calibration curve of A-001 spiked into pooled mouse plasma obtained from nondosed animals.

[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. 1999 Mar;288(3):1117-24.

[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.

[3]. Varespladib (A-002), a Secretory Phospholipase A2 Inhibitor, Reduces Atherosclerosis and Aneurysm Formation in ApoE−/− Mice J Cardiovasc Pharmacol. 2009 Jan;53(1):60-5.

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]

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The family of secretory phospholipase A2 (sPLA2) enzymes has been associated with inflammatory diseases and tissue injury including atherosclerosis. A-001 is a novel inhibitor of sPLA2 enzymes discovered by structure-based drug design, and A-002 (varespladib methyl/LY333013/S-3013 is the orally bioavailable prodrug currently in clinical development. A-001 inhibited human and mouse sPLA2 group IIA, V, and X enzymes with IC50 values in the low nM range. A-002 (1 mg/kg) led to high serum levels of A-001 and inhibited PLA2 activity in transgenic mice overexpressing human sPLA2 group IIA in C57BL/6J background. In addition, the effects of A-002 on atherosclerosis in 2 ApoE−/− mouse models were evaluated using en face analysis. (1) In a high-fat diet model, A-002 (30 and 90 mg/kg twice a day for 16 weeks) reduced aortic atherosclerosis by 50% (P < 0.05). Plasma total cholesterol was decreased (P < 0.05) by 1 month and remained lowered throughout the study. (2) In an accelerated atherosclerosis model, with angiotensin II-induced aortic lesions and aneurysms, A-002 (30 mg/kg twice a day) reduced aortic atherosclerosis by approximately 40% (P < 0.05) and attenuated aneurysm formation (P = 0.0096). Thus, A-002 was effective at significantly decreasing total cholesterol, atherogenesis, and aneurysm formation in these 2 ApoE−/− mouse models.[3]

C21H20N2O5

380.39

380.137

C, 66.31; H, 5.30; N, 7.36; O, 21.03

172732-68-2

Varespladib sodium;172733-42-5;Varespladib methyl;172733-08-3

155815

White to yellow solid powder

1.3±0.1 g/cm3

667.9±65.0 °C at 760 mmHg

357.7±34.3 °C

0.0±2.1 mmHg at 25°C

1.630

2.45

111.62

2

5

8

28

589

0

CCC1=C(C2=C(N1CC3=CC=CC=C3)C=CC=C2OCC(=O)O)C(=O)C(=O)N

BHLXTPHDSZUFHR-UHFFFAOYSA-N

InChI=1S/C21H20N2O5/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)

2-((3-(2-amino-2-oxoacetyl)-1-benzyl-2-ethyl-1H-indol-4-yl)oxy)acetic acid

A002; A 002; LY315920; Varespladib; 172732-68-2; LY315920; Varespladib (LY315,920); LY-315,920; S-5920; VAREPLADIB; 2Q3P98DATH; LY-315920; LY 315920; A-002;

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)

配方 1 中的溶解度: 2.5 mg/mL (6.57 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: 30% PEG400+0.5% Tween80+5% propylene glycol:30 mg/mL

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配方 3 中的溶解度: 1.5 mg/mL (3.94 mM) in 17% Polyethylene glycol 12-hydroxystearate in Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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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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;
5、为保证最佳实验结果，工作液请现配现用！
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;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。