sPLA2/secretory phospholipase A2 (IC50 = 9 nM)

Varespladibmethyl 也称为 LY333013，是甲醇的前药 [1]。当用于对抗来自六大洲的 28 种具有医学毒性的蛇毒时，伐瑞拉迪及其侧壁生物可利用的前药甲基伐瑞拉迪（甲基伐瑞拉迪）在纳摩尔和皮摩尔浓度下表现出高水平的致命性 PLA2 (sPLA2)。抑制影响[2]。

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体外抑制sPLA2活性[2]
在研究可用于蛇咬伤的药物时，我们发现varespladib（LY315920）和甲基varespladib（LY333013）使用显色分析在体外抑制了大量蛇毒的sPLA2活性，但对用作标准阳性对照的蜂毒没有显示出很大的活性（表1和图1）。此外，令人惊讶的是，观察到varespladib和甲基varespladib对几乎所有测试的蛇毒的IC50明显低于包括人类在内的哺乳动物sPLA2的抑制值[2]。

疗效。在治疗的第1周，1000mg/天的methyl-varespladib/LY333013在达到ACR20反应的患者比例方面优于安慰剂（p=0.064），并且观察到剂量反应关系（p=0.058）（图1）。在第1周观察到CRP具有统计学意义的剂量反应关系（p=0.046）。然而，在研究第4周和第8周，所有治疗组的ACR20反应率都有所增加，与安慰剂相比没有差异。探索性分析显示，65岁以上患者的ACR20反应呈剂量依赖关系（p=0.077）。次要终点的分析，包括达到ACR20反应的时间、治疗失败的时间以及患者报告的疼痛或整体关节炎状态改善20%的时间，在治疗组之间没有差异。在56名接受安慰剂治疗的患者中，有9名（16.1%）达到了ACR50反应，而在60名接受methyl-varespladib/LY33301350、250和1000mg/天治疗的患者当中，分别有10名（16.7%）、5名（8.5%）和5名（8.2%）达到了反应。ACR反应成分的治疗组之间的变化通常很小或不一致，没有统计学意义。LY333013、250和1000mg/天组的TJC在第8周和第12周增加，SJC在第12周也增加。研究人员的总体评估反映了这些发现，但患者的总体和疼痛评估没有。探索性分析表明，在研究开始时，RF的存在、使用的DMARD数量或使用的特定DMARD方面没有出现治疗反应。值得注意的是，在研究开始时，只有18%的患者服用了非甾体抗炎药，而使用低剂量皮质类固醇的患者为48%。在LY333013 1000 mg/天组中，皮质类固醇的使用似乎降低了ACR20的实现[1]。

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治疗组的人口统计学特征相似。在第1周，ACR20反应（p=0.058）和C反应蛋白降低（p=0.558）之间存在剂量-反应关系。随后，在第4周和第8周，包括安慰剂组在内的所有研究组中，ACR20反应的患者比例都有所增加，并且失去了最初的治疗效果。不良事件的严重程度通常较轻，与治疗无关。 结论：使用methyl-varespladib/LY333013治疗12周耐受良好，但作为DMARD治疗活动性RA的辅助手段无效[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）。

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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）。

体外实验[2]
使用二庚酰基磷脂酰胆碱的1,2-二硫代类似物进行实验以评估sPLA2活性。sPLA2催化sn-2位磷脂的水解，产生游离脂肪酸和溶血磷脂。PLA从膜磷脂中释放花生四烯酸被认为是控制细胞内类二十烷生成的关键步骤。蜂毒PLA2对照品是一种100μg/mL的蜂毒溶液，PLA2作为阳性对照从试剂盒中提供。耶鲁分子发现中心进行了检测优化、筛选和剂量反应测量。实验在含有25 mM Tris-HCl、pH 7.5、10 mM CaCl2、100 mM KCl、0.3%Triton X-100（Fluka）和454µM DTNB的测定缓冲液中进行，并镀入透明的未处理384孔板中。毒液在1倍磷酸盐缓冲盐水中复溶至10000µg/mL的浓度。所有病例均使用购自Sigma（E.carinatus和D.russelli）或Miami Serpentarium（所有其他）的粗、普通冻干毒液。Varespladib和甲基Varespladib/LY333013购自Chemietek，溶于DMSO用于体外实验，溶于碳酸氢盐/葡萄糖用于体内实验。基于在室温下进行的动力学酶测定，选择了含有0.375 mM 1,2-双（庚酰基）甘油磷胆碱（sPLA2底物）的静脉的活性。选择毒液浓度进行筛选和效力研究，在这些研究中，相对于没有毒液对照孔的任何背景活性，观察到较高的sPLA2活性，并且在60分钟时底物消耗可以忽略不计。用于检测的毒液的最终浓度范围为0.0037–5µg/mL，表明不同毒液在该底物的比例或相对sPLA2活力方面存在很大差异。对于13种蛇毒，最终浓度范围从黄颡鱼的0.0037µg/mL到多叶石斛的100µg/mL（平均浓度7.8±28µg/mL；中位浓度0.1µg/mL），对于15种蝰蛇毒，其最终浓度范围为0.033µg/mL，例如白蛉和25µg/mL红细胞肉瘤（平均浓度2.47±6.35µg/mL；中值浓度1µg/mL）。筛选第一阶段使用的试验性集合由发明人选择，或从选定毒液实验时可用的已知化合物和天然产物库中选择，包括：美国国立卫生研究院临床收藏、GenPlus、Pharmakon、生物活性脂质、蛋白酶抑制剂、采购药物和美国食品药品监督管理局批准的药物库。MicroSource Discovery Systems的GenPlus（NINDS定制系列）含有960种化合物。Varespladib的表现优于文库中的所有化合物，这证实了（在筛选文库的范围内）其作为sPLA2抑制剂的优越效力。在这些总计4000多个不同化学实体的集合中，没有发现任何分子的效力在varespladib的两个数量级以内，因此结果不在本手稿的范围内。对于抑制剂和剂量反应测试，使用多通道移液管或多滴分配器将10µL蛇毒或蜂毒（+对照）添加到分析板中。使用针工具将来自化学文库或制备的溶解在DMSO中的连续稀释母版的化合物加入到分析板中，以转移20nL的化合物。测定中DMSO的最终浓度为0.1%。然后加入10µL底物，最终测定体积为20µL。在复制孔的每个板上都包括对照组。阴性对照孔是不含小分子化合物的载体（仅DMSO）。模拟完全毒液活性抑制的阳性对照是没有添加毒液的孔，并在其位置添加了测定缓冲液。在起始时和在室温下反应60分钟后测量测定信号。在Tecan infiniTe M1000平板读数器上量化信号，测量405nm处的吸光度。从60分钟的信号中减去启动时的信号。将这些背景校正值归一化为板内重复阴性和阳性对照孔的平均值。为了定义标准化尺度，代表完全毒液活性的阴性对照孔信号的平均值被标准化为100%效果，代表完全抑制毒液活性的阳性对照孔信号平均值被归一化为0%效果。板块内的井也相应地进行了缩放。这些计算是在MicroSoft Excel中进行的。将数据转移到GraphPad Prism（2014年第6版，美国加利福尼亚州拉霍亚）中，绘制并拟合模型，从而可以确定IC50或EC50值。显著性检验由Student的t计算，所有其他检验都是描述性的。

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sPLA2抑制[3]
根据别处描述的显色法测量A-001对sPLA2 V和X组酶抑制的内在活性。

Two hundred and fifty-one patients with active RA despite treatment with one or more disease modifying antirheumatic drugs (DMARD) received oral doses of LY333013 (50, 250, and 1000 mg) or placebo once daily for 12 weeks. Concomitant low-dose glucocorticoids (< or = 10 mg/day prednisone equivalent) were allowed. Clinical improvement was assessed using the response criteria of the American College of Rheumatology (ACR20), and safety was evaluated with respect to adverse events and laboratory test abnormalities. [1]

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Animal in Vivo Studies [2]
M. fulvius and V. berus venoms had the highest sPLA2 activity in vivo and and after successful pilot survival studies was chosen for the Non-GLP study in rats and V. berus for mouse studies. CD-1 mice and Sprague-Dawley Rats with implanted jugular venous catheters were used. 18 Sprague-Dawley rats weighing between 183 and 214 g at the time of the study had jugular vein cannulas surgically implanted by the supplier. Rats were randomly assigned to six treatment groups (n = 3 each) and received snake venom with and without varespladib. Animals were monitored for signs of toxicity for approximately 24 h. Blood samples (without anticoagulant) were collected from each rat prior to dose administration and post dose administration at approximately 30 min, 1 h and 4 h. Per protocol, nominal blood collection times were pre-dose administration and post-varespladib administration at 30 min ± 1 min., 1 h ± 1 min., and 4 h ± 5 min. There were no deviations from these specifications except for animals that died prior to the last scheduled blood collect ion at 4 h. Blood was processed to serum and analyzed by the AILAC certified contract research organization to determine sPLA2 activity validated beforehand with rat serum for quality control. Surviving animals were euthanized following the 24-h observation. Tissues were grossly examined but not collected for further processing. Justification for the use of the mouse in this study is based on the premise that animal testing is an appropriate and ethical prerequisite to testing new drugs in humans, and that data obtained from nonclinical animal models will have relevance to the behavior of the test material in humans. Because of the complex interactions that occur in vivo, an in vitro system does not provide sufficient information for evaluation of a compound’s in vivo activities. It was expected that the number of animals used in this study would provide a large enough sample for scientifically meaningful results while using the fewest possible animals to achieve that result. The intravenous route was chosen to maximize the bioavailability of varespladib. All experiments were designed to insure 100% mortality in control animals within the expected ½ life of the test drug in order to produce clear results using the lowest number of animal.

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sPLA2 Group IIA Transgenic Model [3]
A transgenic mouse model with human sPLA2 group IIA under inducible mouse metallothionein promoter control in a C57BL/6J ApoE+/+ background was used to evaluate the inhibition of phospholipase A2 activity in serum from animals dosed with methyl-varespladib/LY333013/A-002 as described by Fox et al.21 Animals were administered a single oral dose of methyl-varespladib/LY333013/A-002 (1 or 3 mg/kg) or vehicle (5% acacia), and enzymatic activity22 was measured in samples at baseline before treatment and 0.5, 2, and 4 hours after dosing.

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ApoE−/− Standard Model [3]
The ApoE−/− model was developed as described elsewhere.23 Male ApoE−/− mice (129/Ola × C57BL/6J, multiple generations) were used at an age of 6-8 weeks. Mice were fed ad libitum a high-fat diet (21% fat: 0.15% cholesterol and 19.5% casein) for 2 weeks to allow time to adjust to the diet. Mice were randomized into groups based on plasma triglyceride and total cholesterol levels using the GroupOptimizer V211.xls program. Animals were treated for 16 weeks with 30 mg/kg twice a day or 90 mg/kg twice a day methyl-varespladib/LY333013/A-002. Compounds were formulated in 0.2 mL of 5% acacia (10 mL/kg body weight) and administered by oral gavage twice a day at 7 am and 3 pm.

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ApoE−/− Angiotensin II Accelerated Model [3]
Male ApoE−/− mice (129/Ola × C57BL/6J, multiple generations) were used at an age of 6-8 weeks. In this model, angiotensin II was administered by 0.7 mg kg−1 d−1 subcutaneous infusion to induce faster atherosclerosis development associated with aneurysm formation.24 Mice were fed ad libitum a high-fat diet (21% fat: 0.15% cholesterol and 19.5% casein) for 2 weeks to allow time to adjust to the diet. The animals were then randomized into groups based on plasma triglyceride and total cholesterol levels using the GroupOptimizer V211.xls program and were treated for 4 weeks with 30 mg/kg twice a day of methyl-varespladib/LY333013/A-002 as described in the standard ApoE−/− model.

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Procedures. Patients meeting the inclusion and exclusion criteria at an initial entry visit were reassessed at their baseline visit to assure that they continued to meet the entry criteria for active RA. The patients completed the Stanford Health Assessment Questionnaire (HAQ)20, including a 100 mm horizontal visual analog scale (VAS) for pain (0 = no pain; 100 = extreme pain), and assessed the global activity of their arthritis on a 100 mm VAS (0 = very well; 100 = very poorly) at each visit. The investigators were instructed in a standardized 28-joint technique21 for counting the number of swollen (SJC) and tender joints (TJC) at each visit, and they judged global arthritis activity on a 100 mm VAS (0 = very well; 100 = very poorly). Safety was assessed on the basis of adverse events reported at each visit (baseline and after 1, 4, 8, and 12 weeks of treatment), findings on physical examination, and laboratory evaluations. Samples for population pharmacokinetics and/or CRP were obtained at baseline and subsequent visits. Study medications. The study medications were prepared in identicalappearing white tablets containing 0, 50, 125, or 250 mg of methyl-varespladib/LY333013. Patients took 2 tablets twice daily from blister cards that corresponded to assigned dose levels of 0 (placebo), 50, 250, and 1000 mg/day of methyl-varespladib/LY333013. The 50 and 250 mg/day dose groups received active medication only in the morning dose. Randomization to treatment was blocked and stratified by site. Treatment assignments were managed by an interactive telephone voice response system. Treatment identities were provided to the investigators in a sealed envelope, but except for emergencies warranting it, unblinding did not occur until after the last study visit. Unblinded patients were discontinued from study participation unless there was a compelling ethical reason for their continuation in the study. Compliance to the treatment regimen was monitored by counts of medication in the returned blister cards. In the event that the blister cards were lost, medication use was assessed by patient report. Statistical analysis. The p values for the demographic, RA baseline disease features and medications, and adverse event data (Tables 1-4) were computed for continuous variables from the rank-transformed analysis of variance model with terms for dose and pooled investigative site; p values for categorical variables were computed from the Cochran-Mantel-Haenszel test stratified by pooled investigative site. The primary efficacy evaluation utilized the ACR Definition of Improvement22 at the 20% level (ACR20). Treatment was deemed a failure if, at the end of 4 weeks of treatment, the patient’s condition had worsened by > 20% relative to baseline or, after 8 weeks, there had been < 10% improvement or, after 12 weeks, there had been < 20% improvement. These patients were discontinued from study treatment and were classified as non-responders with respect to any definition of improvement. Efficacy analysis was performed on all randomized patients who received at least one dose of study medication and who had at least one efficacy assessment at the 4, 8, or 12 week visits [intention-totreat (ITT) group]. Missing data were handled using a last observation carried forward analysis. Adverse event analysis was performed on all patients who received at least one dose of study medication. Assuming that 30% of patients receiving placebo would achieve an ACR20 response compared to 50% of patients receiving LY333013, 1000 mg/day, 59 patients per treatment group would provide 80% probability of detecting a linear doseresponse relationship at a one-sided α = 0.1 using a logistic regression model with dose as the explanatory variable. Pharmacokinetic measurements. High performance liquid chromatography/mass spectroscopic methods were used to measure the active metabolite of LY333013. Results were evaluated according to the reported time of the last 4 doses of study medication and the assigned dose. A 2- compartment distribution model was used with assumption of first-order elimination. Based on previous studies, it was known that plasma clearance is affected by bioavailability, which in turn is reduced by increasing dose. These factors were included in the model evaluating individual apparent oral clearance and inter-individual variability in this parameter. Systemic exposure was estimated based on dose and apparent oral clearance for all patients with available pharmacokinetics data.[1]

Treatment exposure. The mean plasma steady-state concentration of the active metabolite of methyl-varespladib/LY333013 (LY315920) was estimated to be 222, 627, and 1445 ng/ml for the 50, 250, and 1000 mg/day dose groups, respectively. There were no apparent interactions between LY315920 concentrations and co-therapy with MTX, sulfasalazine, and hydroxychloroquine. Ninety-one percent of the efficacy analysis patients took 80-119% of their prescribed doses of study drug. [1]

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Methyl-varespladib/LY333013, a rapidly absorbed, orally bioavailable prodrug of varespladib could be administered orally (e.g., as an elixir) so that a person with no or very limited skill could potentially initiate treatment outside a hospital setting. Methyl-varespladib/LY333013is metabolized to varespladib, so the parent compound was the focus of our proof-of-concept studies though field use of an IV formulation is an unlikely scenario. The use of 3-substituted indoles for snake venom sPLA2 inhibition represents a possible springboard for the genesis of effective field treatments for snakebites; either could be rapidly developed with programmatic support and industry cooperation. Our findings warrant further investigation into the efficacy of veraspladib and methyl-varespladib in an even wider diversity of snakes to determine if either could be an essential component of the long sought-after venom antagonistic, first-line field-treatment for snakebite.[2]

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Plasma Levels and Dose Selection [3]
Figure 1 shows plasma levels of A-001 in serum after a single oral dose of methyl-varespladib/LY333013/A-002. Levels of 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, reached concentrations of 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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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]

Safety. Of the 251 patients who received at least one dose of study medication, 182 reported at least one adverse event (AE). The most commonly reported AE were: rhinitis (21.9%), headache (19.5%), cough (13.5%), diarrhea (9.2%), nausea (8.8%), fever (8.4%), dizziness (6.4%), abdominal pain (6.0%), sinusitis (6.0%), and pharyngitis (5.6%), with no statistically significant differences across treatment groups for any of these events (Table 4). In general, the type, severity, and distribution of AE were similar across all treatment groups. The overall proportion of patients with AE was somewhat lower in the placebo group (60.3%) than in the methyl-varespladib/LY333013 50 (69.4%), 250 (79.0%), or 1000 mg/day groups (81.3%, overall p = 0.043), but an analysis of AE by organ system indicated that only the cardiovascular system showed a statistical difference among treatment groups (p = 0.033). Cardiovascular AE did not appear to be dose-related; they occurred in more patients in the methyl-varespladib/LY333013 50 and 1000 mg/day treatment groups (16.1%; 15.6%, respectively) than in the methyl-varespladib/LY333013 250 mg/day (3.2%) and placebo groups (4.3%). Upper respiratory tract infections were more common in LY333013-treated patients, whereas urinary tract infections were more common among patients receiving placebo. These differences were not thought to be clinically significant. Six patients with non-serious AE discontinued study treatment: all were receiving LY333013 (Table 5). These AE were vertigo (50 mg); dyspepsia, nausea, rash (250 mg); asthenia and somnolence (1000 mg). Serious AE were observed in 3 placebo- and 4 LY333013-treated patients; these included septic arthritis, pneumonia, and stroke in the placebo group, and chest pain (50, 250, 1000 mg groups) and perforated colonic diverticulum (50 mg) in the LY333013 groups. The latter patient had a history of diverticulitis, and all 3 patients with chest pain had prior diagnoses of cardiovascular disease. One patient (250 mg group) underwent coronary angioplasty. Study treatment was discontinued in patients with serious AE, i.e., pneumonia and colonic perforation. Only the latter serious AE was assessed by the investigator to be possibly related to study treatment. No patients died during study participation. There were no clinically relevant changes in laboratory variables, vital signs, or electrocardiogram recordings. Using the National Cancer Institute Common Toxicity Criteria (CTC) grades, only serum AST and calcium differed between placebo and LY333013 (p = 0.049). No patient had a CTC grade 4 abnormality of any analyte; the highest CTC grade for serum calcium was 2. Serum AST was more frequently elevated in the LY333013 1000 mg/day group; the highest value in this group was 122 IU. [1]

[1]. A randomized, double-blinded, placebo-controlled clinical trial of LY333013, a selective inhibitor of group II secretory phospholipase A2, in the treatment of rheumatoid arthritis. J Rheumatol. 2005 Mar;32(3):417-23.

[2]. Varespladib (LY315920) Appears to Be a Potent, Broad-Spectrum, Inhibitor of Snake Venom Phospholipase A2 and a Possible Pre-Referral Treatment for Envenomation. Toxins (Basel). 2016 Aug 25;8(9). pii: E248.

[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 methyl or methyl-varespladib/LY333013 is a methyl ester resulting from the formal condensation of the carboxy group of varespladib with methanol. It is a potential therapy for the treatment of snakebite envenomings in which toxicity depends on the action of PLA2s. It has a role as a prodrug, an anti-inflammatory drug, an antidote and an EC 3.1.1.4 (phospholipase A2) inhibitor. It is a methyl ester, an aromatic ether, a member of benzenes, a member of indoles and a primary carboxamide. It is functionally related to a varespladib.
Varespladib methyl has been investigated for the treatment of Acute Coronary Syndrome. Studies showed that Varespladib methyl treatment resulted in significant positive changes on lipoproteins and inflammation.

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Drug Indication
Investigated for use/treatment in atherosclerosis and coronary artery disease.

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Mechanism of Action
A–002 is an orally administered‚ potent inhibitor of secretory phospholipase spla2(spla2)‚ including groups IIA‚ V‚ and X. Atherosclerosis is a disease of the arteries that results from inflammation and the build-up of plaque under the lining of the blood vessel. This build-up can cause vascular swelling and eventual rupture. spla2 levels have been shown to be elevated in patients with both stable and unstable coronary artery disease. Higher levels of the enzyme have been shown to predict an increased risk for future cardiovascular events such as heart attacks and stroke.

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Varespladib methyl is a methyl ester resulting from the formal condensation of the carboxy group of varespladib with methanol. It is a potential therapy for the treatment of snakebite envenomings in which toxicity depends on the action of PLA2s. It has a role as a prodrug, an anti-inflammatory drug, an antidote and an EC 3.1.1.4 (phospholipase A2) inhibitor. It is a methyl ester, an aromatic ether, a member of benzenes, a member of indoles and a primary carboxamide. It is functionally related to a varespladib.
Varespladib methyl has been investigated for the treatment of Acute Coronary Syndrome. Studies showed that Varespladib methyl treatment resulted in significant positive changes on lipoproteins and inflammation.
VARESPLADIB METHYL is a small molecule drug with a maximum clinical trial phase of III (across all indications) and has 3 investigational indications.

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Snakebite remains a neglected medical problem of the developing world with up to 125,000 deaths each year despite more than a century of calls to improve snakebite prevention and care. An estimated 75% of fatalities from snakebite occur outside the hospital setting. Because phospholipase A2 (PLA2) activity is an important component of venom toxicity, we sought candidate PLA2 inhibitors by directly testing drugs. Surprisingly, varespladib and its orally bioavailable prodrug, methyl-varespladib/LY333013 showed high-level secretory PLA2 (sPLA2) inhibition at nanomolar and picomolar concentrations against 28 medically important snake venoms from six continents. In vivo proof-of-concept studies with varespladib had striking survival benefit against lethal doses of Micrurus fulvius and Vipera berus venom, and suppressed venom-induced sPLA2 activity in rats challenged with 100% lethal doses of M. fulvius venom. Rapid development and deployment of a broad-spectrum PLA2 inhibitor alone or in combination with other small molecule inhibitors of snake toxins (e.g., metalloproteases) could fill the critical therapeutic gap spanning pre-referral and hospital setting. Lower barriers for clinical testing of safety tested, repurposed small molecule therapeutics are a potentially economical and effective path forward to fill the pre-referral gap in the setting of snakebite.[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 methyl-varespladib/LY333013/A-002 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/methyl-varespladib/LY333013 (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]

C22H22N2O5

394.427

394.153

C, 66.99; H, 5.62; N, 7.10; O, 20.28

172733-08-3

Varespladib;172732-68-2; 172733-42-5 (Varespladib sodium)

9886917

White to off-white solid powder

3.172

100.62

1

5

9

29

604

0

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

VJYDOJXJUCJUHL-UHFFFAOYSA-N

InChI=1S/C22H22N2O5/c1-3-15-20(21(26)22(23)27)19-16(24(15)12-14-8-5-4-6-9-14)10-7-11-17(19)29-13-18(25)28-2/h4-11H,3,12-13H2,1-2H3,(H2,23,27)

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

A002; LY-333013; S-3013; A002; A-002; LY333013; Varespladib methyl; 172733-08-3; LY-333,013; Varespladib methyl [USAN]; LY333,013; Varespladib methyl ester; S3013

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 (~253.54 mM)

配方 1 中的溶解度: ≥ 5 mg/mL (12.68 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 50.0 mg/mL澄清DMSO储备液加入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 (6.34 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；
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。