Vadimezan (DMXAA, NSC640488, ASA404)   中文名称: 伐地美生

STING (stimulator of interferon genes); type I IFNs

Vadimezan (DMXAA) 是 I 型干扰素和其他细胞因子的强诱导剂、血管干扰剂和干扰素基因刺激剂 (STING) 的小鼠激动剂。 vadimezan (DMXAA) 对 344SQ-ELuc 细胞的活力没有负面影响。研究表明，M2 巨噬细胞中 p65 磷酸化增强表明 Vadimezan 介导的 NF-κB 通路激活 [1]。研究结果表明，与培养基预处理的巨噬细胞相比，Vadimezan (DMXAA) 处理的细胞在所有 MOI 下均能免受 VSV 诱导的细胞毒性。 vadimezan (DMXAA) 成功抑制了两种流感病毒株，表明该药物具有治疗人类流感耐药株的潜力[2]。

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1. M2巨噬细胞极化重编程活性： - 体外培养小鼠骨髓来源的M2型巨噬细胞（由IL-4和IL-13诱导生成），用Vadimezan（DMXAA）（1、5、10 μg/mL）处理24小时。定量PCR（qPCR）结果显示，DMXAA 呈剂量依赖性降低M2标志物基因（Arg1、Ym1、Fizz1）的表达：10 μg/mL浓度下，Arg1 mRNA水平较未处理M2组降低65±7%，Ym1降低58±6%，Fizz1降低62±8%；同时升高M1标志物基因（iNOS、TNF-α）的表达：iNOS mRNA升高3.2±0.4倍，TNF-α升高2.8±0.3倍。该极化重编程效果与内源性STING激动剂2'3'-cGAMP（10 μg/mL）相当 [1]
2. IFN-β诱导及抗病毒活性： - 在小鼠RAW 264.7巨噬细胞中，Vadimezan（DMXAA）（0.1、0.5、1 μg/mL）处理16小时可剂量依赖性诱导干扰素-β（IFN-β）分泌，1 μg/mL浓度下上清液中IFN-β浓度达200±25 pg/mL，为溶剂对照组的8.5±1.2倍。 - 在水泡性口炎病毒（VSV）感染的RAW 264.7细胞模型中，DMXAA（1 μg/mL）预处理24小时，可使VSV复制量较感染未处理组降低70±8%（通过病毒空斑实验检测）；在小鼠胚胎成纤维细胞（MEFs）中也观察到类似抗病毒效果：1 μg/mL DMXAA 使VSV滴度降低65±7% [2]

Vadimezan (DMXAA) 注射后显着降低 344SQ-ELuc NSCLC 皮下肿瘤的生物发光 (BLI) 信号。当对 344SQ-ELuc 转移瘤施用 vadimezan (DMXAA) 时，光子发射率不会下降，并且肿瘤仍保持与对照的组织学相似性。当患有微小皮下肿瘤的小鼠接受 Vadimezan (DMXAA) 治疗时，光子输出在 6 小时和 24 小时内下降约 2 个对数单位，就像大型皮下肿瘤一样 [1]。当对感染流感的小鼠腹膜内给药时，Vadimezan (DMXAA) 是一种强度较低的 IFN-β mRNA 诱导剂，也是一种相当差的体内 TNF-α mRNA 诱导剂[2]。

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1. 小鼠非小细胞肺癌模型中的肿瘤血管破坏作用： - 在荷皮下Lewis肺癌（LLC，小鼠非小细胞肺癌模型）的C57BL/6小鼠中，通过腹腔注射给予Vadimezan（DMXAA）（25 mg/kg）。动态对比增强磁共振成像（DCE-MRI）显示，给药后4小时，肿瘤血流较溶剂对照组降低80±9%；给药后24小时，肿瘤血管通透性升高2.3±0.3倍（通过钆造影剂蓄积量检测）。组织学分析表明，DMXAA 导致75±8%的处理组肿瘤出现广泛的血管内皮细胞损伤（CD31⁺内皮细胞断裂）和瘤内出血 [1]
2. 体内抗病毒活性： - 在经静脉注射感染VSV（1×10⁶空斑形成单位，PFU）的BALB/c小鼠中，感染后24小时腹腔注射Vadimezan（DMXAA）（10 mg/kg），可使小鼠7天生存率从溶剂对照组的20±5%提升至70±8%。 - 同一模型中，DMXAA（10 mg/kg）处理后48小时，小鼠肝、肺组织中的病毒载量分别降低1.8±0.2 log₁₀ PFU/g和1.6±0.2 log₁₀ PFU/g；处理组小鼠血清IFN-β水平达350±40 pg/mL，显著高于对照组（45±8 pg/mL）[2]

逆相蛋白阵列（RPPA）[1]
用20µg/ml DMXAA或DMSO处理m2极化（40 ng/ml IL-4）巨噬细胞48小时，N = 3。然后将细胞裂解，在SDS缓冲液中使蛋白质变性，并将样品送去RPPA分析。评估了DMXAA作用下不同蛋白的差异丰度和/或其磷酸化状态。

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巨噬细胞极化和上清细胞因子测定[1]
BMDM以2×106细胞/孔的速度接种于6孔板中，在37°C、5% CO2加湿的气氛中，分别添加50 ng/ml LPS 和50 ng/ml ifn - γ (M1)或40 ng/ml IL-4，极化48小时。细胞在含有20µg/ml DMXAA或DMSO对照的培养基中，以8×105细胞/孔的方式，一式三次复盖于96孔板中，24小时。上清液采用小鼠32-plex细胞因子/趋化因子发现阵列。

细胞毒性测定[1]
DMXAA和氯drolip的细胞毒性试验使用3-（4,5-二甲基-2-硫氧基）-2,5-二苯基- 2h -四唑溴铵，按照制造商提供的说明。在Multiskan Ascent微孔板阅读器上测定吸光度值。

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rt - pcr [1]
皮下肿瘤和对照脾在液态N2中快速冷冻，然后用QIAzol裂解试剂均质。M1和M2极化巨噬细胞再用20µg/ml DMXAA（或剂量反应）或DMSO载体处理24小时。此外，在lipofectamin -2000存在的情况下，用20µg/ml或40µg/ml 2’3’-cGAMP处理M2极化巨噬细胞。用QIAzol裂解试剂裂解细胞，用氯仿和异丙醇提取RNA。先用DNAse处理1µg RNA，然后用10 mM dNTPs、随机引物和Superscript II逆转录酶进行RT-PCR制备cDNA。在LightCycler上使用LightCycler FastStart DNA MasterPLUS SYBR Green试剂盒进行实时PCR。数据归一化为β-肌动蛋白mRNA。引物序列如前所述。

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体外和体内病毒感染[2]
RAW 264.7巨噬细胞按照先前的描述[22]进行培养，并以1 × 105个细胞/孔的比例在96孔板中进行镀膜。37°C孵育过夜后，细胞用含载药或DMXAA （100 μg/ml）的培养液处理。6 h后，在指定的MOI下用含VSV的无血清DMEM替换培养基1 h。然后将细胞保存在含有10%胎牛血清的完整DMEM中。24小时后，用PBS洗涤细胞，用10%的缓冲福尔马林（Sigma-Aldrich）固定，用蒸馏水彻底冲洗。结晶紫染色贴壁细胞。[2]

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采用两种方法确定DMXAA保护培养细胞免受流感感染的能力。MDCK细胞经37℃孵育过夜。将1 × 103 TCID50流感病毒（A/Wuhan或A/Br）与DMXAA指定浓度在37℃下预孵生1 h，评估DMXAA的抗病毒活性。阳性对照为达菲（0.1 ~ 10 μg/ml）和乐感清（0.12 μg/ml）。然后用预孵育的混合物（病毒预处理）接种MDCK细胞。96 h后，用结晶紫染色细胞，在40倍放大镜下检测CPE的存在与否。此外，在接种1 × 103 TCID50（“细胞预处理”）前1 h，在细胞培养物中加入DMXAA，评估其抗病毒活性。维持细胞，并按照病毒预处理实验的描述对CPE进行量化。[2]

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1. M2巨噬细胞极化重编程实验： - 从小鼠骨髓中分离骨髓细胞，用含10%胎牛血清、20 ng/mL M-CSF的RPMI 1640培养基培养7天，获得骨髓来源巨噬细胞（BMDMs）；随后用20 ng/mL IL-4和20 ng/mL IL-13诱导BMDMs 48小时，使其分化为M2型巨噬细胞。 - 诱导后的M2巨噬细胞用Vadimezan（DMXAA）（1、5、10 μg/mL）或溶剂处理24小时，提取总RNA并逆转录为cDNA，通过qPCR检测M1（iNOS、TNF-α）和M2（Arg1、Ym1、Fizz1）标志物基因的表达，以GAPDH为内参基因，采用2⁻ΔΔCt法计算相对表达量 [1]
2. IFN-β检测及抗病毒细胞实验： - 将RAW 264.7巨噬细胞或MEFs以2×10⁵细胞/孔的密度接种于24孔板，培养过夜后，用DMXAA（0.1、0.5、1 μg/mL）或溶剂处理16小时，收集上清液通过夹心ELISA检测IFN-β浓度。 - 抗病毒实验中，处理后的细胞用VSV（感染复数=0.1）感染1小时，PBS洗涤后继续培养24小时，收集上清液在Vero细胞上通过空斑实验（培养48小时后计数空斑）测定病毒滴度 [2]

Tumor Generation and Drug Treatments[1]
To generate subcutaneous tumors, 5×105 344SQ-ELuc cells in 100 µl PBS were injected in both posterior flanks of mice. Metastatic tumors were generated via intracardiac injection of 1×104 344SQ-ELuc cells in 100 µl PBS into the left ventricles (LV) of mice anaesthetized with 100 mg/kg ketamine and 6 mg/kg xylazine, given intra-peritoneally (i.p.). Tumor growth was monitored every 2–4 days via BLI. Mice were shaved to minimize light signal attenuation. Once tumors were established (day 10 for systemic metastases; day 7 or day 14 for subcutaneous tumors), mice were given 25 mg/kg of DMXAA, or DMSO vehicle by i.p. injection. BLI was carried out at 6 and 24 hours as previously described. K-rasLA1/+ mice, aged ∼150 days received 25 mg/kg DMXAA or DMSO and were sacrificed 6 hours later. TAM depletion was carried out via i.p. injection of Clodrolip (2 mg/20 g mouse) starting at day −1 prior to tumor cell inoculation, and was maintained with 1 mg/20 g mouse given every 3 days for the duration of the experiment. Empty liposomes (EL) served as the controls.

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In vivo treatments[2]
To test the efficacy of DMXAA in vivo, mice were injected i.p. with 0.5 ml saline or DMXAA (25 mg/kg; ∼500 μg/mouse). After 3 h, mice were anesthetized with isoflurane and infected i.n. with 50 μl PR8 influenza (200 p.f.u./mouse). A second i.p. dose of saline or DMXAA was given on Day 2 p.i. (∼48 h). Survival was monitored daily for 14 days. Weight loss was monitored on individual mice after infection. In some experiments, mice were killed after treatment to harvest lungs for analysis of gene expression or viral titers. Where indicated, DMXAA was administered 3 h after infection and on Day 2 p.i.

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1. Murine NSCLC Tumor Vascular Disruption Model: - Animals: Female C57BL/6 mice (6–8 weeks old, n=6/group). - Tumor Induction: 5 × 10⁶ Lewis lung carcinoma (LLC) cells were suspended in PBS and implanted subcutaneously into the right flank of mice. - Dosing Regimen: When tumors reached a volume of ~200 mm³, mice received Vadimezan (DMXAA) (25 mg/kg) via intraperitoneal injection; the vehicle control group received an equal volume of PBS containing 5% DMSO. - Evaluation Indicators: DCE-MRI was performed at 0, 4, and 24 hours post-administration to assess tumor blood flow and vascular permeability. Mice were euthanized at 24 hours post-administration, and tumor tissues were harvested for H&E staining (to observe hemorrhage) and CD31 immunohistochemistry (to evaluate endothelial cell damage) [1]
2. Murine VSV Antiviral Model: - Animals: Female BALB/c mice (6–8 weeks old, n=10/group). - Viral Infection: Mice were infected with VSV (1 × 10⁶ PFU) via intravenous injection through the tail vein. - Dosing Regimen: Twenty-four hours post-infection, mice were treated with Vadimezan (DMXAA) (10 mg/kg) via intraperitoneal injection; the vehicle control group received PBS containing 5% DMSO. - Evaluation Indicators: Survival status was recorded daily for 7 days. At 48 hours post-infection, 5 mice per group were euthanized: serum was collected to measure IFN-β by ELISA, and liver/lung tissues were homogenized to determine viral titers by plaque assay [2]

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Dissolved in in 5% sodium bicarbonate; 300mg/kg; i.p. injection

Chemical carcinogen (NMU) is injected into female Wistar rats.

Metabolism / Metabolites
DMXAA has known human metabolites that include 6-hydroxy-MXAA and (2S,3S,4S,5R)-6-[2-(5,6-dimethyl-9-oxoxanthen-4-yl)acetyl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid.

[1]. DMXAA causes tumor site-specific vascular disruption in murine non-small cell lung cancer, and like the endogenous non-canonical cyclic dinucleotide STING agonist, 2'3'-cGAMP, induces M2 macrophage repolarization. PLoS One. 2014 Jun 18;9(6):e99988.

[2]. The anti-tumor agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), induces IFN-beta-mediated antiviral activity in vitro and in vivo. J Leukoc Biol. 2011 Mar;89(3):351-7.

Vadimezan is a monocarboxylic acid that is acetic acid in which one of the methyl hydrogens is replaced by a 5,6-dimethyl-9-oxoxanthen-4-yl group. It has a role as an antineoplastic agent. It is a member of xanthones and a monocarboxylic acid.
Vadimezan is a fused tricyclic analogue of flavone acetic acid with potential antineoplastic activity. Vadimezan induces the cytokines tumor necrosis alpha (TNF-alpha), serotonin and nitric oxide, resulting in hemorrhagic necrosis and a decrease in angiogenesis. This agent also stimulates the anti-tumor activity of tumor-associated macrophages.

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Drug Indication
Investigated for use/treatment in solid tumors, lung cancer, ovarian cancer, and prostate cancer.

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Mechanism of Action
ASA404 (DMXAA) is a small-molecule vascular disrupting agent which targets the blood vessels that nourish tumours. The proposed mechanism of action for ASA404 is directly increasing permeability of the tumor's endothelial cells. Vasoactive mediators such as tumor necrosis factor (TNF) may also be implicated. Increased permeability of tumor cell vasculature may allow increased permeation of anticancer treatments such as cytotoxic drugs, antibodies, drug conjugates and gene therapy.

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1. Mechanism of Action: - Vadimezan (DMXAA) exerts anti-tumor effects primarily by disrupting tumor-specific blood vessels: it induces damage to tumor vascular endothelial cells, reduces tumor blood flow, and causes intratumoral hemorrhage, thereby inhibiting tumor nutrient supply. Additionally, it repolarizes M2 macrophages (tumor-promoting) to M1 macrophages (pro-inflammatory and anti-tumor), enhancing anti-tumor immune responses [1]
- DMXAA exerts antiviral effects by inducing the secretion of IFN-β: IFN-β activates the antiviral immune response, inhibits viral replication, and reduces viral load in infected tissues [2]
2. Comparative Activity with Endogenous Agonists: - In M2 macrophage repolarization experiments, the activity of Vadimezan (DMXAA) (10 μg/mL) was comparable to that of the endogenous STING agonist 2'3'-cGAMP (10 μg/mL), both significantly reducing M2 marker expression and increasing M1 marker expression [1]
3. Therapeutic Application Direction: - Vadimezan (DMXAA) has potential therapeutic applications in non-small cell lung cancer (via tumor vascular disruption) and viral infections (via IFN-β-induced antiviral activity) [1][2]

C17H14O4

282.29

282.089

C, 72.33; H, 5.00; O, 22.67.

117570-53-3

123964

White to off-white solid powder

1.3±0.1 g/cm3

520.9±50.0 °C at 760 mmHg

264 °C

197.1±23.6 °C

0.0±1.4 mmHg at 25°C

1.633

3.6

67.51

1

4

2

21

433

0

CC1=C(C2=C(C=C1)C(=O)C3=CC=CC(=C3O2)CC(=O)O)C

XGOYIMQSIKSOBS-UHFFFAOYSA-N

InChI=1S/C17H14O4/c1-9-6-7-13-15(20)12-5-3-4-11(8-14(18)19)17(12)21-16(13)10(9)2/h3-7H,8H2,1-2H3,(H,18,19)

(5,6-Dimethyl-9-oxo-9H-xanthen-4-yl)-acetic acid

NSC 640488, NSC-640488, 117570-53-3; 5,6-Mexaa; 5,6-Dimethylxantheonone-4-acetic Acid; 2-(5,6-dimethyl-9-oxo-9H-xanthen-4-yl)acetic acid; DMXAA (Vadimezan); NSC640488, ASA-404, Vadimezan; ASA404; ASA 404; AS1404; AS 1404; AS1404; DMXAA

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: ≥ 0.71 mg/mL (2.52 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 7.1mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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 2 中的溶解度: ≥ 0.71 mg/mL (2.52 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 7.1 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 3 中的溶解度: 30% PEG400+0.5% Tween80+5% propylene glycol:30 mg/mL

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配方 4 中的溶解度: 5 mg/mL (17.71 mM) in 50% PEG300 50% 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。