Beta-Lapachone (ARQ-501)   中文名称: Beta-兰帕科恩

Topoisomerase I

体外活性：Beta-Lapachone 以剂量依赖性方式抑制 DNA 拓扑异构酶 I 诱导的 DNA 松弛。与 DMSO 对照相比，β-拉帕酮（100 nM 或更高）处理可抑制 >95% 的 Topo I DNA 解旋活性。 β-拉帕酮 (1-5 μM) 可阻断细胞周期的 G0/G1 期，并通过将 Topo I 锁定在 DNA 上并阻断 HL-60 和三种人类前列腺癌（DU-145、PC- 3和LNCaP）细胞。 Beta-Lapachone 通过不同的 MAPK 信号通路促进小鼠 3T3 成纤维细胞和人内皮 EAhy926 细胞的迁移，从而加速体外擦伤伤口的愈合。此外，β-拉帕酮通过非竞争性抑制抑制纯化的重组 IDO1 活性，IC50 为 0.44 μM，β-拉帕酮还表现出优异的细胞内 IDO1 抑制活性保留，IC50 为 1.0 μM，部分依赖于 NQO1 的生物转化。 Beta-lapachone 通过 NQO1 依赖性活性氧 (ROS) 形成和 PARP1 过度激活诱导 NQO1+ 癌细胞程序性坏死。激酶测定：拓扑异构酶I催化活性测定：通过DNA解旋测定来分析酶活性。来自 TopoGEN 的 DNA 拓扑异构酶 I（1 单位，定义为在 37 °C 下 30 分钟内将 0.5 μg 超螺旋 DNA 转化为松弛状态的酶量）与 0.5 μg 6x174 RF DNA 一起孵育，在存在或不存在 Beta-Lapachone，在 20 μL 松弛缓冲液（50 mM Tris (pH 7.5)、50 mM KCI、10 mM MgCl2、0.5 mM 二硫苏糖醇、0.5 mM EDTA、30 μg/mL 牛血清白蛋白）中处理 30 分钟37°C。添加 1% SDS 和蛋白酶 K (50 μg/mL) 终止反应。在 37 °C 下再孵育 1 小时后，通过在 TAE 缓冲液（0.04 M 三乙酸盐、0.001 M EDTA）中的 1% 琼脂糖凝胶中电泳分离产物。电泳后用溴化乙锭对凝胶进行染色。使用 NIH 图像分析系统扫描照相底片。细胞测定：每个细胞系的 IC50 计算由 DNA 量 (IS) 和贴壁依赖性集落形成 (CF) 测定确定。对于 CF 测定，细胞以 500 个活细胞/孔接种在 6 孔板中并孵育过夜，然后用等体积的含有 β-拉帕酮的培养基进行处理，终浓度范围为 0.005 至 50 μM，以半对数增量（对照）用 0.25% DMSO（相当于所使用的最高剂量的 β-lapachonc）处理 4 小时或连续暴露 12 小时。将板（3孔/条件）用结晶紫染色，并计数>50个外观正常的细胞的集落。使用药物剂量计算各种细胞的 IC50 值，其中集落数约为对照的 50%。对于 DNA 测定，使用 CytoFluor 2350 荧光测量系统处理后 8 天收获板进行 IC50 测定。六孔采样包含在每个剂量的 DNA 荧光单位的计算中。使用 β-拉帕酮剂量与荧光单位百分比对照 DNA 的关系图来计算每个 IC50。所有实验至少重复两次，每次一式两份。

β-拉帕酮治疗（50 mg/kg）可有效抑制人卵巢癌异种移植小鼠模型中的体内肿瘤生长，并且β-拉帕酮和紫杉醇的组合可协同诱导细胞凋亡。在正常和糖尿病 (db/db) 小鼠中，β-拉帕酮治疗比仅用载体治疗导致更快的愈合过程。β-lapachone对体内伤口愈合的影响。[3]
为了确定β-拉帕酮是否对伤口愈合有治疗作用，将单独或含有29.8μg/gβ-拉帕酮的软膏涂抹在C57BL/6或db/db小鼠背部的伤口上21天，从受伤第0天到受伤后第21天每5天检查一次伤口愈合情况（图5和图6）。在受伤后第3、7、14或21天切下伤口中心的皮肤组织（约1×1 cm2），并进行苏木精和伊红染色（图6）。使用Imagescope软件测量愈合皮肤下的血管密度（图6E）。显微镜观察显示，db/db小鼠伤口愈合所需的时间明显长于C57BL/6小鼠（图5，A和C），在5至20天内，用含β-lapachone的软膏治疗的C57BL-6或db/db鼠的伤口面积（图5、B和D）明显小于用对照软膏治疗的小鼠（图五，A和C）。与用对照软膏治疗的小鼠相比，C57BL/6和db/db小鼠的β-lapachone治疗伤口面积显著减小（图5E）。与用不含β-拉帕酮的软膏治疗的伤口相比，β-拉帕酮治疗的伤口愈合恢复过程在C57BL/6或db/db小鼠中更快（图6，A-D）。第14天，在C57BL/6小鼠中，用对照软膏治疗的伤口瘢痕组织较厚，真皮出现紊乱（图6A3）；然而，在同一天，β-lapachone治疗的伤口的皮肤层完全修复（图6B3）。同样，在db/db小鼠中，瘢痕组织相对较薄，在14天时，β-lapachone治疗的伤口真皮中出现了毛囊（图6D3），但用对照软膏治疗的伤口中的毛囊仅在21天时观察到。

DNA 拓扑异构酶 I 在含有或不含药物（包括 β-Lapachone）的 20 μL 松弛缓冲液（50 mM Tris，pH 7.5）中培养。 （30 μg/mL 牛血清白蛋白、50 mM KCl、10 mM MgCl₂、0.5 mM 二硫苏糖醇、0.5 mM EDTA）37°C 30 分钟。添加蛋白酶 K (50 μg/mL) 和 1% SDS 以终止反应。在 37°C 下额外孵育 1 小时后，通过在 TAE 缓冲液（0.04 M 三乙酸盐、0.001 M EDTA）中的 1% 琼脂糖凝胶中电泳分离产物。电泳后，使用溴化乙锭对凝胶进行染色。利用 NIH 图像分析系统扫描底片。

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生化测定[4]
纯化的人重组IDO1（hrIDO1）酶在大肠杆菌中表达，并如前所述纯化。IDO1酶测定使用之前发表的磷酸钾缓冲系统进行。25 Ehrlich试剂用于分光光度法检测犬尿氨酸（Kyn），如上所述。25首先混合包括底物和抑制剂在内的试剂，最后加入enyme以在T=0时引发反应。为了确定hrIDO1制剂的酶动力学，在1 mL体积中进行了不同L-Trp浓度（0-400μM）的酶测定，并在多个时间点（0-90分钟）收集了100μL等分试样进行Kyn分析。结果证实，hrIDO1酶遵循之前发表的Michaelis-Menten动力学26，Km为110μM，Vmax为5.9μM/min（补充图S1）。随后，在hrIDO1酶反应中，在固定底物浓度（100μM L-Trp）下，使用不同浓度的抑制剂（0-50μM）测定IC50，或在不同浓度的两种抑制剂（0-800 nM）和底物（0-400μM）进行Ki测定时，评估了β-拉帕酮的抑制活性。反应在100μL体积内进行，并在酶活性在线性范围内时停止15分钟。使用Prism v.5.0进行数据分析和绘图。

MTT 测定用于量化细胞毒性。在添加不同浓度的拓扑替康或 β-拉帕酮前两天，将 IMR-32 和 JCI 细胞以 5.0 × 10⁴（拓扑替康）或 2.5 × 的浓度接种在 96 孔微量滴定板中。 10⁴ (β-lapachone) 细胞/孔/100 µL 培养基。之后，将细胞在37℃的CO₂培养箱中保存72小时。细胞增殖试剂盒 I 用于测量细胞增殖。实验中使用了四种不同的培养物。

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细胞处理和细胞活力测定。[3]
将100μl培养基中的HS68细胞（103个）、3T3细胞（103）或EAhy926细胞（104个）在37°C下在96孔培养板中在加湿的5%CO2气氛中接种24小时。由于生长速率较低，将HEKn细胞（104）、XB-2细胞（104”和HUVEC（104）接种48小时。对于3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑（MTT）试验，在细胞活力试验前24小时向培养基中加入不同浓度的β-lapachone。简而言之，向每个孔中加入10μl MTT（0.5mg/ml），将平板在37°C下孵育4小时。然后将甲赞产物溶解在100μl DMSO中，在37°℃下孵育30分钟，用微孔板读数器测量570nm处的吸光度。 为了测试MAPK抑制剂的作用，将3T3细胞或EAhy926细胞（103个细胞在100μl培养基/孔中）与0、5或10μM ERK抑制剂或p38抑制剂（SB-203580）或0、50或100 nM JNK抑制剂（SP-600125）一起孵育1小时；然后将细胞换成含有相同MAPK抑制剂的培养基，加入或不加入1μMβ-lapachone。使用MTT法测量处理后活细胞的数量。对于所有研究，至少进行了三组独立实验，每组三份。

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细胞周期分析。[3]
细胞用1μMβ-lapachone 处理3、6、9、12或24小时，用0.5%胰蛋白酶-EDTA收获，用冷80%乙醇固定。用PBS洗涤三次后，将细胞在37°C下与RNase A（1μg/ml）一起孵育1小时，然后在37°℃下与碘化丙啶（50μg/ml）孵育15分钟。使用FL-2参数通过流式细胞术检测染色细胞，并使用Cell Quest Pro软件分析数据。

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免疫荧光染色。 将细胞与1μM的β-lapachone孵育0至24小时，然后在4%的多聚甲醛中固定15分钟。在室温下用10%的正常山羊血清（NGS）封闭1小时后，将细胞在4°C下用抗PCNA单克隆抗体（1:1000）染色过夜，在室温下与罗丹明偶联的二抗和赫斯特染料孵育1小时，并使用徕卡荧光显微镜进行检查和拍照。

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蛋白质印迹分析。[3]
用1μMβ-拉帕酮处理0-24小时的细胞用裂解缓冲液（0.25 mM HEPES，pH 7.4，14.9 mM NaCl，10 mM NaF，2 mM MgCl2，0.5%NP-40，0.1 mM PMSF，20μM pepstatin A和20μM亮肽）裂解。然后将裂解物在4°C下以1000 g离心15分钟，收集上清液进行免疫印迹。使用ELISA阅读器通过Bradford测定法测量样品中的蛋白质量。通过10-12%SDS-PAGE从每个样品中分离出约25-50μg蛋白质，然后转移到电泳转移池中的Immobilon-P膜上（在200V下2小时）。所有后续步骤均在室温下进行。用含有0.05%吐温20（PBST）的PBS中的5%脱脂乳将膜封闭1小时，用1%BSA中的抗磷酸化ERK、抗ERK、反磷酸化JNK、抗JNK、反磷酸化p38、抗p38或抗肌动蛋白抗体（1:1000稀释）孵育2小时，用PBST洗涤30分钟，然后用辣根过氧化物酶偶联的二抗孵育1小时。用ECL-Western印迹试剂检测结合抗体，用富士医用X射线胶片检测化学发光。使用Scion软件定量每种蛋白质的量。

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刮伤愈合试验。[3]
细胞在24孔培养皿上生长至融合，吸出培养基，加入单独或与ERK抑制剂、p38抑制剂或JNK抑制剂一起含有或不含有1μMβ-lapachone的新培养基。用200-μl一次性塑料移液管尖端在细胞涂层表面刮取一条（约150μm宽）条纹，在37°C下使伤口愈合24小时（内皮细胞）或48小时（成纤维细胞）。通过测量伤口的宽度来评估伤口闭合的平均程度。

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Transwell迁移分析。[3]
使用改良的Millicell室（8-μm孔）评估细胞迁移。在0.2 ml培养基中以1×104个细胞/孔的速度接种到上室中的细胞单独用β-拉帕酮或用β-拉帕酮加ERK抑制剂、p38抑制剂或JNK抑制剂处理，并将0.6 ml培养基加入到下室中。在37°C下24小时后，机械去除膜上表面的细胞，固定膜下表面的迁移细胞，并用考马斯亮蓝染色。计数膜下表面上迁移的细胞总数。每个实验进行三次。

Male Balb/c mice are fed a commercial pellet diet and given unlimited access to water. Following one week of acclimation, the mice are divided into five groups at random and placed in the following groups: control, β-lapachone, cisplatin (18 mg/kg, ip), and β-lapachone + cisplatin (18 mg/kg, ip). Two weeks before receiving an injection of cisplatin, the β-lapachone groups are given a diet containing the medication (0.066). Three days following their injection of cisplatin, all mice are killed while sedated with carbon dioxide. Analysis of the serum BUN and CRE is performed on the blood samples. For histopathological and immunohistochemical (IHC) research, the kidney is promptly removed in half. The remaining half is kept cold until the western blot test.

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Wound biopsy and measurement of wound closure. [3]
Mice (C57BL/6 or db/db) were anesthetized with 2% Rompun solution (0.1 ml/20 g body wt; Bayer, Leverkusen, Germany). The back of the mouse was shaved and then sterilized using an alcohol swab. A sterile biopsy punch (6-mm diameter) was used to punch through the full thickness of the back skin below the shoulder blades. A wound placed in this area cannot be reached by the mouse and therefore prevents self-licking. Ointment [100 mg pure white petrolatum jelly (Vaseline)] alone (control ointment) or containing 29.8 μg/g β-lapachone was applied to the wound and changed every 2 days. Wounds from individual mice were digitally photographed every 5 days, beginning on the day of wounding. For all measurements, the wound area was quantified using Scion software.

[1]. J Biol Chem . 1993 Oct 25;268(30):22463-8.

[2]. Cancer Res . 1995 Sep 1;55(17):3706-11.

[3]. Am J Physiol Cell Physiol . 2008 Oct;295(4):C931-43.

[4]. Int J Tryptophan Res . 2013 Aug 19:6:35-45.

[5]. Cancer Res . 2012 Jun 15;72(12):3038-47.

[6]. Proc Natl Acad Sci U S A . 1999 Nov 9;96(23):13369-74.

Beta-lapachone is a benzochromenone that is 3,4-dihydro-2H-benzo[h]chromene-5,6-dione substituted by geminal methyl groups at position 2. Isolated from Tabebuia avellanedae, it exhibits antineoplastic and anti-inflammatory activities. It has a role as an antineoplastic agent, an anti-inflammatory agent and a plant metabolite. It is a benzochromenone and a member of orthoquinones.
Lapachone has been used in trials studying the treatment of Cancer, Carcinoma, Advanced Solid Tumors, Head and Neck Neoplasms, and Carcinoma, Squamous Cell.
beta-Lapachone has been reported in Catalpa longissima, Handroanthus guayacan, and other organisms with data available.
Lapachone is a poorly soluble, ortho-naphthoquinone with potential antineoplastic and radiosensitizing activity. Beta-lapachone (b-lap) is bioactivated by NAD(P)H:quinone oxidoreductase-1 (NQO1), creating a futile oxidoreduction that generates high levels of superoxide. In turn, the highly reactive oxygen species (ROS) interact with DNA, thereby causing single-strand DNA breaks and calcium release from endoplasmic reticulum (ER) stores. Eventually, the extensive DNA damage causes hyperactivation of poly(ADP-ribose) polymerase-1 (PARP-1), an enzyme facilitating DNA repair, accompanied by rapid depletion of NAD+/ATP nucleotide levels. As a result, a caspase-independent and ER-stress induced mu-calpain-mediated cell death occurs in NQO1-overexpressing tumor cells. NQO1, a flavoprotein and two-electron oxidoreductase, is overexpressed in a variety of tumors.

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beta-Lapachone is a plant product that has been found to have many pharmacological effects. To date, very little is known about its biochemical target. In this study, we found that beta-lapachone inhibits the catalytic activity of topoisomerase I from calf thymus and human cells. But, unlike camptothecin, beta-lapachone does not stabilize the cleavable complex, indicating a different mechanism of action. beta-Lapachone inhibits topoisomerase I-mediated DNA cleavage induced by camptothecin. Incubation of topoisomerase I with beta-lapachone before adding DNA substrate dramatically increases this inhibition. Incubation of topoisomerase I with DNA prior to beta-lapachone makes the enzyme refractory, and treatment of DNA with beta-lapachone before topoisomerase has no effect. These results suggest a direct interaction of beta-lapachone with topoisomerase I rather than DNA substrate. beta-Lapachone does not inhibit binding of enzyme to DNA substrate. In cells, beta-lapachone itself does not induce a SDS-K(+)-precipitable complex, but it inhibits complex formation with camptothecin. We propose that the direct interaction of beta-lapachone with topoisomerase I does not affect the assembly of the enzyme-DNA complex but does inhibit the formation of cleavable complex. [1]

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beta-Lapachone and certain of its derivatives directly bind and inhibit topoisomerase I (Topo I) DNA unwinding activity and form DNA-Topo I complexes, which are not resolvable by SDS-K+ assays. We show that beta-lapachone can induce apoptosis in certain cells, such as in human promyelocytic leukemia (HL-60) and human prostate cancer (DU-145, PC-3, and LNCaP) cells, as also described by Li et al. (Cancer Res., 55: 0000-0000, 1995). Characteristic 180-200-bp oligonucleosome DNA laddering and fragmented DNA-containing apoptotic cells via flow cytometry and morphological examinations were observed in 4 h in HL-60 cells after a 4-h, > or = 0.5 microM beta-lapachone exposure. HL-60 cells treated with camptothecin or topotecan resulted in greater apoptotic DNA laddering and apoptotic cell populations than comparable equitoxic concentrations of beta-lapachone, although beta-lapachone was a more effective Topo I inhibitor. beta-Lapachone treatment (4 h, 1-5 microM) resulted in a block at G0/G1, with decreases in S and G2/M phases and increases in apoptotic cell populations over time in HL-60 and three separate human prostate cancer (DU-145, PC-3, and LNCaP) cells. Similar treatments with topotecan or camptothecin (4 h, 1-5 microM) resulted in blockage of cells in S and apoptosis. Thus, beta-lapachone causes a block in G0/G1 of the cell cycle and induces apoptosis in cells before, or at early times during, DNA synthesis. These events are p53 independent, since PC-3 and HL-60 cells are null cells, LNCaP are wild-type, and DU-145 contain mutant p53, yet all undergo apoptosis after beta-lapachone treatment. Interestingly, beta-lapachone treatment of p53 wild type-containing prostate cancer cells (i.e., LNCaP) did not result in the induction of nuclear levels of p53 protein, as did camptothecin-treated cells. Like other Topo I inhibitors, beta-lapachone may induce apoptosis by locking Topo I onto DNA, blocking replication fork movement, and inducing apoptosis in a p53-independent fashion. beta-Lapachone and its derivatives, as well as other Topo I inhibitors, have potential clinical utility alone against human leukemia and prostate cancers.[2]

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Impaired wound healing is a serious problem for diabetic patients. Wound healing is a complex process that requires the cooperation of many cell types, including keratinocytes, fibroblasts, endothelial cells, and macrophages. beta-Lapachone, a natural compound extracted from the bark of the lapacho tree (Tabebuia avellanedae), is well known for its antitumor, antiinflammatory, and antineoplastic effects at different concentrations and conditions, but its effects on wound healing have not been studied. The purpose of the present study was to investigate the effects of beta-lapachone on wound healing and its underlying mechanism. In the present study, we demonstrated that a low dose of beta-lapachone enhanced the proliferation in several cells, facilitated the migration of mouse 3T3 fibroblasts and human endothelial EAhy926 cells through different MAPK signaling pathways, and accelerated scrape-wound healing in vitro. Application of ointment with or without beta-lapachone to a punched wound in normal and diabetic (db/db) mice showed that the healing process was faster in beta-lapachone-treated animals than in those treated with vehicle only. In addition, beta-lapachone induced macrophages to release VEGF and EGF, which are beneficial for growth of many cells. Our results showed that beta-lapachone can increase cell proliferation, including keratinocytes, fibroblasts, and endothelial cells, and migration of fibroblasts and endothelial cells and thus accelerate wound healing. Therefore, we suggest that beta-lapachone may have potential for therapeutic use for wound healing.[3]

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β-lapachone is a naturally occurring 1,2-naphthoquinone-based compound that has been advanced into clinical trials based on its tumor-selective cytotoxic properties. Previously, we focused on the related 1,4-naphthoquinone pharmacophore as a basic core structure for developing a series of potent indoleamine 2,3-dioxygenase 1 (IDO1) enzyme inhibitors. In this study, we identified IDO1 inhibitory activity as a previously unrecognized attribute of the clinical candidate β-lapachone. Enzyme kinetics-based analysis of β-lapachone indicated an uncompetitive mode of inhibition, while computational modeling predicted binding within the IDO1 active site consistent with other naphthoquinone derivatives. Inhibition of IDO1 has previously been shown to breach the pathogenic tolerization that constrains the immune system from being able to mount an effective anti-tumor response. Thus, the finding that β-lapachone has IDO1 inhibitory activity adds a new dimension to its potential utility as an anti-cancer agent distinct from its cytotoxic properties, and suggests that a synergistic benefit can be achieved from its combined cytotoxic and immunologic effects.[4]

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Agents, such as β-lapachone, that target the redox enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), to induce programmed necrosis in solid tumors have shown great promise, but more potent tumor-selective compounds are needed. Here, we report that deoxynyboquinone kills a wide spectrum of cancer cells in an NQO1-dependent manner with greater potency than β-lapachone. Deoxynyboquinone lethality relies on NQO1-dependent futile redox cycling that consumes oxygen and generates extensive reactive oxygen species (ROS). Elevated ROS levels cause extensive DNA lesions, PARP1 hyperactivation, and severe NAD+ /ATP depletion that stimulate Ca2+ -dependent programmed necrosis, unique to this new class of NQO1 "bioactivated" drugs. Short-term exposure of NQO1+ cells to deoxynyboquinone was sufficient to trigger cell death, although genetically matched NQO1- cells were unaffected. Moreover, siRNA-mediated NQO1 or PARP1 knockdown spared NQO1+ cells from short-term lethality. Pretreatment of cells with BAPTA-AM (a cytosolic Ca2+ chelator) or catalase (enzymatic H2O2 scavenger) was sufficient to rescue deoxynyboquinone-induced lethality, as noted with β-lapachone. Investigations in vivo showed equivalent antitumor efficacy of deoxynyboquinone to β-lapachone, but at a 6-fold greater potency. PARP1 hyperactivation and dramatic ATP loss were noted in the tumor, but not in the associated normal lung tissue. Our findings offer preclinical proof-of-concept for deoxynyboquinone as a potent chemotherapeutic agent for treatment of a wide spectrum of therapeutically challenging solid tumors, such as pancreatic and lung cancers.[5]

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Ablation of tumor colonies was seen in a wide spectrum of human carcinoma cells in culture after treatment with the combination of beta-lapachone and taxol, two low molecular mass compounds. They synergistically induced death of cultured ovarian, breast, prostate, melanoma, lung, colon, and pancreatic cancer cells. This synergism is schedule dependent; namely, taxol must be added either simultaneously or after beta-lapachone. This combination therapy has unusually potent antitumor activity against human ovarian and prostate tumor prexenografted in mice. There is little host toxicity. Cells can commit to apoptosis at cell-cycle checkpoints, a mechanism that eliminates defective cells to ensure the integrity of the genome. We hypothesize that when cells are treated simultaneously with drugs activating more than one different cell-cycle checkpoint, the production of conflicting regulatory signaling molecules induces apoptosis in cancer cells. beta-Lapachone causes cell-cycle delays in late G(1) and S phase, and taxol arrests cells at G(2)/M. Cells treated with both drugs were delayed at multiple checkpoints before committing to apoptosis. Our findings suggest an avenue for developing anticancer therapy by exploiting apoptosis-prone "collisions" at cell-cycle checkpoints.[6]

C15H14O3

242.27

242.094

C, 74.36; H, 5.82; O, 19.81

4707-32-8

3885

Brown to red solid powder

1.3±0.1 g/cm3

381.4±42.0 °C at 760 mmHg

>110ºC (dec.)

169.7±27.9 °C

0.0±0.9 mmHg at 25°C

1.595

2.82

43.37

0

3

0

18

445

0

O1C2C3=C([H])C([H])=C([H])C([H])=C3C(C(C=2C([H])([H])C([H])([H])C1(C([H])([H])[H])C([H])([H])[H])=O)=O

QZPQTZZNNJUOLS-UHFFFAOYSA-N

InChI=1S/C15H14O3/c1-15(2)8-7-11-13(17)12(16)9-5-3-4-6-10(9)14(11)18-15/h3-6H,7-8H2,1-2H3

2,2-dimethyl-3,4-dihydrobenzo[h]chromene-5,6-dione

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 (10.32 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 (10.32 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 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，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 3 中的溶解度: ≥ 2.5 mg/mL (10.32 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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配方 4 中的溶解度: 2.86 mg/mL (11.81 mM) in 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; 通过加热和超声助溶。
*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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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。