AM580   中文名称: 4-[(5,6,7,8-四氢-5,5,8,8-四甲基-2-萘基)甲酰氨基]苯甲酸

RARα（IC50 = 8 nM; EC50 = 0.36 nM）

在 G-CSF 存在的情况下，AM580（10-8 M）显着增加 NB4 细胞中 LAP mRNA 的表达。当与 G-CSF 结合时，AM580 和 ATRA 在 10-5 M 浓度下诱导几乎相同数量的 LAP 转录本。在 NB4 细胞中，AM580（10-8 M）增加编码 G-CSF 转录本的稳态水平受体大约增加六倍[1]。在所有集落中，AM580 (50 nM) 都会升高 caspase-3 的表达，并且在 30% 的集落中，它会导致腺泡空化 [2]。使用shRARη1和Am580处理原代Myc细胞中RARη1敲低后观察到更高水平的CRBP1表达，表明RARη对这些细胞中的RARα靶基因CRBP1具有抑制作用。 Am580 (200 nM) 增强了 MCF-10A 和 MCF-7 细胞系中 RARγ 敲除的抗增殖作用，而 MDA-MB-231 细胞不受影响 [3]。

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我们用RARγ激动剂BMS961、RARγ/β激动剂CD347和RARγ拮抗剂SR11253单独或与ATRA或AM580联合治疗三种人类细胞系和表达Myc的小鼠乳腺细胞。与使用RARγ敲除获得的结果类似（图3），用RARγ拮抗剂SR11253和RARα激动剂Am580共同处理这些细胞系，包括小鼠Myc细胞，导致强烈的生长抑制（图4）。正如预期的那样，Am580抑制了MCF-10A-和MCF-7细胞的生长（图4，上图），而RARγ激动剂BMS961增加了它们的增殖。拮抗剂SR11253抑制MDA-MB-231细胞的增殖（图4，左下图），与AM580联合使用不会进一步抑制增殖，因为它们的RARα表达较低。Myc小鼠细胞对治疗的一般反应模式与人类细胞相似，除了CD437/AHN（RARγ/β激动剂）及其与AM580的组合最有效地损害了细胞增殖（图4，右下图）。这些结果表明，RARγ的促增殖作用是配体依赖性的，更重要的是，它可以被RARγ特异性拮抗剂如SR11253单独或与Am580联合靶向。[3]

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全反式维甲酸（ATRA）已成功用于急性早幼粒细胞白血病（APL）的细胞分化治疗。矛盾的是，APL细胞表达PML-RAR，这是一种异常形式的维甲酸受体α型（RARα），来源于白血病特异性t（15；17）染色体易位。我们在这里表明，AM580是一种稳定的视黄苯甲酸衍生物，最初是作为RARα激动剂合成的，是APL衍生细胞系NB4和新鲜分离的APL母细胞中粒细胞成熟的强大诱导剂。用AM580单独或与粒细胞集落刺激因子（G-CSF）联合治疗APL细胞后，该化合物诱导粒细胞成熟，通过测定白细胞碱性磷酸酶、CD11b、CD33和G-CSF受体mRNA的水平来评估，其浓度比产生类似效果所需的ATRA低10至100倍。相比之下，AM580作为ATRA在调节HL-60细胞系和在疾病稳定期从慢性粒细胞白血病患者外周血中获得的新鲜分离粒细胞中这些分化标志物的表达方面无效。在NB4细胞中，另外两种合成的非选择性RAR配体能够诱导与AM580一样多的LAP，而RARβ或RARγ特异性配体则完全无效。这些结果表明，AM580在仅在存在PML-RAR的细胞中调节分化抗原的表达方面比ATRA更强大。使用瞬时转染PML-RAR和正常RARα的COS-7细胞进行的结合实验表明，AM580对这两种受体的亲和力均低于ATRA。然而，在PML-RAR存在的情况下，合成的维甲酸是一种比天然维甲酸更好的含维甲酸反应元件启动子的反式激活剂，而在RARα存在的条件下，AM580和ATRA具有相似的活性。这可能解释了AM580在含PML-RAR的白血病细胞中具有很强的细胞分化潜力[1]。

AM580（0.3 mg/kg/天）治疗对 MMTV-wnt1 小鼠的无瘤生存有更显着的影响；在肝、肺、肾、脾以及早期出现的肿瘤中都观察到了影响。在这两种转基因腺体中，Am580 治疗均显着且同等地降低了增生水平 [2]。当给予MMTV-Myc小鼠时，RARα选择性激动剂Am580可以有效抑制63%小鼠的乳腺肿瘤、肺转移瘤的生长，并延长肿瘤潜伏期[3]。

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我们假设，在与人类癌症相关的小鼠模型中，单一视黄酸受体α（RARα）的特异性激活，而不直接和同时激活RARβ和γ，将抑制乳腺肿瘤的发生。共有50只单周小鼠乳腺肿瘤病毒（MMTV）-neu和50只无胎MMTV-wnt1转基因小鼠分别用添加到饮食中的RARalpha激动剂（维生素A苯甲酸，AM580）治疗40周（neu）和35周（wnt1）。共同的抗肿瘤作用包括抑制上皮增生，显著提高无瘤生存率（P<0.05），降低肿瘤发病率和已建立肿瘤的生长。在这两种模型中，导致这些效应的机制涉及抑制增殖和存活途径，以及诱导细胞凋亡。该治疗在MMTV-wnt1模型中更有效，其中Am580在体内和三维（3D）培养中也诱导分化。在这些肿瘤中，AM580抑制了wnt通路，这是通过核β-catenin的缺失来衡量的，表明部分癌基因依赖于治疗。Am580治疗增加了RARbeta并降低了RARgamma的水平，RARgamma是一种同种型，其表达与肿瘤增殖有关。RAR-alpha的抗癌作用，以及新发现的RAR-gamma的促增殖作用，表明RAR-alpha特异性激活和RAR-gama抑制可能在乳腺癌症治疗中有效。[2]

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通过改变RAR靶基因表达来调节正常小鼠、癌小鼠和人类乳腺细胞系乳腺中RARα/β对RARγ的表达，影响细胞增殖、存活和肿瘤生长。用RARα选择性激动剂AM580治疗MMTV-Myc小鼠，可显著抑制63%小鼠的乳腺肿瘤生长（~90%，P<0.001）、肺转移（P<0.01）和延长肿瘤潜伏期。免疫细胞化学分析显示，在这些小鼠中，RARα反应基因如Cyp26A1、E-cadherin、细胞视黄醇结合蛋白1（CRBP1）和p27上调。相比之下，对Am580治疗反应不佳的小鼠乳腺肿瘤（37%）表达的RARγ水平显著升高。[3]

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AM580激活RARα对肿瘤潜伏期、生长和肺转移的影响[3]
基于我们之前的工作和上述结果，我们推断使用RARα选择性激动剂可能会绕过RARγ激活诱导的促增殖和pRb抑制作用，从而预防肿瘤发展。为了实现这一目标，我们选择了RARα激动剂AM580，据报道，AM580对RARα的结合亲和力比RARβ高10倍，对RARγ没有可检测到的亲和力。单胎、15周龄MMTV-Myc雌性小鼠，每组30只，喂食含有Am580的标准饮食或单独的标准饮食（Myc-Ctrl组），每周触诊两次肿瘤的外观。两个实验组（对照组和Am580组）在第16周首次发现肿瘤。在第50周，通过Kaplan-Meier图分析，两组之间的肿瘤发展没有显著差异。当时，80%的Am580治疗小鼠和100%的对照小鼠患有肿瘤（图6A，Myc-Ctrl与AM580治疗；NR+R，其中NR是Am580无反应者，R是Am580反应者）。初步肿瘤检测后的肿瘤生长率分析显示，Am580治疗组有两个不同的亚群，即快速生长肿瘤的亚群（图6B，Am580无反应者用正方形表示）和缓慢生长肿瘤的亚群（图6C，Am580反应者用三角形表示）。在第50周可供评估的27只接受治疗的小鼠中，17只（63%）对AM580治疗有反应，与未经治疗的对照组相比，肿瘤大小减小了90%，而10只（37%）没有显示出肿瘤大小减小（无反应者）（图6B）。Kaplan-Meier分析显示，Am580应答者与Myc-Ctrl组的比较显示，应答者的肿瘤潜伏期显著延长（P=0.0465，log-rank检验，Am580 R vs.Myc-Ctrl），35%的接受治疗的Am580应答小鼠在50周时没有发生肿瘤（图6B）。

肿瘤细胞的转移扩散是癌症死亡的最终原因，肺是发生转移的主要靶器官之一。对每只小鼠的肺转移发生率和转移性病变数量的分析表明，与66.6%发生肺转移的未治疗小鼠（Myc-Ctrl）相比，只有16.6%的AM580反应小鼠发生了转移。AM580无反应小鼠的转移发生率为71.4%，而与该组相比，Am580有反应的小鼠的转移率为36.8%。Am580也减少了每只应答小鼠的转移数量（图6C）。这些结果共同证明了Am580在MMTV-Myc肿瘤模型中减少肿瘤生长和侵袭性的能力。

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AM580反应的差异与肿瘤中RARα/RARγ的平衡有关[3]
由于MMTV-Myc小鼠是近交系种群，我们没有预料到对AM580的反应差异是宿主依赖性的。然而，为了正式排除这种可能性，将每个Am580治疗组的两个肿瘤切除、切碎，然后接种到单独的同基因FVB雌性组的乳腺脂肪垫中，这些雌性组与MMTV-Myc转基因模型具有相同的遗传背景。接种的小鼠被喂食含有Am580的饮食20天。移植的肿瘤再现了原始肿瘤的个体反应和非反应模式（附加文件2，图S2），表明对Am580的反应独立于宿主，是肿瘤固有的。

因此，我们测试了RAR同种型表达的不平衡是否定义了对AM580治疗的反应。RAR同种型蛋白水平分析表明，Am580 NR肿瘤的RARγ蛋白水平高于Am580 R肿瘤（图7A）。为了证实RARγ的高表达与对Am580缺乏反应性相关，通过蛋白质印迹和免疫组织化学检查了未治疗（对照）和治疗反应性和非反应性肿瘤的切片中RARα靶基因的蛋白水平。如图7A所示，与Am580无反应性肿瘤相比，用Am580治疗的Am580反应性小鼠的肿瘤裂解物显示出生长停滞（p27）和分化（E-cadherin）标志物水平升高。Am580治疗的无反应小鼠的肿瘤裂解物中p-Erk1/2水平也升高（图7A），这也表明增殖反应增加。两个Am580治疗组的RARα蛋白水平均较低，而AM580非反应性肿瘤的RARγ水平要高得多。肿瘤切片的免疫组织化学（图7B）证实了蛋白质印迹结果，显示Am580反应性肿瘤中E-cadherin和p27水平升高，并且还显示，与Am580无反应性和Myc-Ctrl肿瘤相比，参与ATRA分解代谢的RARα靶基因细胞质Cyp26A1在这些肿瘤中被强烈诱导。免疫组织化学（图7C）和蛋白质印迹（图8A）均表明，Am580治疗仅在反应性肿瘤中诱导了CRBP1的强烈表达。

核提取物的制备和结合实验。[1]
在常规结合试验中，在存在或不存在不同浓度的CD367、AM580或ATRA作为冷竞争对手的情况下，将核提取物（200至400μL）与1 nmol/L[3H]CD367（CIRD Galderma；比活性，52.8 Ci/mmol）一起孵育。将制剂在4°C下孵育18小时，并直接装载在Superose 6HR 10/30上。在等度条件下，使用含有0.4 mol/L KC1的PTG缓冲液在室温下以0.4 mL/min的流速进行色谱分析。收集各组分（各0.4 mL），并在加入5 mL Filtercount后通过液体闪烁计数测定各组分中的放射性。尽管大多数饱和或竞争结合分析都是用这种方法进行的，但我们也用一种更快的技术进行了实验，这种技术可以给出类似的结果，并允许同时处理大量样品。37简而言之，核提取物被装载在PD10脱盐柱上，之前用15 mL磷酸盐缓冲盐水和10 mL含有0.4 mol/L KC1的PTG缓冲液连续洗涤，并在室温下用相同的缓冲液洗脱。丢弃前2mL洗脱缓冲液，收集后2.5mL，如上所述通过液体闪烁计数测量放射性。借助计算机程序BDATA-EMF对scatchard图进行了线性最小二乘分析。使用非线性最小二乘回归分析程序ALLFIT计算抑制50%总特异性CD367结合的维甲酸浓度。

NB4和HL-60细胞内AM580和ATRA水平的测定。[1]
加入内标后，用乙腈从NB4和HL-60细胞中提取ATRA和AM580，并用高效液相色谱法进行定量，如所述进行紫外检测。每组样品同时分析40,41无药物细胞和含有已知量ATRA或AM580的细胞。标准校准曲线是通过化合物和内标之间的峰高比与其在生物样品中的浓度的线性最小二乘回归分析构建的。检测限、精密度和再现性如前所述，用于血浆分析。40曲线下面积（AUC）值是通过计算机辅助积分在将两种化合物加入培养基后不同时间测量的AM580或ATRA的细胞内水平来计算的。

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反式激活实验。[1]
Simian COS-7成纤维细胞从ATCC获得，并在含有10%FCS的Dulbecco改良Eagle培养基（DMEM）中常规传代。根据标准磷酸钙共沉淀程序进行瞬时转染实验。在使DNA共沉淀物与细胞接触16小时后，加入单独的新鲜培养基（10%木炭剥离的胎牛血清，以消除内源性维甲酸）或含有ATRA或AM580的新鲜培养液，并将细胞进一步孵育36小时。在处理结束时，收获细胞并处理以测定CAT和β-半乳糖苷酶活性。

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增殖试验[3]
将原代Myc细胞（2×104）一式三份接种在6孔培养皿中，并允许其附着过夜。然后用培养基洗涤，用RARγ拮抗剂SR11253（2-（4-羧基苯基）-2-（5,6,7,8-四氢-5,5,8,8-四甲基-2-萘基）-1,3-二硫戊环）以递增剂量（10、50和250 nM）或单独用DMSO（0.001%终浓度）处理，然后用0.05%胰蛋白酶分离，每24小时计数一次，持续4天。统计显著性通过t检验确定。按照上述Myc的相同方案，MCF-10A、MCF-7和MDA-MB-231细胞分别用1μM ATRA（RAR泛激动剂）、200 nMAM580（RARα激动剂），100 nM CD437（RARγ/β激动剂6-[3-（1-金刚烷基）-4-羟基苯基]-2-萘甲酸，AHPN）、30 nM BMS961（RARβ激动剂3-氟-4-[2-羟基-2-（5,5,8,8-四甲基-5,6,7,8-四氢-2-萘基）乙酰基]氨基]苯甲酸）和SR11253或DMSO（终浓度0.001%）单独处理。作为控制。

AM580 treatment [2]
Four months old uniparous (1 pregnancy/lactation cycle) MMTV-neu and 6 weeks old nulliparous MMTV-wnt1 female mice (50 mice/group) were treated with the RARα agonist AM580 (0.3 mg/kg body weight per mouse per day) in the diet, by adding 1.5mg AM580 per kg of Purina 5053 diet. Mice that developed tumors within the first month of treatment were removed from the study. Mice were palpated twice a week and tumor appearance was recorded. Once palpable, the size of the tumors was measured weekly. Tumor-free survival was calculated from Kaplan-Meier curves and statistical significance was determined by the Log-rank test for the survival studies and t-test for the tumor growth studies. Metastasis was evaluated by removing the lungs of all the anesthetized mice, selecting randomly 20 mice per group and inspecting the lung surface for lesions using a stereoscope.

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In vivo studies [3]
Three-month-old uniparous MMTV-Myc female mice (30 mice/group) were fed with 0.3 mg/kg/day of the RARa agonist AM580 (4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carboxamido]benzoic acid). AM580 was mixed into their regular diet. Food consumption was measured to calculate the amount of AM580 to be added to the diet to achieve the daily dose as described previously. Regular diet was used as the control. Because the objective was to study the effect of AM580 on tumor initiation and development, mice that developed tumors within the first month were removed from the study on the assumption that their tumors had developed before treatment began. Mice were palpated twice weekly and the onset of tumor development was recorded. Once palpable, the tumor sizes were measured weekly in two dimensions and volumes calculated using the equation Vol = Dxd2/2 (where D = major diameter and d = minor diameter). Tumor-free survival was calculated from Kaplan-Meier curves, and statistical significance was determined using the log-rank test for survival and the t-test for tumor growth. Metastasis dissemination was evaluated by dissecting the lungs from euthanized mice and inspecting the Bouin-fixed lung surface for lesions using a stereoscope. For xenograft experiments, 8-week-old syngenic FVB mice were used. Cells or tumor fragments were inoculated into the mammary fat pad of the inguinal mammary glands (gland numbers 4 and 8) under soft anesthesia and analgesia in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines.

[1]. AM580, a stable benzoic derivative of retinoic acid, has powerful and selective cyto-differentiating effects on acute promyelocytic leukemia cells. Blood. 1996 Feb 15;87(4):1520-31.

[2]. Mechanism of inhibition of MMTV-neu and MMTV-wnt1 induced mammary oncogenesis by RARalpha agonist AM580. Oncogene. 2010 Jun 24;29(25):3665-76.

[3]. Reversal by RARα agonist Am580 of c-Myc-induced imbalance in RARα/RARγ expression during MMTV-Myc tumorigenesis. Breast Cancer Res. 2012 Aug 24;14(4):R121.

4-{[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)carbonyl]amino}benzoic acid is an amidobenzoic acid obtained by formal condensation of the carboxy group of (5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)benzoic acid with the anilino group of 4-aminobenzoic acid. A selective RARalpha agonist. It has a role as a retinoic acid receptor alpha/beta agonist and an antineoplastic agent. It is an amidobenzoic acid and a member of tetralins.

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Regardless of the mechanism of action, AM580 is much more interesting than CD367 and TTNPB from a perspective therapeutical point of view. In vivo, the retinoic acid-mimetic activity of the two latter compounds is very strong; however, both CD367 and TTNPB show significant systemic toxicity1,, probably as a consequence of their promiscous ability to interact with RARα, β, and γ. The toxicologic profile of AM580 in animals and humans is not yet completely known, although it looks favorable. This probably reflects the ability of the compound to significantly interact only with RARα in normal animals. The results obtained with AM580 show that it is possible to develop retinoic acid derivatives that preferentially interact with PML-RAR and are more effective than ATRA in causing granulocytic differentiation of APL cells. Such compounds may show lower toxicity and a higher therapeutic index than ATRA in the treatment of this type of leukemia. [1]

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Taken together, our data document that two mammary carcinoma models, MMTV-neu and MMTV-wnt1 are responsive to AM580 treatment and that this response is only partially oncogene-dependent. The fact that AM580, a RARα agonist insensitive to P450 cytochrome degradation is effective in 2 cancer models relevant to the human disease and in a human breast cell line, has potentially important clinical implications. Based on these observations and the novel role of RARγ, we suggest that better understanding of the specific roles of individual RARs in cancer cell differentiation, proliferation and apoptosis will help develop rational chemopreventive and, possibly, chemotherapeutic retinoid-based approaches to breast cancer. Combinations of selective RARα agonists with RARγ antagonists may prove to be one such successful approach. [2]

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Introduction: Retinoic acid signaling plays key roles in embryonic development and in maintaining the differentiated status of adult tissues. Recently, the nuclear retinoic acid receptor (RAR) isotypes α, β and γ were found to play specific functions in the expansion and differentiation of the stem compartments of various tissues. For instance, RARγ appears to be involved in stem cell compartment expansion, while RARα and RARβ are implicated in the subsequent cell differentiation. We found that over-expressing c-Myc in normal mouse mammary epithelium and in a c-Myc-driven transgenic model of mammary cancer, disrupts the balance between RARγ and RARα/β in favor of RARγ.
Methods: The effects of c-Myc on RAR isotype expression were evaluated in normal mouse mammary epithelium, mammary tumor cells obtained from the MMTV-Myc transgenic mouse model as well as human normal immortalized breast epithelial and breast cancer cell lines. The in vivo effect of the RARα-selective agonist 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carboxamido]benzoic acid (AM580) was examined in the MMTV-Myc mouse model of mammary tumorigenesis.
Results: Modulation of the RARα/β to RARγ expression in mammary glands of normal mice, oncomice, and human mammary cell lines through the alteration of RAR-target gene expression affected cell proliferation, survival and tumor growth. Treatment of MMTV-Myc mice with the RARα-selective agonist AM580 led to significant inhibition of mammary tumor growth (~90%, P<0.001), lung metastasis (P<0.01) and extended tumor latency in 63% of mice. Immunocytochemical analysis showed that in these mice, RARα responsive genes such as Cyp26A1, E-cadherin, cellular retinol-binding protein 1 (CRBP1) and p27, were up-regulated. In contrast, the mammary gland tumors of mice that responded poorly to AM580 treatment (37%) expressed significantly higher levels of RARγ. In vitro experiments indicated that the rise in RARγ was functionally linked to promotion of tumor growth and inhibition of differentiation. Thus, activation of the RARα pathway is linked to tumor growth inhibition, differentiation and cell death.[3]

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AM580: 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carboxamido]benzoic acid; ATCC: American Type Culture Collection; ATRA: All-trans retinoic acid; BSA: bovine serum albumin; CRBP1: cellular retinol-binding protein 1; DAB: 3,3'-diaminobenzidine; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; EGF: epithelial growth factor; EMT: epithelial to mesenchymal transition; ER: estrogen receptor; FBS: fetal bovine serum; FVB: Friend Virus B wild type mice; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H & E: haematoxylin and eosin; IACUC: Institutional Animal Care and Use Committee; IgG: immunoglobulin G; MEC: mammary epithelial cells; MMTV: mouse mammary tumor virus; NR: nonresponders; PBS: phosphate buffered saline; PCR: polymerase chain reaction; R: responders; RA: retinoic acid; RAR: retinoic acid receptor; RARE: retinoic acid response element; RIPA: radioimmunoprecipitation assay; RXR: retinoid × receptor; SDS: sodium dodecyl sulfate. [3]

C22H25NO3

351.438806295395

351.183

C, 75.19; H, 7.17; N, 3.99; O, 13.66

102121-60-8

AM580;102121-60-8

2126

White to off-white solid powder

1.2±0.1 g/cm3

461.0±45.0 °C at 760 mmHg

232.6±28.7 °C

0.0±1.2 mmHg at 25°C

1.593

6.48

66.4

2

3

3

26

546

0

O=C(C1C=CC2=C(C=1)C(C)(C)CCC2(C)C)NC1C=CC(C(=O)O)=CC=1

SZWKGOZKRMMLAJ-UHFFFAOYSA-N

InChI=1S/C22H25NO3/c1-21(2)11-12-22(3,4)18-13-15(7-10-17(18)21)19(24)23-16-8-5-14(6-9-16)20(25)26/h5-10,13H,11-12H2,1-4H3,(H,23,24)(H,25,26)

4-[(5,5,8,8-tetramethyl-6,7-dihydronaphthalene-2-carbonyl)amino]benzoic acid

AM-580; AM 580; AM580; CD336; NSC608001; Ro406055; 102121-60-8; 4-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-carboxamido)benzoic acid; 4-{[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)carbonyl]amino}benzoic acid; Ro-406055; CD-336; CD 336; NSC 608001 Ro 40-6055; CD-336; NSC-608001 Ro-40-6055;

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 (7.11 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 (7.11 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。