T0901317 (T-1317)   中文名称: N-(2,2,2-三氟乙基)-N-[4-[2,2,2-三氟-1-羟基-1-(三氟甲基)乙基]苯基]-苯磺酰胺

LXR (EC50 = 20 nM for LXRα); FXR (EC50 = 5 μM); RORα (Ki = 132 nM); RORγ (Ki = 51nM)

T0901317（5-50 μM；72 小时）以剂量和时间依赖性方式有效减少人卵巢癌细胞系 CaOV3、SKOV3 和 A2780 的细胞生长 [5]。 T0901317（10 μM；24-72 小时）表明细胞周期停滞在 G1-S 检查点，这会降低 S 期细胞的百分比并增加 G0/G1 期细胞的百分比。随着时间的推移，处于 G0/G1 期的细胞比例会上升 [5]。 T0901317 在 10–40 μM 浓度下 24 小时可显着增加早期细胞凋亡 [5]。 48 小时后，T0901317（5–40 μM；48 小时）以剂量依赖性方式增加蛋白质 p21 和 p27 的表达 [5]。

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CDCA激活嵌合受体，预期EC50在40μM范围内；然而，令人惊讶的是，我们还注意到T0901317激活了FXR，EC50为约5μM。尽管T0901317的FXR效力远低于LXR所描述的效力（约50 nM），但其效力约为天然FXR配体CDCA的10倍。为了确定T0901317是否作为FXR的直接配体，我们利用了生化配体传感分析，其中我们评估了T0901317诱导FXRLBD构象变化的能力，足以引起辅活化蛋白SRC-1或SRC-2的募集。与基于细胞的转染试验一致，T0901317诱导SRC-1（图1B）或SRC-2（图1C）的FXR募集，其效力大于天然FXR配体CDCA。SRC-1和SRC-2的CDCA EC50值分别为37和18μM，而T0901317的值分别为7和4μM。两种配体的最大效率相似。在这些测定中还评估了结构独特的LXR配体GW3965的活性，发现其无活性（在10μM下的功效<10%），表明LXR/FXR双重活性对T0901317具有特异性（数据未显示）[2]。
为了确定T0901317是否在天然FXR靶基因的背景下表现出FXR激动剂活性，我们检查了该化合物诱导Huh7细胞中胆汁盐输出蛋白（BSEP）或短异二聚体伴侣（SHP）表达的能力。如图2A和B所示，CDCA以剂量依赖的方式诱导BSEP和SHP mRNA的表达。与我们之前的数据一致，T0901317也以剂量依赖的方式增加FXR靶基因的表达，其最大疗效与CDCA相似（图2C和D）。因此，我们的数据表明，高亲和力LXR配体T0901317也表现出FXR激动剂活性，尽管效力较低[2]。 在针对所有48种人类核受体的筛选中，苯磺酰胺肝X受体（LXR）激动剂N-（2,2,2-三氟乙基）-N-[4-[2,2,2-二氟-1-羟基-1-（三氟甲基）乙基]苯基]-苯磺酰胺（T0901317）抑制了RORα和RORγ的反式激活活性，但不抑制RORβ。T0901317被发现以高亲和力（Ki=132和51 nM）直接结合RORα和RORγ，从而调节受体与转录辅因子蛋白相互作用的能力。T0901317抑制ROR反应报告基因的RORα/γ依赖性转录激活，并在HepG2细胞中减少RORα在内源性ROR靶基因（G6Pase）上对类固醇受体辅激活因子-2的募集。使用小干扰RNA，我们证明T0901317对HepG2细胞中葡萄糖异生酶葡萄糖-6-磷酸酶的抑制是ROR依赖性的，而不是由于该化合物的LXR活性。总之，T0901317代表了一种检测RORα/γ功能的新型化学探针，也是开发ROR选择性调节剂的良好起点。更重要的是，我们的研究结果表明，小分子可用于靶向ROR，以治疗代谢和免疫疾病。[3]

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T0901317处理导致CaOV3、SKOV3和A2780细胞以时间和剂量依赖的方式显著抑制细胞增殖（P<0.001）。Western blot分析显示p21和p27被诱导，磷酸RB蛋白水平同时降低。细胞周期分析表明，G1期细胞周期明显停滞（P<0.001）。治疗后Caspase-3和BAX基因表达显著诱导。FACS分析、caspase-glo测定、BAX蛋白诱导的caspase活性显著升高（P<0.001），Western blot分析的caspase 3前体蛋白表达降低，证实了凋亡的诱导。LXRα/β敲除实验没有逆转T0901317的抗增殖和细胞毒性作用。
结论： LXR激动剂T0901317以剂量和时间依赖的方式显著抑制细胞增殖并诱导程序性细胞死亡。我们的结果表明，T0901317通过LXR非依赖性机制诱导其抗增殖和细胞毒性作用[4]。

T0901317（10 mg/kg/天；口服；12 周）可减缓动脉粥样硬化进展的速度 [5]。 T0901317（腹腔注射；50 mg/kg；每周两次，持续 7 天）可预防高脂饮食喂养的雄性 C57BL/6 小鼠的胰岛素抵抗和肥胖 [6]。

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我们研究了BMS-852927对脂质、血浆CETP和血液mRNA终点的剂量和暴露反应关系，并将其与全泛LXR激动剂T0901317进行了比较。在14天的口服给药研究中，BMS-852927在升高血浆TG和LDL-C方面的效果远低于T0901317，血浆暴露反应曲线向右偏移≥40倍（图1A）。血浆CETP质量暴露反应曲线同样右移（图1A）。相比之下，这两种化合物在诱导血液ABCG1 mRNA方面具有相似的效力（图1A），这是RCT刺激的替代指标。虽然血液ABCA1 mRNA也被这两种化合物诱导，但由于该基因是通过动物口服灌胃人工诱导的，因此没有用于这些治疗窗口分析，这在图S1A中可见的载体治疗动物的效果中很明显，可在线获得。重要的是，BMS-852927诱导的最大血液ABCG1（16倍）发生在对血浆TG或LDL-C没有影响的暴露下（是BMS-852928 WBA EC50的4.9倍）。BMS-852927治疗还导致HDL-C的暴露依赖性增加，最高达到基线以上38%（图1A）。在14天治疗的第4天，化合物对上述所有终点的影响均处于或接近稳定状态（图S1A-S1F）。作为食蟹猴毒理学研究的一部分，还评估了BMS-852927和另一种LXR激动剂BMS-779788对肝脏脂肪生成基因诱导的影响。T0901317未进行测试；然而，BMS-779788的LXRα活性比BMS-852927高约2倍（38%对20%），总体激动剂活性更高（50%对26%）（Kick等人，2015），因此是一个有信息量的比较物。BMS-852927在诱导脂肪生成基因SREBP1c、FAS和SCD1方面的活性远低于BMS-779788，导致仅诱导2至3倍，肝脏暴露量是其WBA EC50的36倍（图1B）。相比之下，BMS-779788在其EC50的12倍暴露下诱导了相同基因的5至21倍。与此一致，通过磁共振波谱（MRS）测量，BMS-852927治疗在3 mg/kg/天的剂量下连续7天对肝脏TG没有影响，而BMS-779788引起的剂量依赖性增加高达基线以上75%（图1C）。[5]

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在C57BL/6小鼠中研究了N-（2,2,2-三氟乙基）-N-[4-[2,2,2-二氟-1-羟基-1（三氟甲基）乙基]苯基]苯磺酰胺（T0901317）激活肝脏X受体对高脂饮食（HFD）诱导的肥胖和胰岛素抵抗的影响。当连续服用HFD 10周时，C57BL/6小鼠变得肥胖，平均体重为42克，胰岛素抵抗和葡萄糖不耐受。在相同饮食的动物中，每周两次腹腔注射50mg/kg的T0901317完全阻断了肥胖的发展、肥胖相关的胰岛素抵抗和葡萄糖不耐受。定量实时PCR分析显示，T0901317处理的动物具有显著更高的能量代谢相关基因mRNA水平，包括Ucp-1、Pgc1a、Pgc1b、Cpt1a、Cpt1b、Acadm、Acadl、Aox和Ehhadh。在T0901317处理的动物中也观察到Cyp7a1、Srebp-1c、Fas、Scd-1和Acc-1基因的转录激活。T0901317治疗诱导肝脏中脂质的可逆聚集。这些结果表明，肝脏X受体可能是预防肥胖和肥胖相关胰岛素抵抗的潜在靶点[6]。

放射配体受体结合分析[3]
将45或90 ng纯化的GST-RORα或GST-RORγ与不同浓度的[3H]25-羟基胆固醇在测定缓冲液（50 mM HEPES，pH 7.4，0.01%牛血清白蛋白，150 mM NaCl和5 mM MgCl2）中孵育，以确定Kd值。非特异性结合是在没有蛋白质和过量非放射性25-羟基胆固醇的情况下定义的，并且被证明是相同的。通过多筛板中的预浸Whatman GF/B过滤器（0.5%聚乙烯亚胺在磷酸盐缓冲盐水中）快速过滤终止测定，并用冰冷的测定缓冲液洗涤（3×0.1 ml）。使用Prism软件分析放射性配体结合结果。对于竞争试验，在3 nM[3H]25-羟基胆固醇的存在下，将不同浓度的T0901317与受体一起孵育。

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AlphaScreen[3]
在白色不透明384孔板上进行三次测定。用于生成化合物剂量反应曲线（0.5-7.5μM）的最终体积为20μl。所有稀释液均在测定缓冲液（100 mM NaCl、25 mM HEPES和0.1%牛血清白蛋白，pH 7.4）中制备。DMSO的最终浓度为0.25%。将12μl His-RORα-LBD（75 nM）、珠粒（每粒30μg/ml）和4μl浓度递增的化合物（0.02-8μM）的混合物加入孔中，密封板并在室温下在黑暗中孵育1小时。在此预孵育步骤后，加入4μl生物素-RIP140B（25 nM），密封板，并在室温在黑暗中进一步孵育2小时。在PerkinElmer Envision 2104上读取板，并使用Prism软件分析数据。

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半胱氨酸天冬氨酸蛋白酶-3和-7测定[4]
Vybrant FAM Caspase-3和-7检测试剂盒V35118用于根据制造商的说明定量测定主动凋亡的细胞百分比。简而言之，卵巢癌细胞以每孔2×105的密度在6孔板中接种过夜。然后用T0901317（10μM）或0.1%DMSO作为阴性对照处理细胞24小时。然后将细胞胰蛋白酶消化并收集，每个样本1×105个细胞用10μl FLICA试剂和7-AAD染色，并在37°C的5%CO2中孵育一小时。然后用1倍洗涤缓冲液洗涤细胞，以1500RPM离心5分钟。丢弃上清液，加入400μL 1×洗涤缓冲液，根据制造商的建议通过流式细胞术分析样品。

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Caspase-3/7激活试验[4]
根据制造商的说明，使用Caspase-Glo™3/7检测试剂盒进行Caspase-3/7激活检测。简而言之，卵巢癌细胞以1×104个细胞/孔的密度接种在96孔板中。24小时后，用不同浓度的T0901317（5、10、20、40和50μM）或0.1%DMSO作为阴性对照处理细胞。然后将Caspase Glo 3/7试剂（100μl）加入到每个孔中，包括单独的培养基、未处理的对照细胞或用T0901317处理6小时的细胞。然后将平板在室温下孵育1小时，用Veritas微孔板光度计测量每个样品的发光。

细胞增殖测定[5]
细胞类型： A2780、CaOV3 和 SKOV3 卵巢癌细胞系
测试浓度： 5、10、20、40 或 50 μM
孵育持续时间：72小时
实验结果：以剂量依赖性和时间依赖性方式抑制所有细胞系中的细胞增殖。

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细胞周期分析[5]
细胞类型： A2780、CaOV3 和 SKOV3 细胞
测试浓度： 10 μM
孵育时间：24、48或72小时
实验结果：减少S期细胞百分比，增加G0/G1期细胞百分比阶段。

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细胞凋亡分析[5]
细胞类型： CaOV3 细胞
测试浓度： 10 至 40 μM
孵育持续时间：24小时
实验结果：导致早期细胞凋亡显着增加。

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蛋白质印迹分析[5]
细胞类型： CaOV3 细胞
测试浓度： 5 至 40 μM
孵育持续时间：48小时
实验结果：导致p21和p27蛋白表达以剂量依赖性方式增加。

Animal/Disease Models: 8- to 10weeks old LDL receptor null mice[5]
Doses: 10 mg/kg
Route of Administration: po (oral gavage) daily; for 12 weeks
Experimental Results: Inhibited the progression of atherosclerosis.

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Cynomolgus Monkey Studies [5]
In a PD study, animals were randomized into six treatment groups (n = 3/group) and dosed once daily with vehicle, 10 mg/kg/day T0901317, and 0.1, 0.3, 1, or 3 mg/kg/day BMS-852927 for 14 days. Blood RNA and plasma lipids were determined at baseline and days 1, 4, 7, and 14 of dosing for the PD study, and on days 1 and 7 for the liver TG MRS study (see below). In a cynomolgus monkey liver mRNA study conducted as part of a larger toxicology study, animals were randomized into four treatment groups (n = 5/group) and dosed daily for 28 days with vehicle, and 0.3, 3, or 30 mg/kg/day BMS-852927. A similar study was conducted with BMS-779788 in which animals were treated for 14 days with vehicle and 1, 10, or 30 mg/kg/day BMS-779788. Liver samples from both studies were taken at 24 hr after the final dose for compound concentration and mRNA determinations. All blood and liver mRNAs were quantitated as described in detail in the RNA preparation and analysis section of the Supplemental Experimental Procedures.

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Mouse Studies [5]
To study effects of LXR agonists on neutrophils, C57BL/6 mice pre-acclimated to oral dosing (n = 8/group) were randomly assigned to vehicle; 0.03, 0.1, 1, or 3 mg/kg/day BMS-852927; and 0.3 or 3 mg/kg/day GW3965 and dosed orally for 3 days. Following anesthesia with isoflurane, blood was collected by retro-orbital bleeding and analyzed for neutrophil levels using an Advia hematology instrument employing peroxidase staining. In an atherosclerosis prevention study, 8- to 10-week-old LDL receptor null mice fed a western diet were orally gavaged daily with vehicle, BMS-852927 (0.1, 1, or 3 mg/kg/day), or 10 mg/kg/day T0901317 for 12 weeks. At the end of treatment, mice were euthanized and atherosclerosis was quantitated en face in Oil Red O-stained aortas by image analysis. Lesion area was expressed as percent of total aortic area.

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Animals and Animal Treatments [6]
Male C57BL/6 mice were housed under a 12-h light–dark cycle. The mice were divided into two groups (n = 5) and fed with HFD from Bio-Serv for 10 weeks. Beginning in week 1, one group of animals (treated group) was intraperitoneally (i.p.) given T0901317 solubilized in DMSO twice weekly at a dose of 50 mg/kg. The second group was given the same volume of DMSO as a control, and named as control group. The food intake and body weight of the mice in both groups were determined twice weekly. The BMI value was calculated as body weight (grams) divided by the square of the anal–nasal length (centimeters). The body composition was analyzed using EchoMRI-100 from Echo Medical Systems. Animals were sacrificed at the end of 10 weeks for histological and biochemical analysis.

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To test the reversibility of T0901317-induced lipid accumulation in the liver, the mice were divided into three groups (n = 5), including a control, T0901317 treated, and T0901317 withdrawal group. On day 1, the T0901317-treated group was put on high fat diet and started daily treatment with T0901317 (i.p., 50 mg/kg) for 7 days, while the control group was on high fat diet and treated with DMSO. Mice in the T0901317 withdrawal group were first given T0901317 (i.p., 50 mg/kg) daily for 7 days, stayed treatment free for additional 7 days, and then sacrificed.

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IPGTT and ITT During the last week of the experiment, the intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were performed after the last T0901317 treatment. For IPGTT, mice were fasted overnight before the injection of glucose (2 g/kg, i.p.), and the blood glucose level was measured at the predetermined time points using glucose test strips and glucose meters. For ITT, the mice fasted for 4 h before the injection of insulin (Humulin, 0.5 U/kg), and the blood glucose level was measured at predetermined time points using the same method as above. The Homeostasis Model of Assessment–Insulin Resistance (HOMA-IR) value was calculated using previously established formula: HOMA-IR = [fasting insulin (nanograms per milliliter) × fasting plasma glucose (milligrams per deciliter)/405].

[1]. Role of LXRs in Control of Lipogenesis. Genes Dev. 2000 Nov 15;14(22):2831-8.

[2]. T0901317 Is a Dual LXR/FXR Agonist. Mol Genet Metab. Sep-Oct 2004;83(1-2):184-7.

[3]. The Benzenesulfoamide T0901317 [N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide] Is a Novel Retinoic Acid Receptor-Related Orphan Receptor-Alpha/Gamma Inverse Agonist. Mol Pharmacol. 2010 Feb;77(2):228-36.

[4]. Anti-proliferative effect of LXR agonist T0901317 in ovarian carcinoma cells. J Ovarian Res. 2010 May 26;3:13.

[5]. Beneficial and Adverse Effects of an LXR Agonist on Human Lipid and Lipoprotein Metabolism and Circulating Neutrophils. Cell Metab. 2016 Aug 9;24(2):223-33.

[6]. The Liver X Receptor Agonist T0901317 Protects Mice From High Fat Diet-Induced Obesity and Insulin Resistance. AAPS J. 2013 Jan;15(1):258-66.

TO-901317 is an LXRalpha and LXRbeta agonist.
N-(2,2,2-Trifluoroethyl)-N-{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl}benzenesulfonamide has been reported in Aspergillus puniceus with data available.

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We characterize the ability of the liver X receptor (LXRalpha [NR1H3] and LXRbeta [NR1H2]) agonist, T0901317, to activate the farnesoid X receptor (FXR [NR4H4]). Although T0901317 is a much more potent activator of LXR than FXR, this ligand actually activates FXR more potently than a natural bile acid FXR ligand, chenodeoxycholic acid. Thus, the FXR activity of T0901317 must be considered when utilizing this agonist as a pharmacological tool to investigate LXR function.[2]

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Initial biological characterization of T0901317 indicated that it was specific for LXR or retained slight FXR activity relative to LXR. In the latter study, which indicated some limited FXR activity, no potency data were provided. Our results indicate that T0901317 acts as an FXR agonist with efficacy similar to a natural bile acid ligand, CDCA. The EC50 for T0901317 ranged from 4 to 7 μM within the various FXR assays, which is within a range that is significant given that 1 μM concentrations are often used as a standard in various in vitro assays assessing LXR activity. Since T0901317 has been the primary pharmacological tool for elucidating the physiological role of the LXRs, it is apparent that the concentration of this ligand must be carefully monitored so as to avoid FXR activation and conclusions that may be erroneous due to activation of both receptors.[2]

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RORs regulate a variety of physiological processes, including hepatic gluconeogenesis, lipid metabolism, circadian rhythm, and immune function. Here we demonstrate that T0901317 represents the first synthetic ligand for RORα and RORγ, and this compound is a potent inverse agonist of these two orphan nuclear receptors. This was demonstrated by competitive radioligand binding assay and cell-based assays in which T0901317 repressed RORα/γ-dependent transactivation of reporter genes driven by the ROR-responsive promoters from the G6Pase and Cyp7b1 genes. Moreover, repression of G6Pase by T0901317 was relieved after knockdown of both RORs, concluding that this compound's effects on this gluconeogenic enzyme are ROR-dependent. Finally, we show that T0901317 reduces recruitment of the p160 coactivator SRC2 by RORα at the G6Pase promoter, thus providing a mechanism for control of this important enzyme by the RORs. The pharmacology of T0901317 has been extensively studied in animal models, with the compound exhibiting acceptable pharmacokinetic properties. More importantly, the benzenesulfonamide scaffold is amenable to a modular synthetic chemistry optimization (Michael et al., 2005). Therefore, T0901317 represents a novel chemical tool to examine RORα/γ function, and our findings offer an excellent starting point for the design of potent and selective ROR ligands with potential application in the treatment of metabolic and immune disorders. [3]

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To our knowledge, this is the first study to report the anti-proliferative and pro-apoptotic activity of T0901317 on ovarian cancer cells mediated via an LXR-independent pathway. We believe that based on our results that synthetic LXR agonists warrant further studies as anti-neoplastic agents in the treatment of ovarian cancer.[4]

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The development of LXR agonists for the treatment of coronary artery disease has been challenged by undesirable properties in animal models. Here we show the effects of an LXR agonist on lipid and lipoprotein metabolism and neutrophils in human subjects. BMS-852927, a novel LXRβ-selective compound, had favorable profiles in animal models with a wide therapeutic index in cynomolgus monkeys and mice. In healthy subjects and hypercholesterolemic patients, reverse cholesterol transport pathways were induced similarly to that in animal models. However, increased plasma and hepatic TG, plasma LDL-C, apoB, apoE, and CETP and decreased circulating neutrophils were also evident. Furthermore, similar increases in LDL-C were observed in normocholesterolemic subjects and statin-treated patients. The primate model markedly underestimated human lipogenic responses and did not predict human neutrophil effects. These studies demonstrate both beneficial and adverse LXR agonist clinical responses and emphasize the importance of further translational research in this area.[5]

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C17H12F9NO3S

481.332715034485

481.039

C, 42.42; H, 2.51; F, 35.52; N, 2.91; O, 9.97; S, 6.66

293754-55-9

447912

White to off-white solid powder

1.5±0.1 g/cm3

470.5±55.0 °C at 760 mmHg

116-122° C

238.4±31.5 °C

0.0±1.2 mmHg at 25°C

1.491

4.82

65.99

1

13

5

31

684

0

S(C1C=CC=CC=1)(N(CC(F)(F)F)C1C=CC(=CC=1)C(C(F)(F)F)(C(F)(F)F)O)(=O)=O

SGIWFELWJPNFDH-UHFFFAOYSA-N

InChI=1S/C17H12F9NO3S/c18-14(19,20)10-27(31(29,30)13-4-2-1-3-5-13)12-8-6-11(7-9-12)15(28,16(21,22)23)17(24,25)26/h1-9,28H,10H2

N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide

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: ≥ 3 mg/mL (6.23 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 30.0 mg/mL 澄清的 DMSO 储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: ≥ 3 mg/mL (6.23 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 30.0 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: ≥ 3 mg/mL (6.23 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 30.0 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: 2.5 mg/mL (5.19 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 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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配方 5 中的溶解度: ≥ 2.5 mg/mL (5.19 mM) (饱和度未知) in 5% DMSO + 95% (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。
*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。