Decitabine (NSC-127716)   中文名称: 5-氮杂-2-脱氧胞苷

DNMT1; DNMT3A; DNMT3B

接触地西他滨 96 小时后，地西他滨治疗显着降低了 SNU719、NCC24 和 KATOIII 的细胞增殖。地西他滨导致 EBVaGC 中 G2/M 期停滞和死亡，降低侵袭能力，并上调 EBVaGC 中 E-钙粘蛋白表达 [1]。只有高剂量 (10 μM) 地西他滨（0.1-1 μM；24-72 小时）才会产生 G2 期停滞，并伴有 G1 细胞减少 [3]。地西他滨上调 HeLa 细胞中的 DCTPP1 和 dUTPase 表达 [4]。

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本研究探讨了DNA去甲基化剂地西他滨对eb病毒相关胃癌（EBVaGC）的作用。地西他滨抑制EBVaGC细胞生长，诱导G2/M阻滞和凋亡。地西他滨处理后，E-cadherin表达上调，细胞运动明显受到抑制。p73和RUNX3启动子区域去甲基化，地西他滨上调其表达。它们增强p21的转录，通过下调c-Myc诱导G2/M阻滞和细胞凋亡。地西他滨还诱导了BZLF1在SNU719中的表达。诱导EBV裂解感染是引起宿主细胞凋亡的另一种方法。本研究首次报道了去甲基化剂在EBVaGC中抑制肿瘤细胞增殖和上调E-cadherin的有效性。 [1]

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DNA甲基转移酶抑制剂阿扎胞苷和地西他滨是表观遗传癌症治疗的典型药物。为了表征阿扎胞苷和地西他滨的去甲基化活性，我们用这两种药物治疗结肠癌和白血病细胞，并对超过14,000个基因启动子进行了基于阵列的DNA甲基化分析。此外，将药物诱导的去甲基化与缺乏DNA甲基转移酶1 （DNMT1）和DNMT3B的等基因结肠癌细胞的甲基化模式进行了比较。我们发现药物诱导的去甲基化模式具有高度特异性、非随机性和可重复性，表明在复制后特异性位点发生了靶向再甲基化。相应地，我们发现CG岛中的CG二核苷酸优先重新甲基化，表明DNA序列背景的作用。我们还在结肠癌或白血病细胞系中发现了一个从未被药物治疗去甲基化的基因子集。这些去甲基化抗性基因在胚胎干细胞中富集了Polycomb suppression Complex 2组分，并富集了去甲基化基因中不存在的转录因子结合基序。我们的研究结果为阿扎胞苷和地西他滨诱导的DNA甲基化模式提供了详细的见解，并表明药物诱导的DNA去甲基化涉及复杂的调节机制。[3]

在雌性 CD-1 小鼠中，地西他滨（1.0 mg/kg，口服）与四氢尿苷（THU）联合使用会导致严重毒性，并增加与地西他滨血浆水平相关的地西他滨毒性的易感性[5]。已建立 EL4 肿瘤的 C57BL/6 小鼠在给予地西他滨（1.0 mg/kg；腹腔注射；每天一次，持续 5 天）后显示出消退 [7]。

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癌细胞缺乏免疫原性被认为是其无法诱导肿瘤特异性T细胞反应的主要原因。在本文中，我们提出证据表明，地西他滨（DAC）是一种DNA甲基化抑制剂，目前用于治疗骨髓增生异常综合征（MDS）、急性髓系白血病（AML）和其他恶性肿瘤，能够在小鼠EL4肿瘤模型中引发抗肿瘤细胞毒性T淋巴细胞（CTL）反应。建立EL4肿瘤的C57BL/6小鼠给予DAC （1.0 mg/kg体重）治疗，每天1次，连用5 d。我们发现DAC治疗导致产生IFN-γ的T淋巴细胞浸润到肿瘤中并引起肿瘤排斥反应。CD8(+) T细胞耗竭，而CD4(+) T细胞不耗竭，肿瘤恢复生长。dac诱导的CTL反应似乎是通过诱导肿瘤细胞上CD80的表达而引起的。表观遗传学证据表明，DAC通过CD80基因启动子中CpG二核苷酸位点的去甲基化诱导CD80在EL4细胞中的表达。此外，我们还发现，短暂的低剂量DAC处理可以诱导多种人类癌细胞中的CD80基因表达。本研究首次证明了表观遗传调控可以诱导肿瘤细胞上一种主要的T细胞共刺激分子的表达，从而克服免疫耐受，诱导有效的抗肿瘤CTL反应。这些结果对设计基于dac的癌症免疫疗法具有重要意义。[7]

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Decitabine (5-aza-2脱氧胞苷;DAC)联合四氢吡啶（THU）是一种治疗镰状细胞病和β-地中海贫血的潜在口服疗法。在小鼠中进行了一项研究，以评估这种联合治疗的安全性，方法是在DAC前1小时口服DAC和THU，连续2天/周，持续9周，然后恢复28天，以支持其临床试验长达9周的持续时间。四氢吡啶，胞苷脱氨酶的竞争性抑制剂，被用于联合提高DAC的口服生物利用度。剂量为167 mg/kg THU，随后是0,0.2,0.4或1.0 mg/kg DAC；THU载药后加入1.0 mg/kg DAC；或者车辆本身。评估的终点是临床观察、体重、食物消耗、临床病理、大体/组织病理、骨髓微核和毒性动力学。在体重、食物消耗、血清化学或尿液分析参数方面没有发现与治疗相关的影响。血浆DAC水平的剂量和性别依赖性变化在1小时内达到Cmax。在测试的1mg /kg剂量下，与单独使用DAC相比，THU增加了DAC的血浆浓度（~ 10倍）。接受高剂量1 mg/kg DAC + THU的女性出现严重毒性，需要在第5周停止治疗。显微镜检查结果的严重程度和发生率呈剂量依赖性增加；结果包括骨髓细胞减少（伴随相应的血液学改变，白细胞、红细胞、血红蛋白、红细胞压积、网状细胞、中性粒细胞和淋巴细胞减少）、胸腺/淋巴细胞减少、肠上皮细胞凋亡和睾丸变性。骨髓微核分析证实骨髓细胞毒性，抑制红细胞生成和遗传毒性。在恢复期之后，观察到这些影响完全或有缓解的趋势。总之，联合治疗导致DAC毒性敏感性增加，与DAC血浆水平相关，女性比男性更敏感。[5]

掺入测定[8]
处理前24小时，将细胞一式三份接种在12孔板中，密度为每孔2×105个细胞，然后与100nM[3H]-地西他滨（或其他浓度，如有指示）一起孵育。孵育24小时（或其他时间段，如果需要）后，用磷酸盐缓冲盐水（PBS）洗涤细胞。对于掺入测量，分别使用DNeasy血液和组织试剂盒或RNeasy试剂盒纯化DNA或RNA，并通过紫外线（UV）光度计进行定量。将纯化的样品与液体闪烁混合物混合，并通过液体闪烁计数测量其放射性。将测量值标准化为DNA（或RNA）的量。如前所述，地西他滨对胞嘧啶的取代百分比计算为作为总胞嘧啶的一部分引入的地西他宾的量。对于竞争实验，细胞要么用100nM[3H]-地西他滨加上增加浓度（100nM，500nM，1μM，2μM）的[14C]-脱氧胞苷处理，要么用100nm[14C]--脱氧胞苷加上增加的[3H]--地西他宾浓度（100nm，500nM，1μM，2μM）处理。24小时后，用PBS洗涤细胞，使用DNeasy血液和组织试剂盒提取DNA，并使用液体闪烁计数进行测量。

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DNA甲基化分析[8]
使用DNeasy血液和组织试剂盒从细胞中分离基因组DNA。如前所述，通过毛细管电泳测定全局DNA甲基化水平。

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地西他滨（5-aza-2'-脱氧胞苷，aza-dCyd）是一种抗癌药物，临床上用于治疗骨髓增生异常综合征和急性髓性白血病，可以作为一种剂量依赖性的dna去甲基化或基因毒性药物。另一方面，DCTPP1 （dCTP焦磷酸酶1）和dUTPase是两种参与消除非典型核苷酸的“大扫除”核苷酸水解酶。在本研究中，我们发现，暴露于地西他滨的HeLa细胞上调了几种嘧啶代谢酶的表达，包括DCTPP1、dUTPase、dCMP脱氨酶和胸苷酸合成酶，从而表明它们参与了细胞对这种抗癌核苷的反应。我们提出了几条证据支持，除了形成aza-dCTP （5-aza-2'-脱氧胞苷-5'-三磷酸）外，地西他滨的另一种细胞毒性机制可能涉及形成aza-dUMP，一种潜在的胸苷酸合成酶抑制剂。事实上，dUTPase或DCTPP1的下调增强了地西他滨的细胞毒性作用，产生了含有尿嘧啶的三磷酸核苷的积累，以及尿嘧啶在基因组DNA中的错误掺入和双链断裂。此外，DCTPP1以与其天然底物dCTP相似的动力学效率水解地西他滨的三磷酸形式，并阻止地西他滨诱导的整体DNA去甲基化。这些数据表明，核苷酸水解酶DCTPP1和dUTPase是参与地西他滨作用模式的因素，作为改善地西他滨化疗的酶靶点具有潜在价值。[3]

细胞周期分析[1]
细胞类型： HCT116 细胞
测试浓度： 0.1、1、10 µM
孵育时间： 24、48、72 小时
实验结果： 只有高药物浓度 (10 µM) 才会导致 G2 期停滞，同时伴随着 G1 期细胞的减少阶段。

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转运试验[8]
如前所述进行运输分析。治疗前24小时，细胞三次接种于12孔板，每孔密度为2 × 105个细胞，然后用100 nM [3H]-地西他滨孵育。经过指定时间的孵育后，用PBS洗涤细胞，用0.2%十二烷基硫酸钠（SDS）裂解。分离样品与液体闪烁鸡尾酒混合，用闪烁计数法测定放射性。与此同时，用比辛胆酸（BCA）蛋白质测定法测量蛋白质浓度，使细胞总摄取与总蛋白质浓度正常化。

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细胞周期分析[8]
100 nM 地西他滨处理约1 × 106个细胞。在指定的时间点收集细胞并用冷冻无水乙醇固定。固定后，用PBS洗涤细胞，离心，在染色液（0.1% Triton X-100, 0.2 mg/ml RNase A和20 μg/ml碘化丙啶）中重悬15分钟，37℃，黑暗。流式细胞术分析使用FACS Canto II （BD Biosciences）检测了1万个细胞，并使用FlowJo软件分析数据。

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全基因组测序[8]
实验前24 h播种细胞。细胞用100 nM 地西他滨处理24 h，使用dnasy Blood and Tissue kit提取基因组DNA。将DNA剪切成约300 bp的片段。然后连接转接头，选择片段大小并纯化。在Illumina cBot上进行聚类生成。从8个样本（4个对照，4个处理）中生成的聚类在Illumina HiSeq 2000平台上使用101 bp的配对末端reads在一个通道上同时测序。使用FastQC对生成的序列进行质量控制。使用Bowtie2和hg19作为参考完成映射。采用SAMtools进行点突变分析。基因组重排的数量是通过确定不一致对齐的读对的比例来估计的。所有实验均采用独立的生物重复。映射效率和覆盖范围见补充表S2。测序数据已存入SRA数据库，登录号为SRP040672。

Animal/Disease Models: C57BL/6 mice (bearing EL4 cells)[6]
Doses: 1.0 mg/kg
Route of Administration: intraperitoneal (ip)injection; one time/day for 5 days consecutive
Experimental Results: Caused continuous tumor regression even after Decitabine treatment was stopped.

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Experimental Design [5]
Mice were assigned to four dose groups and a vehicle control group as shown in Table 1. Animals were gavaged with Decitabine (5-aza-2'-deoxycytidine; DAC) or its vehicle 1 hour ± 5 minutes after administration of THU or its vehicle at a dose volume of 10 mL/kg. The DAC doses were selected based on the range finding study in which the mice tolerated six oral doses (2x/week) of 0.1, 0.2 and 0.4 mg/kg DAC in combination with a fixed dose of 167 mg/kg THU. A fixed THU dose (500 mg/m2) and the optimal timing between THU and DAC administration (60 min) were selected based on previous studies11. Conversion of milligrams per body surface area dose in mice into milligrams per kilogram body weight dose estimation was based on Michaelis constant (km) values for mice obtained from US Food and Drug Administration published guidelines. In brief, the mouse dose in milligrams per body surface area (500 mg/m2) was divided by the km of 3 to convert the dose to milligrams per kilogram body weight (167 mg/kg). The working body weight range of mice in the guideline is 11-34 gram; the body weight range of mice used in this study was 24-38 gram.

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Toxicokinetics [5]
Sample collection tubes were prepared prior to each collection day by adding 10 μL/tube of a 10 mg/mL THU solution. Blood samples (~0.5 mL) were collected via intra-cardiac puncture from non-fasted, anesthetized toxicokinetic animals on study day 1 (Groups 2 to 5) and 58 (Groups 2 to 5 with the exception of Group 4 females) at 15, 30, 60, 90, 120 and 180 minutes after administration of Decitabine (5-aza-2'-deoxycytidine; DAC) from 3 animals/sex/group at each time point. Due to mortality in the Group 4 females, the first 5 surviving animals were necropsied on study day 38 and blood samples were collected from three females per time point at 15, 30, and 120 minutes following administration of DAC. All samples were collected within 5 minutes of the target time.

Absorption, Distribution and Excretion
Decitabine administered intravenously at 15 mg/m2 for three hours every eight hours over three days resulted in a Cmax of 73.8 ng/mL (66% coefficient of variation, CV), an AUC0-∞ of 163 ng\*h/mL (62% CV), and a cumulative AUC of 1332 ng\*h/mL (95% CI of 1010-1730). Similarly, decitabine at 20 mg/m2 for one hour once daily over five days resulted in a Cmax of 147 ng/mL (49% CV), an AUC0-∞ of 115 ng\*h/mL (43% CV), and a cumulative AUC of 570 ng\*h/mL (95% CI of 470-700).
Less than 1% of administered decitabine is excreted in the urine.
Decitabine as an apparent volume of distribution of 4.59 ± 1.42 L/kg.
Decitabine has a clearance of 125 L/hr/m² (53% CV) when administered intravenously at 15 mg/m² for three hours every eight hours over three days, and a clearance of 210 L/hr/m² (47% CV) at 20 mg/m² for one hour once daily over five days.

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Metabolism / Metabolites
Decitabine is phosphorylated inside cells by the sequential action of deoxycytidine kinase, nucleotide monophosphate kinase, and nucleotide diphosphate kinase, prior to being incorporated into newly synthesized DNA by DNA polymerase. Decitabine not incorporated into cellular DNA undergoes deamination by cytidine deaminase followed by additional degradation prior to excretion.

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Biological Half-Life
Decitabine has a half-life of 0.62 hours (49% CV) when administered intravenously at 15 mg/m² for three hours every eight hours over three days, and a half-life of 0.54 hours (43% CV) at 20 mg/m² for one hour once daily over five days.

Hepatotoxicity
In early clinical trials using high doses of decitabine, serum enzyme elevations occurred in up to 16% of patients with underlying liver disease or liver metastases, but rarely in persons without hepatic illness. In subsequent studies, serum ALT elevations were reported in 5% to 15% of treated patients, but all were self-limited and no clinically apparent liver injury was reported. Recent studies have reported elevations in serum bilirubin levels in 7% to 12% of treated patients, but the elevations resolved rapidly and were not associated with other clinical or laboratory evidence of liver injury. Monitoring of serum enzyme levels during treatment is recommended only in patients with concurrent liver disease.
The oral combination therapy of decitabine with cedazuridine appears to have a similar frequency and pattern of adverse events as iv decitabine alone. In several prospective clinical trials, single cycles of oral vs iv decitabine had similar rates of aminotransferase elevations and long term, multi-course regimens of the oral fixed dose combination therapy resulted in serum aminotransferase elevations in 20% to 37% of patients, which were above 5 times the upper limit of normal (ULN) in 2% to 3% with no cases of clinically apparent liver injury attributable to the chemotherapeutic agent.
Thus, despite widescale use as therapy of MDS, decitabine has not been convincingly linked to cases of clinically apparent liver injury. Nevertheless, the frequency of serum enzyme elevations with therapy make it difficult to say that decitabine is totally without potential for causing drug induced liver injury.
Likelihood score: E* (unproven but suspected, rare cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Most sources consider breastfeeding to be contraindicated during maternal antineoplastic drug therapy. It might be possible to breastfeed safely during intermittent decitabine therapy with an appropriate period of breastfeeding abstinence; the manufacturer recommends an abstinence period of 1 week after the last dose. Chemotherapy may adversely affect the normal microbiome and chemical makeup of breastmilk. Women who receive chemotherapy during pregnancy are more likely to have difficulty nursing their infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
A telephone follow-up study was conducted on 74 women who received cancer chemotherapy at one center during the second or third trimester of pregnancy to determine if they were successful at breastfeeding postpartum. Only 34% of the women were able to exclusively breastfeed their infants, and 66% of the women reported experiencing breastfeeding difficulties. This was in comparison to a 91% breastfeeding success rate in 22 other mothers diagnosed during pregnancy, but not treated with chemotherapy. Other statistically significant correlations included: 1) mothers with breastfeeding difficulties had an average of 5.5 cycles of chemotherapy compared with 3.8 cycles among mothers who had no difficulties; and 2) mothers with breastfeeding difficulties received their first cycle of chemotherapy on average 3.4 weeks earlier in pregnancy. Of the 9 women who received a fluorouracil-containing regimen, 8 had breastfeeding difficulties.

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Protein Binding
Decitabine exhibits negligible (< 1%) plasma protein binding.

[1]. Decitabine inhibits tumor cell proliferation and up-regulates E-cadherin expression in Epstein-Barr virus-associated gastric cancer. J Med Virol. 2017 Mar;89(3):508-517.
[2]. Enzymology of purine and pyrimidine antimetabolites used in the treatment of cancer. Chem Rev. 2009 Jul;109(7):2880-93.
[3]. Azacytidine and decitabine induce gene-specific and non-random DNA demethylation in human cancer cell lines. PLoS One. 2011 Mar 7;6(3):e17388.
[4]. The nucleotidohydrolases DCTPP1 and dUTPase are involved in the cellular response to decitabine. Biochem J. 2016 Sep 1;473(17):2635-43.
[5]. Subchronic oral toxicity study of decitabine in combination with tetrahydrouridine in CD-1 mice. Int J Toxicol. 2014 Mar-Apr;33(2):75-85.
[6]. DNA methyltransferase expression in triple-negative breast cancer predicts sensitivity to decitabine. J Clin Invest. 2018 Jun 1;128(6):2376-2388.
[7]. Low dose decitabine treatment induces CD80 expression in cancer cells and stimulates tumorspecific cytotoxic T lymphocyte responses. PLoS One. 2013 May 9;8(5):e62924.
[8]. Quantitative determination of decitabine incorporation into DNA and its effect on mutation rates in human cancer cells. Nucleic Acids Res. 2014 Oct 29; 42(19): e152.

Pharmacodynamics
Decitabine is a prodrug analogue of the natural nucleotide 2’-deoxycytidine, which, upon being phosphorylated intracellularly, is incorporated into DNA and exerts numerous effects on gene expression. The use of decitabine is associated with neutropenia and thrombocytopenia. In addition, decitabine can cause fetal harm in pregnant women; effective contraception and avoidance of pregnancy are recommended during treatment with decitabine.

C8H12N4O4

228.21

228.085

C, 42.10; H, 5.30; N, 24.55; O, 28.04

2353-33-5

451668

White to off-white solid

1.9±0.1 g/cm3

485.8±55.0 °C at 760 mmHg

~200 °C (dec.)

247.6±31.5 °C

0.0±2.8 mmHg at 25°C

1.780

-1.93

123.49

3

4

2

16

356

3

O1[C@]([H])(C([H])([H])[C@@]([H])([C@@]1([H])C([H])([H])O[H])O[H])N1C([H])=NC(N([H])[H])=NC1=O

XAUDJQYHKZQPEU-KVQBGUIXSA-N

InChI=1S/C8H12N4O4/c9-7-10-3-12(8(15)11-7)6-1-4(14)5(2-13)16-6/h3-6,13-14H,1-2H2,(H2,9,11,15)/t4-,5+,6+/m0/s1

4-amino-1-((2S,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,3,5-triazin-2(1H)-one

5-Aza-2'-deoxycytidine; deoxyazacytidine; 2353-33-5; Dacogen; 2'-Deoxy-5-azacytidine; 5-Azadeoxycytidine; AzadC; 5-aza-CdR; 5-aza-dCyd; Deoxycytidine; NSC127716; NSC 127716; NSC-127716; dezocitidine; Brand name: Dacogen. Abbreviations: 5AZA; DAC

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

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 (10.95 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 (10.95 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 (10.95 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 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 30% propylene glycol, 5% Tween 80, 65% D5W:30mg/mL

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配方 5 中的溶解度: 10 mg/mL (43.82 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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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。