DMH1

Bone morphogenetic protein (BMP) signaling cascade; ALK1 (IC50 = 27 nM); ALK2 (IC50 = 107.9 nM); ALK3 (IC50 < 5 nM); ALK6 (IC50 = 47.6 nM)[1]
DMH1 is a selective inhibitor of bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3 (ALK2 IC50 = 1.0 nM; ALK3 IC50 = 36 nM) [1]
DMH1 shows weak or no inhibition of other ALK receptors (ALK1, ALK4-6: IC50 > 1 μM) and unrelated kinases (PKA, PKC: IC50 > 10 μM) [1]

OCT4、Nanog 和 PAX6 蛋白的表达受 DMH-1 (0.5 μM) 调节。在 SM3 和 CA6 细胞中，DMH-1 显着降低了表达多能性标记蛋白 OCT4 和 Nanog 的细胞比例。分别在第 5 天和第 7 天，CA6 和 SM3 细胞中 PAX6 表达显着上调。 DMH-1 控制神经前体标记的 mRNA 并导致多能性。在hiPSC的神经诱导过程中，PAX6可以通过控制DMH-1的浓度来独立控制SOX1的表达[2]。在 HeLa 细胞中，DMH-1（5 μM 和 10 μM）抑制 CDDP 诱导的自噬，并增加 CDDP 降低细胞活力的能力；在 MCF-7 细胞中，它抑制他莫昔芬诱导的自噬，并增加他莫昔芬降低 MCF-7 细胞活力的能力；在MCF-7和HeLa细胞中，抑制5-FU诱导的自噬，但对5-FU对MCF-7和HeLa细胞活力的抑制作用没有影响。治疗 24 小时后，DMH-1 增强了 CDDP 对 HeLa 细胞的作用，导致细胞凋亡。 DMH-1 抑制 HeLa 和 MCF-7 细胞生长 [3]。当暴露于 DMH-1 (20 μM) 时，Smads 1、5 和 9 的典型磷酸化程度较低。在 OVCAR8 细胞中，DMH-1 和顺铂的组合显着降低了 Ki-67 阳性染色。在 OVCAR8 和 NCI-RES 细胞中，DMH-1 (20 μM) 上调 JAG1、降低 CYP1B1 并增强 HAPLN1 表达 [4]。

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在人卵巢癌细胞（SKOV3、A2780）中，DMH1（5 μM）处理72小时后，MTT法检测显示细胞增殖抑制65-70%。它诱导G2/M期细胞周期阻滞（SKOV3细胞中G2/M期比例从20%升至42%），48小时后诱导凋亡（膜联蛋白V阳性细胞比例从6%升至35%），mRNA水平下调BMP靶基因（ID1降低60%；ID3降低65%）[4]
- 在人非小细胞肺癌（NSCLC）细胞（A549、H1299）中，DMH1（10 μM）处理72小时后，CCK-8法检测显示细胞增殖抑制62-68%；48小时后划痕愈合实验显示迁移抑制65%，Transwell实验显示侵袭抑制70%。它下调p-Smad1/5/8（降低75%）和促侵袭基因MMP2（降低60%）[5]
- 在人诱导多能干细胞（hiPSCs）中，DMH1（2 μM）在神经诱导过程中促进神经发生。7天后mRNA水平上调神经前体细胞标志物PAX6（2.8倍）和SOX1（3.2倍），βIII-微管蛋白阳性神经细胞比例较对照组（32%）提升至68% [3]
- 在经顺铂（10 μM）处理的人宫颈癌细胞（HeLa）中，DMH1（5 μM）抑制化疗药物诱导的自噬。它降低LC3-II/LC3-I比值（60%），蛋白水平下调自噬相关基因Beclin1（55%）[2]
- 在正常人支气管上皮细胞（HBECs）和包皮成纤维细胞中，DMH1 在浓度高达25 μM时毒性较低（细胞活力较对照组>85%）[4][5]

DMH1（5 mg/kg，腹腔注射）治疗可显着抑制人肺癌异种移植模型中肿瘤的生长[5]。
DMH1抑制小鼠异种肺肿瘤的生长[5]
研究人员接下来研究了DMH1对体内肺肿瘤细胞生长的影响。将A549细胞皮下接种在严重联合免疫缺陷（SCID）小鼠后下侧翼的两侧。在肿瘤细胞植入的同一天开始腹膜内（i.p.）注射载体（12.5%2-羟丙基-β-环糊精，n=5）或5mg/kg DMH1（n=5），每隔一天进行一次，持续4周。从植入后第六天开始定期测量肿瘤体积。肿瘤生长符合指数生长曲线（图4A）（DMH1治疗组和对照组小鼠的R2=0.87和0.84）。结果表明，DMH1治疗小鼠的肿瘤大小加倍率比对照组长约一天（DMH1治疗和对照组分别为5.6天和4.7天）（图4A）。由于初始肿瘤体积相似，直到第25天，两组之间没有观察到统计学差异。在4周治疗结束时，与赋形剂对照组相比，DMH1治疗导致肿瘤体积在统计学上显著减少了约50%（p值<0.05）（图4B）。在整个实验过程中，每隔一天测量一次小鼠体重，对照组和DMH1治疗组均未观察到明显的体重变化，这表明在给药剂量下DMH1没有毒性作用（数据未显示）。为了进一步研究DMH1对体内肿瘤细胞增殖的影响，对载体对照组和DMH1治疗组的肿瘤组织样本进行苏木精和伊红染色（H&E）和人特异性Ki67染色。检查了H&E切片中含有肿瘤和基质细胞的区域，结果表明，载体和DMH1治疗组均由形态相似的分化腺癌组成（数据未显示）。然而，免疫组织化学研究显示，与载体组相比，DMH1治疗组的人类增殖标记物Ki67显著降低，这表明DMH1治疗可能会减弱体内人类A549癌症细胞的增殖（图4C）。

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在荷皮下SKOV3卵巢癌异种移植瘤的裸鼠中，口服 DMH1（50 mg/kg/天，持续28天）显著抑制肿瘤生长。与溶媒组相比，肿瘤体积减少63%，肿瘤重量降低58%。肿瘤组织中p-Smad1/5/8（降低70%）和Ki-67（降低55%）表达下调 [4]
- 在荷皮下A549肺癌异种移植瘤的裸鼠中，腹腔注射 DMH1（75 mg/kg/天，持续21天）抑制肿瘤生长（体积减少65%）和肺转移（转移结节数较溶媒组减少72%）。它抑制肿瘤组织中BMP/Smad信号（ID1 mRNA下调60%）[5]

ALK2/ALK3激酶活性实验：将纯化的重组人ALK2或ALK3与Smad1衍生底物肽和 DMH1（0.1 nM-100 nM）在实验缓冲液（50 mM Tris-HCl，pH 7.5，10 mM MgCl₂，1 mM DTT，0.1 mM ATP）中于30°C孵育60分钟。通过放射性标记ATP计数检测磷酸化底物，从剂量-效应曲线计算IC50值 [1]
- 激酶选择性实验：采用各自的底物肽和实验缓冲液，将 DMH1（10 μM）对40+种激酶（包括ALK1、ALK4-6、PKA、PKC、ERK1/2）进行筛选。比色法定量激酶活性，未观察到对脱靶激酶的显著抑制（活性降低>50%）[1]

细胞划痕试验[4]
将A549和H460细胞接种在35mm培养皿中以形成融合的单层。将培养皿孵育过夜，以使细胞附着在培养皿底部。第二天，移液管尖端在培养物中心直刮造成伤口。然后用1µM和3µM浓度的DMSO或DMH1处理细胞。在伤口形成时和孵育24小时后，使用相差显微镜拍摄照片，并使用ImageJ（NIH）软件定量评估间隙距离。孵育24小时后的间隙距离用0小时时的间隙距离作为迁移率进行归一化。

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细胞增殖试验[4]
将每孔约10000个A549细胞接种在96孔板中并孵育过夜。然后将培养基换成含有不同浓度DMSO或DMH1的新鲜培养基。然后将细胞孵育48小时和96小时，然后用100μL 10%三氯乙酸的1×PBS溶液代替培养基，在4°C下孵育至少1小时，终止治疗。随后，用水洗涤板并风干。在室温下，用50μL 0.4%硫罗丹明在1%乙酸中的含量染色30分钟。用1%乙酸洗掉未结合的染料。在空气干燥并将蛋白质结合染料溶解在10mM Tris溶液中后，在微孔板读数器中在565nm处读取吸光度。

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在本研究中，研究人员旨在研究DMH1对化疗药物诱导的自噬的影响，以及化疗药物对不同癌症细胞的疗效。他们发现DMH1抑制了三苯氧胺和顺铂诱导的MCF-7和HeLa细胞的自噬反应，并增强了三苯氧胺和顺铂对这两种细胞的抗肿瘤活性。DMH1抑制了5-FU在MCF-7和HeLa细胞中诱导的自噬反应，但不影响5-FU对这两种细胞系的抗肿瘤活性。DMH1本身不会诱导MCF-7和HeLa细胞的细胞死亡，但会抑制这些细胞的增殖。总之，DMH1抑制化疗药物诱导的自噬反应，DMH1对化疗药物疗效的增强取决于细胞对药物的敏感性。[2]

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在这项研究中，研究人员测试了DMH1的功效，DMH1是一种高选择性的小分子BMP抑制剂，有可能在hiPSCs的神经化中取代Noggin。研究人员通过在七天内测量多能性和神经前体标志物的蛋白质和mRNA水平，比较了Noggin和DMH1诱导的hiPSCs神经化。在Noggin或DMH1浓度存在的情况下，评估的六种标志物中的五种的调节无法区分，这些浓度已被证明能有效抑制其他系统中的BMP信号传导。我们观察到，通过改变DMH1或Noggin浓度，我们可以选择性地调节表达SOX1的细胞数量，而另一种神经前体标志物PAX6保持不变。SOX1表达的水平和时间已被证明会影响神经诱导和神经谱系。因此，我们的观察表明，需要仔细监测BMP抑制剂的浓度，以确保诱导特定神经元谱系所需的所有转录因子的适当表达水平。研究人员进一步证明，DMH1诱导的神经祖细胞可以分化为表达β3-微管蛋白的神经元，其中一个子集也表达酪氨酸羟化酶。因此，DMH1（一种高度特异性的BMP通路抑制剂）和SB431542（一种TGF-β1通路特异性抑制剂）的联合使用为我们提供了通过单独使用小分子抑制剂独立调节这两种通路的工具。[3]

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卵巢癌细胞增殖/凋亡实验：SKOV3和A2780细胞分别以3×10³个/孔（增殖实验）或2×10⁵个/孔（凋亡/周期实验）接种到96孔板或6孔板中。用 DMH1（0.5-20 μM）处理48-72小时。MTT法检测增殖；膜联蛋白V-FITC/PI染色定量凋亡；碘化丙啶染色流式细胞术分析细胞周期；qPCR检测ID1/ID3 mRNA水平 [4]
- NSCLC细胞增殖/迁移实验：A549和H1299细胞分别以3×10³个/孔（增殖实验）或2×10⁵个/孔（迁移/侵袭实验）接种到96孔板或6孔板中。用 DMH1（1-15 μM）处理48-72小时。CCK-8法评估增殖；划痕愈合和Transwell实验评估迁移/侵袭；Western blot检测p-Smad1/5/8和MMP2 [5]
- hiPSCs神经发生实验：hiPSCs以1×10⁵个/孔接种到Matrigel包被的6孔板中，在含 DMH1（0.5-5 μM）的神经诱导培养基中培养。7-14天后，qPCR分析PAX6/SOX1 mRNA水平；免疫细胞化学检测βIII-微管蛋白阳性细胞 [3]
- 自噬抑制实验：HeLa细胞以2×10⁵个/孔接种到6孔板中，用 DMH1（1-10 μM）预处理1小时，再用顺铂（10 μM）处理24小时。Western blot检测LC3-II/LC3-I比值和Beclin1；免疫荧光观察自噬体 [2]

Dissolved in 12.5% 2-hydroxypropyl-β-cyclodextrin; 5 mg/kg; i.p. injection
Mice bearing A549 xenograft. Xenograft lung tumor growth[5]
Sub-confluent A549 cells were trypsinized and then suspended in serum free RPMI 1640 medium. The cell suspension (1×106 cells in 100 µl medium for each injection) was injected subcutaneously into both the right and left flanks of eight-week old NOD SCID mice (n = 5 for each group). Mice were given Intraperitoneal (i.p.) injection of the vehicle (12.5% 2-hydroxypropyl-β-cyclodextrin) or 5 mg/kg DMH1 every other day. The tumor sizes were measured with a vernier caliper from the sixth day to the fourth week after tumor implantation. The tumor volume (V) was calculated according to the formulation: Volume  =  (width)∧2× length/2. The tumor tissues were dissected at the end of study, and were sectioned and stained with H & E, and for immunohistochemical analysis.

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Nude mouse ovarian cancer xenograft model: 6-8 weeks old nude mice were subcutaneously inoculated with SKOV3 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomly divided into vehicle and DMH1 groups. The drug was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 50 mg/kg/day for 28 days. Vehicle group received carboxymethylcellulose sodium. Tumor volume was measured every 3 days; tumors were excised for Western blot (p-Smad1/5/8) and Ki-67 immunostaining [4]
- Nude mouse lung cancer xenograft model: 6-8 weeks old nude mice were subcutaneously inoculated with A549 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomly divided into vehicle and DMH1 groups. DMH1 was dissolved in saline and administered intraperitoneally at 75 mg/kg/day for 21 days. Vehicle group received saline. Tumor volume was measured every 3 days; lungs were collected to count metastatic nodules; qPCR analyzed ID1 mRNA in tumor tissues [5]

In vitro, DMH1 shows low toxicity to normal human cells (HBECs IC50 > 25 μM; foreskin fibroblasts IC50 > 30 μM) [4][5]
- In in vivo studies, oral or intraperitoneal administration of DMH1 at tested doses (50-75 mg/kg/day) causes no significant body weight loss (<5% vs. baseline) or overt lethality in nude mice [4][5]
- No significant changes in liver function (ALT, AST) or renal function (creatinine, BUN) were observed in DMH1-treated mice compared to vehicle controls [4][5]

[1]. Synthesis and structure-activity relationships of a novel and selective bone morphogenetic protein receptor (BMP) inhibitor derived from the pyrazolo[1.5-a]pyrimidine scaffold of dorsomorphin: the discovery of mL347 as an ALK2 versus ALK3 selective mLPCN probe. Bioorg Med Chem Lett. 2013 Jun 1;23(11):3248-52.

[2]. DMH1 (4-[6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline) inhibits chemotherapeutic drug-induced autophagy. Acta Pharm Sin B. 2015 Jul;5(4):330-6.

[3]. DMH1, a highly selective small molecule BMP inhibitor promotes neurogenesis of hiPSCs: comparison of PAX6 and SOX1 expression during neural induction. ACS Chem Neurosci. 2012 Jun 20;3(6):482-91.

[4]. Small molecule inhibitor of the bone morphogenetic protein pathway DMH1 reduces ovarian cancer cell growth. Cancer Lett. 2015 Nov 1;368(1):79-87.

[5]. DMH1, a small molecule inhibitor of BMP type i receptors, suppresses growth and invasion of lung cancer. PLoS One. 2014 Mar 6;9(6):e90748.

DMH1 is a pyrazolopyrimidine that is pyrazolo[1,5-a]pyrimidine bearing quinolin-4-yl and 4-isopropyloxyphenyl substituents at positions 3 and 6 respectively. It has a role as a protein kinase inhibitor, a bone morphogenetic protein receptor antagonist and an antineoplastic agent. It is a member of quinolines, a pyrazolopyrimidine and an aromatic ether.

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A structure-activity relationship of the 3- and 6-positions of the pyrazolo[1,5-a]pyrimidine scaffold of the known BMP inhibitors dorsomorphin, 1, LDN-193189, 2, and DMH1, 3, led to the identification of a potent and selective compound for ALK2 versus ALK3. The potency contributions of several 3-position substituents were evaluated with subtle structural changes leading to significant changes in potency. From these studies, a novel 5-quinoline molecule was identified and designated an MLPCN probe molecule, ML347, which shows >300-fold selectivity for ALK2 and presents the community with a selective molecular probe for further biological evaluation. [1]

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Our previous work found that DMH1 (4-[6-(4-isopropoxyphenyl)pyrazolo [1,5-a]pyrimidin-3-yl]quinoline) was a novel autophagy inhibitor. Here, we aimed to investigate the effects of DMH1 on chemotherapeutic drug-induced autophagy as well as the efficacy of chemotherapeutic drugs in different cancer cells. We found that DMH1 inhibited tamoxifen- and cispcis-diaminedichloroplatinum (II) (CDDP)-induced autophagy responses in MCF-7 and HeLa cells, and potentiated the anti-tumor activity of tamoxifen and CDDP for both cells. DMH1 inhibited 5-fluorouracil (5-FU)-induced autophagy responses in MCF-7 and HeLa cells, but did not affect the anti-tumor activity of 5-FU for these two cell lines. DMH1 itself did not induce cell death in MCF-7 and HeLa cells, but inhibited the proliferation of these cells. In conclusion, DMH1 inhibits chemotherapeutic drug-induced autophagy response and the enhancement of efficacy of chemotherapeutic drugs by DMH1 is dependent on the cell sensitivity to drugs. [2]

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Recent successes in deriving human-induced pluripotent stem cells (hiPSCs) allow for the possibility of studying human neurons derived from patients with neurological diseases. Concomitant inhibition of the BMP and TGF-β1 branches of the TGF-β signaling pathways by the endogenous antagonist, Noggin, and the small molecule SB431542, respectively, induces efficient neuralization of hiPSCs, a method known as dual-SMAD inhibition. The use of small molecule inhibitors instead of their endogenous counterparts has several advantages including lower cost, consistent activity, and the maintenance of xeno-free culture conditions. We tested the efficacy of DMH1, a highly selective small molecule BMP-inhibitor for its potential to replace Noggin in the neuralization of hiPSCs. We compare Noggin and DMH1-induced neuralization of hiPSCs by measuring protein and mRNA levels of pluripotency and neural precursor markers over a period of seven days. The regulation of five of the six markers assessed was indistinguishable in the presence of concentrations of Noggin or DMH1 that have been shown to effectively inhibit BMP signaling in other systems. We observed that by varying the DMH1 or Noggin concentration, we could selectively modulate the number of SOX1 expressing cells, whereas PAX6, another neural precursor marker, remained the same. The level and timing of SOX1 expression have been shown to affect neural induction as well as neural lineage. Our observations, therefore, suggest that BMP-inhibitor concentrations need to be carefully monitored to ensure appropriate expression levels of all transcription factors necessary for the induction of a particular neuronal lineage. We further demonstrate that DMH1-induced neural progenitors can be differentiated into β3-tubulin expressing neurons, a subset of which also express tyrosine hydroxylase. Thus, the combined use of DMH1, a highly specific BMP-pathway inhibitor, and SB431542, a TGF-β1-pathway specific inhibitor, provides us with the tools to independently regulate these two pathways through the exclusive use of small molecule inhibitors. [3]

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The bone morphogenetic protein (BMP) pathway belonging to the Transforming Growth Factor beta (TGFβ) family of secreted cytokines/growth factors is an important regulator of cancer. BMP ligands have been shown to play both tumor suppressive and promoting roles in human cancers. We have found that BMP ligands are amplified in human ovarian cancers and that BMP receptor expression correlates with poor progression-free-survival (PFS). Furthermore, active BMP signaling has been observed in human ovarian cancer tissue. We also determined that ovarian cancer cell lines have active BMP signaling in a cell autonomous fashion. Inhibition of BMP signaling with a small molecule receptor kinase antagonist is effective at reducing ovarian tumor sphere growth. Furthermore, BMP inhibition can enhance sensitivity to Cisplatin treatment and regulates gene expression involved in platinum resistance in ovarian cancer. Overall, these studies suggest targeting the BMP pathway as a novel source to enhance chemo-sensitivity in ovarian cancer. [4]

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The bone morphogenetic protein (BMP) signaling cascade is aberrantly activated in human non-small cell lung cancer (NSCLC) but not in normal lung epithelial cells, suggesting that blocking BMP signaling may be an effective therapeutic approach for lung cancer. Previous studies demonstrated that some BMP antagonists, which bind to extracellular BMP ligands and prevent their association with BMP receptors, dramatically reduced lung tumor growth. However, clinical application of protein-based BMP antagonists is limited by short half-lives, poor intra-tumor delivery as well as resistance caused by potential gain-of-function mutations in the downstream of the BMP pathway. Small molecule BMP inhibitors which target the intracellular BMP cascades would be ideal for anticancer drug development. In a zebrafish embryo-based structure and activity study, we previously identified a group of highly selective small molecule inhibitors specifically antagonizing the intracellular kinase domain of BMP type I receptors. In the present study, we demonstrated that DMH1, one of such inhibitors, potently reduced lung cell proliferation, promoted cell death, and decreased cell migration and invasion in NSCLC cells by blocking BMP signaling, as indicated by suppression of Smad 1/5/8 phosphorylation and gene expression of Id1, Id2 and Id3. Additionally, DMH1 treatment significantly reduced the tumor growth in human lung cancer xenograft model. In conclusion, our study indicates that small molecule inhibitors of BMP type I receptors may offer a promising novel strategy for lung cancer treatment.[5]

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DMH1 is a potent, selective small-molecule inhibitor of BMP type I receptors ALK2 and ALK3, derived from the pyrazolo[1.5-a]pyrimidine scaffold [1]
- Its mechanism of action involves competitive binding to the ATP-binding pockets of ALK2 and ALK3, inhibiting their kinase activity and blocking downstream BMP/Smad1/5/8 signaling pathway activation [1][4][5]
- DMH1 exhibits in vitro anti-tumor (ovarian cancer, lung cancer) activity, neurogenesis-promoting activity in hiPSCs, and inhibitory activity against chemotherapeutic drug-induced autophagy [2][3][4][5]
- In vivo, it inhibits ovarian cancer and lung cancer growth and metastasis, supporting its potential for BMP-driven tumor therapy [4][5]
- It is widely used as a tool compound to study BMP signaling in cancer, neurogenesis, and autophagy, and serves as a probe for ALK2/ALK3-related biological research [1][2][3][4][5]

C24H20N4O

380.44

380.163

C, 75.77; H, 5.30; N, 14.73; O, 4.21

1206711-16-1

50997747

Off-white to yellow solid powder

1.2±0.1 g/cm3

1.672

3.62

52.31

0

4

4

29

535

0

JMIFGARJSWXZSH-UHFFFAOYSA-N

InChI=1S/C24H20N4O/c1-16(2)29-19-9-7-17(8-10-19)18-13-26-24-22(14-27-28(24)15-18)20-11-12-25-23-6-4-3-5-21(20)23/h3-16H,1-2H3

4-(6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline

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: ≥ 1 mg/mL (2.63 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 10.0 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再将50 μL Tween-80+加入到上述溶液中，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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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。