Proteasome (IC50 = 100 nM); Calpain (IC50 = 1.2 μM)

体外活性：MG-132 在抑制 20S 蛋白酶体 ZLLL-MCA 降解活性方面比 ZLLal 高 1000 倍以上，IC50 分别为 100 nM 和 110 μM。 MG-132 还抑制钙蛋白酶，IC50 为 1.2 μM。 MG-132 在 20 nM 的最佳浓度下诱导 PC12 细胞中的神经突生长，其效力是 ZLLal 的 500 倍。 MG-132 (10 μM) 通过抑制蛋白酶体介导的 IκBα 降解，有效抑制 A549 细胞中 TNF-α 诱导的 NF-κB 激活、白介素 8 (IL-8) 基因转录和 IL-8 蛋白释放。 MG-132 治疗通过 26S 蛋白酶体抑制有效诱导 KIM-2 细胞中 p53 依赖性细胞凋亡。与 BzLLLCOCHO 或 PS-341 不同，MG-132 处理会弱抑制 26S 蛋白酶体的胰凝乳蛋白酶样 (CT-L) 和肽酰谷氨酰肽水解 (PGPH) 活性，而多发性骨髓瘤细胞（U266 和 OPM-2）则更敏感MG-132 诱导细胞凋亡的能力强于 BzLLLCOCHO。 MG-132 (1 μM) 通过激活 AP-1 家族成员 c-Fos 和 c-Jun 使 TRAIL 耐药性前列腺癌细胞变得敏感，进而抑制抗凋亡分子 c-FLIP(L)。 MG-132 显着增强肌醇六磷酸 (IP6) 降低 PC3 和 DU145 雄激素非依赖性前列腺癌 (AIPCa) 细胞系中细胞代谢活性的能力。激酶测定：20S 蛋白酶体抑制测定的反应混合物含有 0.1 M Tris-乙酸盐、pH 7.0、20S 蛋白酶体、MG-132 和 25 μM 底物，溶解在二甲亚砜中，最终体积为 1 mL。 37°C 孵育 15 分钟后，加入 0.1 mL 10% SDS 和 0.9 mL 0.1M Tris 醋酸盐（pH 9.0）终止反应。测量反应产物的荧光。为了确定针对 20S 蛋白酶体的 IC50，测定混合物中包含不同浓度的 MG-132。细胞测定：将细胞（KIM-2、HC11 和 ES）暴露于不同浓度的 MG-132 24 和 48 小时。通过离心收获上清液和单层细胞，并固定在含 70% 乙醇的 PBS 中，用于用吖啶橙染色。将等体积的细胞和吖啶橙（5 mg/mL，溶于 PBS）在显微镜载玻片上混合，并通过荧光显微镜检查。对于膜联蛋白 V 分析，通过离心收获细胞并用膜联蛋白 V 和碘化丙啶染色。对于细胞周期分析，将细胞在室温下在 PBS 中再水合 10 分钟，然后用碘化丙啶 (5 mg/mL) 染色。所有样品均使用 Coulter Epics XL 流式细胞仪进行分析。

给予MG-132可有效挽救mdx小鼠骨骼肌纤维中肌营养不良蛋白、β-肌营养不良聚糖、α-b肌营养不良聚糖和α-肌肌聚糖的表达水平和质膜定位，减少肌膜损伤，并改善肌营养不良症的组织病理学体征。 MG-132 治疗通过下调肌肉特异性泛素连接酶 atrogin-1/MAFbx 和 MuRF-1 mRNA，显着减少固定诱导的小鼠骨骼肌萎缩。

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肌营养不良蛋白是杜氏肌营养不良症（DMD）基因的蛋白质产物，在DMD患者和mdx小鼠的骨骼肌中不存在。在骨骼肌纤维的质膜上，肌营养不良蛋白与多聚体蛋白复合物结合，称为肌营养不良素糖蛋白复合物（DGC）。这种复合物的蛋白质成员通常在肌营养不良蛋白缺乏的骨骼肌纤维中缺失或大大减少，并且被认为是通过未知途径降解的。因此，我们推断，抑制蛋白酶体降解途径可能会挽救肌营养不良蛋白相关蛋白的表达和亚细胞定位。为了验证这一假设，我们用特征明确的蛋白酶体抑制剂MG-132治疗mdx小鼠。首先，我们将MG-132局部注射到腓肠肌中，并在24小时后观察结果。接下来，我们使用渗透泵进行全身治疗，使我们能够在8天内输送不同浓度的蛋白酶体抑制剂。通过免疫荧光和蛋白质印迹分析，我们表明施用蛋白酶体抑制剂MG-132有效地挽救了mdx小鼠骨骼肌纤维中肌营养不良蛋白、β-肌营养不良聚糖、α-肌营养异常聚糖和α-肌氨酸聚糖的表达水平和质膜定位。此外，我们表明，蛋白酶体抑制剂的全身治疗1）减少了肌肉膜损伤，如从治疗过的mdx小鼠中分离出的膈肌和腓肠肌的活体染色（用伊文思蓝染料）所示，2）改善了肌肉营养不良的组织病理学征状，如从处理过的mdx小鼠中取出的肌肉活检的苏木精和伊红染色所判断的。因此，目前的研究为我们理解DMD的发病机制开辟了新的重要途径。最重要的是，这些新发现可能对DMD患者的药物治疗具有临床意义。[7]

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在本研究中，我们发现蛋白酶体抑制剂MG132显著抑制了IκBα的降解，从而在体外阻止了NFκB的激活。MG132通过在体内下调肌肉特异性泛素连接酶atrogin-1/MAFbx和MuRF-1 mRNA来保护肌肉和肌纤维的横截面积。这种影响导致康复期缩短。 结论：这些发现表明，蛋白酶体抑制剂在开发预防肌肉萎缩的药物疗法方面具有潜力，从而有利于肌肉康复[8]。

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复发或晚期宫颈癌症的治疗仍然有限，需要新的治疗选择来改善患者的预后和生活质量。由于人乳头状瘤病毒（HPV）感染在宫颈癌发生中至关重要，HPV的E6和E7癌基因通过泛素-蛋白酶体系统降解肿瘤抑制蛋白，因此抑制泛素-蛋白质体系统似乎是抑制宫颈肿瘤生长的理想靶点。在此，我们重点研究了泛素蛋白酶体抑制剂MG132（碳苯氧基-Leu-Leu-leucinal）作为抗宫颈癌症细胞的抗癌剂，并将其物理掺入胶束纳米药物中，以实现对实体瘤的选择性递送并提高其体内功效。这些负载MG132的聚合物胶束（MG132/m）对HeLa和CaSki细胞的HPV阳性肿瘤，甚至对C33A细胞的HPV阴性肿瘤显示出强烈的体内肿瘤抑制作用。根据体重变化或组织病理学分析，重复注射MG132/m对小鼠没有明显毒性。此外，与游离MG132治疗的肿瘤相比，用MG132/m治疗的肿瘤显示出更高水平的肿瘤抑制蛋白、hScrib和p53以及凋亡程度。MG132/m的疗效增强归因于其在血液中的循环延长，这使得它们能够逐渐外渗并渗透到肿瘤组织内，如活体显微镜所示。这些结果支持将MG132掺入聚合物胶束中作为一种安全有效的治疗宫颈肿瘤的策略[10]。

MG-132、20S 蛋白酶体、pH 7.0、0.1 M Tris 乙酸盐和 25 μM 底物溶解在二甲亚砜中，最终体积为 1 mL，组成用于 20S 蛋白酶体抑制测定的反应混合物。 37°C 孵育 15 分钟后，加入 0.1 mL 10% SDS 和 0.9 mL 0.1M Tris 醋酸盐（pH 9.0）终止反应。测量反应产物的荧光。将不同浓度的 MG-132 添加到测定混合物中，以计算针对 20S 蛋白酶体的 IC50。

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26S蛋白酶体是一种多催化蛋白酶，负责调节细胞内蛋白质的降解。其功能由三种主要催化活性介导：（a）胰凝乳蛋白酶样（CT-L），（b）胰蛋白酶样，（c）肽基谷氨酰肽水解（PGPH）。蛋白酶体抑制是许多癌症的一种新兴疗法，也是多发性骨髓瘤的一种新疗法。在这里，我们分析了三种催化活性在多发性骨髓瘤细胞系中的作用，并比较了新型蛋白酶体抑制剂BzLLLCOCHO和抑制剂PS-341（Velcade，硼替佐米）和MG-132的特异性和细胞毒性。使用荧光底物和蛋白酶体催化亚基特异性的活性位点导向探针，我们显示了每种抑制剂的不同亚基特异性。添加BzLLLCOCHO强烈抑制了所有三种催化活性，用PS-341处理完全抑制了CT-L和PGPH活性，用MG-132处理导致CT-L与PGPH活性的弱抑制。多发性骨髓瘤细胞对PS-341和MG-132诱导的凋亡比BzLLLCOCHO更敏感。这项研究强调，需要进一步研究这些化合物对细胞中基因和蛋白质表达的影响，以开发更特异和靶向的抑制剂[4]。

将 MG-132 以不同浓度添加到细胞中，持续 24 和 48 小时。离心收集上清液和单层细胞，然后将其保存在含70%乙醇的PBS中，然后用吖啶橙染色。吖啶橙（PBS 中 5 mg/mL）和等体积的细胞在显微镜载玻片上混合，并使用荧光显微镜检查混合物。通过离心收集细胞并用碘化丙啶和膜联蛋白 V 染色以进行膜联蛋白 V 分析。在室温下将细胞在 PBS 中再水化 10 分钟后进行碘化丙啶 (5 mg/mL) 染色，以分析细胞周期。使用 Coulter Epics XL 流式细胞仪检查每个样品。

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细胞活力测定[1]
细胞类型：C6神经胶质瘤细胞
测试浓度：10、20、30、40μM
培养时间：24小时
实验结果：从6小时开始，C6神经胶质瘤细胞的生存能力以时间和浓度依赖的方式显著降低，24小时时IC50为18.5μM

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细胞活力测定[1]
细胞类型：A549细胞
测试浓度：10μM
培养时间：1小时
实验结果：逆转TNF-α对IκB降解的影响，并导致TNF-α诱导的NF-κB活化的逆转。

Animal/Disease Models: Female athymic nude mice bearing EC9706 xenograft (5- to 6-weeks old) [10]
Doses: 10 mg/kg
Route of Administration: i.p.; daily for 25 days starting 5 days after inoculation of EC9706 tumors
Experimental Results: Dramatically suppressed tumor growth of the EC9706 xenograft without apparrent toxicity to mice.

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Animal/Disease Models: Female C.B-17/lcr-scid/scidJcl mice bearing HeLa tumors (5- to 6-weeks old) [10]
Doses: 10 mg/kg
Route of Administration: Intravenous/i.v. injection; twice a week for 4 weeks
Experimental Results: The growth inhibition rates in HeLa tumors was 49% compared to the control. Dramatically suppressed tumor growth of HeLa tumors with a TGI of 49%.

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Male mdx (C57BL/10ScSn DMD mdx) mice
~10 μg/kg/day
Injection

[1]. J Biochem. 1996 Mar;119(3):572-6.

[2]. Am J Respir Cell Mol Biol. 1998 Aug;19(2):259-68.

[3]. Cell Death Differ. 2001 Mar;8(3):210-8.

[4]. Cancer Res. 2006 Jun 15;66(12):6379-86.

[5]. J Med ChemCancer Res. 2007 Mar 1;67(5):2247-55.

[6]. Br J Cancer. 2008 Nov 18;99(10):1613-22.

[7]. Am J Pathol. 2003 Oct;163(4):1663-75.

[8]. BMC Musculoskelet Disord. 2011 Aug 15:12:185.

[9]. J Med Chem. 2010 Feb 25;53(4):1509-18.

[10]. Cancer Sci. 2016 Jun;107(6):773-81.

N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal is a tripeptide that is L-leucyl-L-leucyl-L-leucine in which the C-terminal carboxy group has been reduced to the corresponding aldehyde and the N-terminal amino group is protected as its benzyloxycarbonyl derivative. It has a role as a proteasome inhibitor. It is a tripeptide, an amino aldehyde and a carbamate ester.
Z-Leu-leu-leu-al has been reported in Tricholoma pardinum, Glycyrrhiza glabra, and Glycyrrhiza inflata with data available.

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To explore membrane-permeable synthetic inhibitors that discriminate between endogenous calpain and proteasome in cells, we examined the inhibition of profiles against calpain and proteasome in vitro and in vivo of peptidyl aldehydes possessing di-leucine and tri-leucine. The tripeptide aldehyde benzyloxycarbonyl-leucyl-leucinal (ZLLLal) strongly inhibited calpain and proteasome activities in vitro. The concentration required for 50% inhibition (IC50) of the casein-degrading activity of calpain was 1.25 microM, and the IC50s for the succinyl-leucyl-leucyl-valyl-tyrosine-4-methylcoumaryl-7-amide (Suc-LLVY-MCA)- and benzyloxycarbonyl-leucyl-leucyl-leucine-4-methylcoumaryl -7-amide (ZLLL-MCA)-degrading activities of proteasome were 850 and 100 nM, respectively. On the other hand, the synthetic dipeptide aldehyde benzyloxycarbonyl-leucyl-leucinal (ZLLal) strongly inhibited the casein degrading activity of calpain (IC50 1.20 microM), but the inhibition of proteasome was weak (IC50S for SucLLVY-MCA- and ZLLL-MCA-degrading activities were 120 and 110 microM, respectively). Thus, while calpain was inhibited by similar concentrations of ZLLal and ZLLLal, the inhibitory potencies of ZLLLal against the ZLLL-MCA- and Suc-LLVY-MCA-degrading activities in proteasome were 1,100 and 140 times stronger than those of ZLLal, respectively. To evaluate the effectiveness of these inhibitors on intracellular proteasome, the induction of neurite outgrowth in PC12 cells caused by proteasome inhibition was examined. ZLLLal and ZLLal initiated neurite outgrowth with optimal concentrations of 20 nM and 10 microM, respectively, again showing a big difference in the effective concentrations for the proteasome inhibition as in vitro. As for the effect on intracellular calpain, the concentration of ZLLLal and ZLLal required for the inhibition of the autolytic activation of calpain in rabbit erythrocytes were 100 and 100 microM or more, respectively. The almost equal inhibitory potencies of ZLLLal and ZLLal were in agreement with the inhibition of calpain in vitro. These differential effects of inhibitors against calpain and proteasome are potentially useful for identifying the functions of calpain and proteasome in cell physiology and pathology. [1]

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The working hypothesis of the studies described herein was that inhibition of proteasome-mediated IkappaB degradation would inhibit TNF-alpha-induced nuclear factor-kappaB (NF-kappaB) activation, interleukin-8 (IL-8) gene transcription, and IL-8 protein release in A549 cells. Mutational analysis of the 5' flanking region of the IL-8 gene confirmed that an intact NF-kappaB site is necessary for TNF-alpha-induced IL-8 gene transcription. The addition of TNF-alpha to A549 cells resulted in rapid loss of IkappaB from the cytoplasm of cells, associated with a corresponding increase in NF-kappaB-binding activity in nuclear extracts from the cells. However, pretreatment of the cells with the proteasome inhibitor N-cbz-Leu-Leu-leucinal (MG-132, 10 microM) reversed the effects of TNF-alpha on IL-8 release from A549 cells (as determined with an enzyme-linked immunosorbent assay [ELISA]) and on IL-8 gene transcription (as determined with reporter-gene assays). MG-132 reversed the effects of TNF-alpha on IkappaB degradation as determined by Western blot analysis. IkappaB phosphorylation and ubiquination were not altered by MG-132, which implies that the effects of MG-132 were secondary to proteasome inhibition. MG-132 also reversed the increase in NF-kappaB binding in nuclear extracts from TNF-alpha-treated cells. These studies show that inhibition of proteasome-mediated IkappaB degradation results in inhibition of TNF-alpha induced IL-8 production in A549 cells by limiting NF-kappaB-mediated gene transcription.[2]

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We have examined the effects of inhibition of the 26S proteasome in a murine mammary cell line, KIM-2 cells using the peptide aldehyde inhibitor MG132. These studies have demonstrated a clear requirement for proteasome function in cell viability. Induction of apoptosis was observed following MG132 treatment in KIM-2 cells and this death was shown to be dependent on the cell actively traversing the cell cycle. KIM-2 cells were generated using a temperature sensitive T-antigen (Tag) and studies at the permissive temperature (33 degrees C) have shown that a Tag binding protein was essential for this apoptotic response. Studies in two additional cell lines, HC11, which is a mammary epithelial cell line carrying mutant p53 alleles and p53 null ES cells suggest that p53 is actively required for the apoptosis induced as a consequence of proteasome inhibition. These results suggest a pivotal role for the 26S proteasome degradation pathway in progression through the cell cycle in proliferating cells. [3]

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Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent because it induces apoptosis in cancer cells but not in normal cells. Unfortunately, some cancer cells develop resistance to TRAIL-induced apoptosis. Therefore, it is clinically relevant to determine the molecular mechanisms that differentiate between TRAIL-sensitive and TRAIL-resistant tumors. Previously, we have shown that the antiapoptotic molecule cellular-FLICE-inhibitory protein long isoform [c-FLIP(L)] is necessary and sufficient to maintain resistance to TRAIL-induced apoptosis. We have found that c-FLIP(L) is transcriptionally regulated by the activator protein-1 (AP-1) family member protein c-Fos. Here, we report that MG-132, a small-molecule inhibitor of the proteasome, sensitizes TRAIL-resistant prostate cancer cells by inducing c-Fos and repressing c-FLIP(L). c-Fos, which is activated by MG-132, negatively regulates c-FLIP(L) by direct binding to the putative promoter region of the c-FLIP(L) gene. In addition to activating c-Fos, MG-132 activates another AP-1 family member, c-Jun. We show that c-Fos heterodimerizes with c-Jun to repress transcription of c-FLIP(L). Therefore, MG-132 sensitizes TRAIL-resistant prostate cancer cells by activating the AP-1 family members c-Fos and c-Jun, which, in turn, repress the antiapoptotic molecule c-FLIP(L).[5]

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Effective treatments for androgen-independent prostate cancer (AIPCa) are lacking. To address this, emerging therapeutics such as proteasome inhibitors are currently undergoing clinical trials. Inositol hexakisphosphate (IP6) is an orally non-toxic phytochemical that exhibits antitumour activity against several types of cancer including PCa. We have previously shown that treatment of PC3 cells with IP6 induces the transcription of a subset of nuclear factor-kappaB (NF-kappaB)-responsive and pro-apoptotic BCL-2 family genes. In this study, we report that although NF-kappaB subunits p50/p65 translocate to the nucleus of PC3 cells in response to IP6, inhibition of NF-kappaB-mediated transcription using non-degradable inhibitor of kappaB (IkappaB)-alpha does not modulate IP6 sensitivity. Treatment with IP6 also leads to increased protein levels of PUMA, BIK/NBK and NOXA between 4 and 8 h of treatment and decreased levels of MCL-1 and BCL-2 after 24 h. Although blocking transcription using actinomycin D does not modulate PC3 cell sensitivity to IP6, inhibition of protein translation using cycloheximide has a significant protective effect. In contrast, blocking proteasome-mediated protein degradation using MG-132 significantly enhances the ability of IP6 to reduce cellular metabolic activity in both PC3 and DU145 AIPCa cell lines. This effect of combined treatment on mitochondrial depolarisation is particularly striking and is also reproduced by another proteasome inhibitor (ALLN). The enhanced effect of combined MG132/IP6 treatment is almost completely inhibited by cycloheximide and correlates with changes in BCL-2 family protein levels. Altogether these results suggest a role for BCL-2 family proteins in mediating the combined effect of IP6 and proteasome inhibitors and warrant further pre-clinical studies for the treatment of AIPCa.[6]

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MG-132 is a tripeptide aldehyde (Z-l-leu-l-leu-l-leu-H, 2) proteasome inhibitor that exerts antitumor activity and enhances cytostatic/cytotoxic effects of chemo- and radiotherapy. Because of a troublesome synthesis of tripeptides with a non-natural configuration and modified side chains of amino acids, only two stereoisomers of MG-132 have been reported. Here, we propose a new approach to the synthesis of tripeptide aldehydes based on the Ugi reaction. Chiral, enantiomerically stable 2-isocyano-4-methylpentyl acetates were used as substrates for Ugi reaction resulting in a formation of tripeptide skeletons. Further functionalization of the obtained products led to a synthesis of tripeptide aldehydes. All stereoisomers of MG-132 were synthesized and studied as potential inhibitors of chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities of proteasome. These studies demonstrated the influence of absolute configuration of chiral aldehydes on the cytostatic/cytotoxic effects of the synthesized compounds and revealed that only (S,R,S)-(-)-2 stereoisomer is a more potent proteasome inhibitor than MG-132. [9]

C26H41N3O5

475.62

475.304

C, 65.66; H, 8.69; N, 8.83; O, 16.82

133407-82-6

(R)-MG-132;1211877-36-9;MG-132 (negative control)

462382

Z-Leu-Leu-Leu-al; N-benzoxycarbonyl-L-leucyl-L-leucyl-L-leucinal

Cbz-Leu-Leu-Leu-al; LLL

White to yellow solid powder

1.1±0.1 g/cm3

682.0±55.0 °C at 760 mmHg

80-84℃ (DEC.)

366.3±31.5 °C

0.0±2.1 mmHg at 25°C

1.506

5.75

113.6

3

5

15

34

644

3

O=C([C@]([H])(C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])N([H])C([C@]([H])(C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])N([H])C(=O)OC([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O)N([H])[C@]([H])(C([H])=O)C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H]

TZYWCYJVHRLUCT-VABKMULXSA-N

InChI=1S/C26H41N3O5/c1-17(2)12-21(15-30)27-24(31)22(13-18(3)4)28-25(32)23(14-19(5)6)29-26(33)34-16-20-10-8-7-9-11-20/h7-11,15,17-19,21-23H,12-14,16H2,1-6H3,(H,27,31)(H,28,32)(H,29,33)/t21-,22-,23-/m0/s1

benzyl N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]carbamate

MG-132; MG 132; MG132; MGI132; MGI 132; Z-Leu-leu-leu-al; Zlllal; Z-Leu-leu-leucinal; Z-LLL-CHO; MGI-132

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: ≥ 1.67 mg/mL (3.51 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 16.7 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: 1.67 mg/mL (3.51 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 16.7mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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: ≥ 1.67 mg/mL (3.51 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 16.7 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: 4% DMSO+30% PEG 300+20% propylene glycol+ddH2O: 2 mg/mL

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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。