Cyclopamine (11-Deoxojervine)   中文名称: 环巴胺

Human Endogenous Metabolite

体外活性：环巴明抑制 Hedgehog 信号通路，IC50 为 46 nM，并在 [3H]Hh-Ag 结合测定中阻断 CHO-K1 细胞中表达的人 Smo 受体的活性，IC50 为 280 nM。 Cyclopamine 在表达 Patched (PTCH) mRNA 的肠源性肿瘤细胞系中以剂量依赖性方式显着抑制 Hedgehog 通路活性，并在 3 μM 浓度下诱导这些肿瘤细胞系生长抑制 75-95%，但对结肠肿瘤细胞没有 PTCH mRNA 表达，表明 Cyclopamine 治疗的效果与 Hedgehog 通路相关，而不是一般的细胞毒性。通过与 Smo 直接相互作用阻断 Hedgehog 信号传导，Cyclopamine (10 μM) 可抑制 SMOhigh Cyclopamine 反应细胞系 L3.6sl 和 Panc 05.04 的增殖 75-80%，并使细胞凋亡增加 2.5 至 3.5 倍，且不产生任何影响。影响 BxPC3-SMOlow 细胞系。环巴明处理显着降低 E3LZ10.7 细胞系中的 Snail mRNA 并增加 E-钙粘蛋白转录物。与细胞生长的抑制无关，Cyclopamine 处理可显着抑制 Hedgehog 依赖性 L3.6pl 细胞的侵袭表型，导致迁移细胞数量减少 500 倍以上，但不影响 Hedgehog 依赖性细胞系 Panc-1 。激酶测定：该测定使用荧光素酶作为读数，测量 Hh 信号通路的末期，即 Gli 的转录调节（Gli-Luc 测定）。环巴明通过在 DMSO 中连续稀释来制备用于测定，然后添加到空测定板中。将 TM3Hh12 细胞（含有 Hh 响应报告基因构建体 pTA-8xGli-Luc 的 TM3 细胞）重悬于含有 5% FBS 和 15 mM Hepes pH 7.3 的 F12 Hams/DMEM (1:1) 中，添加到测定板中并与环巴明一起孵育在 37 °C、5% CO2 下大约 30 分钟。然后将 1 nM Hh-Ag 1.5 添加到测定板中，并在 5% CO2 存在下于 37 °C 下孵育。 48 小时后，将 Bright-Glo 或 MTS 试剂添加到测定板中，并测定 492 nm 处的发光或吸光度。 IC50 值定义为逻辑曲线的拐点，通过使用 R 统计软件包对来自 MTS 测定的 Gli 驱动的荧光素酶发光或吸光度信号与环巴明的 log10（浓度）进行非线性回归来确定。细胞测定：将细胞（SEG1、OE33、KYAE、KYSE180、SNU1、AGS、SNU16、NCI-N-87、HUCCT1、PANC1、PL5、PL6、BXPC3、HS766T、KYSE150、GBD1、DLD1 和 HCT116）暴露于环巴明在 96 孔板中。通过 MTS（可溶性四唑盐）测定法测量细胞活力。使用 CellTiter96 比色测定法在第 2 天和第 4 天在 490 nm (OD490) 处测量光密度来确定活细胞质量。相对生长的计算公式为 OD（第 4 天）﹣OD（第 2 天）/OD（第 2 天）。

以 50 mg/kg/天的剂量给予环巴明 22 天，可根除小鼠体内的 HUCCT1 异种移植物，且没有明显的副作用。在Panc 05.04和L3.6sl衍生的肿瘤中，环巴明以1.2 mg的剂量治疗7天可诱导肿瘤细胞显着凋亡，并使肿瘤质量分别减少50-60%，但在BxPC3-SMOlow肿瘤中则不然。 [3] 单独使用环巴明可显着抑制 E3LZ10.7 和 L3.6pl 异种移植物中的肿瘤转移，并且与吉西他滨联合使用时可完全消除转移。

Gli-Luc 测定使用荧光素酶作为读数，测量 Gli（Hh 信号通路的最后阶段）的转录调节。在 DMSO 中连续稀释后，环巴明即可用于测定，并添加到空的测定板中。重悬于含有 5% FBS 和 15 mM Hepes pH 7.3 的 F12 Ham's/DMEM (1:1) 中后，将 TM3Hh12 细胞（具有 Hh 反应报告基因构建体 pTA-8xGli-Luc 的 TM3 细胞）添加到测定板中并孵育与环巴明在 37°C、5% CO₂ 中反应约 30 分钟。之后，测定板中充满 1 nM Hh-Ag 1.5，并在 37 °C、5% CO₂ 下孵育。 48 小时后，用 Bright-Glo 或 MTS 试剂重新填充测定板，并测量 492 nm 处的吸光度或发光度。 Logistic 曲线的拐点或 IC50 值是通过使用 R 统计软件包对 MTS 测定中的 Gli 驱动的荧光素酶发光或吸光度信号与 log10（环巴明浓度）进行非线性回归来找到的。

在 96 孔板中，细胞暴露于环巴明。可溶性四唑盐（MTS）测定用于测量细胞活力。使用 CellTiter96 比色测定法，第 2 天和第 4 天在 490 nm (OD₄₉₀) 处进行光密度测量以确定活细胞质量。计算相对生长的公式为 OD（第 4 天）⋣OD（第 2 天）/OD（第 2 天）。

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Hh反应性报告分析[2]
如前所述，在亚融合的三重培养物上进行了Hh反应性萤火虫荧光素酶和对照SV40肾荧光素酶报告基因检测。转染后两天，将培养基替换为检测培养基，为期两天：RPMI-1640，补充0.5%（已建立的细胞系）或20%（第一代异种移植物）FBS，并含有5E1抗Hh单克隆抗体、重组双脂修饰的Sonic hedgehog（ShhNp）肽、从藜芦提取物纯化的Cyclopamine 或番茄碱的组合，浓度如正文所示。按照其他地方的描述制备和分析裂解物。

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增殖试验[2]
细胞在96孔板中在试验培养基中培养三次，试验培养基在0小时时加入5E1单克隆抗体、ShhNp和/或Cyclopamine，浓度如正文所示。使用CellTiter96比色测定法在2天和4天内在490 nm（OD490）下通过光密度测量确定活细胞质量。相对生长计算为OD（第4天）-OD（第2天）/OD（第二天）。

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细胞培养[3]
人胰腺癌细胞系HPAC、SW1990、Mpanc-96、SU86.86、PL45、Panc 10.05、Panc 8.13和Panc 2.03购自美国组织培养物保藏中心；细胞系MiaPaCa2、Panc-1、CFPAC1、HPAFII、Capan-2、AsPC1、Hs766T和BxPC3是先灵葆雅赠送的礼物。COLO357、L3.3、L3.6sl和L3.6pl细胞系是I.Fidler赠送的；细胞系Panc 3.07、Panc 5.04、Panc 2.13、Panc 6.03、Panc 4.21和Panc 1.28是E.Jaffee的礼物。BxPC3和所有Panc细胞系在添加了10%胎牛血清、l-谷氨酰胺和青霉素/链霉素的RPMI培养基中生长；Panc细胞系的培养基也补充了胰岛素、转铁蛋白和硒。CFPAC、Panc1、L3.6sl和L3.6pl细胞系在不含酚红的DMEM中生长，并补充了10%的胎牛血清。为了测试Cyclopamine反应性，细胞在单独含有番茄碱或DMSO的对照培养基或含有Cyclopamine（10µM，Cyclopamine剂量-反应如补充图4b所示）的实验培养基中生长7天。我们每两天更换一次介质。显示细胞形态的照片是用尼康Eclipse TE300拍摄的。

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BrdU掺入试验[3]
细胞在含有番茄碱（对照）或Cyclopamine（10µM）的培养基中生长3或4天。每48小时更换一次培养基。在培养的最后2小时，用10µM BrdU对细胞进行脉冲处理。用异硫氰酸荧光素偶联的抗BrdU抗体检测BrdU；总DNA用7-AAD染色。根据BD Biosciences BrdU流动试剂盒说明书进行FACS分析。S期细胞被定义为已掺入BrdU的细胞群，其DNA含量在2N至4N之间。根据手册，凋亡细胞被定义为DNA含量低于二倍体的G0/G1细胞亚群。

Mice: Subcutaneous injections of 0.1 mL Hanks balanced salt solution and matrigel (1:1) containing 2×10⁶ cells are administered to CD-1 nude mice. All subjects receive treatment at the same time after the tumors are grown for four days to a minimum volume of 125 mm³. Mice receive subcutaneous injections of either a vector (triolein:ethanol 4:1 v/v) or a suspension of cyclopamine (1.2 mg per mouse in triolein:ethanol 4:1 v/v) every day for seven days. Tumors removed from mice at the conclusion of treatment are weighed, fixed for three hours at 4°C using 4% paraformaldehyde, embedded in paraffin wax, and sectioned (6 µm). Apoptotic cells are detected with recombinant Tdt via TUNEL. Eosin is then used as a counterstain on the sections. Random selection is used to select eight ×20-magnified fields from regions representing the outside, middle, and inside of two control and two cyclopamine-treated tumors. The quantity of TUNEL-positive nuclei was manually tallied. Staining with hematoxylin and eosin is done.

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Rats: A total of 15 normal male SD rats and 50 SD rats with BPH (6-8 weeks, weighing 400-450 g) were purchased from the Hunan SLAC Laboratory Animal Co., Ltd. and housed under a 12 h light/dark cycle at 22±2°C with relative humidity at 50±10%. Following 1 week of acclimatization, rats were fasted overnight with free access to water prior to experiments. Cyclopamine (0, 10, 20 and 30 mg/kg) was intraperitoneally injected into rats with BPH (n=5), and BPH tissues was collected for western blot analysis of Smo protein. The remaining 45 rats were assigned into the normal group (normal rats, n=15), the BPH group (BPH rats, n=15) and the cyclopamine group (BPH rats, n=15). Rats in the cyclopamine group were intraperitoneally injected with 20 mg/kg cyclopamine. Rats in the normal and BPH groups were fed normally. After 1 week, rats were sacrificed via CO2 overdose; prostate tissues were obtained to determine the indexes described below. Wet weight was measured using an analytical balance, prostate volume was measured by the volumetric method (20), and prostate index (PI) was calculated using the formula: PI=wet weight of prostate/total body weight. All rats were fed in specific-pathogen-free grade chambers, which was compliant with the Laboratory Animal Requirements of Environment and Housing Facilities Guidelines (GB 14925-2010).[5] Xenograft treatment [2]
HUCCT1 tumours (n = 18) were grown in athymic (nude) mice and treated with cyclopamine (50 mg kg-1 d-1, subcutaneous injection) or control vehicle as described previously.

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Allograft treatment in vivo [3]
Allograft treatment in vivo was performed according to ref. 19 with minor modifications. A total of 0.1 ml Hanks’ balanced salt solution and matrigel (1:1) containing 2 × 106 cells was injected subcutaneously into CD-1 nude mice. Tumours were grown for 4 days to a minimum volume of 125 mm3; treatment was initiated simultaneously for all subjects. Mice were injected subcutaneously with vector alone (triolein:ethanol 4:1 v/v) or a cyclopamine suspension (1.2 mg per mouse in triolein:ethanol 4:1 v/v) daily for 7 days. At the end of the treatment period, tumours were excised from mice, weighed and then fixed for 3 h at 4 °C with 4% paraformaldehyde, embedded in paraffin wax and sectioned (6 µm). Apoptotic cells were identified by TUNEL using recombinant Tdt as previously described29. Sections were then counterstained with eosin. Eight ×20-magnified fields from regions corresponding to the exterior, middle and interior of two control and two cyclopamine-treated tumours were chosen at random. We counted the number of TUNEL-positive nuclei manually. Haematoxylin/eosin staining was done as previously described

Metabolism / Metabolites
Conversion to teratogen of cyclopamine by rumen microorganisms is not essential. Indeed, ruminant sheep and non-ruminant rabbits were about equally susceptible to cyclopamine on a body weight basis.

Toxicity Summary
Cyclopamine causes usually fatal birth defects. It can prevent the fetal brain from dividing into two lobes (holoprosencephaly) and cause the development of a single eye (cyclopia). It does so by inhibiting the hedgehog pathway (Hh). Cyclopamine inhibits the Hh by influencing the balance between the active and inactive forms of the smoothened protein. Cyclopamine is useful in studying the role of Hh in normal development, and as a potential treatment for certain cancers in which Hh is overexpressed. Cyclopamine acts as a primary inhibitor of the hedgehog signaling pathway in cells. This pathway named for the ligand for the signal protein, is used by cells to help them react to external chemical signals. The pathway carries out important functions in embryonic development and when it goes awry, deformities can occur. However, errant activation of the pathway can also trigger cancer in adult humans, leading to basal cell carcinoma, medulloblastoma, rhabdomyosarcoma, and prostate, pancreatic and breast cancers. A way of controlling the pathway using cyclopamine could turn this problem on its head and provide a way to treat cancer. (Wikipedia) Cyclopamine inhibits the Hh pathway by binding to and preventing the activation of Smoothened (Smo), preventing downstream target gene regulation. (A15437)

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Non-Human Toxicity Excerpts
CYCLOPAMINE FED TO PREGNANT EWES DURING DAY 28, 29, AND 30 OF GESTATION, PRODUCED CONGENITAL DEFORMITIES OF LIMBS. THESE DEFORMITIES INCLUDED SHORTENING OF THE METACARPAL OR METATARSAL BONES. KEELER RF; TERATOGENIC COMPOUNDS OF VERATRUM CALIFORNICUM (DURAND): XIV. LIMB DEFORMITIES PRODUCED BY CYCLOPAMINE; PROC SOC EXP BIOL MED 142(4) 1287 (1973)

MALFORMATIONS IN HATCHED CHICKS WERE PRODUCED BY DIRECT APPLICATION OF 1-2 MG OF CYCLOPAMINE TO THE EMBRYONIC SHIELD OF WINDOWED CHICKEN EGGS. ...INTRAUTERINE INJECTION OF AS LITTLE AS 1-2 MG OF CYCLOPAMINE PRODUCED DEFORMITIES /IN SHEEP/. Keeler, R.F., A. T. Tu (eds.). Handbook of Natural Toxins. Volume 1. Plant and Fungal Toxins. New York: Marcel Dekker, Inc., 1983., p. 175

FETAL RABBITS BECAME MALFORMED GROSSLY SIMILAR TO LAMBS UPON MATERNAL INGESTION OF CYCLOPAMINE. THE CYCLOPIA AND RELATED CEPHALIC MALFORMATIONS OCCURRED WHEN INGESTION TOOK PLACE ON THE 7TH DAY OF GESTATION. KEELER RF; TERATOGENIC COMPOUNDS OF VERATRUM CALIFORNICUM. XI. GESTATIONAL CHRONOLOGY AND COMPOUND SPECIFICITY IN RABBITS; PROC SOC EXP BIOL MED 136(3) 1174 (1971)

Cyclopamine produced deformities in rats, mice, and hamsters gavaged during the primitive streak/neural plate stage of development with as little as 240, 180, and 170 mg/kg, respectively, but cyclopics were not evident. In rats, microphthalmia and cebocephalia predominated; in mice, a few exencephalics resulted; whereas in hamsters, cebocephalia, encephalocele (cranial bleb), exencephaly, and hare lip resulted. Keeler RF; Cyclopamine and Related Steroidal Alkaloid Teratogens: Their Occurrence, Structural Relationship, and Biologic Effects; Lipids 13 (10): 708-15 (1978)

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442972 mouse LDLo oral 180 mg/kg Proceedings of the Society for Experimental Biology and Medicine., 149(302), 1975 [PMID:1144444]
442972 hamster LDLo oral 170 mg/kg Proceedings of the Society for Experimental Biology and Medicine., 149(302), 1975 [PMID:1144444]

[1]. Bioorg Med Chem Lett. 2009 Jan 15;19(2):328-31.

[2]. Nature. 2003 Oct 23;425(6960):846-51.

[3]. Nature. 2003 Oct 23;425(6960):851-6.

[4]. Cancer Res. 2007 Mar 1;67(5):2187-96.

[5]. Int J Mol Med. 2020 Jul; 46(1): 311–319.

Cyclopamine is a member of piperidines. It has a role as a glioma-associated oncogene inhibitor.
Cyclopamine has been reported in Veratrum dahuricum, Veratrum grandiflorum, and Veratrum californicum with data available.
Cyclopamine is a naturally occurring chemical that belongs to the group of steroidal jerveratrum alkaloids. It is a teratogen isolated from the corn lily (Veratrum californicum) that causes usually fatal birth defects. It can prevent the fetal brain from dividing into two lobes (holoprosencephaly) and cause the development of a single eye (cyclopia). It does so by inhibiting the hedgehog signaling pathway (Hh). Cyclopamine is useful in studying the role of Hh in normal development, and as a potential treatment for certain cancers in which Hh is overexpressed.

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Mechanism of Action
A NUMBER OF VERATRUM ALKALOIDS WERE TESTED IN PREGNANT SHEEP FOR TERATOGENICITY. THE COMPOUNDS JERVINE, CYCLOPAMINE...AND CYCLOPOSINE...PRODUCED DEFORMITIES SIMILAR TO NATURAL CASES. THE 3 TERATOGENIC COMPOUNDS ARE CLOSELY RELATED STEROIDAL FURANOPIPERIDINES, BUT CYCLOPAMINE IS THE TERATOGEN OF NATURAL IMPORTANCE BECAUSE OF PLANT CONCN. CLOSELY RELATED COMPD DEVOID OF THE FURAN RING DID NOT PRODUCE CYCLOPIA IN SHEEP, SUGGESTING THAT AN INTACT FURAN RING WAS REQUIRED FOR ACTIVITY, PERHAPS CONFERRING SOME ESSENTIAL CONFIGURATION ON THE MOLECULE.

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Therapeutic Uses
Plants have and continue to provide medicine with an abundance of pharmacologically interesting and useful chemicals. In recent years, cyclopamine, a steroidal alkaloid isolated from Veratrum californicum, has been instrumental in dissecting the sonic hedgehog pathway. This brief report outlines cyclopamine's discovery with discussion of its potential application to clinical dermatology.

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Activation of the Hedgehog (Hh) signalling pathway by sporadic mutations or in familial conditions such as Gorlin's syndrome is associated with tumorigenesis in skin, the cerebellum and skeletal muscle. Here we show that a wide range of digestive tract tumours, including most of those originating in the oesophagus, stomach, biliary tract and pancreas, but not in the colon, display increased Hh pathway activity, which is suppressible by cyclopamine, a Hh pathway antagonist. Cyclopamine also suppresses cell growth in vitro and causes durable regression of xenograft tumours in vivo. Unlike in Gorlin's syndrome tumours, pathway activity and cell growth in these digestive tract tumours are driven by endogenous expression of Hh ligands, as indicated by the presence of Sonic hedgehog and Indian hedgehog transcripts, by the pathway- and growth-inhibitory activity of a Hh-neutralizing antibody, and by the dramatic growth-stimulatory activity of exogenously added Hh ligand. Our results identify a group of common lethal malignancies in which Hh pathway activity, essential for tumour growth, is activated not by mutation but by ligand expression. [2]

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Hedgehog signalling--an essential pathway during embryonic pancreatic development, the misregulation of which has been implicated in several forms of cancer--may also be an important mediator in human pancreatic carcinoma. Here we report that sonic hedgehog, a secreted hedgehog ligand, is abnormally expressed in pancreatic adenocarcinoma and its precursor lesions: pancreatic intraepithelial neoplasia (PanIN). Pancreata of Pdx-Shh mice (in which Shh is misexpressed in the pancreatic endoderm) develop abnormal tubular structures, a phenocopy of human PanIN-1 and -2. Moreover, these PanIN-like lesions also contain mutations in K-ras and overexpress HER-2/neu, which are genetic mutations found early in the progression of human pancreatic cancer. Furthermore, hedgehog signalling remains active in cell lines established from primary and metastatic pancreatic adenocarcinomas. Notably, inhibition of hedgehog signalling by cyclopamine induced apoptosis and blocked proliferation in a subset of the pancreatic cancer cell lines both in vitro and in vivo. These data suggest that this pathway may have an early and critical role in the genesis of this cancer, and that maintenance of hedgehog signalling is important for aberrant proliferation and tumorigenesis. [3]

C27H41NO2

411.62

411.313

C, 78.78; H, 10.04; N, 3.40; O, 7.77

4449-51-8

442972

White to off-white solid powder

1.1±0.1 g/cm3

550.8±50.0 °C at 760 mmHg

236-238ºC

286.9±30.1 °C

0.0±3.4 mmHg at 25°C

1.583

5.44

41.49

2

3

0

30

801

10

O1[C@]2([H])C([H])([H])[C@]([H])(C([H])([H])[H])C([H])([H])N([H])[C@@]2([H])[C@@]([H])(C([H])([H])[H])[C@@]21C(C([H])([H])[H])=C1C([H])([H])[C@]3([H])[C@@]4(C([H])([H])[H])C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])C4=C([H])C([H])([H])[C@@]3([H])[C@]1([H])C([H])([H])C2([H])[H])O[H]

QASFUMOKHFSJGL-LAFRSMQTSA-N

InChI=1S/C27H41NO2/c1-15-11-24-25(28-14-15)17(3)27(30-24)10-8-20-21-6-5-18-12-19(29)7-9-26(18,4)23(21)13-22(20)16(27)2/h5,15,17,19-21,23-25,28-29H,6-14H2,1-4H3/t15-,17+,19-,20-,21-,23-,24+,25-,26-,27-/m0/s1

(3S,3'R,3'aS,6'S,6aS,6bS,7'aR,9R,11aS,11bR)-3',6',10,11b-tetramethylspiro[2,3,4,6,6a,6b,7,8,11,11a-decahydro-1H-benzo[a]fluorene-9,2'-3a,4,5,6,7,7a-hexahydro-3H-furo[3,2-b]pyridine]-3-ol

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 (4.06 mM) (饱和度未知) in 10% EtOH + 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 澄清乙醇储备液添加到 900 μL 玉米油中并充分混合。

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配方 2 中的溶解度: ≥ 1 mg/mL (2.43 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 10.0 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 3 中的溶解度: ≥ 0.5 mg/mL (1.21 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 5.0 mg/mL澄清DMSO储备液加入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 4 中的溶解度: ≥ 0.5 mg/mL (1.21 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，将100 μL 5.0 mg/mL澄清DMSO储备液加入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 5 中的溶解度: 10% DMSO+30% PEG 300+5% Tween 80+ddH2O: 1mg/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。