Size | Price | Stock | Qty |
---|---|---|---|
5mg |
|
||
10mg |
|
||
25mg |
|
||
50mg |
|
||
100mg |
|
||
250mg |
|
||
500mg |
|
||
Other Sizes |
|
Purity: ≥98%
Cyclopamine (formerly HSDB3505; HSDB-3505; 11-deoxojervine), a naturally occuring steroid alkaloid found in Veratrum plant, is reported to be a potent and specific inhibitor of Hedgehog (Hh) signaling pathway with both anticancer and teratogenic activities. In TM3Hh12 cells, it inhibits Smoothened (Smo) at an IC50 of 46 nM.
Targets |
Human Endogenous Metabolite
|
|
---|---|---|
ln Vitro |
|
|
ln Vivo |
|
|
Enzyme Assay |
Using Luciferase as a readout, the Gli-Luc assay measures the transcriptional modulation of Gli, the final stage of the Hh signaling pathway. After serial dilution in DMSO, cyclopamine is ready for assay and added to assay plates that are empty. After being resuspended in F12 Ham's/DMEM (1:1) containing 5% FBS and 15 mM Hepes pH 7.3, TM3Hh12 cells (TM3 cells with the Hh-responsive reporter gene construct pTA-8xGli-Luc) are added to assay plates and incubated with Cyclopamine for about 30 minutes at 37°C in 5% CO2. After that, assay plates are filled with 1 nM Hh-Ag 1.5 and allowed to incubate at 37 °C with 5% CO2. Following 48 hours, the assay plates are refilled with either Bright-Glo or MTS reagent, and the absorbance or luminescence at 492 nm is measured. The logistic curve's inflection point, or IC50 value, is found by non-linearly regressing the Gli-driven luciferase luminescence or absorbance signal from the MTS assay against log10 (cyclopamine concentration) using the R statistical software package.
|
|
Cell Assay |
In 96-well plates, cells are exposed to cyclopamine. Soluble tetrazolium salt, or MTS, assay is used to measure cell viability. Using the CellTiter96 colorimetric assay, optical density measurements at 490 nm (OD490) at 2 and 4 days determine the viable cell mass. The formula for calculating relative growth is OD (day 4)⋣OD (day 2)/OD (day 2).
|
|
Animal Protocol |
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.
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] |
|
References |
Molecular Formula |
C27H41NO2
|
|
---|---|---|
Molecular Weight |
411.62
|
|
Exact Mass |
411.31
|
|
Elemental Analysis |
C, 78.78; H, 10.04; N, 3.40; O, 7.77
|
|
CAS # |
4449-51-8
|
|
Related CAS # |
|
|
Appearance |
Solid powder
|
|
LogP |
5.4
|
|
tPSA |
41.49
|
|
SMILES |
C[C@H]1C[C@@H]2[C@H]([C@H]([C@]3(O2)CC[C@H]4[C@@H]5CC=C6C[C@H](CC[C@@]6([C@H]5CC4=C3C)C)O)C)NC1
|
|
InChi Key |
QASFUMOKHFSJGL-LAFRSMQTSA-N
|
|
InChi Code |
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
|
|
Chemical Name |
(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
|
|
Synonyms |
|
|
HS Tariff Code |
2934.99.9001
|
|
Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
|
Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
Solubility (In Vitro) |
|
|||
---|---|---|---|---|
Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.67 mg/mL (4.06 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well. Solubility in Formulation 2: ≥ 1 mg/mL (2.43 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. View More
Solubility in Formulation 3: ≥ 0.5 mg/mL (1.21 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: ≥ 0.5 mg/mL (1.21 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 5: 10% DMSO+30% PEG 300+5% Tween 80+ddH2O: 1mg/mL |
Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 2.4294 mL | 12.1471 mL | 24.2943 mL | |
5 mM | 0.4859 mL | 2.4294 mL | 4.8589 mL | |
10 mM | 0.2429 mL | 1.2147 mL | 2.4294 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration: mg/mL;
Method for preparing DMSO stock solution: mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation::Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
A, viable cell masses, determined using MTT assays, were reduced by nearly 100% to 0% by Hh inhibition with 6 μmol/L cyclopamine for 4 d as compared with solvent-treated cells in vitro. Cancer Res . 2007 Mar 1;67(5):2187-96. td> |
A, using the Guava Multicaspase assay, an increase of cells undergoing apoptosis was detected on treatment with cyclopamine. Cancer Res . 2007 Mar 1;67(5):2187-96. td> |
Effects of cyclopamine treatment on pancreatic adenocarcinoma cells. Nature . 2003 Oct 23;425(6960):851-6. td> |
Cyclopamine treatment blocks tumour formation of human pancreatic adenocarcinoma cells after transplantation into nude mice. Nature . 2003 Oct 23;425(6960):851-6. td> |