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Cyclopamine (11-Deoxojervine)

Alias: HSDB3505; 11-deoxojervine; CYCLOPAMINE; 4449-51-8; 11-Deoxyjervine; Jervine, 11-deoxo-; [3H]-Cyclopamine; UNII-ZH658AJ192; DTXSID6043709; HSDB 3505; HSDB-3505
Cat No.:V1332 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.
Cyclopamine (11-Deoxojervine)
Cyclopamine (11-Deoxojervine) Chemical Structure CAS No.: 4449-51-8
Product category: Hedgehog(Smoothened) ROCK
This product is for research use only, not for human use. We do not sell to patients.
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Purity & Quality Control Documentation

Purity: ≥98%

Purity: ≥98%

Purity: ≥98%

Purity: ≥98%

Product Description

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.

Biological Activity I Assay Protocols (From Reference)
Targets
Human Endogenous Metabolite
ln Vitro

In vitro activity: Cyclopamine blocks the activity of the human Smo receptor expressed in CHO-K1 cells in the [3H]Hh-Ag binding assay with an IC50 of 280 nM and inhibits the Hedgehog signaling pathway with an IC50 of 46 nM.[1] Cyclopamine suppresses the Hedgehog pathway activity in gut-derived tumor cell lines that express Patched (PTCH) mRNA in a dose-dependent manner, and causes growth inhibition of those tumor cell lines by 75-95% at 3 μM. However, it is ineffective against colon tumor cells that do not express PTCH mRNA, indicating that the effects of cyclopamine treatment are related to the Hedgehog pathway rather than being generally cytotoxic.[2] Cyclopamine (10 μM) inhibits the proliferation of SMOhigh Cyclopamine-responsive cell lines L3.6sl and Panc 05.04 by 75–80% and increases apoptosis by 2.5–3.5 times, while having no effect on the BxPC3-SMOlow cell line. This is achieved by blocking Hedgehog signaling through direct interaction with Smo.[3] In the E3LZ10.7 cell line, cyclopamine treatment dramatically reduces Snail mRNA and increases E-cadherin transcripts. Cyclopamine treatment significantly inhibits the invasive phenotype of Hedgehog-dependent L3.6pl cells, causing a >500-fold reduction in the number of transmigrating cells, independent of inhibition of cell growth. However, this effect is not observed for the Hedgehog-independent cell line Panc-1.[4]

ln Vivo
In mice, HUCCT1 xenografts are eliminated after 22 days of administration of cyclopamine at a dose of 50 mg/kg/day with no discernible side effects.[2] Cyclopamine treatment at a dose of 1.2 mg for 7 days causes a significant amount of tumor cell apoptosis and reduces the tumor mass by 50–60% in tumors derived from Panc 05.04 and L3.6sl, respectively, but not in tumors derived from BxPC3-SMOlow.[3] Cyclopamine administration by itself significantly reduces tumor metastases in xenografts of E3LZ10.7 and L3.6pl, and when combined with gemcitabine, completely eliminates metastases.[4]
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).
Hh-responsive reporter assays [2]
Hh-responsive firefly luciferase and control SV40 Renilla luciferase reporter assays were performed on subconfluent triplicate cultures as described previously. Two days after transfection, culture medium was replaced for a 2-day culture period with assay medium: RPMI-1640 supplemented with 0.5% (established cell lines) or 20% (first-passage xenografts) FBS and containing combinations of 5E1 anti-Hh monoclonal antibody, recombinant doubly lipid-modified Sonic hedgehog (ShhNp) peptide, Cyclopamine purified from Veratrum extract or tomatidine at the concentrations indicated in the main text. Lysates were prepared and analysed as described elsewhere.
Proliferation assays [2]
Cells were cultured in triplicate in 96-well plates in assay media to which 5E1 monoclonal antibody, ShhNp and/or Cyclopamine were added at 0 h at concentrations indicated in the main text. Viable cell mass was determined by optical density measurements at 490 nm (OD490) at 2 and 4 days using the CellTiter96 colorimetric assay. Relative growth was calculated as OD (day 4) - OD (day 2)/OD (day 2).
Cell culture [3]
Human pancreatic adenocarcinoma cell lines HPAC, SW1990, Mpanc-96, SU86.86, PL45, Panc 10.05, Panc 8.13 and Panc 2.03 were obtained from the American Tissue Culture Collection; cell lines MiaPaCa2, Panc-1, CFPAC1, HPAFII, Capan-2, AsPC1, Hs766T and BxPC3 were a gift from Schering Plough. The cell lines COLO357, L3.3, L3.6sl and L3.6pl were a gift from I. Fidler; cell lines Panc 3.07, Panc 5.04, Panc 2.13, Panc 6.03, Panc 4.21 and Panc 1.28 were a gift from E. Jaffee. BxPC3 and all the Panc cell lines were grown in RPMI medium supplemented with 10% fetal bovine serum, l-glutamine and penicillin/streptomycin; medium for Panc cell lines was also supplemented with insulin–transferrin–selenium. The CFPAC, Panc1, L3.6sl and L3.6pl cell lines were grown in DMEM without phenol red, supplemented with 10% fetal calf serum. To test for Cyclopamine responsiveness, cells were grown for 7 days in control medium containing tomatidine or DMSO alone or experimental medium containing cyclopamine (10 µM, the cyclopamine dose–response is shown in Supplementary Fig. 4b). We changed the medium every 2 days. Pictures showing cell morphology were taken with a Nikon Eclipse TE300.
BrdU incorporation assay [3]
Cells were grown for 3 or 4 days in medium containing tomatidine (control) or Cyclopamine (10 µM). The medium was changed every 48 h. Cells were pulsed with 10 µM BrdU during the final 2 h of culture. BrdU was detected with a fluorescein isothiocyanate-conjugated anti-BrdU antibody; total DNA was stained with 7-AAD. FACS analysis was performed according to the BD Biosciences BrdU flow kit instruction manual. Cells in S phase were defined as a cell population that had incorporated BrdU, with DNA content comprising between 2N and 4N. According to the manual, apoptotic cells were defined as a subpopulation of G0/G1 cells with DNA content lower than the diploid amount.
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] 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.
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
ADME/Pharmacokinetics
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/Toxicokinetics
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)
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)
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]
References

[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.

Additional Infomation
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.
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.
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.
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]
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]
These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C27H41NO2
Molecular Weight
411.62
Exact Mass
411.313
Elemental Analysis
C, 78.78; H, 10.04; N, 3.40; O, 7.77
CAS #
4449-51-8
Related CAS #
4449-51-8
PubChem CID
442972
Appearance
White to off-white solid powder
Density
1.1±0.1 g/cm3
Boiling Point
550.8±50.0 °C at 760 mmHg
Melting Point
236-238ºC
Flash Point
286.9±30.1 °C
Vapour Pressure
0.0±3.4 mmHg at 25°C
Index of Refraction
1.583
LogP
5.44
Hydrogen Bond Donor Count
2
Hydrogen Bond Acceptor Count
3
Rotatable Bond Count
0
Heavy Atom Count
30
Complexity
801
Defined Atom Stereocenter Count
10
SMILES
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]
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
HSDB3505; 11-deoxojervine; CYCLOPAMINE; 4449-51-8; 11-Deoxyjervine; Jervine, 11-deoxo-; [3H]-Cyclopamine; UNII-ZH658AJ192; DTXSID6043709; HSDB 3505; HSDB-3505
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 Data
Solubility (In Vitro)
DMSO: <1 mg/mL
Water: <1 mg/mL
Ethanol: 2~20 mg/mL (4.9~48.6 mM)
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.

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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.
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 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

 (Please use freshly prepared in vivo formulations for optimal results.)
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.

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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.

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             (2) Be sure to add the solvent(s) in order.

Biological Data
  • 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.
  • 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.
  • Effects of cyclopamine treatment on pancreatic adenocarcinoma cells. Nature . 2003 Oct 23;425(6960):851-6.
  • Cyclopamine treatment blocks tumour formation of human pancreatic adenocarcinoma cells after transplantation into nude mice. Nature . 2003 Oct 23;425(6960):851-6.
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