Human Endogenous Metabolite

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]

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.

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).

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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.

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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).

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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.

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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.

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
It is not necessary for rumen microbes to convert cyclopamine into a teratogen. In fact, ruminant sheep and non-ruminant rabbits have roughly the same sensitivity to cyclopamine based on body weight.

Toxicity Summary
Cyclopamine commonly causes fatal birth defects. It can prevent the fetal brain from splitting into two lobes (holoneencephaly) and cause monocular development (monophthalmos). Its mechanism of action is through inhibition of the Hedgehog signaling pathway (Hh). Cyclopamine inhibits Hh by affecting the balance between the active and inactive forms of the smoothed protein. Cyclopamine can be used to study the role of Hh in normal development and may serve as a potential therapeutic approach for certain cancers with Hh overexpression. Cyclopamine is a major inhibitor of the Hedgehog signaling pathway in cells. This pathway, named after the ligands of signaling proteins, is used by cells to respond to external chemical signals. This pathway plays a crucial role in embryonic development, and abnormalities can lead to malformations. However, aberrant activation of this pathway can also induce cancers in adults, leading to basal cell carcinoma, medulloblastoma, rhabdomyosarcoma, as well as prostate, pancreatic, and breast cancer. Utilizing cyclopamine to control this pathway holds promise for reversing this trend and providing new avenues for cancer treatment. (Wikipedia) Cyclopamine inhibits the Hh pathway by binding to and preventing the activation of Smoothened (Smo), thereby preventing the regulation of downstream target genes. (A15437)

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Excerpt of non-human toxicity
Feeding pregnant ewes with cyclopamine on days 28, 29 and 30 of gestation can cause congenital limb deformities. These deformities include metatarsal or shortened metatarsal bones. KEELER RF; Teratogenic compounds of California veratrum (Duland): XIV. Limb deformities caused by cyclopamine; Journal of the Society for Experimental Biology and Medicine 142(4) 1287 (1973)
Applying 1-2 mg of cyclopamine directly to the embryonic membrane of open-windowed chicken eggs can cause deformities in chicks. ...Intrauterine injection of as little as 1-2 mg of cyclopamine can cause deformities in sheep. Keeler, RF, AT Tu (eds.). Handbook of Natural Toxins. Volume 1: Plant and Fungal Toxins. New York: Marcel Dekker, 1983, p. 1287. 175
After female rabbits ingested cyclopamine, the degree of fetal malformation was very similar to that of lambs. Cyclopsis and related head malformations occurred when cyclopamine was ingested on day 7 of gestation. KEELER RF; Teratogenic compounds of veratrum davidii. XI. Timing of gestation in rabbits and compound specificity; PROC SOC EXP BIOL MED 136(3) 1174 (1971)
During the development of the protostome/neural plate, cyclopamine was administered by gavage to rats, mice and hamsters at doses as low as 240, 180 and 170 mg/kg, respectively, resulting in malformations, but cyclopamine was not observed. In rats, microphthalmia and cranial malformations are relatively common; in mice, encephalocele is rare; and in hamsters, cranial malformations, encephalocele (cerebrovascular bullae), encephalocele, and cleft lip are observed. Keeler RF; Cyclopamine and related steroidal alkaloid teratogenic agents: their presence, structural relationships and biological effects; Lipids 13 (10): 708-15 (1978)

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442972 Mice LDLo orally 180 mg/kg Proceedings of the Society for Experimental Biology and Medicine., 149(302), 1975 [PMID:1144444]
442972 Hamster LDLo orally 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 belongs to the piperidine class of compounds and has the ability to inhibit glioma-related oncogenes. It has been reported that cyclopamine is present in Veratrum dahuricum, Veratrum grandiflorum, and Veratrum californicum, and relevant data are available. Cyclopamine is a naturally occurring chemical substance belonging to the steroidal veratrine alkaloid class. It is a teratogen isolated from Veratrum californicum, which typically causes fatal birth defects. It can prevent the fetal brain from splitting into two lobes (holoneencephaly) and cause unilateral development (monophthalmos). Its mechanism of action is through inhibition of the Hedgehog signaling pathway (Hh). Cyclopamine can be used to study the role of Hh in normal development and may serve as a potential drug for treating certain Hh-overexpressing cancers. We conducted teratogenicity tests on various veratrine alkaloids in pregnant sheep. Compounds javen, cyclopamine, and cyclopamine all produce malformations similar to those in natural cases. These three teratogenic compounds are closely related steroidal furanopiperidine compounds, but cyclopamine exhibits significant natural teratogenicity due to its higher concentrations in plants. Compounds closely related to these compounds, lacking the furan ring, do not cause unilateral cytopia in sheep, suggesting that the intact furan ring is essential for their activity, perhaps conferring a necessary configuration to the molecule.

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Therapeutic Uses
Plants have long provided a wealth of pharmacologically interesting and useful chemicals for medicine. In recent years, the steroidal alkaloid cyclopamine, isolated from Veratrum californicum, has played a significant role in elucidating the Hedgehog factor signaling pathway. This brief report outlines the discovery of cyclopamine and explores its potential applications in clinical dermatology.

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Sporadic mutations or familial diseases (such as Goring syndrome) activate the Hedgehog (Hh) signaling pathway and are associated with tumorigenesis of the skin, cerebellum, and skeletal muscle. We found that a variety of gastrointestinal tumors, including most tumors originating from the esophagus, stomach, bile ducts and pancreas (but excluding colon tumors), exhibit enhanced Hh pathway activity, which can be inhibited by the Hh pathway antagonist cyclopamine. Cyclopamine also inhibits cell growth in vitro and induces persistent regression of xenograft tumors in vivo. Unlike Göring syndrome tumors, signaling pathway activity and cell growth in these gastrointestinal tumors are driven by the expression of endogenous Hh ligands, as evidenced by the presence of Sonic hedgehog and Indian hedgehog transcripts, the pathway and growth-inhibiting activity of Hh neutralizing antibodies, and the significant growth-stimulating activity of exogenous Hh ligands. Our findings reveal a common class of deadly malignancies in which the Hh signaling pathway activity, which is crucial for tumor growth, is activated not by mutations but by ligand expression. [2] The Hedgehog signaling pathway—an important pathway in embryonic pancreatic development, whose dysregulation is associated with a variety of cancers—may also be an important mediator of human pancreatic cancer. This article reports that the secretory hedgehog ligand Sonic hedgehog (Shh) is abnormally expressed in pancreatic adenocarcinoma and its precancerous lesion, pancreatic intraepithelial neoplasia (PanIN). The pancreas of Pdx-Shh mice (where Shh is abnormally expressed in the pancreatic endoderm) forms abnormal tubular structures, which are similar in phenotype to human PanIN-1 and -2. In addition, these PanIN-like lesions also have K-ras gene mutations and overexpress HER-2/neu, which are common in the early stages of human pancreatic cancer progression. Moreover, the hedgehog signaling pathway remains active in both primary and metastatic pancreatic adenocarcinoma cell lines. Notably, cyclopamine inhibition of the hedgehog signaling pathway can induce apoptosis in some pancreatic cancer cell lines and inhibit their proliferation both in vitro and in vivo. These data suggest that this pathway may play a key role in the early stages of cancer development, and that maintaining Hedgehog signaling is essential for abnormal 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

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)

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.

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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.

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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.

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Solubility in Formulation 5: 10% DMSO+30% PEG 300+5% Tween 80+ddH2O: 1mg/mL

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