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Purity: ≥98%
Temsirolimus (also known as CCI-779, NSC-683864; Torisel), an ester analog of rapamycin, is a potent and specific inhibitor of mTOR (mammalian target of rapamycin) with potential anticancer activity. It inhibits mTOR with an IC50 of 1.76 μM in a cell-free assay. Temsirolimus is an ester derivative of rapamycin and has improved pharmacodynamic and pharmacokinetic properties. Temsirolimus, an mTOR inhibitor, reduced cell proliferation in cancer cells where mTOR dysregulates the cell cycle targets. Temsirolimus demonstrated strong antigrowth activity against a panel of eight human breast cancer cell lines, with IC50 values of 0.6, 0.7, and 0.7nM for BT-474, MDA-MB-468, and SKBR-3 cells, respectively. Temsirolimus (trade name: Torisel) was approved by the FDA in May 2007 for the treatment of renal cell carcinoma (RCC).
Targets |
mTOR (IC50 = 1.76 μM)
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ln Vitro |
In the absence of FKBP12, Temsirolimus potently inhibits mTOR kinase activity with IC50 of 1.76 μM, similar to that of rapamycin with IC50 of 1.74 μM. Temsirolimus treatment at nanomolar concentrations (10 nM to <5 μM) exhibits a modest and selective antiproliferative activity via FKBP12-dependent mechanism, but at low micromolar concentrations (5-15 μM), it can completely inhibit the proliferation of a wide panel of tumor cells by suppressing mTOR signaling in a manner that is FKBP12-independent. Treatment with temsirolimus at micromolar (20 μM), but not nanomolar, concentrations results in a marked reduction in overall protein synthesis and polyribosome disassembly, which is accompanied by a sharp rise in the phosphorylation of the translation elongation factor eEF2 and the translation initiation factor eIF2A.[1] Temsirolimus inhibits cell growth and clonogenic survival in both cells in a concentration-dependent manner, but more potently in PTEN-positive DU145 cells than in PTEN-negative PC-3 cells. It also inhibits the phosphorylation of ribosomal protein S6. [2] Primary human lymphoblastic leukemia (ALL) cells are potently inhibited from proliferating and are induced to undergo apoptosis by temsirolimus (100 ng/mL).[3]
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ln Vivo |
In the NOD/SCID xenograft models with human ALL, Temsirolimus treatment at 10 mg/kg/day produces a decrease in peripheral blood blasts and in splenomegaly.[3] Temsirolimus (20 mg/kg i.p. 5 days/week) significantly slows down the growth of DAOY xenografts compared to controls, delaying it by 160% after 1 week and 240% after 2 weeks. One week of treatment with a single high-dose of temsirolimus (100 mg/kg i.p.) causes a 37% reduction in tumor volume. The growth of rapamycin-resistant U251 xenografts is also 148% delayed by temsirolimus treatment for 2 weeks.[4] Temsirolimus's inhibition of mTOR enhances performance on four distinct behavioral tasks and reduces aggregate formation in a mouse model of Huntington disease. [5] Temsirolimus administration results in significant dose-dependent, antitumor responses against subcutaneous growth of 8226, OPM-2, and U266 xenografts, with ED50 values of 20 mg/kg and 2 mg/kg for 8226 and OPM-2, respectively. These responses are linked to decreased tumor cell growth and inhibition of angiogenesis as well as increased apoptosis and inhibition of proliferation.[6]
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Enzyme Assay |
Transiently transfecting HEK293 cells with Flag-tagged wild-type human mTOR (Flag-mTOR) DNA constructs. 48 hours later, Flag-mTOR protein is extracted and purified. Purified Flag-mTOR in vitro kinase assays are carried out in 96-well plates in the presence of various Temsirolimus concentrations without FKBP12, and the results are detected using the dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) method with His6-S6K1 as the substrate. Enzymes are first diluted in kinase assay buffer (10 mM Hepes (pH 7.4), 50 mM NaCl, 50 mM β-glycerophosphate, 10 mM MnCl2, 0.5 mM DTT, 0.25 μM microcystin LR, and 100 μg/mL BSA). 12 μL of the diluted enzyme and 0.5 μL of temsirolimus are quickly combined in each well. The kinase reaction is started by adding 12.5 μL of ATP and His6-S6K-containing kinase assay buffer to create a final reaction volume of 25 μL that contains 800 ng/mL FLAG-mTOR, 100 μM ATP, and 1.25 μM His6-S6K. The reaction plate is incubated for 2 hours (linear at 1-6 hours) at room temperature with gentle shaking before being stopped by adding 25 μL of stop buffer (20 mM Hepes (pH 7.4), 20 mM EDTA, and 20 mM EGTA). A monoclonal anti-P(T389)-p70S6K antibody labeled with Europium-N1-ITC (Eu) (10.4 Eu per antibody) is used for the DELFIA detection of the phosphorylated (Thr-389) His6-S6K at room temperature. Transfer 45 μL of the terminated kinase reaction mixture to a MaxiSorp plate containing 55 μL PBS. Eu-P(T389)-S6K antibody is added to 100 μL of DELFIA buffer at a concentration of 40 ng/mL. With minimal agitation, the antibody binding is continued for an additional hour. The wells are then aspirated and cleaned using PBS with 0.05% Tween 20 (PBST). Each well receives 100 L of DELFIA Enhancement solution before the plates are read using a PerkinElmer Victor model plate reader.
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Cell Assay |
Temsirolimus is applied to cells in a range of concentrations for 72 hours. Viable cell densities are assessed following treatment using the CellTiter AQ assay kit to measure MTS dye conversion.
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Animal Protocol |
Cells are implanted in matrigel for the creation of xenografts; matrigel is stored at −20°C and thawed on ice at 4°C for 3 hours prior to use. After being gently resuspended in 1 mL of PBS, the cells are incubated for 5 minutes on ice. Cells are transferred to the tube containing 1 mL of matrigel using a prechilled pipette, and the cell concentration is adjusted to 3×107/mL. Using a 25-gauge needle, the cells (3×106 in 0.1 mL) are injected s.c. into the mice's flanks. When xenografts grew to a size of about 5 mm in diameter, animals are assorted randomLy into groups of 10 mice. The following experiments are conducted: Mice bearing PC-3 tumors are treated with CCI-779 (1, 5, 10, and 20 mg per kg per day), or vehicle solution for 3 or 5 days per week for 3 weeks. Mice bearing DU145 tumors are only treated with CCI-779 (20 mg per kg per day) or vehicle solution for 3 weeks. Mice bearing PC-3 tumors receive the following treatments: (a) control, vehicle solution for CCI-779; (b) chemotherapy alone, mitoxantrone 1.5 mg/kg or docetaxel 10 mg/kg is injected i.p. weekly for 3 doses; (c) CCI-779 alone, 5 or 10 mg/kg is injected i.p. daily, three times a week for 3 weeks; (4) chemotherapy followed by CCI-779.
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References |
Molecular Formula |
C56H87NO16
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Molecular Weight |
1030.29
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Exact Mass |
1029.60249
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Elemental Analysis |
C, 65.28; H, 8.51; N, 1.36; O, 24.85
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CAS # |
162635-04-3
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Appearance |
Solid powder
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SMILES |
C[C@@H]1CC[C@H]2C[C@@H](/C(=C/C=C/C=C/[C@H](C[C@H](C(=O)[C@@H]([C@@H](/C(=C/[C@H](C(=O)C[C@H](OC(=O)[C@@H]3CCCCN3C(=O)C(=O)[C@@]1(O2)O)[C@H](C)C[C@@H]4CC[C@H]([C@@H](C4)OC)OC(=O)C(C)(CO)CO)C)/C)O)OC)C)C)/C)OC
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InChi Key |
CBPNZQVSJQDFBE-FUXHJELOSA-N
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InChi Code |
InChI=1S/C56H87NO16/c1-33-17-13-12-14-18-34(2)45(68-9)29-41-22-20-39(7)56(67,73-41)51(63)52(64)57-24-16-15-19-42(57)53(65)71-46(30-43(60)35(3)26-38(6)49(62)50(70-11)48(61)37(5)25-33)36(4)27-40-21-23-44(47(28-40)69-10)72-54(66)55(8,31-58)32-59/h12-14,17-18,26,33,35-37,39-42,44-47,49-50,58-59,62,67H,15-16,19-25,27-32H2,1-11H3/b14-12+,17-13+,34-18+,38-26+/t33-,35-,36-,37-,39-,40+,41+,42+,44-,45+,46+,47-,49-,50+,56-/m1/s1
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Chemical Name |
[(1R,2R,4S)-4-[(2R)-2-[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl] 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate
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Synonyms |
CCI-779; CCI779; CCI 779; NSC 683864; NSC683864; NSC-683864; Temsirolimus; Brand name: Torisel
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HS Tariff Code |
2934.99.9001
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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)
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Solubility (In Vitro) |
DMSO: ~75 mg/mL (~72.8 mM)
Water: <1 mg/mL Ethanol: ~75 mg/mL (~72.8 mM) |
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Solubility (In Vivo) |
30% PEG400+0.5% Tween80+5% propylene glycol:10mg/mL (Please use freshly prepared in vivo formulations for optimal results.)
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Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 0.9706 mL | 4.8530 mL | 9.7060 mL | |
5 mM | 0.1941 mL | 0.9706 mL | 1.9412 mL | |
10 mM | 0.0971 mL | 0.4853 mL | 0.9706 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.
NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
NCT02420613 | Active Recruiting |
Drug: Vorinostat Drug: Temsirolimus |
Diffuse Intrinsic Pontine Glioma | M.D. Anderson Cancer Center | October 5, 2015 | Phase 1 |
NCT01375829 | Active Recruiting |
Drug: Ixabepilone Drug: Temsirolimus |
Adult Solid Neoplasm | National Cancer Institute (NCI) |
June 27, 2011 | Phase 1 |
NCT02389309 | Active Recruiting |
Drug: Dasatinib Drug: Temsirolimus |
Recurrent Brain Neoplasm Refractory Brain Neoplasm |
M.D. Anderson Cancer Center | October 5, 2015 | Phase 1 |
NCT01187199 | Active Recruiting |
Drug: Bevacizumab Drug: Temsirolimus |
Advanced Cancer | M.D. Anderson Cancer Center | August 19, 2010 | Phase 1 |
NCT01396408 | Active Recruiting |
Drug: Sunitinib Drug: Temsirolimus |
Advanced Rare Tumours | Canadian Cancer Trials Group | February 9, 2012 | Phase 2 |
Minimal toxicity of CCI-779 (Temsirolimus) in NOD/SCID mice.Blood.2004 Dec 15;104(13):4181-7. td> |
Antitumor effect of CCI-779.Blood.2004 Dec 15;104(13):4181-7. td> |
Antitumor effects of CCI-779.Blood.2004 Dec 15;104(13):4181-7. td> |
Antiangiogenic effects of CCI-779.Blood.2004 Dec 15;104(13):4181-7. |
CCI-779 induces myeloma cell apoptosis. CCI-779''''s effects on p70S6kinase phosphorylation and cell-cycle regulatory proteins in vivo.Blood.2004 Dec 15;104(13):4181-7. |
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