化合物信息

Description

Soft Tissue Sarcoma,Multiple myeloma,Cancer

Description

Temsirolimus (CCI-779, Torisel) is a specific mTOR inhibitor with IC50 of 1.76 μM.

Targets

IC₅₀

In Vitro

Temsirolimus inhibits the phosphorylation of ribosomal protein S6, more potently in PTEN-positive DU145 cells than in PTEN-negative PC-3 cells, and inhibits cell growth and clonogenic survival of both cells in a concentration-dependent manner. ^[5] Temsirolimus (100 ng/mL) potently inhibits proliferation and induces apoptosis in primary human lymphoblastic leukemia (ALL) cells. ^[6] 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)="" displays="" a="" modest="" and="" selective="" antiproliferative="" activity="" via="" fkbp12-dependent="" mechanism,="" but="" can="" completely="" inhibit="" the="" proliferation="" of="" a="" broad="" panel="" of="" tumor="" cells="" at="" low="" micromolar="" concentrations="" (5-15="" μm),="" involving="" fkbp12-independent="" suppression="" of="" mtor="" signaling.="" temsirolimus="" treatment="" at="" micromolar="" but="" not="" nanomolar="" concentrations="" (20="" μm)="" causes="" a="" marked="" decline="" in="" global="" protein="" synthesis="" and="" disassembly="" of="" polyribosomes,="" accompanied="" by="" rapid="" increase="" in="" the="" phosphorylation="" of="" translation="" elongation="" factor="" eef2="" and="" the="" translation="" initiation="" factor="" eif2a.="">^[1]

In Vivo

Administration of Temsirolimus (20 mg/kg i.p. 5 days/week) significantly delays the growth of DAOY xenografts by 160% after 1 week and 240% after 2 weeks, compared with controls. Single high-dose of Temsirolimus (100 mg/kg i.p) treatment induces 37% regression of tumor volume within 1 week. Temsirolimus treatment for 2 weeks also delays the growth of rapamycin-resistant U251 xenografts by 148%. ^[2] Inhibition of mTOR by Temsirolimus improves performance on four different behavioral tasks and decreases aggregate formation in a mouse model of Huntington disease. ^[3] Administration of Temsirolimus induces significant dose-dependent, antitumor responses against subcutaneous growth of 8226, OPM-2, and U266 xenografts with ED50 of 20 mg/kg and 2 mg/kg for 8226 and OPM-2, respectively, which are associated with inhibited proliferation and angiogenesis, induction of apoptosis, and reduction in tumor cell size. ^[4] 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 ^[6]

Clinical Trials

A Phase IV study of two different doses of Temsirolimus in patients with mantle cell lymphoma is currently ongoing.

Features

Description

Administration of Temsirolimus (20 mg/kg i.p., 5 days/week for 2 weeks) in combination with cisplatin (5 mg/kg i.p., day 1) induces 1.3 times greater growth inhibition of DAOY xenografts than cisplatin treatment alone. ^[2] Low doses of Temsirolimus (5 mg/kg or 10 mg/kg, 3 days per week for 3 weeks) given between courses of mitoxantrone (1.5 mg/kg) or docetaxe (10 mg/kg) treatment potently increases the growth delay of PC-3 tumors. ^[5] A Phase I study of Temsirolimus plus capecitabine in patients with advanced cancer is currently ongoing.

In vitro assay of mTOR catalytic activity

The Flag-tagged wild-type human mTOR (Flag-mTOR) DNA constructs are transiently transfected into HEK293 cells. Protein extraction and purification of Flag-mTOR are carried out 48 hours later. In vitro kinase assays of purified Flag-mTOR in the presence of various concentrations of Temsirolimus without FKBP12 are performed in 96-well plate and detected by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) using His6-S6K1 as the substrate. Enzymes is 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). To each well, 12 μL of the diluted enzyme is mixed briefly with 0.5 μL Temsirolimus. The kinase reaction is initiated by adding 12.5 μL kinase assay buffer containing ATP and His6-S6K to give a final reaction volume of 25 μL containing 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 and then terminated by adding 25 μL Stop buffer (20 mM Hepes (pH 7.4), 20 mM EDTA, and 20 mM EGTA). The DELFIA detection of the phosphorylated (Thr-389) His6-S6K is performed at room temperature using a monoclonal anti-P(T389)-p70S6K antibody labeled with Europium-N1-ITC (Eu) (10.4 Eu per antibody). 45 μL of the terminated kinase reaction mixture is transferred to a MaxiSorp plate containing 55 μL PBS. The His6-S6K is allowed to attach for 2 hours after which the wells are aspirated and washed once with PBS. 100 μL of DELFIA buffer with 40 ng/mL Eu-P(T389)-S6K antibody is added. The antibody binding is continued for 1 hour with gentle agitation. The wells are then aspirated and washed four times with PBS containing 0.05% Tween 20 (PBST). 100 μL of DELFIA Enhancement solution is added to each well and the plates are read in a PerkinElmer Victor model plate reader.

Cell Lines

A549, H157, H460, H446, HCT116, HT29, SW480, DLD1, Caco2, LNCap, DU145, MDA468, MDA231, HEK293, and PC3-MM2

Concentrations

Dissolved in DMSO, final concentrations ~20 μM

Incubation Time

72 hours

Methods

Cells are exposed to various concentrations of Temsirolimus for 72 hours. After treatment, viable cell densities are determined by MTS dye conversion using CellTiter AQ assay kit.

Animal Models

Female athymic nude mice injected s.c. with DAOY, or U251 cells

Formulation

Prepared in 100% EtOH as a 50 mg/mL stock solution, and diluted in 5% Tween 80 and 5% polyethylene glycol 400

Doses

20 mg/kg

Administration

Injection daily 5 times per week