Temsirolimus

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

Rapamycin, 42-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]

Dissolved in DMSO, final concentrations ca.20 uM

20 mg/kg

1.76 M [1], 1.76 M [1], 1.76 M [1], 1.76 M [1], 1.76 M [1], 1.76 M [1]

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] 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%. [4] Inhibition of mTOR by Temsirolimus improves performance on four different behavioral tasks and decreases aggregate formation in a mouse model of Huntington disease. [5] 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. [6]

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 beta-glycerophosphate, 10 mM MnCl2, 0.5 mM DTT, 0.25 uM microcystin LR, and 100 ug/mL BSA). To each well, 12 uL of the diluted enzyme is mixed briefly with 0.5 uL Temsirolimus. The kinase reaction is initiated by adding 12.5 uL kinase assay buffer containing ATP and His6-S6K to give a final reaction volume of 25 uL containing 800 ng/mL FLAG-mTOR, 100 uM ATP, and 1.25 uM 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 uL 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 uL of the terminated kinase reaction mixture is transferred to a MaxiSorp plate containing 55 uL PBS. The His6-S6K is allowed to attach for 2 hours after which the wells are aspirated and washed once with PBS. 100 uL 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 uL of DELFIA Enhancement solution is added to each well and the plates are read in a PerkinElmer Victor model plate reader.

1030, 29

Data independently produced by, , , Dr. Zhang, of Tianjin Medical University, Temsirolimus (Torisel)purchased from Selleck, Breast cancer cells were pretreated with 100ng/ml EGF for 15 min and then treated with the indicated concentrations of Temsirolimus for 24 hours.

2 years -20CPowder, 2 weeks4Cin DMSO, 2 months-80Cin DMSO