Ridaforolimus (Deforolimus, MK-8669)

Treatment of HT-1080 cells with Deforolimus induces a dose-dependent inhibition of phosphorylation of both S6 and 4E-BP1, with IC50 of 0.2 nM and 5.6 nM, respectively, and leads to a decrease in cell size, an increase in the proportion of cells in the G1 phase of the cell cycle, and inhibition of glucose uptake. Deforolimus displays significant antiproliferative activity a broad panel of cell lines with EC50 of 0.2-2.3 nM. Deforolimus potently and selectively inhibits VEGF production in a dose-dependent manner. ^[1] Deforolimus treatment significantly induces growth suppression in human NSCLC cell lines with IC30 values of 2.45-8.83 nM, with the exception of H157 with IC30 of >20 nM. Deforolimus treatment (2.8-5.9 nM) significantly dephosphorylates p70S6K^Thr389 in A549, H1703 and H157 cells, except H1666 that may express a resistant variant of mTORC1, and causes increased phosphorylation of pAKT^ser473 and pAKT^Thr308 in A549 and H1703 cells. Deforolimus in combination with the MEK inhibitors, CI-1040 or PD0325901 exhibits dose-dependent synergism in lung cancer cell lines, which is associated with the suppression of proliferation rather than enhancement of cell death, involving the inhibition of ribosomal biogenesis by 40% within 24 hours and a decreased polysome/monosome ratio. ^[2]

Administration of Deforolimus exerts significant antitumor effects in mice bearing PC-3 (prostate), HCT-116 (colon), MCF7 (breast), PANC-1 (pancreas) or A549 (lung) xenografts in a dose-dependent manner, and inhibits mTOR signaling in in SK-LMS-1 xenograft model associated with inhibition of tumor growth. ^[1]

• Cell based target inhibition

  HT-1080 cells are treated with increasing concentrations of Deforolimus (0-100 nM) for 2 hours, prior to harvest. Cellular lysates are extracted in denaturing lysis buffer, resolved on SDS-PAGE and transferred to PVDF membranes. After blocking, membranes are incubated with primary antibodies for 1 hour, followed by appropriate HRPconjugated secondary antibodies for 1 hour at room temperature. Immunoreactive proteins are detected using enhanced chemiluminescence and autoradiography performed by exposure to X-ray film. IC50 is determined from the inhibition of levels of phosphorylated ribosomal protein S6 (p-S6) and 4E-BP1 (p-4E-BP1).

• Cell lines

  Colo205, H1755, H1395, H1666, A549, H157, and H1703 cells

• Concentrations

  Dissolved in ethanol, final concentrations ~ 1 μM

• Incubation Time

  72-120 hours

• Method

  Cells are seeded at 2-3 × 10⁴/mL, and serial dilutions of Deforolimus are added after 2 hours, for at least three cell doublings (72-120 hours). Deforolimus effects are measured using the CellTiter 96 Aqueous nonradioactive cell proliferation assay and Sulforhodamine B assays. For Deforolimus, growth effects are described as IC30 because rapamycin and its derivatives do not significantly impede cell proliferation.

• Animal Models

  Male and female athymic NCr-nu mice with xenografts established by subcutaneous implantation of PC-3, A549, HCT-116, MCF7, PANC-1 and SK-LMS-1 tumors

• Dosages

  ~10 mg/kg

• Administration

  Intraperitoneally