Everolimus (RAD001)

Catalog No.S1120 Synonyms: SDZ-RAD

For research use only.

Everolimus (RAD001, SDZ-RAD) is an mTOR inhibitor of FKBP12 with IC50 of 1.6-2.4 nM in a cell-free assay. Everolimus induces cell apoptosis and autophagy and inhibits tumor cells proliferation.

Everolimus (RAD001) Chemical Structure

CAS No. 159351-69-6

Selleck's Everolimus (RAD001) has been cited by 681 publications

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Biological Activity

Description Everolimus (RAD001, SDZ-RAD) is an mTOR inhibitor of FKBP12 with IC50 of 1.6-2.4 nM in a cell-free assay. Everolimus induces cell apoptosis and autophagy and inhibits tumor cells proliferation.
Targets
FKBP12 [1]
(Cell-free assay)
mTOR (FKBP12) [1]
(Cell-free assay)
1.6-2.4 nM 1.6 nM-2.4 nM
In vitro

Everolimus exhibits the immunosuppressive activity which is comparable to that of rapamycin. Everolimus competes with immobilized FK 506 for binding to biotinylated FKBP12 and shows the inhibitory effect on a two-way MLR performed with spleen cells from BALB/c and CBA mice with IC50 of 0.12-1.8 nM. [1] Everolimus also shows antiangiogenic/vascular effects in VEGF-induced HUVEC proliferation with IC50 of 0.12 nM and bFGF-induced HUVEC proliferation with IC50 of 0.8 nM, respectively. [2] A recent study shows that Everolimus shows a dose-dependent inhibitory effects on both the total cells and the stem cells from the BT474 cell line and the primary breast cancer cells with IC50 of 156 nM in total cells of primary breast cancer cells and 71 nM in total cells of BT474 cells. In addition, combination treatment with Everolimus and trastuzumab produces the significantly increased inhibition on the growth of cancer stem cells with the inhibition rate increased by more than 50 %. [3]

Cell Data
Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
SQ20B NIjkSmZEgXSxdH;4bYMhSXO|YYm= MXe3NkBp NGDzbWlFVVOR MljXTWM2OD13LkWg{txO NYjufmNPRGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC|Oj:vdJVjdWWmLn7jZokvdmyvLn7pbE5od3ZxMkS0OFU{OTFpPkK0OFQ2OzFzPD;hQi=>
Colo205 MmXnR5l1d3SxeHnjJGF{e2G7 MVO3NkBp M2Lp[2ROW09? MUTJR|UxRTJyIN88US=> M4j1UFxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJ2NES1N|EyLz5{NES0OVMyOTxxYU6=
ColoR NH7nSmlEgXSxdH;4bYMhSXO|YYm= NFHydXY4OiCq MnXFSG1UVw>? M1LFZ2lEPTB;OD63JO69VQ>? MYW8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zPDR2NUOxNUc,OjR2NEWzNVE9N2F-
HCT116 MoDTR5l1d3SxeHnjJGF{e2G7 MXW3NkBp NEXaZnVFVVOR NW[3dYFtUUN3ME2xNkDPxE1? M4nJUlxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJ2NES1N|EyLz5{NES0OVMyOTxxYU6=
HT29 M{n4[2N6fG:2b4jpZ{BCe3OjeR?= MV23NkBp NULLSFFHTE2VTx?= Ml3nTWM2OD1zNTFOwG0> M4HLWlxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJ2NES1N|EyLz5{NES0OVMyOTxxYU6=
CAKI1 NEDqV4xEgXSxdH;4bYMhSXO|YYm= NF;mSIU4OiCq MXjEUXNQ NEPKN|ZKSzVyPUG0JO69VQ>? MUK8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zPDR2NUOxNUc,OjR2NEWzNVE9N2F-
SK-HEP1 MV7DfZRwfG:6aXOgRZN{[Xl? NH;JNmo4OiCq MVLEUXNQ M1jMTGlEPTB;MUKg{txO MWW8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zPDR2NUOxNUc,OjR2NEWzNVE9N2F-
DU145 MYXDfZRwfG:6aXOgRZN{[Xl? NFH3cGM4OiCq NVGxT4N3TE2VTx?= M4DH[WlEPTB;ODFOwG0> NYjFTFJ[RGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC|Oj:vdJVjdWWmLn7jZokvdmyvLn7pbE5od3ZxMkS0OFU{OTFpPkK0OFQ2OzFzPD;hQi=>
OVCAR3 MWHDfZRwfG:6aXOgRZN{[Xl? NVXmN3B{PzJiaB?= MVnEUXNQ M2CwN2lEPTB;MU[g{txO MYq8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zPDR2NUOxNUc,OjR2NEWzNVE9N2F-
HOP62 MonPR5l1d3SxeHnjJGF{e2G7 MlToO|IhcA>? M1qxZmROW09? M2\ZUmlEPTB;MUmg{txO M1jaN|xiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJ2NES1N|EyLz5{NES0OVMyOTxxYU6=
Colo205 NWGyZ41yTnWwY4Tpc44hSXO|YYm= MX[yOEBp NXSxUlRRTE2VTx?= NI\IN|NKdmirYnn0d{BuXE:UQ{GgbY4hcHWvYX6gR29NVzJyNTDj[YxteyCjc4Pld5Nm\CCjczDy[YR2[3Srb36gc4YhWzZicHjvd5Bpd3K7bHH0bY9vKGG2IECuNUB1dyB6IIXN NVzRW|NORGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC|Oj:vdJVjdWWmLn7jZokvdmyvLn7pbE5od3ZxMkS4N|YxPzBpPkK0PFM3ODdyPD;hQi=>
Colo205 M1LUVGZ2dmO2aX;uJGF{e2G7 MVWyOEBp NEey[|dFVVOR MYrJcohq[mm2czDtWG9TSzFiaX6gbJVu[W5iQ1;MU|IxPSClZXzsd{Bie3Onc4Pl[EBieyC{ZXT1Z5Rqd25ib3[gOE1GSlBzIIDoc5NxcG:{eXzheIlwdiCjdDCwMlEhfG9iODD1US=> NWPYXWk2RGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC|Oj:vdJVjdWWmLn7jZokvdmyvLn7pbE5od3ZxMkS4N|YxPzBpPkK0PFM3ODdyPD;hQi=>
SK-HEP1 MXzGeY5kfGmxbjDBd5NigQ>? NFjzTpMzPCCq MWXEUXNQ M4n2TmlvcGmkaYTzJI1VV1KFMTDpckBpfW2jbjDTT{1JTVBzIHPlcIx{KGG|c3Xzd4VlKGG|IILl[JVkfGmxbjDv[kBUPiCyaH;zdIhwenmuYYTpc44h[XRiMD6xJJRwKDhidV2= NXTRWGV2RGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC|Oj:vdJVjdWWmLn7jZokvdmyvLn7pbE5od3ZxMkS4N|YxPzBpPkK0PFM3ODdyPD;hQi=>
SK-HEP1 MVvGeY5kfGmxbjDBd5NigQ>? M370NFI1KGh? M1\tNmROW09? M2XRfmlvcGmkaYTzJI1VV1KFMTDpckBpfW2jbjDTT{1JTVBzIHPlcIx{KGG|c3Xzd4VlKGG|IILl[JVkfGmxbjDv[kA1NUWEUEGgdIhwe3Cqb4L5cIF1cW:wIHH0JFAvOSC2bzC4JJVO M3LaeVxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJ2OEO2NFcxLz5{NEizOlA4ODxxYU6=
Sf9 MofCSpVv[3Srb36gRZN{[Xl? NInqVZUyPSC2bzCyNEBucW6| NInNTXNKdmirYnn0bY9vKG:oIHj1cYFvKEKVRWCgc5ZmemW6cILld5Nm\CCrbjDT[lkh[2WubDDt[Y1jemGwZTD2[ZNq[2ynczDhd5Nme3OnZDDhd{B2eHSja3Wgc4YhYzOKXT30ZZVzd2Oqb3zheIUhcW5icILld4Vv[2Vib3[gRXRRKG2nYYP1doVlKGGodHXyJFE2KHSxIEKwJI1qdnNiYomgcYVu[nKjbnWgeoV{cWOuZTD0doFve3CxcoSgZZN{[XluIFnDOVAhRSB{IN88UU4> NI\JPYE9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{M{m1OlExOSd-MkO5OVYyODF:L3G+
Sf9 NIrXRWNHfW6ldHnvckBCe3OjeR?= NFfHdVUzOCCvaX7z NV72PIl6UW6qaXLpeIlwdiCxZjDoeY1idiCPUmCyJI93\XKneIDy[ZN{\WRiaX6gV4Y6KGOnbHygcYVu[nKjbnWgeoV{cWOuZYOgZZN{\XO|ZXSgZZMhfXC2YXvlJI9nKFt|SG2t[ZN1emGmaX;sMVE4[mW2YT3EMYdtfWO3cn;ubYRmKGmwIIDy[ZNmdmOnIH;mJGFVWCCjbnSgS3NJKG2nYYP1doVlKGGodHXyJFIxKG2rboOgZpkhdWWvYoLhcoUhfmW|aXPs[UB1emGwc4DvdpQh[XO|YYmsJGlEPTBiPTCxNU4{KM7:TT6= NUXGVpFJRGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC|Oj:vdJVjdWWmLn7jZokvdmyvLn7pbE5od3ZxMkO5OVYyODFpPkKzPVU3OTBzPD;hQi=>
A549 NHPiNXdCfXSxcHjh[5kh[XO|YYm= NGHm[nI2KHWP MX62JIg> M2XCVGlv\HWldHnvckBw\iCjdYTvdIhi\3liaX6gbJVu[W5iQUW0PUBk\WyuczDhd5Nme3OnZDDhd{BqdmO{ZXHz[UBqdiClb372[ZJ{cW:wIH;mJGxEOy1zIITvJGxEOy1{IHH0JFUhfU1iYX\0[ZIhPiCqcoOgZpkhX2W|dHXyckBjdG:2IHHuZYx6e2m| MXu8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zPjF5NkG2OUc,OjZzN{[xOlU9N2F-
A549 NWO3fWxHTnWwY4Tpc44hSXO|YYm= NFzDdow2KHWP M3[3cFMhfG9iMkSgbC=> NIrqNWlKdmirYnn0bY9vKG:oIH3UU3IheGixc4Doc5J6dGG2aX;uJIF1KHOnckK0OFghcW5iaIXtZY4hSTV2OTDj[YxteyCjdDC1JJVOKGGodHXyJFMhfG9iMkSgbJJ{KGK7IGfld5Rmem5iYnzveEBidmGueYPpdy=> NUP4cZpURGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC|Oj:vdJVjdWWmLn7jZokvdmyvLn7pbE5od3ZxMk[xO|YyPjVpPkK2NVc3OTZ3PD;hQi=>
A549 M{jod2Z2dmO2aX;uJGF{e2G7 NVvYWGRVPSC3TR?= NUPNXY5[OyC2bzCyOEBp M1TPb2lvcGmkaYTpc44hd2ZibWTPVkBqdiCqdX3hckBCPTR7IHPlcIx{KGG|c3Xzd4VlKGG|IHTve45z\We3bHH0bY9vKG:oIIC3NHM3UyCyaH;zdIhwenmuYYTpc44h[XRidHjyN|g6KGG2IEWgeW0h[W[2ZYKgN{B1dyB{NDDodpMh[nliV3XzeIVzdiCkbH;0JIFv[Wy7c3nz NH[4PJg9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{NkG3OlE3PSd-Mk[xO|YyPjV:L3G+
Assay
Methods Test Index PMID
Western blot Mcl-1 / p-ERK / S6K1(T389) ; pS6RP ; cleaved caspase-3 27351224 25014496
Growth inhibition assay Cell counts ; IC50 ; Cell proliferation 27127803 23509629 21135857
Immunofluorescence TERT 27127803
In vivo Everolimus (0.1 to 10 mg/kg) dose-dependently inhibits growth of the primary (ear) and lymph node metastases of B16/BL6 melanoma, with decreased total number of vessels and reduced mature vessels. [2] In a xenograft animal model of BT474 stem cells, Everolimus shows significant reductions in mean tumor sizes (590.6 mm3), compared to the control group with a tumor size of 698 mm3. Furthermore, combination treatment with Everolimus and trastuzumab significantly decreases the xenograft tumor size (410.8 mm3) more than Everolimus treatment alone. [3]

Protocol (from reference)

Kinase Assay:[1]
  • FKBP12 binding assay & Mixed lymphocyte reaction (MLR) :

    FKBP12 binding assay: Binding to the FK 506 binding protein (FKBP12) is indirectly assessed by means of an ELISA-type competition assay. FK 506 is included in each individual experiment as a standard, and the inhibitory activity is expressed as relative IC50 compared to FK 506 (rIC50 = IC50 Everolimus/IC50 FK 506). Mixed lymphocyte reaction (MLR): The immunosuppressive activities of RAP and its derivatives are assessed in a two-way MLR, using spleen cells of BALB/c and CBA mice. RAP is included in each individual experiment as a standard, and the inhibitory activity is expressed as relative IC50 compared to RAP (rIC50 = IC50 Everolimus/IC50 RAP).

Cell Research:[3]
  • Cell lines: BT474 cell line and the primary breast cancer cells
  • Concentrations: 0.001-10 μM
  • Incubation Time: 24 hours
  • Method: The 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction assay (MTT assay) is used to compare the effects of Everolimus or trastuzumab on total breast cancer cells and breast CSCs. The total cells and the stem cells from the BT474 cell line and the primary breast cancer cells are respectively seeded into 96-well plates with different concentrations of the drugs, with five wells for each concentration, and the cells are cultured at 37 °C with 5 % CO2 in an incubator for 24 hours. The concentrations of Everolimus are 1 nM, 10 nM, 100 nM, 1 μM and 10 μM, and the concentrations of trastuzumab are 0.5 μg/mL, 1 μg/mL, 10 μg/mL, 50 μg/mL, and 100 μg/mL. The combinatorial inhibitory effect of Everolimus and Trastuzumab on the in vitro growth of breast CSCs is examined by MTT assay as well using 10 μg/mL trastuzumab in combination of increasing concentrations of everolimus (1 nM, 10 nM, 100 nM and 1 μM). After drug treatment for 24 hours, 20 μL MTT [5 mg/mL in phosphate buffered saline (PBS)] is added to each well, and the cells are incubated at 37 °C with 5 % CO2 and saturated humidity for 4 hours. Following the subsequent removal of the supernatant, 150 μL dimethyl sulfoxide (DMSO) is added to each well, and the cells are vortexed for 10 minutes. The light absorbance (OD value) is measured for each well using an ELISA reader. Each experiment is repeated in triplicate, and dose–response curves are plotted. The probit software of the statistical software package SPSS 17.0 for Windows is used to calculate the inhibitory concentration (IC50) of Everolimus.
Animal Research:[3]
  • Animal Models: Cultured BT474 stem cells are injected beneath the left breast pad of BALB/c nude mice.
  • Dosages: ≤2 mg/kg
  • Administration: Administered via p.o.

Solubility (25°C)

In vitro

In vivo

Add solvents to the product individually and in order
(Data is from Selleck tests instead of citations):
30% Propylene glycol (dissolve first)+5% Tween 80+ddH2O
For best results, use promptly after mixing.

5mg/mL

Chemical Information

Molecular Weight 958.22
Formula

C53H83NO14

CAS No. 159351-69-6
Storage 3 years -20°C powder
2 years -80°C in solvent
Smiles CC1CCC2CC(C(=CC=CC=CC(CC(C(=O)C(C(C(=CC(C(=O)CC(OC(=O)C3CCCCN3C(=O)C(=O)C1(O2)O)C(C)CC4CCC(C(C4)OC)OCCO)C)C)O)OC)C)C)C)OC

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)

mg/kg g μL

Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)

% DMSO % % Tween 80 % ddH2O
%DMSO %

Calculation results:

Working concentration: mg/ml;

Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid 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 master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O, mix and clarify.

Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such
as vortex, ultrasound or hot water bath can be used to aid dissolving.

Molarity Calculator

Mass Concentration Volume Molecular Weight

Clinical Trial Information

NCT Number Recruitment Interventions Conditions Sponsor/Collaborators Start Date Phases
NCT04485559 Recruiting Drug: Everolimus|Drug: Trametinib Recurrent World Health Organization (WHO) Grade II Glioma University of California San Francisco|Novartis Pharmaceuticals|Pediatric Brain Tumor Foundation|The Lilabean Foundation for Pediatric Brain Cancer Research December 9 2020 Phase 1
NCT02860494 Withdrawn Drug: Everolimus|Drug: Placebo Facial Angiofibromas Hospices Civils de Lyon December 2020 Phase 2|Phase 3
NCT04305444 Recruiting Drug: DTRM-555 Relapsed Chronic Lymphocytic Leukemia|Refractory Chronic Lymphocytic Leukemia|Diffuse Large B Cell Lymphoma|Follicular Lymphoma|Richter''s Transformation Zhejiang DTRM Biopharma April 24 2020 Phase 2

(data from https://clinicaltrials.gov, updated on 2022-01-17)

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

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Frequently Asked Questions

Question 1:
For the in vivo work, I know the drug needs to be dissolved in 30% propylene glycol (dilution in water) and 5%Tween 80. Would the final solution be a clear liquid or a turbid suspension?

Answer:
Our S1120 Everolimus (RAD001) in 30% Propylene glycol+5% Tween 80+ddH2O at 5mg/ml is a clear solution. And for oral gavage, there is another common vehicle, 1% CMC Na. Everolimus can be dissolved in it at 30mg/ml as a suspension.

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