Everolimus (RAD001) 化学構造
分子量: 958.22

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製品説明

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製品の説明

生物活性

製品説明 Everolimus (RAD001) は、1.6-2.4nMのIC50によるFKBP12のmTOR阻害剤です。
ターゲット mTOR (FKBP12)
IC50 1.6-2.4 nM [1]
In vitro試験 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 LinesAssay TypeConcentrationIncubation TimeFormulationActivity DescriptionPMID
SQ20B M{TUXWN6fG:2b4jpZ{BCe3OjeR?= NILGRmI4OiCq M1v5SWROW09? MWHJR|UxRTVwNTFOwG0> NHTLbZMzPDR2NUOxNS=>
Colo205 MknrR5l1d3SxeHnjJGF{e2G7 NWjueYhNPzJiaB?= M2\ObWROW09? NF;NNlhKSzVyPUKwJO69VQ>? MWWyOFQ1PTNzMR?=
ColoR NIS1TXJEgXSxdH;4bYMhSXO|YYm= M{TXeFczKGh? NXHDcIpYTE2VTx?= NGXKXm5KSzVyPUiuO{DPxE1? NYroT2hnOjR2NEWzNVE>
HCT116 MX\DfZRwfG:6aXOgRZN{[Xl? NWDGdWJOPzJiaB?= M{TCWmROW09? MVLJR|UxRTF{IN88US=> NEflWYQzPDR2NUOxNS=>
HT29 MlzHR5l1d3SxeHnjJGF{e2G7 MkLiO|IhcA>? MWfEUXNQ Mn\GTWM2OD1zNTFOwG0> NFXIcoQzPDR2NUOxNS=>
CAKI1 MXrDfZRwfG:6aXOgRZN{[Xl? MUK3NkBp M3jhZWROW09? MVjJR|UxRTF2IN88US=> NIjn[WczPDR2NUOxNS=>
SK-HEP1 MmjjR5l1d3SxeHnjJGF{e2G7 NUjlNW01PzJiaB?= NGXhTYNFVVOR MXPJR|UxRTF{IN88US=> NHH1WWEzPDR2NUOxNS=>
DU145 MnvQR5l1d3SxeHnjJGF{e2G7 NIi0RpA4OiCq NY\FW2hDTE2VTx?= NFfFdJFKSzVyPUig{txO NXTQXFczOjR2NEWzNVE>
OVCAR3 MmjKR5l1d3SxeHnjJGF{e2G7 NHHiTmU4OiCq NYXqe|JKTE2VTx?= MlfKTWM2OD1zNjFOwG0> MmHQNlQ1PDV|MUG=
HOP62 M3fTcWN6fG:2b4jpZ{BCe3OjeR?= M3fRSFczKGh? NXzzV3RDTE2VTx?= M1LyNGlEPTB;MUmg{txO MmXCNlQ1PDV|MUG=
Colo205 NXLPeXVQTnWwY4Tpc44hSXO|YYm= NGXofnYzPCCq NXLze2g{TE2VTx?= MWXJcohq[mm2czDtWG9TSzFiaX6gbJVu[W5iQ1;MU|IxPSClZXzsd{Bie3Onc4Pl[EBieyC{ZXT1Z5Rqd25ib3[gV|YheGixc4Doc5J6dGG2aX;uJIF1KDBwMTD0c{A5KHWP NEnoSIQzPDh|NkC3NC=>
Colo205 MUTGeY5kfGmxbjDBd5NigQ>? MX6yOEBp NFXuWI1FVVOR MoDXTY5pcWKrdIOgcXRQWkNzIHnuJIh2dWGwIFPPUG8zODViY3XscJMh[XO|ZYPz[YQh[XNicnXkeYN1cW:wIH;mJFQuTUKSMTDwbI9{eGixconsZZRqd25iYYSgNE4yKHSxIEigeW0> NHzDb3EzPDh|NkC3NC=>
SK-HEP1 NGLyTHRHfW6ldHnvckBCe3OjeR?= M3zHUlI1KGh? M{[xbWROW09? NGmwenVKdmirYnn0d{BuXE:UQ{GgbY4hcHWvYX6gV2suUEWSMTDj[YxteyCjc4Pld5Nm\CCjczDy[YR2[3Srb36gc4YhWzZicHjvd5Bpd3K7bHH0bY9vKGG2IECuNUB1dyB6IIXN MXmyOFg{PjB5MB?=
SK-HEP1 NG\mcGlHfW6ldHnvckBCe3OjeR?= M1Pzb|I1KGh? NWXvfXM1TE2VTx?= M2HLVGlvcGmkaYTzJI1VV1KFMTDpckBpfW2jbjDTT{1JTVBzIHPlcIx{KGG|c3Xzd4VlKGG|IILl[JVkfGmxbjDv[kA1NUWEUEGgdIhwe3Cqb4L5cIF1cW:wIHH0JFAvOSC2bzC4JJVO Mm\VNlQ5OzZyN{C=

... Click to View More Cell Line Experimental Data

In vivo試験 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]
臨床試験 Everolimus is currently in Phase I clinical trials in patients with Unspecified Adult Solid Tumor.
特集

プロトコル (参考用のみ)

キナーゼアッセイ: [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).

細胞アッセイ: [3]

細胞株 BT474 cell line and the primary breast cancer cells
濃度 0.001-10 μM
反応時間 24 hours
実験の流れ 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.

動物実験: [3]

動物モデル Cultured BT474 stem cells are injected beneath the left breast pad of BALB/c nude mice.
製剤 Everolimus is dissolved in saline.
投薬量 ≤2 mg/kg
投与方法 Administered via p.o.

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

SpeciesMouseRatRabbitGuinea pigHamsterDogMonkeyBaboon
Weight (kg)0.020.151.80.40.0810312
Body Surface Area (m2)0.0070.0250.150.050.020.50.240.6
Km factor361285201220
Animal A (mg/kg) = Animal B (mg/kg) multiplied by  Animal B Km
Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Rat dose (mg/kg) = mouse dose (22.4 mg/kg) ×  mouse Km(3)  = 11.2 mg/kg
rat Km(6)
1

参考

化学情報

Download Everolimus (RAD001) SDF
分子量 958.22
化学式

C53H83NO14

CAS No. 159351-69-6
保管 2年-20℃
6月-80℃in solvent
別名 N/A
溶解度 (25°C) * In vitro DMSO 30 mg/mL (31.3 mM)
エタノール 7 mg/mL (7.3 mM)
<1 mg/mL (<1 mM)
In vivo 30% Propylene glycol (dissolve first)+5% Tween 80+ddH2O 5mg/mL
* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
化学名 23,27-Epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine, rapamycin deriv

カスタマーフィードバック (5)


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Rating
Source Cancer Cell, 2012, 21(2), 155-67. Everolimus (RAD001) purchased from Selleck
Method Western blotting
Cell Lines MP tumor cells
Concentrations 1, 5 uM
Incubation Time 3 h
Results To confirm that these compounds were acting on the PI3K/mTOR pathway, it performed western blotting to analyze phosphorylation of critical proteins in the pathway. As shown in figure, MP tumor cells showed substantial amounts of phospho-AKT and phospho-S6 in the absence of inhibitors (DMSO lanes). RAD-001 inhibited S6 phosphorylation but did not affect phospho-AKT. In contrast with RAD-001, treatment with BEZ-235 or BKM-120 inhibited phosphorylation of both AKT and S6.

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Rating
Source Clin Cancer Res, 2013, 19(3),598-609. Everolimus (RAD001) purchased from Selleck
Method Immunoblotting
Cell Lines K028, M34, K029 cells
Concentrations 0.25, 0.5, 1 uM
Incubation Time 30 min
Results Treatment of the BRAFi-resistant melanoma cells with the mTOR inhibitor RAD001 did lead to substantial decrease of S6 phosphorylation in the 3 BRAFi-resistant lines K028PR, M34PR, and K029PR; however, such an inhibition failed to elicit any inhibitory effect on the expression of PD-L1.

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Rating
Source Biochem Pharmacol, 2011, 82, 216-226. Everolimus (RAD001) purchased from Selleck
Method Western blot
Cell Lines H1299 cells
Concentrations 5 μM
Incubation Time 48 h
Results RAD001 and other mTOR inhibitors decreased the levels of survivin protein, as assessed by Western blot analysis, without affecting the levels of other IAP members. In addition, combined treatment with sorafenib and mTOR inhibitor Rapamycin and RAD001 decreased survivin expression to a greater extent than treatment with either alone.

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Rating
Source Br J Cancer, 2014, 111(6), 1168-79. Everolimus (RAD001) purchased from Selleck
Method Fluorescent staining
Cell Lines 786-O SuR cells
Concentrations 1 uM
Incubation Time 24 h
Results Actin-based structures were revealed by phalloidin, whereas localisation of focal adhesion points was achieved by fluorescent staining of p-paxillin. β-actin and p-paxillin were well organised in untreated cells; instead, the RAD-001 treatment was able to mildly disassemble cytoskeleton organisation and focal adhesion points formation. These effects were much more evident with the combination treatment with NVP-LDE225.

Click to enlarge
Rating
Source Aging (Albany NY), 2012, 4(2), 119-32. Everolimus (RAD001) purchased from Selleck
Method Fluorescence microscopy
Cell Lines HGPS fibroblast cells
Concentrations 0.1 uM
Incubation Time 30 min
Results To examine the effects on nuclear morphology, it labeled cells with an antibody for lamin A/C and an antibody specific for progerin. At least 200 randomly selected cells were scored by fluorescence microscopy for cells under each condition. The rapamycin or RAD001 treated HGPS cells exhibited a clear reduction in nuclear blebbin.

文献中の引用 (49)

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Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
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