mTOR (IC50 = 5-6 nM)

依维莫司 (RAD001) 是一种口服活性雷帕霉素衍生物，可抑制 Ser/Thr 激酶 mTOR[1]。抗增殖浓度的依维莫司导致敏感的鼠 B16/BL6 黑色素瘤 (IC50, 0.7 nM) 和不敏感的人宫颈 KB-31 (IC50, 1,778 nM) 中 S6K1 和底物 S6 完全去磷酸化以及活动性的变化4E-BP1，这表明磷酸化状态降低[3]。尽管依维莫司在不同程度上抑制了 BT474 细胞系的总细胞、干细胞和原发性乳腺癌细胞的生长。与细胞总数相比，依维莫司在所有测试浓度下抑制干细胞生长的效果较差（P<0.001）。依维莫司对 BT474 和原代 CSC 的 IC50 分别为 2,054 和 3,227 nM，分别比相应总细胞的 IC50 高 29 和 21 倍[4]。

依维莫司在小鼠和大鼠中均具有口服活性，产生抗肿瘤作用，其特征是肿瘤生长速率显着降低，而不是产生肿瘤消退。依维莫司（0.5 或 2.5 mg/kg）每日治疗以剂量依赖性方式抑制大鼠 CA20498 模型中的肿瘤生长，间歇性施用较高剂量 5 mg/kg（每周一次或两次）也表现出相当的抗肿瘤功效。依维莫司抑制不伴随任何体重减轻，其特点是持续抑制而不是消退[1]。依维莫司治疗（0.1-10 mg/kg/d）具有不同于 PTK/ZK 治疗（100 mg/kg）的选择性作用。当存在任一生长因子时，依维莫司会增加血红蛋白含量，血红蛋白含量是衡量血管数量及其渗漏性的指标，当转换为血液当量时。然而，依维莫司会降低 Tie-2 含量，这对 VEGF 刺激很重要，但对 bFGF 刺激则不重要。根据依维莫司在小鼠体内的药代动力学，血浆水平仅在约 4 小时内达到 1 至 3 μM，而单次给药后在人类肿瘤异种移植物中的最高水平仅为 0.1 M[3]。

FKBP12 结合测定：ELISA 式竞争测定用于无意中测量与 FK 506 结合蛋白 (FKBP12) 的结合。每个实验均使用 FK 506 作为标准，抑制活性表示为与 FK 506 相比的相对 IC50（rIC50 = IC50 依维莫司/IC50 FK 506）。使用 BALB/c 和 CBA 小鼠的脾细胞，观察免疫抑制效果RAP 及其衍生物的检测采用双向混合淋巴细胞反应 (MLR)。由于 RAP 在每个实验中用作标准，因此抑制活性表示为与 RAP 相比的相对 IC50（rIC50 = IC50 依维莫司/IC50 RAP）。

在 96 孔板中，肿瘤细胞的铺板密度为 500 至 5,000/100 μL/孔。然后以最佳细胞密度（通常每孔 1,000 至 2,000 个细胞）进行重复实验，并孵育过夜。在细胞暴露于依维莫司并孵育 4 天后，使用亚甲蓝染色对细胞进行计数。为此，向孔中注入 50 μL [20% (v/v)] 戊二醛，并在室温下静置 10 分钟。吸出培养基、用蒸馏水洗涤细胞并添加染料后，在 37°C 下孵育 100 L 亚甲基蓝 [0.05% (w/v) 水溶液] 10 分钟。

Mice: Everolimus, PTK/ZK, and their respective vehicles are prepared each day just before administration to animals and the administration volume is individually adjusted based on animal body weight. Everolimus is given to C57/BL6 mice at doses ranging from 0.1 to 10 mg/kg/d orally (10 mL/kg), with 2.5 to 10 mg/kg being the most frequently used dose because it has the greatest impact. PTK/ZK is given orally at a dose of 50 to 100 mg/kg/d.
Rats: Based on body weight, Wistar-Furth rats are divided into two equal groups and given either a control dose of the drug or Everolimus (10 mg/kg/d orally in mice and 5 mg/kg three times per week orally in rats). Everolimus or vehicle is given orally by gavage (10 mL/kg) for a maximum of 7 days, with subsequent magnetic resonance measurements taken within 30 minutes of the last dose. This is done immediately after the initial measurement at baseline (day 0).

[1]. Biomarker Development for the Clinical Activity of the mTOR Inhibitor Everolimus (RAD001): Processes,Limitations, and Further Proposals. Transl Oncol. 2010 Apr;3(2):65-79.

[2]. Dual inhibition of the mTORC1 and mTORC2 signaling pathways is a promising therapeutic target for adult T-cell leukemia. Cancer Sci. 2018 Jan;109(1):103-111.

[3]. mTOR inhibitor RAD001 (everolimus) has antiangiogenic/vascular properties distinct from a VEGFR tyrosine kinase inhibitor. Clin Cancer Res, 2009, 15(5), 1612-1622.

[4]. Antitumor effect of the mTOR inhibitor Everolimus on human breast cancer stem cells in vitro and in vivo. Tumour Biol. 2012 Oct;33(5):1349-62.

Everolimus is a macrocyclic lactone that is rapamycin in which the hydroxy group attached to the cyclohexyl moiety has been converted into the corresponding 2-hydroxyethyl ether. It is an immunosuppressant and antineoplastic agent. It has a role as an antineoplastic agent, an immunosuppressive agent, a mTOR inhibitor, an anticoronaviral agent and a geroprotector. It is a primary alcohol, a secondary alcohol, an ether, a cyclic ketone, a cyclic acetal and a macrolide lactam. It is functionally related to a member of sirolimus.

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Everolimus is a derivative of Rapamycin (sirolimus), and works similarly to Rapamycin as an mTOR (mammalian target of rapamycin) inhibitor. It is currently used as an immunosuppressant to prevent rejection of organ transplants. In a similar fashion to other mTOR inhibitors Everolimus' effect is solely on the mTORC1 protein and not on the mTORC2 protein.

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Everolimus is a Kinase Inhibitor and mTOR Inhibitor Immunosuppressant. The mechanism of action of everolimus is as a Protein Kinase Inhibitor and Cytochrome P450 3A4 Inhibitor and Cytochrome P450 2D6 Inhibitor and mTOR Inhibitor. The physiologic effect of everolimus is by means of Decreased Immunologic Activity.

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Everolimus is an inhibitor of cell proliferation and immunosuppressive agent that is used alone or in combination with calcineurin inhibitors to prevent cellular rejection after organ transplantation, and in combination with other anticancer agents as treatment of advanced renal cell and other cancers. Everolimus therapy can be associated with mild serum enzyme elevations, but has yet to be linked to instances of clinically apparent liver injury with jaundice.

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Everolimus is a derivative of the natural macrocyclic lactone sirolimus with immunosuppressant and anti-angiogenic properties. In cells, everolimus binds to the immunophilin FK Binding Protein-12 (FKBP-12) to generate an immunosuppressive complex that binds to and inhibits the activation of the mammalian Target of Rapamycin (mTOR), a key regulatory kinase. Inhibition of mTOR activation results in the inhibition of T lymphocyte activation and proliferation associated with antigen and cytokine (IL-2, IL-4, and IL-15) stimulation and the inhibition of antibody production. (NCI05)

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A derivative of sirolimus and an inhibitor of TOR SERINE-THREONINE KINASES. It is used to prevent GRAFT REJECTION in heart and kidney transplant patients by blocking cell proliferation signals. It is also an ANTINEOPLASTIC AGENT.

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Drug Indication: Everolimus is indicated for the treatment of postmenopausal women with advanced hormone receptor-positive, HER2-negative breast cancer (advanced HR+ BC) in combination with exemestane, after failure of treatment with letrozole or anastrozole. Indicated for the treatment of adult patients with progressive neuroendocrine tumors of pancreatic origin (PNET) with unresectable, locally advanced or metastatic disease. Indicated for the treatment of adult patients with advanced renal cell carcinoma (RCC) after failure of treatment with sunitinib or sorafenib. Indicated for the treatment of adult patients with renal angiomyolipoma and tuberous sclerosis complex (TSC), not requiring immediate surgery. Indicated in pediatric and adult patients with tuberous sclerosis complex (TSC) for the treatment of subependymal giant cell astrocytoma (SEGA) that requires therapeutic intervention but cannot be curatively resected.
Renal angiomyolipoma associated with tuberous sclerosis complex (TSC)Votubia is indicated for the treatment of adult patients with renal angiomyolipoma associated with tuberous sclerosis complex (TSC) who are at risk of complications (based on factors such as tumour size or presence of aneurysm, or presence of multiple or bilateral tumours) but who do not require immediate surgery. The evidence is based on analysis of change in sum of angiomyolipoma volume. Subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis complex (TSC)Votubia is indicated for the treatment of patients with subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis complex (TSC) who require therapeutic intervention but are not amenable to surgery. The evidence is based on analysis of change in SEGA volume. Further clinical benefit, such as improvement in disease�related symptoms, has not been demonstrated.

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LiverTox Summary: Everolimus is an inhibitor of cell proliferation and immunosuppressive agent that is used alone or in combination with calcineurin inhibitors to prevent cellular rejection after organ transplantation, and in combination with other anticancer agents as treatment of advanced renal cell and other cancers. Everolimus therapy can be associated with mild serum enzyme elevations, but has yet to be linked to instances of clinically apparent liver injury with jaundice.

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Absorption: In patients with advanced solid tumors, peak everolimus concentrations are reached 1 to 2 hours after administration of oral doses ranging from 5 mg to 70 mg. Following single doses, Cmax is dose-proportional between 5 mg and 10 mg. At doses of 20 mg and higher, the increase in Cmax is less than dose-proportional, however AUC shows dose-proportionality over the 5 mg to 70 mg dose range. Steady-state was achieved within 2 weeks following once-daily dosing. Dose Proportionality in Patients with SEGA (subependymal giant-cell astrocytomas) and TSC (tuberous sclerosis complex): In patients with SEGA and TSC, everolimus Cmin was approximately dose-proportional within the dose range from 1.35 mg/m2 to 14.4 mg/m2.

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Route of Elimination: After a single dose of radiolabeled everolimus was given to transplant patients receiving cyclosporine, the majority (80%) of radioactivity was recovered from the feces and only a minor amount (5%) was excreted in urine.
Volume of Distribution: The blood-to-plasma ratio of everolimus is 17% to 73%.

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Clearance: Following a 3 mg radiolabeled dose of everolimus, 80% of the radioactivity was recovered from the feces, while 5% was excreted in the urine.
The blood-to-plasma ratio of everolimus is concentration dependent ranging from 17% to 73% over the range of 5 ng/mL to 5000 ng/mL. Plasma protein binding is approximately 74% in healthy subjects and in patients with moderate hepatic impairment. The apparent distribution volume associated with the terminal phase (Vz/F) from a single-dose pharmacokinetic study in maintenance kidney transplant patients is 342 to 107 L (range 128 to 589 L).

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Metabolism / Metabolites: Everolimus is a substrate of CYP3A4 and PgP (phosphoglycolate phosphatase). Three monohydroxylated metabolites, two hydrolytic ring-opened products, and a phosphatidylcholine conjugate of everolimus were the 6 primary metabolites detected in human blood. In vitro, everolimus competitively inhibited the metabolism of CYP3A4 and was a mixed inhibitor of the CYP2D6 substrate dextromethorphan.
Everolimus is a substrate of CYP3A4 and PgP. Following oral administration, everolimus is the main circulating component in human blood. Six main metabolites of everolimus have been detected in human blood, including three monohydroxylated metabolites, two hydrolytic ring-opened products, and a phosphatidylcholine conjugate of everolimus. These metabolites were also identified in animal species used in toxicity studies, and showed approximately 100-times less activity than everolimus itself.

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Biological Half-Life: ~30 hours.

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Mechanism of Action: Everolimus is a mTOR inhibitor that binds with high affinity to the FK506 binding protein-12 (FKBP-12), thereby forming a drug complex that inhibits the activation of mTOR. This inhibition reduces the activity of effectors downstream, which leads to a blockage in the progression of cells from G1 into S phase, and subsequently inducing cell growth arrest and apoptosis. Everolimus also inhibits the expression of hypoxia-inducible factor, leading to a decrease in the expression of vascular endothelial growth factor. The result of everolimus inhibition of mTOR is a reduction in cell proliferation, angiogenesis, and glucose uptake.
Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that functions via two multiprotein complexes, namely mTORC1 and mTORC2, each characterized by different binding partners that confer separate functions. mTORC1 function is tightly regulated by PI3-K/Akt and is sensitive to rapamycin. mTORC2 is sensitive to growth factors, not nutrients, and is associated with rapamycin-insensitivity. mTORC1 regulates protein synthesis and cell growth through downstream molecules: 4E-BP1 (also called EIF4E-BP1) and S6K. Also, mTORC2 is thought to modulate growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases such as Akt and SGK. Recent evidence has suggested that mTORC2 may play an important role in maintenance of normal as well as cancer cells by virtue of its association with ribosomes, which may be involved in metabolic regulation of the cell. Rapamycin (sirolimus) and its analogs known as rapalogues, such as RAD001 (everolimus) and CCI-779 (temsirolimus), suppress mTOR activity through an allosteric mechanism that acts at a distance from the ATP-catalytic binding site, and are considered incomplete inhibitors. Moreover, these compounds suppress mTORC1-mediated S6K activation, thereby blocking a negative feedback loop, leading to activation of mitogenic pathways promoting cell survival and growth. Consequently, mTOR is a suitable target of therapy in cancer treatments. However, neither of these complexes is fully inhibited by the allosteric inhibitor rapamycin or its analogs. In recent years, new pharmacologic agents have been developed which can inhibit these complexes via ATP-binding mechanism, or dual inhibition of the canonical PI3-K/Akt/mTOR signaling pathway. These compounds include WYE-354, KU-003679, PI-103, Torin1, and Torin2, which can target both complexes or serve as a dual inhibitor for PI3-K/mTOR. This investigation describes the mechanism of action of pharmacological agents that effectively target mTORC1 and mTORC2 resulting in suppression of growth, proliferation, and migration of tumor and cancer stem cells.

C53H83NO14

958.22

957.581

C, 66.43; H, 8.73; N, 1.46; O, 23.38

159351-69-6

Everolimus-d4;1338452-54-2

6442177

White to off-white solid powder

1.2±0.1 g/cm3

998.7±75.0 °C at 760 mmHg

NA

557.8±37.1 °C

0.0±0.6 mmHg at 25°C

1.548

3.35

204.66

O=C1C([C@]2([C@@H](CC[C@@]([H])(C[C@@H](C(=CC=CC=C[C@H](C[C@H](C([C@@H]([C@@H](C(=C[C@H](C(C[C@]([H])(OC([C@]3([H])CCCCN31)=O)[C@H](C)C[C@@H]1CC[C@H]([C@@H](C1)OC)OCCO)=O)C)C)O)OC)=O)C)C)C)OC)O2)C)O)=O |t:11,13,15,23|

HKVAMNSJSFKALM-GKUWKFKPSA-N

InChI=1S/C53H83NO14/c1-32-16-12-11-13-17-33(2)44(63-8)30-40-21-19-38(7)53(62,68-40)50(59)51(60)54-23-15-14-18-41(54)52(61)67-45(35(4)28-39-20-22-43(66-25-24-55)46(29-39)64-9)31-42(56)34(3)27-37(6)48(58)49(65-10)47(57)36(5)26-32/h11-13,16-17,27,32,34-36,38-41,43-46,48-49,55,58,62H,14-15,18-26,28-31H2,1-10H3/b13-11+,16-12+,33-17+,37-27+/t32-,34-,35-,36-,38-,39+,40+,41+,43-,44+,45+,46-,48-,49+,53-/m1/s1

(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18- dihydroxy-12-{(1R)-2-[(1S,3R,4R)-4-(2hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl}-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-aza-tricyclo[30.3.1.04,9]hexatriaconta16,24,26,28-tetraene-2,3,10,14,20-pentaone.

SDZ-RAD; RAD-001; RAD001; RAD 001; Everolimus; Brand name Afinitor; Certican; Zortress; Xience V; Zortress; 001, RAD; 40-O-(2-hydroxyethyl)-rapamycin; 40-O-(2-Hydroxyethyl)rapamycin; Afinitor; Certican; Everolimus; RAD;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.5 mg/mL (2.61 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.5 mg/mL (2.61 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (2.61 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 2.5 mg/mL (2.61 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 5 中的溶解度: 2.5 mg/mL (2.61 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 6 中的溶解度: 30% Propylene glycol (dissolve first)+5% Tween 80+ddH2O: 5 mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。