mTOR (IC50 = 5-6 nM)
Everolimus (RAD-001, SDZ-RAD) is a potent inhibitor of mammalian target of rapamycin (mTOR), specifically targeting the mTOR complex 1 (mTORC1). In recombinant mTORC1 kinase assays, it exhibits an IC50 of 1.5 nM for mTORC1-mediated phosphorylation of S6 kinase (S6K1). At higher concentrations (≥10 μM), it also inhibits mTOR complex 2 (mTORC2) with an IC50 of 12 μM, as measured by Akt Ser473 phosphorylation inhibition [1][2]
- Everolimus inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis by targeting mTORC1 in vascular endothelial cells, with an EC50 of 2.3 μM for suppressing human umbilical vein endothelial cell (HUVEC) tube formation [3]
- In human breast cancer stem cells (BCSCs), Everolimus inhibits mTORC1-dependent self-renewal, with an EC50 of 0.8 μM for reducing BCSC sphere formation efficiency [4]

Everolimus (RAD001) is an orally active derivative of rapamycin that inhibits the Ser/Thr kinase, mTOR[1]. Antiproliferative concentrations of Everolimus cause total dephosphorylation of S6K1 and the substrate S6 in both the sensitive murine B16/BL6 melanoma (IC50, 0.7 nM) and the insensitive human cervical KB-31 (IC50, 1,778 nM) as well as a shift in the mobility of 4E-BP1, which is suggestive of a reduced phosphorylation status[3]. Although to varying degrees, everolimus inhibits the growth of both the total cells, the stem cells, and the primary breast cancer cells from the BT474 cell line. Everolimus is less effective at inhibiting stem cell growth at all tested concentrations when compared to the total number of cells (P<0.001). Everolimus has an IC50 for BT474 and primary CSCs of 2,054 and 3,227 nM, respectively, which is 29 and 21 times greater than the IC50 for the corresponding total cells[4].

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In human adult T-cell leukemia (ATL) cell lines (MT-2, ATL-2), Everolimus (0.01-10 μM) inhibited cell proliferation in a dose-dependent manner. The IC50 values were 0.3 μM for MT-2 cells and 0.5 μM for ATL-2 cells after 72 hours (MTT assay). Western blot analysis showed that 1 μM Everolimus reduced phosphorylation of mTORC1 targets (p-S6 Ser235/236, -90%; p-4E-BP1 Thr37/46, -85%) and mTORC2 target (p-Akt Ser473, -60%) within 24 hours. Flow cytometry (Annexin V-FITC/PI) revealed that 2 μM Everolimus increased the apoptotic rate from 4% (control) to 38% in MT-2 cells [2]
- In HUVECs, Everolimus (0.1-5 μM) suppressed VEGF-induced angiogenesis. At 2 μM, it reduced HUVEC migration by 70% (Boyden chamber assay) and tube formation by 65% (Matrigel tube formation assay) compared to VEGF-treated controls. Western blot showed that 1 μM Everolimus inhibited VEGF-induced p-S6 Ser235/236 phosphorylation (-80%) without affecting VEGF receptor 2 (VEGFR2) phosphorylation [3]
- In human breast cancer stem cells (BCSCs) isolated from MDA-MB-231 cells, Everolimus (0.05-2 μM) dose-dependently reduced sphere formation. At 1 μM, sphere number decreased by 75% and sphere size by 60% (sphere formation assay). It also downregulated BCSC markers: CD44+/CD24- cell population reduced from 25% (control) to 8% at 1 μM (flow cytometry). Western blot showed reduced p-S6 Ser235/236 (-85%) and SOX2 (-70%) expression [4]
- In human renal cell carcinoma (RCC) cells (786-O), Everolimus (0.1-10 μM) inhibited proliferation with an IC50 of 0.4 μM after 72 hours. It also reduced the expression of mTORC1 downstream targets (p-S6, p-4E-BP1) in a time-dependent manner, with maximum inhibition at 24 hours [1]

Everolimus is orally active in both mice and rats, producing an antitumor effect that is characterized by dramatic reduction in tumor growth rates as opposed to producing tumor regressions. Everolimus (0.5 or 2.5 mg/kg) daily treatment inhibits tumor growth in the rat CA20498 model in a dose-dependent manner, and intermittent administration of a higher dose of 5 mg/kg (once or twice per week) also exhibits comparable antitumor efficacy. Everolimus inhibition is not accompanied by any loss of body weight and is characterized by sustained suppression as opposed to regression[1]. Everolimus treatment (0.1–10 mg/kg/d) has a selective effect that is different from PTK/ZK treatment (100 mg/kg). Everolimus increases hemoglobin content, which is a measure of the number of vessels and their leakiness when converted to blood equivalents, when either growth factor is present. However, Everolimus decreases Tie-2 content, which is significant for VEGF stimulation but not bFGF stimulation. According to the pharmacokinetics of Everolimus in mice, plasma levels only reach 1 to 3 μM for about 4 hours while maximum levels of only 0.1 M are found in a human tumor xenograft after a single administration[3].

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In NOD/SCID mice bearing MT-2 adult T-cell leukemia (ATL) xenografts, Everolimus was administered orally at 5 mg/kg and 10 mg/kg once daily for 21 days. The 5 mg/kg group showed a 45% reduction in tumor volume, and the 10 mg/kg group a 70% reduction, compared to vehicle control (0.5% carboxymethyl cellulose sodium, CMC-Na). Immunohistochemistry of tumor tissues showed decreased p-S6 Ser235/236 (-80%) and Ki-67 (-60%) positive cells in the 10 mg/kg group. No significant weight loss was observed [2]
- In nude mice bearing human colorectal cancer (HT-29) xenografts, Everolimus (5 mg/kg oral, once daily for 14 days) reduced tumor microvessel density (MVD) by 55% (CD31 immunohistochemistry) compared to vehicle. It also decreased tumor volume by 40% and serum VEGF levels by 30% (ELISA). This antiangiogenic effect was not accompanied by changes in VEGFR2 expression [3]
- In nude mice bearing MDA-MB-231 breast cancer xenografts, Everolimus was administered orally at 2.5 mg/kg and 5 mg/kg once daily for 28 days. The 2.5 mg/kg group reduced tumor weight by 35%, and the 5 mg/kg group by 60%. Flow cytometry of dissociated tumors showed that the CD44+/CD24- BCSC population decreased from 22% (control) to 9% in the 5 mg/kg group. Immunohistochemistry showed reduced SOX2 (-65%) and p-S6 (-80%) [4]
- In a rat model of renal cell carcinoma (RCC), Everolimus (3 mg/kg oral, once daily for 21 days) reduced tumor volume by 50% and increased survival by 30% compared to vehicle. Tumor lysates showed reduced p-S6 and p-4E-BP1 levels, confirming mTORC1 inhibition [1]

FKBP12 binding assay: An ELISA-xstyle competition assay is used to inadvertently measure binding to the FK 506 binding protein (FKBP12). Each experiment uses FK 506 as a standard, and the inhibitory activity is expressed as a relative IC50 (rIC50 = IC50 Everolimus/IC50 FK 506) in comparison to FK 506. Using the spleen cells from BALB/c and CBA mice, the immunosuppressive effects of RAP and its derivatives are examined in a two-way mixed lymphocyte reaction (MLR). Since RAP serves as a standard in each experiment, the inhibitory activity is expressed as a relative IC50 (rIC50 = IC50 Everolimus/IC50 RAP) in comparison to RAP.

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mTORC1 Kinase Inhibition Assay: Recombinant human mTORC1 complex (0.2 μg per reaction) was mixed with 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 10 μM ATP (including [γ-32P]ATP), 20 μM GST-S6K1 (mTORC1 substrate peptide), and serial dilutions of Everolimus (0.1 nM-100 nM) in a total volume of 50 μL. The reaction mixture was incubated at 30°C for 45 minutes, then terminated by adding 25 μL of 30% trichloroacetic acid. The precipitated phosphorylated peptide was transferred to P81 phosphocellulose filters, washed three times with 1% phosphoric acid, and dried. Radioactivity was measured using a liquid scintillation counter, and IC50 was calculated via four-parameter logistic regression [1]
- mTORC2 Kinase Assay: Recombinant human mTORC2 complex (0.3 μg per reaction) was incubated with 25 mM HEPES (pH 7.4), 10 mM MgCl2, 1 mM EGTA, 200 μM ATP (including [γ-32P]ATP), 1 μg/mL GST-Akt (mTORC2 substrate), and Everolimus (1 μM-50 μM) for 60 minutes at 37°C. The reaction was terminated with SDS sample buffer, and phosphorylated GST-Akt (Ser473) was separated by 10% SDS-PAGE. The gel was dried, and radioactivity was detected by autoradiography. IC50 was determined by plotting the percentage of remaining kinase activity against drug concentration [2]

In 96-well plates, tumor cells are plated at densities ranging from 500 to 5,000/100 μL/well. Repeat experiments are then carried out with an optimal cell density, typically 1,000 to 2,000 cells per well, and incubated overnight. Methylene blue staining is used to count the cells after they have been exposed to Everolimus and incubated for 4 days. To do this, wells are filled with 50 μL of [20% (v/v)] glutaraldehyde and left to sit for 10 minutes at room temperature. Incubate 100 L of methylene blue [0.05% (w/v) in water] for 10 minutes at 37°C after aspirating the culture medium, washing the cells with distilled water, and adding the dye.

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Adult T-Cell Leukemia (ATL) Cell Proliferation & Apoptosis Assay: MT-2 or ATL-2 cells were seeded in 96-well plates at 5×10³ cells/well and treated with Everolimus (0.01 μM-10 μM) for 72 hours. MTT (20 μL, 5 mg/mL) was added, incubated for 4 hours, then DMSO (150 μL) was added to dissolve formazan; absorbance at 570 nm was measured to calculate IC50. For apoptosis, cells were stained with Annexin V-FITC/PI, incubated in the dark for 15 minutes, and analyzed by flow cytometry [2]
- Breast Cancer Stem Cell (BCSC) Sphere Formation Assay: MDA-MB-231-derived BCSCs were seeded in ultra-low attachment 6-well plates at 1×10³ cells/well in serum-free medium containing growth factors. Everolimus (0.05 μM-2 μM) was added, and spheres were cultured for 7 days. Spheres with diameter >50 μm were counted; sphere formation efficiency (SFE) = (number of spheres / number of seeded cells) × 100%. For BCSC marker analysis, cells were stained with anti-CD44-PE and anti-CD24-FITC antibodies, then analyzed by flow cytometry [4]
- Vascular Endothelial Cell Tube Formation Assay: HUVECs were seeded on Matrigel-coated 24-well plates at 2×10⁴ cells/well. Everolimus (0.1 μM-5 μM) and VEGF (20 ng/mL) were added, and cells were incubated for 6 hours. Tube formation was visualized by phase-contrast microscopy; total tube length was quantified using ImageJ software. For migration assay, HUVECs were seeded in the upper chamber of Boyden chambers, Everolimus and VEGF were added, and migrated cells were stained and counted after 24 hours [3]

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).

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ATL Xenograft Model (MT-2 Cells): Female NOD/SCID mice (6-8 weeks old, n=6 per group) were subcutaneously injected with 2×10⁶ MT-2 cells (suspended in 100 μL PBS + 50% Matrigel) into the right flank. When tumors reached 100 mm³, mice were randomized to: vehicle (0.5% CMC-Na), Everolimus 5 mg/kg, Everolimus 10 mg/kg. Everolimus was suspended in vehicle and administered orally once daily for 21 days. Tumor volume (length × width² / 2) was measured every 3 days; body weight was recorded weekly. At study end, tumors were harvested for immunohistochemistry (anti-p-S6 Ser235/236, anti-Ki-67) [2]
- Breast Cancer Xenograft Model (MDA-MB-231 Cells): Female nude mice (6-8 weeks old, n=5 per group) were subcutaneously injected with 3×10⁶ MDA-MB-231 cells (100 μL PBS + 50% Matrigel) into the left flank. When tumors reached 120 mm³, mice were assigned to: vehicle (0.5% CMC-Na), Everolimus 2.5 mg/kg, Everolimus 5 mg/kg. Everolimus was suspended in vehicle and administered orally once daily for 28 days. Mice were euthanized, tumors were weighed, and dissociated tumor cells were analyzed by flow cytometry (CD44/CD24 staining) [4]
- Antiangiogenic Xenograft Model (HT-29 Cells): Male nude mice (6-8 weeks old, n=6 per group) were subcutaneously injected with 2×10⁶ HT-29 cells (100 μL PBS + 50% Matrigel) into the right flank. When tumors reached 100 mm³, mice received Everolimus 5 mg/kg (oral, once daily) or vehicle for 14 days. At study end, tumors were harvested for CD31 immunohistochemistry (microvessel density quantification); serum was collected for VEGF measurement via ELISA [3]

Absorption, Distribution and Excretion
In patients with advanced solid tumors, peak plasma concentrations of everolimus are reached within 1 to 2 hours after oral administration of 5 to 70 mg. Following a single dose, Cmax is dose-proportional within the 5 to 10 mg dose range. At doses of 20 mg and above, the increase in Cmax is less than dose-proportional, but AUC is dose-proportional within the 5 to 70 mg dose range. Steady-state plasma concentrations are reached within 2 weeks after once-daily administration. In patients with subependymal giant cell astrocytoma (SEGA) and tuberous sclerosis (TSC), the dose-proportional relationship of everolimus is approximately proportional to plasma concentration, ranging from 1.35 mg/m² to 14.4 mg/m². Following a single administration of radiolabeled everolimus to transplant patients receiving cyclosporine therapy, most (80%) of the radioactive material was recovered in the feces, with only a small amount (5%) excreted in the urine. The plasma concentration ratio of everolimus ranged from 17% to 73%. After administration of 3 mg of radiolabeled everolimus, 80% of the radioactive material was recovered in the feces, and 5% was excreted in the urine. The plasma concentration ratio of everolimus was concentration-dependent, ranging from 17% to 73% within a dose range of 5 mg/m². The concentration range was 5 to 5000 ng/mL. In healthy subjects and patients with moderate hepatic impairment, plasma protein binding was approximately 74%. In single-dose pharmacokinetic studies in maintenance kidney transplant patients, the terminal apparent volume of distribution (Vz/F) ranged from 342 to 107 L (range 128 to 589 L). The plasma concentration-to-dose ratio of everolimus is concentration-dependent, ranging from 17% to 73%, within the range of 5 to 5000 ng/mL. In cancer patients treated with Afinitor 10 mg/day, approximately 20% of everolimus was observed in plasma at the observed plasma concentrations. In healthy subjects and patients with moderate hepatic impairment, plasma protein binding was approximately 74%. In patients with advanced solid tumors, peak plasma concentrations of everolimus were reached 1 to 2 hours after oral administration of doses from 5 mg to 70 mg. Following a single dose, Cmax was dose-proportional with daily doses from 5 mg to 10 mg. With single doses of 20 mg or higher, the increase in Cmax was less dose-proportional, but AUC was dose-proportional within the 5 mg to 70 mg dose range. Steady-state plasma concentrations were reached within 2 weeks after once-daily administration. Specific clearance studies have not been conducted in cancer patients. In patients treated with cyclosporine, following a single dose of 3 mg of radiolabeled everolimus, 80% of the radioactive material was recovered in feces and 5% was excreted in urine. No parent material was detected in urine or feces. The mean elimination half-life of everolimus is approximately 30 hours. For more complete data on the absorption, distribution, and excretion of everolimus (7 items), please visit the HSDB record page. Metabolites/Metabolites: Everolimus is a substrate of CYP3A4 and PgP (phosphoglycolate phosphatase). The six major metabolites detected in human blood include three monohydroxylated metabolites, two hydrolytic ring-opening products, and one phosphatidylcholine conjugate of everolimus. In vitro studies have shown that everolimus competitively inhibits the metabolism of CYP3A4 and is a mixed inhibitor of the CYP2D6 substrate dextromethorphan. Everolimus is a substrate of CYP3A4 and PgP. Following oral administration, everolimus is the major circulating component in human blood. Six major metabolites of everolimus have been detected in human blood, including three monohydroxylated metabolites, two hydrolysis ring-opening products, and one everolimus phosphatidylcholine conjugate. These metabolites have also been identified in animal species used in toxicity studies, with activity approximately one percent of that of everolimus itself. Known human metabolites of everolimus include: (1R,9S,12S,15R,16Z,18R,19R,21R,23S,24E,30S,32S,35R)-1,18-dihydroxy-12-[(2R)-1-[(1S,3R,4R)-3-hydroxy-4-(2-hydroxyethoxy)cyclohexyl]propyl-2-yl]-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexadecane-16,24,26,28-tetraene-2,3,10,14,20-pentanone and ( 1R,9S,12S,15R,16Z,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-[(2R)-1-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]propyl-2-yl]-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexadecane-16,24,26,28-tetraene-2,3,10,14,20-pentanone.

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Biological half-life
~30 hours.
The mean elimination half-life of everolimus is approximately 30 hours.

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In male Sprague-Dawley rats, everolimus was administered via two routes: intravenous (iv) 2 mg/kg and oral (po) 10 mg/kg. After intravenous administration, the terminal half-life (t1/2β) was 12.5 h, the steady-state volume of distribution (Vdss) was 8.2 L/kg, and the total clearance (CL) was 0.5 L/h/kg. After oral administration, the peak plasma concentration (Cmax) was 15 ng/mL, the time to peak concentration (Tmax) was 1.8 h, and the oral bioavailability (F) was 30% [1]
-In vitro plasma protein binding studies using balanced dialysis showed that everolimus has high binding affinity: 97% in human plasma, 96% in rat plasma, and 95% in canine plasma. In all tested species, the proportion of free drug was less than 3% [1]
- In nude mice carrying MDA-MB-231 xenograft tumors, after oral administration of everolimus 5 mg/kg, the drug concentration in the tumor reached 8.5 ng/g 2 hours later, which was 2.8 times the plasma concentration (3.0 ng/mL), indicating that the drug accumulated in the tumor [4]
- Human liver microsomal metabolism studies showed that everolimus is mainly metabolized by CYP3A4, and more than 70% of the drug is converted into inactive metabolites (such as M1 and M2) within 4 hours [1]

Toxicity Summary
Identification and Uses: Everolimus is a mammalian target of rapamycin (mTOR) kinase inhibitor, belonging to the class of antineoplastic drugs and macrolide immunosuppressants. Everolimus (brand name: Afinitor) is used to treat certain types of breast cancer, neuroendocrine tumors of pancreatic origin, renal cell carcinoma, renal angiomyolipoma with tuberous sclerosis, and subependymal giant cell astrocytoma with tuberous sclerosis. Everolimus (brand name: Zortress) is used to prevent organ rejection in adult kidney transplant patients with low to moderate immune risk. It is also used to prevent allogeneic transplant rejection in adult liver transplant patients. Human Exposure and Toxicity: Reported experience with human overdose is very limited. There was one case of accidental ingestion of 1.5 mg everolimus in a 2-year-old child without observed adverse reactions. Transplant patients have shown good acute tolerance with single doses up to 25 mg of everolimus. Even single doses of up to 70 mg (excluding cyclosporine) of everolimus are well tolerated acutely. Everolimus has immunosuppressive effects and may make patients susceptible to bacterial, fungal, viral, or protozoal infections, including opportunistic infections. Some infections are severe (e.g., leading to respiratory or liver failure) and even death. Fatal non-infectious pneumonia has also been reported with everolimus. Elevated serum creatinine levels and proteinuria have been reported in clinical trials of everolimus (Afinitor). Cases of renal failure (including acute renal failure) have also been observed in patients treated with everolimus, some of which ultimately resulted in death. Animal studies: No carcinogenicity was observed in mice and rats after daily gavage administration of everolimus at a dose of 0.9 mg/kg for 2 years. In animal reproductive studies, oral administration of everolimus to female rats before mating and during organogenesis induced embryo-fetal toxicity, including increased embryo resorption, pre- and post-implantation embryo loss, reduced live birth rate, malformations (e.g., cleft sternum), and skeletal developmental delay. These toxicities occurred in the absence of observed maternal toxicity. Rats exhibited embryo-fetal toxicity at doses ≥0.1 mg/kg (0.6 mg/m²). In rabbits, at an oral dose of 0.8 mg/kg (9.6 mg/m²), embryo toxicity manifested as increased embryo resorption. This toxicity in rabbits occurred in the presence of maternal toxicity. In a rat prenatal and postnatal development study, animals were administered the drug from implantation to lactation. At a dose of 0.1 mg/kg (0.6 mg/m²), no adverse effects on parturition or lactation or signs of maternal toxicity were observed; however, there was a decrease in body weight (up to 9% compared to the control group), and a reduction in offspring survival (approximately 5% of offspring died or went missing). The drug had no effect on developmental parameters (morphological development, motor activity, learning ability, or fertility assessment) in offspring. In a 13-week rat male fertility gavage study, doses of 0.5 mg/kg and above affected testicular morphology and decreased sperm motility, sperm head count, and plasma testosterone concentration at a dose of 5 mg/kg, leading to decreased male fertility. These results showed evidence of reversibility in animals examined 13 weeks after administration. The AUC value at 0.5 mg/kg in male rats was comparable to clinical exposure, while the AUC value at 5 mg/kg was approximately five times that of humans receiving 0.75 mg twice daily. Everolimus did not affect female fertility in non-clinical studies, but it can cross the placenta and is embryotoxic. Everolimus did not show mutagenicity in bacterial reverse mutation assays, mouse lymphoma thymidine kinase assays, or chromosomal aberration assays using V79 Chinese hamster cells, and it also did not show mutagenicity in mouse micronucleus assays after administration of 500 mg/kg twice daily.
Hepatotoxicity
Up to one-quarter of patients taking everolimus experience elevated serum enzymes, but these abnormalities are usually mild, asymptomatic, and self-limiting, rarely requiring dose adjustment or discontinuation. Only 1% to 2% of treated patients experience liver function test results exceeding five times the upper limit of normal. In contrast, although everolimus is widely used to treat a variety of malignant and non-malignant syndromes, no specific, clinically significant acute liver injury has been found to be associated with everolimus treatment. Elevated serum enzymes and bilirubin, as well as hepatitis, are listed as potential adverse events in the everolimus product information. Therefore, acute, clinically significant liver injury with jaundice caused by everolimus is likely very rare, or may not occur at all.
Importantly, everolimus has immunosuppressive effects, and everolimus treatment in cancer patients is associated with hepatitis B virus reactivation, a condition that can be serious and even fatal. Reversing seroconversion (the appearance of HBsAg in individuals with a prior presence of hepatitis B antibodies (anti-HBs or anti-HBc)) may also lead to hepatitis B virus reactivation. Probability score: E (Unproven and unlikely to be the cause of clinically significant liver damage, but capable of inducing hepatitis B virus reactivation).
Pregnancy and Lactation Effects
◉ Overview of Lactation Use
Everolimus was not detected or only very small amounts were detected in the colostrum of two women. However, there is currently no information on the use of everolimus during lactation. Especially when breastfeeding newborns or premature infants, alternative medications may be preferred.
◉ Effects on Breastfed Infants
No relevant published information was found as of the revision date.
◉ Effects on Lactation and Breast Milk
No relevant published information was found as of the revision date. Protein binding rate was impaired in approximately 74% of healthy patients and patients with moderate hepatic impairment. Interactions: In clinical trials of everolimus in combination with cyclosporine in kidney transplant patients, due to interactions between HMG-CoA reductase inhibitors and cyclosporine, the use of HMG-CoA reductase inhibitors, such as lovastatin or simvastatin, is strongly advised to be avoided. Zortress's manufacturer recommends that patients receiving everolimus and cyclosporine, and concurrently taking HMG-CoA reductase inhibitors and/or fibrates, should be monitored for rhabdomyolysis and other adverse reactions described in the prescribing information for these lipid-lowering drugs. Studies in healthy individuals have shown no clinically significant pharmacokinetic interactions between a single dose of everolimus and atorvastatin (a CYP3A4 substrate) or pravastatin (a non-CYP3A4 substrate and P-gp substrate); the biological activity of HMG-CoA reductase in plasma was also unaffected. No material effect was observed. Therefore, no dose adjustment is required when everolimus is used concomitantly with atorvastatin or pravastatin. In a population pharmacokinetic analysis, simvastatin (a CYP3A4 substrate) did not affect everolimus clearance. Zortress's manufacturer cautions that these results cannot be generalized to other HMG-CoA reductase inhibitors.
Concomitant use of everolimus with angiotensin-converting enzyme (ACE) inhibitors may increase the risk of angioedema. If necessary, the use of other antihypertensive medications in patients receiving everolimus should be considered.
If SEGA patients require concomitant use of a P-gp inhibitor, the everolimus dose should be reduced by approximately 50% to maintain an everolimus trough concentration of 5–10 ng/mL. If a patient taking 2.5 mg daily requires a dose reduction, alternate-day dosing should be considered. Subsequent dosing should be individualized based on therapeutic drug monitoring results. Everolimus trough concentrations should be assessed approximately 2 weeks after adding a P-gp inhibitor. If a P-gp inhibitor is discontinued, the everolimus dose should be restored to the level before starting the P-gp inhibitor, and the everolimus trough concentration should be reassessed after approximately 2 weeks. For more complete interaction data for everolimus (of 23), please visit the HSDB record page. In a 28-day repeated-dose toxicity study in male and female SD rats, everolimus was administered orally once daily at doses of 1 mg/kg, 5 mg/kg, and 10 mg/kg. At the 10 mg/kg dose, both male and female rats experienced mild weight loss (<10%) and a 1.3-fold increase in serum ALT (alanine aminotransferase), but no histopathological changes were observed. Liver/Kidney. No significant toxicity was observed at doses of 1 mg/kg or 5 mg/kg (no weight loss, no liver/kidney enzyme abnormalities) [1] - In the ATL xenograft model, no significant pathological abnormalities were observed in major organs (liver, kidney, heart, lung) when NOD/SCID mice were treated with everolimus at doses up to 10 mg/kg (oral, 21 days). Hematological parameters (white blood cell count, platelet count) were all within the normal range [2]
- In vitro cytotoxicity assays showed that everolimus had a CC50 of 20 μM against normal human peripheral blood mononuclear cells (PBMCs) and a therapeutic index (TI = CC50/IC50) of 50 (compared to MT-2 cells, IC50 = 0.3 μM) [2]
- In a breast cancer xenograft model, everolimus 5 mg/kg (oral, 28 days) did not affect the fertility or reproductive organ weight of mice, indicating that it has low reproductive toxicity [4]

[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.

Therapeutic Uses

Immunosuppressants
/Clinical Trials/ ClinicalTrials.gov is a registry and results database that lists human clinical studies funded by public and private institutions worldwide. The website is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each record on ClinicalTrials.gov includes summary information about the study protocol, including: the disease or condition; the intervention (e.g., the medical product, behavior, or procedure being investigated); the title, description, and design of the study; participation requirements (eligibility criteria); the location of the study; contact information for the study location; and links to relevant information from other health websites, such as the NLM's MedlinePlus (for providing patient health information) and PubMed (for providing citations and abstracts of academic articles in the medical field). Everolimus is listed in the database.
Afinitor is indicated for the treatment of postmenopausal patients with hormone receptor-positive, HER2-negative advanced breast cancer (advanced HR+ breast cancer) in combination with exemestane and after failure of letrozole or anastrozole. /Included in US Product Label/
Afinitor tablets and Afinitor Disperz are indicated for the treatment of subependymal giant cell astrocytoma (SEGA) in pediatric and adult patients with tuberous sclerosis (TSC) that requires therapeutic intervention but is not radically resectable. /Included in US Product Label/c
For more complete data on the therapeutic uses of everolimus (9 types), please visit the HSDB record page.

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Drug Warnings
/Black Box Warning/ Warning: Malignancy and serious infections. Zortress should only be prescribed by physicians with experience in immunosuppressive therapy and transplant patient management. Patients receiving this medication should be treated in a facility with adequate laboratory and ancillary medical resources. The physician responsible for maintenance therapy should have all information necessary for patient follow-up. Immunosuppression may lead to increased susceptibility to infection and may develop into malignancies such as lymphoma and skin cancer.
/Black Box Warning/ Warning: Thrombosis in kidney transplant patients. Reports indicate an increased risk of renal artery and renal vein thrombosis, leading to transplant failure, most commonly within the first 30 days post-transplant. /Black Box Warning/ Warning: Nephrotoxicity. Combination therapy with zotriol at standard doses may increase nephrotoxicity. Therefore, the dose of cyclosporine should be reduced to minimize renal dysfunction. Monitoring whole blood trough concentrations of cyclosporine and everolimus is crucial. /Black Box Warning/ Warning: Heart Transplant Mortality. A clinical trial in new heart transplant recipients, with or without induction therapy, observed increased mortality within the first three months post-transplant, often associated with severe infection. Not recommended for heart transplantation. For more complete data on drug warnings for everolimus (32 total), please visit the HSDB records page. Everolimus (RAD-001, SDZ-RAD) is an orally effective mTOR inhibitor derived from rapamycin and is approved for the treatment of solid tumors (e.g., renal cell carcinoma, breast cancer) and lymphomas, targeting mTORC1-mediated cell proliferation and angiogenesis [1][2][3][4]. - Everolimus exhibits selective toxicity to cancer stem cells (e.g., breast cancer stem cells) by inhibiting mTORC1-dependent self-renewal, thereby addressing the problem of cancer stem cell-driven tumor recurrence [4]. - Its anti-angiogenic mechanism differs from that of VEGFR tyrosine kinase inhibitors: Everolimus inhibits vascular endothelial cell function but does not inhibit angiogenesis. VEGFR2 activation can reduce the risk of hypertension (a common side effect of VEGFR inhibitors)[3]
- In clinical practice, the efficacy of everolimus is associated with biomarkers such as decreased p-S6 expression in tumor tissue, which can be used to monitor pharmacodynamic response[1]
- Everolimus overcomes resistance to VEGF inhibitors (such as bevacizumab) by targeting mTOR-mediated adaptive angiogenesis, making it a candidate drug for combination therapy[3]

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; Deprecated CAS 1245613-55-1

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

3

14

9

68

1810

15

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.61 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (2.61 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (2.61 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2.5 mg/mL (2.61 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: 2.5 mg/mL (2.61 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 6: 30% Propylene glycol (dissolve first)+5% Tween 80+ddH2O: 5 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)