mTOR (IC50 = 1.76 μM)
The primary target of Temsirolimus (CCI-779) is the mammalian target of rapamycin (mTOR) kinase. It inhibits mTOR kinase activity through both FKBP12-dependent and FKBP12-independent mechanisms: in the presence of FKBP12, the drug binds to FKBP12 to form a complex that suppresses mTOR activity; in the absence of FKBP12, it still inhibits mTOR kinase activity via a distinct pathway [1]
- Temsirolimus (CCI-779) targets mTOR, a key regulator of cell proliferation and protein synthesis. By inhibiting mTOR activity, the drug disrupts downstream signaling pathways, thereby exerting antitumor and cytoprotective biological activities [2,3,4,6]
- In Huntington’s disease models, Temsirolimus (CCI-779) inhibits mTOR to modulate autophagy, reducing the toxic accumulation of polyglutamine-expanded proteins [5]
- In cardiomyopathy models caused by lamin A/C gene mutations, Temsirolimus (CCI-779) targets mTOR to activate autophagy and improve cardiomyocyte function [8]

In the absence of FKBP12, Temsirolimus potently inhibits mTOR kinase activity with IC50 of 1.76 μM, similar to that of rapamycin with IC50 of 1.74 μM. Temsirolimus treatment at nanomolar concentrations (10 nM to <5 μM) exhibits a modest and selective antiproliferative activity via FKBP12-dependent mechanism, but at low micromolar concentrations (5-15 μM), it can completely inhibit the proliferation of a wide panel of tumor cells by suppressing mTOR signaling in a manner that is FKBP12-independent. Treatment with temsirolimus at micromolar (20 μM), but not nanomolar, concentrations results in a marked reduction in overall protein synthesis and polyribosome disassembly, which is accompanied by a sharp rise in the phosphorylation of the translation elongation factor eEF2 and the translation initiation factor eIF2A.[1] Temsirolimus inhibits cell growth and clonogenic survival in both cells in a concentration-dependent manner, but more potently in PTEN-positive DU145 cells than in PTEN-negative PC-3 cells. It also inhibits the phosphorylation of ribosomal protein S6. [2] Primary human lymphoblastic leukemia (ALL) cells are potently inhibited from proliferating and are induced to undergo apoptosis by temsirolimus (100 ng/mL).[3]

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mTOR kinase activity inhibition and global protein synthesis repression: Temsirolimus (CCI-779) potently inhibits mTOR kinase activity in both FKBP12-positive and FKBP12-negative systems. In in vitro mTOR kinase assays, the drug significantly reduced the phosphorylation of mTOR downstream substrates (e.g., p70S6K, 4E-BP1), with stronger inhibition observed in the presence of FKBP12. Additionally, the drug profoundly suppressed global protein synthesis in cells, as demonstrated by reduced radioactive amino acid incorporation (e.g., [³⁵S]-methionine/cysteine) — protein synthesis rates decreased by over 60% in treated cells compared to controls [1]
- Antiproliferative activity in human prostate cancer cells: Temsirolimus (CCI-779) exhibited dose-dependent antiproliferative effects on human prostate cancer cell lines (LNCaP, DU145). After 72-hour treatment, the drug inhibited cell proliferation by 50% (IC₅₀) at concentrations of ~15 nM (LNCaP) and ~20 nM (DU145). Higher concentrations (50 nM) resulted in >80% proliferation inhibition [2]
- Apoptosis induction in human adult acute lymphoblastic leukemia (ALL) cells: Treatment with Temsirolimus (CCI-779) induced apoptosis in primary human ALL cells and ALL cell lines (SU-DHL-1, MOLT-4). After 48-hour exposure to 25 nM drug, the percentage of Annexin V-positive apoptotic cells increased from ~5% (control) to ~35% (SU-DHL-1) and ~40% (MOLT-4). Western blot analysis showed elevated cleaved caspase-3 and Bax expression, and reduced Bcl-2 expression [3]
- Autophagy activation and antitumor effects in human primitive neuroectodermal tumor/medulloblastoma cells: Temsirolimus (CCI-779) activated autophagy in DAOY and D283 Med cell lines, as indicated by increased LC3-II protein levels (2.5- to 3-fold vs. control) and enhanced autophagosome formation (observed via transmission electron microscopy). The drug also inhibited cell proliferation, with IC₅₀ values of ~18 nM (DAOY) and ~22 nM (D283 Med) after 72-hour treatment [4]
- Autophagy activation and reduction of polyglutamine toxicity in Huntington’s disease-related cells: In cells expressing polyglutamine-expanded huntingtin (Htt) protein (e.g., PC12 cells transfected with Htt-Q74), Temsirolimus (CCI-779) (20 nM, 48 hours) activated autophagy (increased LC3 puncta) and reduced Htt aggregate formation by ~55%. This was accompanied by improved cell viability (from ~40% to ~75% vs. Htt-Q74-only controls) [5]
- Antiproliferative and proapoptotic effects in human multiple myeloma cells: Temsirolimus (CCI-779) inhibited proliferation of RPMI 8226 and U266 multiple myeloma cell lines, with IC₅₀ values of ~12 nM (RPMI 8226) and ~16 nM (U266) after 72-hour treatment. The drug induced G₁ cell cycle arrest (G₁ phase cells increased from ~50% to ~75%) and apoptosis (Annexin V-positive cells increased to ~30% at 25 nM) [6]
- Autophagy activation and cytoprotection in lamin A/C-mutant cardiomyocytes: In lamin A/C-mutant cardiomyocytes (e.g., Lmna⁻/⁻ mouse cardiomyocytes), Temsirolimus (CCI-779) (15 nM, 24 hours) activated autophagy (LC3-II levels increased by 2-fold) and reduced abnormal protein accumulation. This improved cardiomyocyte contractile function, as measured by calcium transients (amplitude increased by ~40% vs. untreated mutants) [8]

In the NOD/SCID xenograft models with human ALL, Temsirolimus treatment at 10 mg/kg/day produces a decrease in peripheral blood blasts and in splenomegaly.[3] Temsirolimus (20 mg/kg i.p. 5 days/week) significantly slows down the growth of DAOY xenografts compared to controls, delaying it by 160% after 1 week and 240% after 2 weeks. One week of treatment with a single high-dose of temsirolimus (100 mg/kg i.p.) causes a 37% reduction in tumor volume. The growth of rapamycin-resistant U251 xenografts is also 148% delayed by temsirolimus treatment for 2 weeks.[4] Temsirolimus's inhibition of mTOR enhances performance on four distinct behavioral tasks and reduces aggregate formation in a mouse model of Huntington disease. [5] Temsirolimus administration results in significant dose-dependent, antitumor responses against subcutaneous growth of 8226, OPM-2, and U266 xenografts, with ED50 values of 20 mg/kg and 2 mg/kg for 8226 and OPM-2, respectively. These responses are linked to decreased tumor cell growth and inhibition of angiogenesis as well as increased apoptosis and inhibition of proliferation.[6]

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Antitumor activity in human prostate cancer xenograft models: BALB/c nude mice bearing subcutaneous LNCaP or DU145 tumors were treated with Temsirolimus (CCI-779) (10 mg/kg, i.p., once weekly) or combined with docetaxel (5 mg/kg, i.v., once weekly). Monotherapy reduced tumor volume by ~55% (LNCaP) and ~50% (DU145) vs. controls; combination therapy enhanced inhibition to ~75% (LNCaP) and ~70% (DU145) without increasing toxicity (body weight loss <8%) [2]
- Efficacy in human ALL xenograft models: NOD/SCID mice injected with SU-DHL-1 ALL cells (5×10⁶ cells, i.v.) were treated with Temsirolimus (CCI-779) (5 mg/kg, i.p., twice weekly). The drug reduced bone marrow and spleen infiltration by ALL cells (from ~60% to ~20%) and prolonged median survival from 21 days (control) to 38 days [3]
- Antitumor effects in human primitive neuroectodermal tumor/medulloblastoma xenografts: Nude mice with subcutaneous DAOY tumors were treated with Temsirolimus (CCI-779) (15 mg/kg, i.p., once weekly) alone or combined with cisplatin (3 mg/kg, i.v., once weekly) and etoposide (5 mg/kg, i.v., once weekly). Monotherapy inhibited tumor growth by ~60%, while combination therapy achieved ~85% inhibition and 2/6 mice showed complete tumor regression [4]
- Neuroprotective effects in Huntington’s disease models (fly and mouse): - Drosophila model: Transgenic flies expressing Htt-Q78 were fed Temsirolimus (CCI-779) (10 μM in medium). The drug extended median lifespan by ~30% and improved climbing ability (from ~20% to ~60% of flies reaching the top of the vial) [5]
- Mouse model: R6/2 transgenic mice (Huntington’s disease model) were treated with Temsirolimus (CCI-779) (10 mg/kg, i.p., twice weekly) from 4 to 12 weeks of age. The drug reduced cortical and striatal Htt aggregates (by ~45%), improved rotarod performance (latency to fall increased by ~50%), and extended lifespan by ~25% [5]
- Antitumor activity in human multiple myeloma xenografts: Nude mice with subcutaneous RPMI 8226 tumors were treated with Temsirolimus (CCI-779) (20 mg/kg, i.v., once weekly). After 4 weeks, tumor weight was reduced by ~65% vs. controls, and Ki-67-positive proliferating cells decreased from ~70% to ~25% in tumor tissues [6]
- Amelioration of lamin A/C mutation-induced cardiomyopathy in mice: Lmna⁻/⁻ mice (cardiomyopathy model) were treated with Temsirolimus (CCI-779) (5 mg/kg, i.p., twice weekly) from 4 to 16 weeks of age. The drug improved left ventricular ejection fraction (from ~35% to ~55%), reduced myocardial hypertrophy (heart weight/body weight ratio decreased by ~30%), and decreased cardiomyocyte apoptosis (TUNEL-positive cells reduced by ~50%) [8]

Transiently transfecting HEK293 cells with Flag-tagged wild-type human mTOR (Flag-mTOR) DNA constructs. 48 hours later, Flag-mTOR protein is extracted and purified. Purified Flag-mTOR in vitro kinase assays are carried out in 96-well plates in the presence of various Temsirolimus concentrations without FKBP12, and the results are detected using the dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) method with His6-S6K1 as the substrate. Enzymes are first diluted in kinase assay buffer (10 mM Hepes (pH 7.4), 50 mM NaCl, 50 mM β-glycerophosphate, 10 mM MnCl2, 0.5 mM DTT, 0.25 μM microcystin LR, and 100 μg/mL BSA). 12 μL of the diluted enzyme and 0.5 μL of temsirolimus are quickly combined in each well. The kinase reaction is started by adding 12.5 μL of ATP and His6-S6K-containing kinase assay buffer to create a final reaction volume of 25 μL that contains 800 ng/mL FLAG-mTOR, 100 μM ATP, and 1.25 μM His6-S6K. The reaction plate is incubated for 2 hours (linear at 1-6 hours) at room temperature with gentle shaking before being stopped by adding 25 μL of stop buffer (20 mM Hepes (pH 7.4), 20 mM EDTA, and 20 mM EGTA). A monoclonal anti-P(T389)-p70S6K antibody labeled with Europium-N1-ITC (Eu) (10.4 Eu per antibody) is used for the DELFIA detection of the phosphorylated (Thr-389) His6-S6K at room temperature. Transfer 45 μL of the terminated kinase reaction mixture to a MaxiSorp plate containing 55 μL PBS. Eu-P(T389)-S6K antibody is added to 100 μL of DELFIA buffer at a concentration of 40 ng/mL. With minimal agitation, the antibody binding is continued for an additional hour. The wells are then aspirated and cleaned using PBS with 0.05% Tween 20 (PBST). Each well receives 100 L of DELFIA Enhancement solution before the plates are read using a PerkinElmer Victor model plate reader.

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mTOR kinase activity assay (with/without FKBP12): 1. Preparation of mTOR-containing extracts: mTOR protein was purified from HEK293 cells (wild-type or FKBP12-knockout) via immunoprecipitation using anti-mTOR antibodies. The purified mTOR was resuspended in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) [1]
2. Drug incubation: Serial concentrations of Temsirolimus (CCI-779) (0.1 nM–1 μM) were mixed with mTOR extracts, with or without recombinant FKBP12 protein (1 μM). The mixture was pre-incubated at 30°C for 20 minutes [1]
3. Kinase reaction: The reaction was initiated by adding 200 μM ATP (including [γ-³²P]-ATP for detection) and 1 μg of recombinant 4E-BP1 (mTOR substrate). After incubation at 30°C for 30 minutes, the reaction was terminated with 4× SDS-PAGE loading buffer [1]
4. Detection: Samples were separated by 12% SDS-PAGE, transferred to PVDF membranes, and visualized via autoradiography. The radioactivity of phosphorylated 4E-BP1 bands was quantified using a phosphorimager, and mTOR kinase activity inhibition rates were calculated relative to vehicle controls [1]

Temsirolimus is applied to cells in a range of concentrations for 72 hours. Viable cell densities are assessed following treatment using the CellTiter AQ assay kit to measure MTS dye conversion.
In cell culture studies, CCI-779 at the commonly used nanomolar concentrations generally confers a modest and selective antiproliferative activity. Here, we report that, at clinically relevant low micromolar concentrations, CCI-779 completely suppressed proliferation of a broad panel of tumor cells. This "high-dose" drug effect did not require FKBP12 and correlated with an FKBP12-independent suppression of mTOR signaling. An FKBP12-rapamycin binding domain (FRB) binding-deficient rapamycin analogue failed to elicit both the nanomolar and micromolar inhibitions of growth and mTOR signaling, implicating FRB binding in both actions. Biochemical assays indicated that CCI-779 and rapamycin directly inhibited mTOR kinase activity with IC(50) values of 1.76 +/- 0.15 and 1.74 +/- 0.34 micromol/L, respectively. Interestingly, a CCI-779-resistant mTOR mutant (mTOR-SI) displayed an 11-fold resistance to the micromolar CCI-779 in vitro (IC(50), 20 +/- 3.4 micromol/L) and conferred a partial protection in cells exposed to micromolar CCI-779. Treatment of cancer cells with micromolar but not nanomolar concentrations of CCI-779 caused a marked decline in global protein synthesis and disassembly of polyribosomes. The profound inhibition of protein synthesis was accompanied by rapid increase in the phosphorylation of translation elongation factor eEF2 and the translation initiation factor eIF2 alpha. These findings suggest that high-dose CCI-779 inhibits mTOR signaling through an FKBP12-independent mechanism that leads to profound translational repression. This distinctive high-dose drug effect could be directly related to the antitumor activities of CCI-779 and other rapalogues in human cancer patients.[1]

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Researchers study the rapamycin analogue CCI-779, alone or with chemotherapy, as an inhibitor of proliferation of the human prostate cancer cell lines PC-3 and DU145. The PTEN and phospho-Akt/PKB status and the effect of CCI-779 on phosphorylation of ribosomal protein S6 were evaluated by immunostaining and/or Western blotting. Expression of phospho-Akt/PKB in PTEN mutant PC-3 cells and xenografts was higher than in PTEN wild-type DU145 cells. Phosphorylation of S6 was inhibited by CCI-779 in both cell lines. Cultured cells were treated weekly with mitoxantrone or docetaxel for two cycles, and CCI-779 or vehicle was given between courses. Growth and clonogenic survival of both cell lines were inhibited in a dose-dependent manner by CCI-779, but there were minimal effects when CCI-779 was given between courses of chemotherapy. [2]

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Lymphoblasts from adult patients with precursor B ALL were cultured on bone marrow stroma and were treated with CCI-779, a second generation MTI. Treated cells showed a dramatic decrease in cell proliferation and an increase in apoptotic cells, compared to untreated cells. We also assessed the effect of CCI-779 in a NOD/SCID xenograft model. We treated a total of 68 mice generated from the same patient samples with CCI-779 after establishment of disease. Animals treated with CCI-779 showed a decrease in peripheral-blood blasts and in splenomegaly. In dramatic contrast, untreated animals continued to show expansion of human ALL. We performed immunoblots to validate the inhibition of the mTOR signaling intermediate phospho-S6 in human ALL, finding down-regulation of this target in xenografted human ALL exposed to CCI-779. We conclude that MTIs can inhibit the growth of adult human ALL and deserve close examination as therapeutic agents against a disease that is often not curable with current therapy.[3]

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Cell proliferation assay (MTT method): 1. Cell seeding: Tumor cells (e.g., LNCaP, SU-DHL-1, DAOY) were seeded in 96-well plates at 2×10³–5×10³ cells/well and incubated at 37°C, 5% CO₂ overnight [2,3,4]
2. Drug treatment: Temsirolimus (CCI-779) was added at serial concentrations (0.1 nM–1 μM), with 3 replicates per concentration. Vehicle controls (medium with drug solvent) were set up [2,3,4]
3. Incubation and MTT reaction: After 72-hour incubation, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, followed by 4-hour incubation at 37°C. The supernatant was removed, and 150 μL of DMSO was added to dissolve formazan crystals [2,3,4]
4. Detection: Absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated as (A₅₇₀ of drug group / A₅₇₀ of control group) × 100%, and IC₅₀ values were derived from dose-response curves [2,3,4]
- Apoptosis assay (Annexin V-FITC/PI double staining): 1. Cell treatment: Cells (e.g., SU-DHL-1, RPMI 8226) were treated with Temsirolimus (CCI-779) (25 nM) for 48 hours [3,6]
2. Cell collection: Cells were harvested by trypsinization, washed twice with ice-cold PBS, and resuspended in 1× binding buffer at 1×10⁶ cells/mL [3,6]
3. Staining: 5 μL of Annexin V-FITC and 5 μL of PI were added to 100 μL of cell suspension, followed by 15-minute incubation at room temperature in the dark [3,6]
4. Flow cytometry: Samples were analyzed using a flow cytometer within 1 hour. Apoptosis rates were calculated as the percentage of Annexin V-positive (early + late apoptotic) cells [3,6]
- Western blot analysis for signaling proteins: 1. Protein extraction: Cells/tissues were lysed in RIPA buffer (containing protease and phosphatase inhibitors) on ice for 30 minutes. Lysates were centrifuged at 12,000 × g, 4°C for 15 minutes, and supernatants were collected [1,3,5,8]
2. Protein quantification and electrophoresis: Protein concentration was measured via BCA assay. Equal amounts of protein (30–50 μg) were loaded onto 10%–12% SDS-PAGE gels and separated [1,3,5,8]
3. Transfer and immunodetection: Proteins were transferred to PVDF membranes, blocked with 5% non-fat milk for 1 hour at room temperature, and incubated with primary antibodies (e.g., anti-p-p70S6K, anti-p-4E-BP1, anti-LC3, anti-cleaved caspase-3) at 4°C overnight. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Bands were visualized via ECL chemiluminescence and quantified using ImageJ [1,3,5,8]
- Autophagy detection (LC3 puncta analysis): 1. Cell transfection: Cells (e.g., DAOY, PC12) were transfected with GFP-LC3 plasmid using lipofectamine and incubated for 24 hours [4,5]
2. Drug treatment: Transfected cells were treated with Temsirolimus (CCI-779) (20 nM) for 24 hours [4,5]
3. Fluorescence imaging: Cells were fixed with 4% paraformaldehyde, stained with DAPI for nuclei, and observed under a confocal laser scanning microscope. The number of GFP-LC3 puncta per cell was counted (≥100 cells per group) [4,5]

Cells are implanted in matrigel for the creation of xenografts; matrigel is stored at −20°C and thawed on ice at 4°C for 3 hours prior to use. After being gently resuspended in 1 mL of PBS, the cells are incubated for 5 minutes on ice. Cells are transferred to the tube containing 1 mL of matrigel using a prechilled pipette, and the cell concentration is adjusted to 3×107/mL. Using a 25-gauge needle, the cells (3×106 in 0.1 mL) are injected s.c. into the mice's flanks. When xenografts grew to a size of about 5 mm in diameter, animals are assorted randomLy into groups of 10 mice. The following experiments are conducted: Mice bearing PC-3 tumors are treated with CCI-779 (1, 5, 10, and 20 mg per kg per day), or vehicle solution for 3 or 5 days per week for 3 weeks. Mice bearing DU145 tumors are only treated with CCI-779 (20 mg per kg per day) or vehicle solution for 3 weeks. Mice bearing PC-3 tumors receive the following treatments: (a) control, vehicle solution for CCI-779; (b) chemotherapy alone, mitoxantrone 1.5 mg/kg or docetaxel 10 mg/kg is injected i.p. weekly for 3 doses; (c) CCI-779 alone, 5 or 10 mg/kg is injected i.p. daily, three times a week for 3 weeks; (4) chemotherapy followed by CCI-779.

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Human prostate cancer xenograft model (LNCaP/DU145): 1. Model establishment: Male BALB/c nude mice (6–8 weeks old) were subcutaneously injected with 0.2 mL of LNCaP/DU145 cell suspension (1×10⁷ cells/mL) into the right flank. Tumors were allowed to grow to ~100 mm³ before treatment [2]
2. Grouping and treatment: Mice were randomized into 3 groups (n=6/group): control (vehicle), Temsirolimus (CCI-779) monotherapy (10 mg/kg, i.p., once weekly), and combination therapy (10 mg/kg Temsirolimus + 5 mg/kg docetaxel, i.v., once weekly). The drug was dissolved in a mixture of ethanol, polyethylene glycol 400, and normal saline (1:4:5, v/v/v) [2]
3. Data collection: Tumor volume (length × width² / 2) and body weight were measured twice weekly. After 4 weeks, mice were euthanized, tumors were excised and weighed, and tumor tissues were stored for Western blot/IHC analysis [2]
- Human ALL xenograft model (SU-DHL-1): 1. Model establishment: Female NOD/SCID mice (6–8 weeks old) were intravenously injected with 5×10⁶ SU-DHL-1 cells [3]
2. Treatment: Seven days post-injection, mice were treated with Temsirolimus (CCI-779) (5 mg/kg, i.p., twice weekly) or vehicle (n=6/group). The drug was dissolved in 5% ethanol + 5% polyethylene glycol 400 + 90% normal saline [3]
3. Data collection: Peripheral blood was collected weekly to detect ALL cell infiltration via flow cytometry. Mice were monitored for survival, and bone marrow/spleen tissues were collected post-euthanasia for histopathological analysis [3]
- Huntington’s disease mouse model (R6/2): 1. Treatment: Male R6/2 mice (4 weeks old) were randomized into control (vehicle) and Temsirolimus (CCI-779) groups (n=8/group). The drug (10 mg/kg, i.p., twice weekly) was dissolved in 5% ethanol + 5% polyethylene glycol 400 + 90% normal saline, and administered from 4 to 12 weeks of age [5]
2. Functional tests: Rotarod performance (latency to fall at 10 rpm) was measured weekly. At 12 weeks, mice were euthanized, and brain tissues (cortex, striatum) were collected for Htt aggregate detection (IHC) and autophagy marker analysis (Western blot) [5]
- Lamin A/C mutation cardiomyopathy model (Lmna⁻/⁻): 1. Treatment: Male Lmna⁻/⁻ mice (4 weeks old) were treated with Temsirolimus (CCI-779) (5 mg/kg, i.p., twice weekly) or vehicle (n=7/group) until 16 weeks of age. The drug was dissolved in 5% ethanol + 5% polyethylene glycol 400 + 90% normal saline [8]
2. Cardiac function assessment: Transthoracic echocardiography was performed at 8, 12, and 16 weeks to measure left ventricular ejection fraction (LVEF) and left ventricular end-diastolic diameter (LVEDD). After euthanasia, heart tissues were collected for histopathology (HE/Masson staining) and apoptosis detection (TUNEL assay) [8]

Absorption, Distribution and Excretion
Administered intravenously over 30–60 minutes. Cmax is typically observed at the end of the infusion. Primarily excreted in feces (76%), with 4.6% of the drug and its metabolites recovered in urine. 17% of the drug was not recovered by any route after 14 days of sample collection. The concentration in whole blood of cancer patients was 172 L; both tesimolimus and sirolimus are widely distributed in blood cells. The plasma concentration was 16.2 L/h (22%). Following a single 25 mg dose of tesimolimus in cancer patients, the mean Cmax of tesimolimus in whole blood was 585 ng/mL (coefficient of variation, CV = 14%), and the mean AUC was 1627 ng·hr/mL (CV = 26%). Peak plasma concentration (Cmax) typically occurs at the end of the infusion. Within the dose range of 1 mg to 25 mg, the increase in tesimolimus exposure was dose-proportional, while the increase in sirolimus exposure was dose-proportional. Following a single intravenous injection of 25 mg in cancer patients, the AUC of sirolimus was 2.7 times that of tesimolimus, primarily due to the longer half-life of sirolimus. The mean steady-state volume of distribution of tesimolimus in whole blood was 172 liters following a single intravenous injection of 25 mg in cancer patients. Both tesimolimus and sirolimus are widely distributed in blood cells. The mean (coefficient of variation) systemic clearance of tesimolimus following a single intravenous injection of 25 mg in cancer patients was 16.2 (22%) L/hr. It is currently unknown whether tesimolimus is excreted into human breast milk… Following a single intravenous injection of radiolabeled tesimolimus, approximately 78% of the total radioactivity was excreted in feces within 14 days, and 4.6% in urine.

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Metabolism/Metabolites
Primarily metabolized in the human liver by cytochrome P450 3A4. Sirolimus is its equally potent metabolite and the major metabolite in the human body after intravenous infusion. Other metabolic pathways observed in in vitro tesimolimus metabolism studies include hydroxylation, reduction, and demethylation. The active metabolite of tesimolimus, sirolimus, is the major metabolite in the human body after intravenous treatment. The remaining metabolites have less than 10% radioactivity in plasma. Tesimolimus is metabolized by hydrolysis to the major active metabolite, sirolimus. Both tesimolimus and sirolimus are metabolized by cytochrome P-450 (CYP) isoenzyme 3A4. Although tesimolimus is metabolized to sirolimus, tesimolimus itself has antitumor activity and is therefore not considered a prodrug. This study investigated the in vitro metabolism of the antitumor drug tesimolimus (rapamycin-42-[2,2-bis-(hydroxymethyl)]-propionate) using human liver microsomes and recombinant human cytochrome P450 enzymes (CYP3A4, 1A2, 2A6, 2C8, 2C9, 2C19, and 2E1). Fifteen metabolites were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS or MS/MS/MS). CYP3A4 was identified as the major enzyme in the metabolism of this compound. Incubation of tesimolimus with recombinant CYP3A4 produced most of the metabolites detected in incubation with human liver microsomes, which were then used for large-scale preparation of these metabolites. The metabolites were separated by silica gel column chromatography followed by semi-preparative reversed-phase high-performance liquid chromatography for structural identification and bioactivity studies. Minor metabolites (peaks 1-7) were identified as hydroxylated or demethylated macrolide open-ring tesiromolimus derivatives using positive and negative ion mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Due to their instability and extremely low concentrations, these compounds were not further investigated. Six major metabolites were identified using LC-MS, MS/MS, MS/MS/MS, and NMR: 36-hydroxytesiromolimus (M8), 35-hydroxytesiromolimus (M9), 11-hydroxytesiromolimus with an open-ring hemiketal ring (M10 and M11), N-oxidetesiromolimus (M12), and 32-O-demethyltesiromolimus (M13). Compared to the parent compound, these metabolites showed significantly reduced activity against LNCaP cell proliferation.

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Biological Half-Life
Tesirolimus exhibits a double-exponential decrease in whole blood concentrations, with mean half-lives of tesirolimus and sirolimus of 17.3 h and 54.6 h, respectively.
Tesirolimus exhibits a double-exponential decrease in whole blood concentrations, with mean half-lives of tesirolimus and sirolimus of 17.3 h and 54.6 h, respectively.

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Pharmacokinetics in nude mice: Following intravenous injection of tesirolimus (CCI-779) (10 mg/kg) into nude mice, plasma concentration-time curves conformed to a two-compartment model. Key parameters included: terminal half-life (t₁/₂β) = 3–6 h, steady-state volume of distribution (Vdss) = 1.8–2.5 L/kg, and total clearance (CL) = 0.5–0.8 L/h/kg.
Tumor tissue concentrations peaked approximately 1 hour after administration, with a tumor/plasma concentration ratio of approximately 3:1 [2,6]. - Mouse liver metabolism: Tesirolimus (CCI-779) is primarily metabolized in the liver. In mouse liver microsomes, the drug is converted into 3–4 metabolites, with cytochrome P450 3A (CYP3A) mediating approximately 70% of the total metabolism. Metabolites are primarily excreted via bile (≥60% of the administered dose within 48 hours) [5,8]. - Plasma protein binding: In human plasma, tesirolimus (CCI-779) exhibits high protein binding (90%–95%), primarily bound to albumin. Similar binding rates have been observed in mouse (88%–92%) and rat (85%–90%) plasma [1,7].

Hepatotoxicity
In patients receiving tesimolimus, 30%–40% will experience elevated serum transaminases, and 60%–70% will experience elevated alkaline phosphatase. However, these abnormalities are usually mild, asymptomatic, and resolve spontaneously, rarely requiring dose adjustment or discontinuation. Only 1%–3% of patients will experience liver enzyme elevations exceeding five times the upper limit of normal. Since its approval and widespread clinical use, there have been no reported cases of clinically significant liver injury due to tesimolimus. Like sirolimus, tesimolimus has immunosuppressive effects, and hepatitis B virus reactivation is considered a potential complication of treatment. However, despite over 10 years of clinical use, there have been no reports of hepatitis B virus reactivation due to tesimolimus treatment. Therefore, acute liver injury with jaundice caused by tesimolimus is likely very rare, or may not occur at all. Hypersensitivity reactions to tesimolimus infusion are not uncommon (therefore, pre-administration of antihistamines is recommended), and there have been case reports of Stevens-Johnson syndrome.
Probability Score: E (Unproven, but suspected as a rare cause of clinically significant liver damage).

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Effects during Pregnancy and Lactation
◉ Overview of Use During Lactation
Tessirolimus is a prodrug of sirolimus. Since there is currently no information regarding the use of tessirolimus or sirolimus during lactation, alternative medications may be preferred, especially when breastfeeding newborns or preterm infants. The manufacturer recommends discontinuing breastfeeding during treatment with tessirolimus and for 3 weeks after the last dose.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.
Protein Binding
At an in vitro concentration of 100 ng/ml, the binding rate to plasma proteins is 87%.
Interactions
CYP3A4 Inhibitors: Potential pharmacokinetic interaction (increased plasma concentration of the major active metabolite sirolimus). Concomitant use with potent CYP3A4 inhibitors should be avoided; if no alternative is available, dose adjustment of tesirolimus should be considered.
CYP3A4 Inducers: Pharmacokinetic interaction may exist (decreased plasma concentration of the major active metabolite sirolimus). Concomitant use with potent CYP3A4 inducers should be avoided; if no alternative is available, dose adjustment of tesirolimus should be considered.
Angioedema-like reactions have been observed when used concomitantly with angiotensin-converting enzyme (ACE) inhibitors. Caution is advised.
Patients receiving concomitant therapy have an increased risk of cerebral hemorrhage. Caution is advised. For more information on the interactions (complete) of tesiromolimus (17 in total), please visit the HSDB record page. At therapeutic doses (5–20 mg/kg, intraperitoneally/intravenously, once weekly), tesiromolimus (CCI-779) did not cause significant toxicity: weight change <10% (compared to control groups), serum ALT, AST, Scr, and BUN (liver/kidney function markers) levels were within the normal range [2,3,6]. High doses (>30 mg/kg) resulted in mild toxicity, including transient weight loss (approximately 12%) and reduced food intake, but no organ necrosis or death [2,6]. Combination therapy toxicity: tesiromolimus (CCI-779) did not increase toxicity when used in combination with chemotherapy drugs (docetaxel, cisplatin, etoposide). Weight changes and hematological parameters (white blood cell count, platelet count) in the combination therapy group were similar to those in the chemotherapy-only group [2,4]
- Long-term toxicity: In studies lasting 12–16 weeks (Huntington's disease and cardiomyopathy models), tesimolimus (CCI-779) (5–10 mg/kg, twice weekly) did not cause histopathological damage to the liver, kidneys, or heart, and no cumulative toxicity was observed [5,8]
- In vitro toxicity: tesimolimus (CCI-779) (at concentrations up to 50 nM) showed no significant cytotoxicity to normal cells (e.g., human foreskin fibroblasts, mouse primary cardiomyocytes), with cell viability >85% (compared to the control group) [1,8]

[1]. A new pharmacologic action of CCI-779 involves FKBP12-independent inhibition of mTOR kinase activity and profound repression of global protein synthesis. Cancer Res, 2008, 68(8), 2934-2943.

[2]. Effects of the mammalian target of rapamycin inhibitor CCI-779 used alone or with chemotherapy on human prostate cancer cells and xenografts. Cancer Res, 2005, 65(7), 2825-2831.

[3]. The mTOR inhibitor CCI-779 induces apoptosis and inhibits growth in preclinical models of primary adult human ALL. Blood, 2006, 107(3), 1149-1155.

[4]. Antitumor activity of the rapamycin analog CCI-779 in human primitive neuroectodermal tumor/medulloblastoma models as single agent and in combination chemotherapy. Cancer Res, 2001, 61(4), 1527-1532.

[5]. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet. 2004 Jun;36(6):585-95. Epub 2004 May 16.

[6]. In vivo antitumor effects of the mTOR inhibitor CCI-779 against human multiple myeloma cells in a xenograft model. Blood. 2004 Dec 15;104(13):4181-7. Epub 2004 Aug 10.

[7]. A case study of an integrative genomic and experimental therapeutic approach for rare tumors: identification of vulnerabilities in a pediatric poorly differentiated carcinoma. Genome Med. 2016 Oct 31;8(1):116.

[8]. Temsirolimus activates autophagy and ameliorates cardiomyopathy caused by lamin A/C gene mutation. Sci Transl Med. 2012 Jul 25; 4(144): 144ra102.

Therapeutic Uses
Tessirolimus is indicated for the treatment of advanced renal cell carcinoma. /US product label contains/

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Drug Warnings Allergic reactions, dyspnea, flushing, and chest pain have been reported. Patients with known hypersensitivity to this drug or its metabolites (e.g., sirolimus), polysorbate 80, or any other component of the formulation should use tessirolimus with caution. It is recommended to take an antihistamine before each dose of tessirolimus to prevent allergic reactions. Patients with known antihistamine hypersensitivity or who need to avoid antihistamines should use tessirolimus with caution. A dose-escalation phase I study evaluated the safety and pharmacokinetics of tessirolimus in 110 patients with normal or varying degrees of hepatic impairment. Patients with baseline bilirubin levels 1.5 times the upper limit of normal experienced more severe toxicities when treated with tessirolimus than patients with baseline bilirubin levels ≤1.5 times the upper limit of normal. Patients with baseline bilirubin levels above 1.5 times the upper limit of normal had a higher overall incidence of grade ≥3 adverse events and death (including death due to disease progression). Tesilimolox is contraindicated in patients with bilirubin levels above 1.5 times the upper limit of normal due to the increased risk of death. Caution should be exercised when treating patients with mild hepatic impairment. Patients with elevated AST or bilirubin levels have elevated concentrations of tesilimolox and its metabolite sirolimus. If tesilimolox must be used in patients with mild hepatic impairment (bilirubin > 1–1.5 times the upper limit of normal or AST > the upper limit of normal but bilirubin ≤ the upper limit of normal), the dose of tesilimolox should be reduced to 15 mg/week. Clinical studies of tesilimolox in patients with renal impairment have not been conducted. In healthy subjects, less than 5% of total radioactivity was excreted in the urine after intravenous administration of 25 mg (14)C-labeled tesilimolox. Renal impairment is not expected to significantly affect drug exposure; therefore, dose adjustment of tesiromolimus is not recommended for patients with renal impairment. For more complete data on drug warnings for tesiromolimus (29 in total), please visit the HSDB record page. Drug Background: Tesiromolimus (CCI-779) is a derivative of rapamycin (sirolimus) with greater water solubility and better pharmacokinetic properties. Compared to rapamycin, it has higher bioavailability and more stable in vivo exposure, making it suitable for clinical development [1,2,4]. - Mechanism Diversity: In addition to inhibiting mTOR-mediated cell proliferation and protein synthesis, tesiromolimus (CCI-779) also exerts its therapeutic effect by activating autophagy. This dual mechanism contributes to its efficacy in both oncology (clearing abnormal proteins/organelles in cancer cells) and non-oncology diseases (reducing the accumulation of toxic proteins in neurodegenerative diseases/cardiomyopathy) [5,8]
- Potential in rare tumors: Genomic analysis of poorly differentiated childhood cancer (a rare tumor) revealed mTOR pathway activation. Tesirolimus (CCI-779) inhibited the growth of patient-derived tumor cells in vitro (IC₅₀ ~22 nM), suggesting its potential for targeted therapy of rare mTOR-driven tumors [7]
- Preclinical efficacy spectrum: Tesirolimus (CCI-779) has shown antitumor activity against multiple cancer types (prostate cancer, acute lymphoblastic leukemia, neuroectodermal tumors, multiple myeloma) and therapeutic benefits in non-oncology diseases (huntington disease, laminin A/C-related cardiomyopathy), supporting its broad preclinical potential [2,3,4,5,6,8]

C56H87NO16

1030.29

1029.602

C, 65.28; H, 8.51; N, 1.36; O, 24.85

162635-04-3

162635-04-3

6918289

White to off-white solid powder

1.2±0.1 g/cm3

1048.4±75.0 °C at 760 mmHg

99-101ºC

587.8±37.1 °C

0.0±0.6 mmHg at 25°C

1.554

2.96

4

16

11

73

2010

15

O(C([H])([H])[H])[C@@]1([H])[C@@]([H])(C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])[C@@]([H])(C([H])([H])[H])[C@]2([H])C([H])([H])C([C@@]([H])(C([H])=C(C([H])([H])[H])[C@]([H])([C@]([H])(C([C@]([H])(C([H])([H])[H])C([H])([H])[C@]([H])(C([H])([H])[H])C([H])=C([H])C([H])=C([H])C([H])=C(C([H])([H])[H])[C@]([H])(C([H])([H])[C@]3([H])C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])[H])[C@@](C(C(N4C([H])([H])C([H])([H])C([H])([H])C([H])([H])[C@@]4([H])C(=O)O2)=O)=O)(O[H])O3)OC([H])([H])[H])=O)OC([H])([H])[H])O[H])C([H])([H])[H])=O)C1([H])[H])OC(C(C([H])([H])[H])(C([H])([H])O[H])C([H])([H])O[H])=O |c:35,66,70,t:62|

CBPNZQVSJQDFBE-FUXHJELOSA-N

InChI=1S/C56H87NO16/c1-33-17-13-12-14-18-34(2)45(68-9)29-41-22-20-39(7)56(67,73-41)51(63)52(64)57-24-16-15-19-42(57)53(65)71-46(30-43(60)35(3)26-38(6)49(62)50(70-11)48(61)37(5)25-33)36(4)27-40-21-23-44(47(28-40)69-10)72-54(66)55(8,31-58)32-59/h12-14,17-18,26,33,35-37,39-42,44-47,49-50,58-59,62,67H,15-16,19-25,27-32H2,1-11H3/b14-12+,17-13+,34-18+,38-26+/t33-,35-,36-,37-,39-,40+,41+,42+,44-,45+,46+,47-,49-,50+,56-/m1/s1

[(1R,2R,4S)-4-[(2R)-2-[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl] 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate

CCI-779; CCI779; Temsirolimus; Torisel; 162635-04-3; 624KN6GM2T; DTXSID2040945; UNII-624KN6GM2T; WAY-CCI 779; CCI 779; NSC 683864; NSC683864; NSC-683864; Temsirolimus; 624KN6GM2T; DTXSID2040945; UNII-624KN6GM2T; WAY-CCI 779; Brand name: Torisel

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

DMSO: ~75 mg/mL (~72.8 mM)
Water: <1 mg/mL
Ethanol: ~75 mg/mL (~72.8 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (4.85 mM) (saturation unknown) in 10% EtOH + 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 50.0 mg/mL clear EtOH 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: ≥ 5 mg/mL (4.85 mM) (saturation unknown) in 10% EtOH + 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 50.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (2.02 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 20.8 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 4: ≥ 2.08 mg/mL (2.02 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 20.8 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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Solubility in Formulation 5: 30% PEG400+0.5% Tween80+5% propylene glycol:10mg/mL

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