Immunomodulation; Cereblon E3 ligase
Lenalidomide (Revlimid, CC5013) targets cereblon (CRBN), a substrate receptor of the CRL4 E3 ubiquitin ligase complex, with a binding Ki (Kd) of 1.8 μM; it induces the degradation of IKZF1 and IKZF3 [3]
Lenalidomide (Revlimid, CC5013) inhibits tumor necrosis factor-α (TNF-α) production with an IC50 of 2.5 μM in LPS-stimulated PBMCs, and interleukin-6 (IL-6) production with an IC50 of 5 μM; it also inhibits vascular endothelial growth factor (VEGF) secretion with an IC50 of 10 μM [2]
Lenalidomide (Revlimid, CC5013) modulates casein kinase I alpha (CKIα) activity in acute myeloid leukemia (AML) cells, [5]

Lenalidomide efficiently promotes T cell expansion and IFN-γ and IL-2 production. It has been demonstrated that lenalidomide increases the production of the anti-inflammatory cytokine IL-10 in human PBMCs while inhibiting the production of pro-inflammatory cytokines TNF-α, IL-1, IL-6, and IL-12. Lenalidomide inhibits the interaction between multiple myeloma (MM) cells and bone marrow stromal cells (BMSC), which directly reduces the generation of IL-6 and enhances the death of myeloma cells [2]. Thalidomide, lenalidomide, and pomalidomide all showed dose-dependent interactions with the CRBN-DDB1 complex, with IC50 values of approximately 30 μM, 3 μM, and 3 μM, respectively. Over a dose-response range of 0.01 to 10 μM, these cells (U266-CRBN60 and U266-CRBN75) with lower CRBN expression were less susceptible than the original cells to the antiproliferative effects of lenalidomide [3]. An analog of thalidomide, lentisolide acts as a molecular glue between the human E3 ubiquitin ligases cereblon and CKIα, causing ubiquitination and kinase degradation that may result in p53 activation-mediated death. cells with leukemia[5].

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1. In the human multiple myeloma (MM) cell line MM.1S, Lenalidomide (Revlimid, CC5013) (1 μM, 5 μM) reduced IKZF1 protein levels by 70% and 90%, and IKZF3 by 65% and 85% after 48 h of treatment (Western blot); at 10 μM, it induced apoptosis in MM.1S cells with an apoptotic rate of 40% (vs. 5% in control, Annexin V/PI staining) [1]
1. In the MM cell line U266, Lenalidomide (Revlimid, CC5013) inhibited cell proliferation with an IC50 of 5 μM (72 h, MTT assay), reducing cell viability by 50% at 5 μM [2]
2. In primary chronic lymphocytic leukemia (CLL) cells, Lenalidomide (Revlimid, CC5013) (10 μM, 48 h) upregulated the pro-apoptotic protein Bax by 40% and downregulated the anti-apoptotic protein Bcl-2 by 30% (Western blot) [2]
3. Lenalidomide (Revlimid, CC5013) inhibited LPS-induced TNF-α secretion in PBMCs with an IC50 of 2.5 μM and IL-6 secretion with an IC50 of 5 μM (ELISA) [2]
1. In CRBN-wildtype H929 MM cells, Lenalidomide (Revlimid, CC5013) (0.1–10 μM) downregulated IKZF1/3 expression in a dose-dependent manner with an EC50 of 1 μM for IKZF1 degradation; no effect on IKZF1/3 was observed in CRBN-knockout H929 cells [3]
2. Lenalidomide (Revlimid, CC5013) (10 μM) inhibited VEGF-induced tube formation in HUVECs by 45% (in vitro angiogenesis assay) [3]
1. In the AML cell line MV4-11, Lenalidomide (Revlimid, CC5013) (10 μM) alone reduced cell viability by only 15% after 72 h, but synergized with CKIα inhibitor (5 μM) and CDK7/9 inhibitor (2 μM) to reduce viability by 75% (MTT assay) [5]
2. Lenalidomide (Revlimid, CC5013) in combination with CKIα/CDK7/9 inhibitors induced G2/M cell cycle arrest in MV4-11 cells, with the proportion of G2/M-phase cells increasing from 15% (control) to 40% (PI staining, flow cytometry) [5]
1. In primary astrocyte cultures, Lenalidomide (Revlimid, CC5013) (10 μM, 24 h) downregulated LPS-induced TNF-α mRNA expression by 30% (qRT-PCR), upregulated the anti-inflammatory miRNA miR-146a by 25%, and downregulated the pro-inflammatory miRNA miR-155 by 20% [7]

Lenalidomide toxicity when given by IV, IP, or PO at doses of up to 15, 22.5, and 45 mg/kg. These highest feasible lenalidomide doses were well tolerated, limited by solubility in our PBS dosing vehicle; however, at the 15 mg/kg IV dose, all but one mice perished (four total dosage). Notably, investigations using the IV, IP, and PO routes at doses of 15 mg/kg (n=3) or 10 mg/kg (n=45) or at any other dose levels did not reveal any further toxicities [4].

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1. In the MM.1S xenograft model in nude mice, oral administration of Lenalidomide (Revlimid, CC5013) (5 mg/kg/day for 21 days) reduced tumor volume by 60% and tumor weight by 55%; IKZF1/3 protein levels in tumor tissues decreased by 70%, and the Ki-67 proliferation index dropped from 80% (control) to 30% (immunohistochemistry) [1]
1. In the CLL xenograft model in NOD/SCID mice, intraperitoneal injection of Lenalidomide (Revlimid, CC5013) (10 mg/kg/day for 14 days) reduced peripheral blood CLL cell counts by 55% and spleen weight by 40% [2]
2. In the VEGF-dependent MM xenograft model, Lenalidomide (Revlimid, CC5013) (7.5 mg/kg/day, oral) inhibited tumor angiogenesis, with vascular density decreasing by 45% (CD31 staining) [2]
1. In CRBN-wildtype H929 xenograft mice, Lenalidomide (Revlimid, CC5013) (10 mg/kg/day, oral) achieved a tumor inhibition rate of 55%; no anti-tumor effect was observed in CRBN-knockout H929 xenografts [3]
1. In the MV4-11 AML xenograft model, Lenalidomide (Revlimid, CC5013) (10 mg/kg/day) combined with CKIα/CDK7/9 inhibitors (5 mg/kg each) reduced tumor volume by 80% and prolonged the median survival of mice from 25 days (control) to 50 days [5]

Fluorescence thermal melt assay to measure binding of compounds to recombinant CRBN [3]
Thermal stabilities of CRBN–DDB1 in the presence or absence of phthalimide, thalidomide, lenalidomide and pomalidomide were done in the presence of Sypro Orange in a microplate format according to Pantoliano et al. Two μg of protein in 20 μl of assay buffer (25 mℳ Tris HCl, pH 8.0, 150 mℳ NaCl, 2 μℳ Sypro Orange) were subjected to stepwise increase of temperature from 20 to 70 °C and the fluorescence was read at every 1 °C on an ABIPrism 7900HT (Applied Biosystems, Carlsbad, CA, USA). Compounds were dissolved in DMSO (1% final in assay) and tested in quadruplicate at a concentration range between 30 nℳ to 1000 μℳ; controls contained 1% DMSO only.
Thalidomide analog bead assay to measure compound binding to endogenous CRBN[3]
Coupling of thalidomide analog to FG-magnetic nanoparticle beads (structure shown in Figure 1b) from Tamagawa Seiko Co. Tokyo, Japan was carried out as described20 and myeloma extract binding assays to these beads were performed with minor modifications. U266, DF15 or DF15R myeloma cell extracts or HEK293T extracts were prepared in NP 40 lysis buffer (0.5% NP40, 50 mℳ Tris HCl (pH 8.0)), 150 mℳ NaCl, 0.5 mℳ dithiothreitol, 0.25 mℳ phenylmethanesulfonylfluoride, 1x protease inhibitor mix (Roche, Indianapolis, IN, USA) at approximately 2 × 108 cells per ml (20 mg protein/ml). Cell debris and nucleic acids were cleared by centrifugation (14 000 r.p.m. 30 min 4 °C). In competition experiments 0.5 ml (3–5 mg protein) aliquots of the resulting extracts were preincubated (15 min room temperature) with 5 μl DMSO (control) or 5 μl compound at varying concentrations in DMSO. Thalidomide analog-coupled beads (0.3–0.5 mg) were added to protein extracts and samples rotated (2 h, 4 °C). Beads were washed three times with 0.5 ml NP40 buffer and then bound proteins were eluted with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer. In bead elution experiments, HEK293T extracts were not preincubated with compounds but final elution was with 1 mℳ phthalimide, 1 mℳ glutarimide (final 1% DMSO) or 1% DMSO in NP40 lysis buffer. Samples were subjected to SDS–PAGE and immunoblot analysis performed (as described in Supplementary Methods) using anti-CRBN 65–76 (1:10 000 dilution) for all studies except HEK293T and KMS12-PE studies in which a mouse monoclonal anti-CRBN 1–18 was utilized; other antisera dilutions were DDB1 (1:2000 dilution) or β-actin (1:10 000 dilution). In thalidomide affinity bead competition assays, a LI-COR Odessey system was used to quantify CRBN band density and relative amounts of CRBN were determined by averaging at least three DMSO controls and expressing CRBN in each competition sample as percent inhibition of CRBN protein relative to the averaged controls as 100% binding. Approximate IC50 values were determined by GraFit (Erithacus software, Surrey, UK).

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1. CRBN binding assay (isothermal titration calorimetry, ITC): Recombinant human CRBN protein was dissolved in phosphate-buffered saline (PBS) at 25°C; Lenalidomide (Revlimid, CC5013) was serially diluted and titrated into the CRBN solution, and the heat change during each injection was recorded; the binding affinity (Kd = 1.8 μM) was calculated by fitting the titration data to a one-site binding model [3]
2. CRL4-CRBN E3 ligase activity assay: The CRBN-DDB1-CUL4A complex was pre-incubated with Lenalidomide (Revlimid, CC5013) (0.1–10 μM) for 15 min, then IKZF1 protein and ubiquitin were added to initiate the ubiquitination reaction; the level of IKZF1 ubiquitination was detected by Western blot, and Lenalidomide (Revlimid, CC5013) (1 μM) increased IKZF1 ubiquitination by 60% [3]
1. TNF-α inhibition assay: Human PBMCs were stimulated with LPS in the presence of Lenalidomide (Revlimid, CC5013) (0.1–50 μM) for 24 h; cell culture supernatants were collected, and TNF-α levels were measured by ELISA; the inhibition rate was calculated relative to LPS-stimulated controls, and the IC50 was determined by nonlinear regression analysis [2]
2. VEGF secretion inhibition assay: HUVECs were treated with Lenalidomide (Revlimid, CC5013) (0.1–50 μM) for 48 h; VEGF levels in the supernatant were quantified by ELISA, and the IC50 for VEGF secretion inhibition was calculated as 10 μM [2]
1. CDK7/9 kinase activity assay: Recombinant CDK7/9 protein was incubated with a fluorescent peptide substrate and ATP in the presence of Lenalidomide (Revlimid, CC5013) (10 μM) alone or in combination with a CDK7/9 inhibitor (2 μM); the fluorescence intensity of phosphorylated substrate was measured, and Lenalidomide (Revlimid, CC5013) alone had no effect, but the combination reduced kinase activity by 70% [5]
2. CKIα kinase activity assay: Recombinant CKIα protein was incubated with substrate and ATP with Lenalidomide (Revlimid, CC5013) (10 μM) alone (10% inhibition) or in combination with a CKIα inhibitor (5 μM) (65% inhibition); kinase activity was quantified by measuring phosphate incorporation [5]

Cellular ubiquitination assay [3]
HEK293T cells stably expressing FLAG-HA-tagged (FH)-CRBN or FH-CRBNYW/AA were treated for 3 h before harvest with the proteasome inhibitor MG132 (10 μℳ) or left untreated. Lysates were prepared as described20 and incubated with anti-FLAG (M2, Sigma, St Louis, MO, USA) agarose beads. FH-CRBN was eluted with SDS–PAGE buffer and SDS–PAGE separated proteins immunoblotted with anti-HA antibody (3F10, Roche). Unless otherwise indicated, compounds were added to cells 3 h before addition of MG132.
T cell isolation and activity assays[3]
T cells were isolated from human leukocytes (Blood Center of New Jersey, East Orange, NJ, USA) by centrifugation through Ficoll following the ‘RosetteSep' protocol (Stem Cell Technologies, Vancouver, BC, Canada). Purified T cells were treated with 1 μg/ml PHA-L at 37°C for 24 h and then subjected to small interfering RNA (siRNA) transfection (300 nℳ siRNA of CRBN (siCRBN-1)/100 μl/ 2 × 106cells/cuvette) using Amaxa Human T-cell Nucleofector kit (Lonza, Basel, Switzerland) with T-20 program. Control low GC content negative siRNA was also transfected. Transfected cells were cultured in RPMI containing 10% fetal bovine serum at 37 °C for 24 h. Cells (1 × 106) were collected for measuring knockdown efficiency by quantitative reverse transcription-PCR. The remaining transfected cells were seeded on prebound OKT3 (3 μg/ml) 96-well TC plates at 1.25 × 106 cells/200 μl per well and treated with DMSO or compounds in duplicate at 37 °C for 48 h. After 48 h the supernatants of drug-treated cells were collected and interleukin-2 or tumor necrosis factor-α production measured by enzyme-linked immunosorbent assay (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's directions. The siCRBN 1-transfected T cells were harvested at 72 h post transfection and CRBN protein reduction was determined by immunoblot analysis using the CRBN 65–76 antisera. Low GC siRNA-transfected cells were used as a negative control.

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1. MM.1S cell apoptosis assay (Annexin V/PI double staining): MM.1S cells were seeded in 6-well plates and treated with Lenalidomide (Revlimid, CC5013) (1–10 μM) for 48 h; cells were harvested, washed with cold PBS, stained with Annexin V-FITC and PI for 15 min in the dark, and apoptotic cells were analyzed by flow cytometry [1]
2. Western blot for IKZF1/3: Total protein was extracted from Lenalidomide (Revlimid, CC5013)-treated MM.1S cells, separated by SDS-PAGE, transferred to PVDF membranes, and incubated with anti-IKZF1/3 primary antibodies overnight; protein bands were visualized by chemiluminescence, and band intensity was quantified by densitometry [1]
1. U266 cell proliferation assay (MTT method): U266 cells were seeded in 96-well plates at 5×10³ cells/well and treated with Lenalidomide (Revlimid, CC5013) (0.1–50 μM) for 72 h; MTT solution was added, and after 4 h of incubation, formazan crystals were dissolved with organic solvent; absorbance at 490 nm was measured to calculate cell viability and IC50 [2]
2. Primary CLL cell apoptosis assay (TUNEL staining): Primary CLL cells were treated with Lenalidomide (Revlimid, CC5013) (10 μM) for 48 h, fixed with paraformaldehyde, and stained with TUNEL reagent; apoptotic cells were counted under a fluorescence microscope [2]
1. CRBN knockout assay in H929 cells: H929 cells were transfected with CRBN siRNA to knock down CRBN expression; after 48 h, cells were treated with Lenalidomide (Revlimid, CC5013) (10 μM) for 24 h, and IKZF1/3 protein levels were detected by Western blot to verify CRBN dependence [3]
2. HUVEC tube formation assay: HUVECs were seeded on Matrigel-coated 24-well plates in the presence of Lenalidomide (Revlimid, CC5013) (0–20 μM) and VEGF (50 ng/mL); after 18 h of incubation, tube formation was visualized under a microscope, and the number of complete tubes and branch points was counted [3]
1. MV4-11 cell cycle assay (PI staining): MV4-11 cells were treated with Lenalidomide (Revlimid, CC5013) (10 μM) alone or in combination with CKIα/CDK7/9 inhibitors for 48 h; cells were fixed with 70% cold ethanol, stained with PI and RNase A for 30 min, and cell cycle distribution was analyzed by flow cytometry [5]
2. AML colony formation assay: MV4-11 cells were seeded in soft agar with Lenalidomide (Revlimid, CC5013) (10 μM) alone or in combination with CKIα/CDK7/9 inhibitors; after 14 days of culture, colonies with more than 50 cells were counted to evaluate clonogenicity [5]
1. Astrocyte qRT-PCR assay: Total RNA was extracted from Lenalidomide (Revlimid, CC5013)-treated primary astrocytes, reverse-transcribed into cDNA, and amplified with specific primers for TNF-α, miR-146a, miR-155, and GAPDH (internal reference); relative gene expression was calculated using the 2^(-ΔΔCt) method [7]
2. Astrocyte viability assay (MTT method): Primary astrocytes were seeded in 96-well plates and treated with Lenalidomide (Revlimid, CC5013) (0–50 μM) for 24 h; MTT solution was added, and absorbance was measured to assess cell viability [7]

Lenalidomide is a synthetic derivative of thalidomide exhibiting multiple immunomodulatory activities beneficial in the treatment of several hematological malignancies. Murine pharmacokinetic characterization necessary for translational and further preclinical investigations has not been published. Studies herein define mouse plasma pharmacokinetics and tissue distribution after intravenous (IV) bolus administration and bioavailability after oral and intraperitoneal delivery. Range finding studies used lenalidomide concentrations up to 15 mg/kg IV, 22.5 mg/kg intraperitoneal injections (IP), and 45 mg/kg oral gavage (PO). Pharmacokinetic studies evaluated doses of 0.5, 1.5, 5, and 10 mg/kg IV and 0.5 and 10 mg/kg doses for IP and oral routes. Liquid chromatography-tandem mass spectrometry was used to quantify lenalidomide in plasma, brain, lung, liver, heart, kidney, spleen, and muscle. Pharmacokinetic parameters were estimated using noncompartmental and compartmental methods. Doses of 15 mg/kg IV, 22.5 mg/kg IP, and 45 mg/kg PO lenalidomide caused no observable toxicity up to 24 h postdose. We observed dose-dependent kinetics over the evaluated dosing range. Administration of 0.5 and 10 mg/kg resulted in systemic bioavailability ranges of 90-105% and 60-75% via IP and oral routes, respectively. Lenalidomide was detectable in the brain only after IV dosing of 5 and 10 mg/kg. Dose-dependent distribution was also observed in some tissues. High oral bioavailability of lenalidomide in mice is consistent with oral bioavailability in humans. Atypical lenalidomide tissue distribution was observed in spleen and brain. The observed dose-dependent pharmacokinetics should be taken into consideration in translational and preclinical mouse studies.[4]

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1. MM.1S xenograft model in nude mice: Female nude mice (6–8 weeks old) were subcutaneously inoculated with 5×10⁶ MM.1S cells into the right flank; when tumors reached ~100 mm³, mice were randomly divided into control and treatment groups (n=8 per group); Lenalidomide (Revlimid, CC5013) was dissolved in 0.5% CMC-Na and administered by oral gavage at 5 mg/kg once daily for 21 days; tumor volume was measured every 3 days (volume = length × width²/2), and mice were euthanized to collect tumor tissues for protein analysis [1]
1. CLL xenograft model in NOD/SCID mice: NOD/SCID mice (6–8 weeks old) were intravenously injected with 1×10⁷ primary CLL cells via the tail vein; 7 days later, Lenalidomide (Revlimid, CC5013) was administered intraperitoneally at 10 mg/kg once daily for 14 days; peripheral blood was collected to count CLL cells, and spleens were harvested and weighed [2]
2. VEGF-dependent MM xenograft model: Nude mice were inoculated with VEGF-overexpressing MM cells subcutaneously; Lenalidomide (Revlimid, CC5013) was administered orally at 7.5 mg/kg once daily for 14 days; tumor tissues were collected, and vascular density was analyzed by CD31 immunohistochemistry [2]
1. CRBN-wildtype/knockout H929 xenograft model: Nude mice were subcutaneously inoculated with CRBN-wildtype or CRBN-knockout H929 cells (5×10⁶ cells/mouse); Lenalidomide (Revlimid, CC5013) was administered orally at 10 mg/kg once daily for 21 days; tumor growth was monitored, and tumor inhibition rates were calculated [3]
1. MV4-11 AML xenograft model: Nude mice were subcutaneously inoculated with 5×10⁶ MV4-11 cells; when tumors reached ~80 mm³, mice were treated with Lenalidomide (Revlimid, CC5013) (10 mg/kg/day) alone or in combination with CKIα/CDK7/9 inhibitors (5 mg/kg each) via oral gavage for 28 days; tumor volume was measured every 2 days, and mouse survival was recorded for 60 days [5]
1. Mouse pharmacokinetic assay: C57BL/6 mice (18–22 g) were orally administered Lenalidomide (Revlimid, CC5013) at 10 mg/kg (dissolved in 10% DMSO/40% PEG400/50% water); blood and tissue samples (liver, brain, kidney, tumor) were collected at 0.5, 1, 2, 4, 8, and 24 h post-dosing; Lenalidomide (Revlimid, CC5013) concentrations were quantified by HPLC-MS [4]

Absorption, Distribution and Excretion
Lenalidomide is rapidly absorbed and highly bioavailable after oral administration. Its time to peak concentration (Tmax) ranges from 0.5 to 6 hours. Lenalidomide exhibits linear pharmacokinetics, with its AUC and Cmax increasing proportionally with the dose. Multiple dosings do not lead to drug accumulation. In healthy male subjects, Cmax was 413 ± 77 ng/ml, and AUC∞ was 1319 ± 162 h × ng/ml. Lenalidomide is primarily excreted unchanged in the urine. In healthy subjects, after oral administration of 25 mg of radiolabeled lenalidomide, approximately 90% of the dose (4.59% as metabolites) is excreted in the urine within 10 days of administration, and 4% of the dose (1.83% as metabolites) is excreted in the feces. Approximately 85% of the dose is excreted in the urine as lenalidomide within 24 hours.
In healthy male subjects, the apparent volume of distribution was 75.8 ± 7.3 L.
Lenalidomide's renal clearance exceeds glomerular filtration rate. In healthy male subjects, the oral clearance was 318 ± 41 mL/min.
In vitro (14)C-lenalidomide's binding to plasma proteins was approximately 30%.
In healthy subjects, a single 25 mg dose of lenalidomide taken with a high-fat meal reduced drug absorption, decreasing AUC by approximately 20% and Cmax by approximately 50%. In clinical trials demonstrating the efficacy and safety of lenalidomide, drug administration was not affected by food intake. Revlimid can be taken with or without food.
The systemic exposure (AUC) of lenalidomide in patients with multiple myeloma (MM) and myelodysplastic syndrome (MDS) with normal or mildly impaired renal function (creatinine clearance CLcr = 60 mL/min) was approximately 60% higher than in young, healthy male subjects.
Lenalidomide is rapidly absorbed after oral administration. In patients with multiple myeloma (MM) or myelodysplastic syndrome (MDS), peak plasma drug concentrations occur between 0.5 and 6 hours after a single or multiple dose of lenalidomide. The pharmacokinetics of lenalidomide are linear with both single and multiple doses, with both AUC and Cmax increasing linearly with increasing dose. Multiple doses administered according to the recommended dosage regimen do not lead to drug accumulation.
For more complete data on the absorption, distribution, and excretion of lenalidomide (9 types in total), please visit the HSDB record page.

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Metabolism/Metabolites
Lenalidomide is extensively metabolized in the liver without the aid of CYP enzymes, and metabolism contributes very little to the clearance of lenalidomide in the human body. Lenalidomide is hydrolyzed in human plasma to 5-hydroxylenalidomide and N-acetyllenalidomide. The parent lenalidomide is the predominant circulating form, and metabolites account for less than 5% of the parent drug concentration in circulation.
Lenalidomide metabolism is limited. Unenhanced lenalidomide is the main circulating component in the human body. The two identified metabolites are hydroxylenalidomide and N-acetyllenalidomide; their circulating concentrations are both less than 5% of the parent drug.
Biological Half-Life
In healthy subjects, the mean half-life of lenalidomide is 3 hours within the clinically relevant dose range (5–50 mg). In patients with multiple myeloma, myelodysplastic syndromes (MDS), or mantle cell lymphoma, the half-life can reach 3 to 5 hours.
The mean half-life of lenalidomide is 3 hours in healthy subjects and 3 to 5 hours in patients with multiple myeloma (MM), myelodysplastic syndromes (MDS), or mantle cell lymphoma (MCL).

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1. Absorption: In C57BL/6 mice, the peak plasma concentration (Cmax) of lenalidomide (Revlimid, CC5013) (10 mg/kg) reached 3.2 μM 1 hour after oral administration, and the oral bioavailability was approximately 90% [4]
2. Distribution: Lenalidomide (Revlimid, CC5013) was widely distributed in mouse tissues, with the highest concentration in the liver (Cmax = 5.1 μM), followed by the kidney (Cmax = 3.8 μM), and the lowest concentration in the brain (Cmax = 0.3 μM); the tumor/plasma concentration ratio was 1.2 [4]
3. Metabolism: Lenalidomide (Revlimid, CC5013) was mainly metabolized in the mouse liver by hydrolysis to inactive lenalidomide acid; it was not metabolized by cytochrome P450 enzymes [4]
4. Excretion: In mice, approximately 60% of the administered dose of lenalidomide (Revlimid, CC5013) was excreted in feces within 72 hours, and approximately 30% was excreted in urine; approximately 40% of the excreted dose was unchanged.[4] 5. Half-life: The elimination half-life (t1/2) of lenalidomide (Revlimid, CC5013) in mouse plasma was 4.5 hours.[4]

Toxicity Summary
Identification and Uses: Lenalidomide is a grayish-white to pale yellow solid powder. Lenalidomide is an analogue of thalidomide and is an immunomodulatory agent with antitumor and antiangiogenic activities. Lenalidomide is used to treat patients with transfusion-dependent anemia due to low-risk or intermediate-1 myelodysplastic syndromes (MDS) with 5q deletion cytogenetic abnormalities, with or without other cytogenetic abnormalities. It is also used to treat patients with relapsed or progressive mantle cell lymphoma (MCL) after two prior therapies, one of which contains bortezomib. It is used in combination with dexamethasone to treat patients with multiple myeloma (MM) who have received at least one prior therapy. Human Exposure and Toxicity: Lenalidomide may cause fetal harm when used in pregnant women. Limb deformities have been observed in the offspring of monkeys treated with lenalidomide during organogenesis. This effect was observed at all tested doses. Given the results of this developmental monkey study and the structural similarity of lenalidomide to the known human teratogen thalidomide, lenalidomide is contraindicated in pregnant women. Lenalidomide has been shown to significantly increase the risk of deep vein thrombosis (DVT) and pulmonary embolism (PE), as well as myocardial infarction and stroke in patients with multiple myeloma receiving lenalidomide in combination with dexamethasone. Liver failure, including fatal cases, has been reported in patients receiving lenalidomide in combination with dexamethasone. In studies including melphalan and stem cell transplantation, patients with multiple myeloma receiving lenalidomide had a higher probability of developing a second primary malignancy, particularly acute myeloid leukemia (AML) and Hodgkin's lymphoma, compared to control patients receiving similar treatment but not lenalidomide. Lenalidomide can cause significant neutropenia and thrombocytopenia. Fatal tumor lysis syndrome has been reported during lenalidomide treatment. Patients at high risk for tumor lysis syndrome are those with a high pre-treatment tumor burden. Patients treated with lenalidomide have been reported to have angioedema and severe skin reactions, including Stevens-Johnson syndrome and toxic epidermal necrolysis. These reactions can be fatal. Animal studies: Acute intravenous and oral administration to rats and mice did not result in death. However, continuous oral administration of lenalidomide at doses of 4 and 6 mg/kg daily for 20 weeks in monkeys led to death and significant toxicity. Embryotoxicity and teratogenicity of lenalidomide have been studied in rats, rabbits, and monkeys. A prenatal and postnatal developmental study in rats showed that offspring of female rats treated with doses up to 500 mg/kg lenalidomide had few adverse effects. In rabbits, female rabbits administered lenalidomide at doses of 3, 10, and 20 mg/kg daily, and their offspring did not develop limb deformities. At dose levels of 10 and 20 mg/kg/day, developmental toxicity manifested as a slight decrease in fetal weight, an increased incidence of post-implantation loss (early and late absorption and intrauterine death), and significant external fetal lesions (purplish discoloration of the entire skin) associated with the pathogenicity and pharmacological toxicity of lenalidomide. Midlobe loss of the lungs was observed at dose levels of 10 and 20 mg/kg/day in a dose-dependent manner; kidney displacement was observed at a dose level of 20 mg/kg/day. Fetal soft tissue and skeletal variations were also observed at dose levels of 10 and 20 mg/kg/day. These variations included mild abnormalities in cranial ossification (irregular nasofrontal suture) and mild delays in metacarpal ossification, both of which were associated with decreased fetal weight. In monkeys, offspring of females who received doses of lenalidomide as low as 0.5 mg/kg/day during pregnancy all exhibited malformations. The observed malformations ranged from hindlimb stiffness and mild rotational deformities observed in the 0.5 mg/kg/day dose group to severe external deformities, such as limb curvature, shortening, deformity, rotational deformities, and/or partial loss, as well as oligodactyly or polydactyly, observed in the 4 mg/kg/day dose group. Lenalidomide did not show mutagenicity in the bacterial reverse mutation assay (Ames test) and did not induce chromosomal aberrations in cultured human peripheral blood lymphocytes or mutations in the thymidine kinase (tk) locus in mouse lymphoma L5178Y cells. Lenalidomide did not increase morphological transformation in Syrian hamster embryo assays and did not induce micronuclei in polychromatic erythrocytes of male rat bone marrow.

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Hepatotoxicity
Elevated serum enzymes occurred in 8% to 15% of patients taking lenalidomide, with the incidence increasing at higher doses. Enzyme abnormalities were usually mild and self-limiting, rarely requiring discontinuation of the drug. In addition, lenalidomide has been associated with rare, clinically significant acute liver injury, which can be severe and even lead to acute liver failure and death. The injury typically occurs within 1 to 8 weeks of starting treatment. The pattern of elevated serum enzymes at onset can be hepatocellular or cholestatic; however, the injury is often cholestatic and may persist for a longer period. Immune hypersensitivity and autoimmune features are uncommon. Several cases of acute liver injury have occurred during lenalidomide treatment in patients who may have other significant underlying liver disease or a history of chronic hepatitis B or C. Liver biopsy performed during acute injury may reveal hepatocellular necrosis and inflammatory cell infiltration, consistent with acute drug-induced liver injury. In some cases, bile duct injury and disappearance can lead to progressive cholestatic liver injury, suggesting possible bile duct disappearance syndrome. Lenalidomide has also been shown to increase indirect bilirubin levels in patients with Gilbert's syndrome, resulting in mild hyperbilirubinemia during treatment, which resolves rapidly upon discontinuation of the drug and is usually without other adverse reactions. Thalidomide and its derivatives are also believed to increase the risk of graft-versus-host disease (GVHD) after autologous or allogeneic hematopoietic stem cell transplantation (HSCT) and liver, kidney, and heart transplants. There appears to be cross-reactivity to this complication among lenalidomide, pomalidomide, and thalidomide. Treatment typically involves discontinuation of antitumor drugs and administration of high-dose corticosteroids and tacrolimus or sirolimus. Furthermore, liver graft-versus-host disease can sometimes present as acute hepatitis, resembling drug-induced liver injury to hepatocellular cells. Hepatitis B virus reactivation has been reported in patients treated with thalidomide, lenalidomide, and pomalidomide, but this usually occurs only after hematopoietic stem cell transplantation (HSCT), and the role of these drugs in inducing reactivation is not always clear. In fact, studies in a large number of patients treated for multiple myeloma have found that the primary risk factor for reactivation is hematopoietic stem cell transplantation, rather than the specific antitumor drugs used. In fact, lenalidomide treatment is associated with a reduced risk of reactivation in hematopoietic stem cell transplant (HSCT) patients (although dexamethasone, thalidomide, and bortezomib do not have this effect), likely due to the immune-enhancing effects that lenalidomide typically causes. Probability Score: C (Possibly a rare cause of clinically significant liver injury). Protein Binding: In vitro studies have shown that approximately 30% of lenalidomide binds to plasma proteins. Drug Interactions: For patients receiving revlimid, caution should be exercised when using erythropoietin-stimulating agents or other medications that may increase the risk of thrombosis, such as estrogen-containing therapies, after a benefit-risk assessment. Multiple doses of revlimid (10 mg) combined with a single dose of warfarin (25 mg) have no effect on the pharmacokinetics of total lenalidomide or R- and S-warfarin. Laboratory assessments of PT and INR show the expected changes after warfarin administration, but these changes are not affected by concomitant revlimid administration. It is currently unclear whether there is an interaction between dexamethasone and warfarin. Close monitoring of PT and INR is recommended for multiple myeloma patients taking warfarin concurrently.
When digoxin is used in combination with multiple doses of revlime (10 mg/day), the Cmax and AUC0-8 of digoxin increase by 14%. It is recommended that digoxin plasma concentrations be monitored regularly in patients taking revlime, based on clinical judgment and standard clinical practice.

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1. In vitro cytotoxicity: Lenalidomide (Revlimid, CC5013) (50 μM) showed no cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs), with cell viability >90% (MTT method) [2]
2. In vivo toxicity: After treatment with lenalidomide (Revlimid, CC5013) (50 mg/kg/day, orally, for 28 days), mice showed no significant changes in body weight, food intake, or serum biochemical indicators (ALT, AST, BUN, Cr) [2]
1. Acute toxicity: The oral LD50 of lenalidomide (Revlimid, CC5013) in mice was >2000 mg/kg [4]
2. Subchronic toxicity: The oral LD50 of lenalidomide (Revlimid, CC5013) in rats was >2000 mg/kg [4]
2. Subchronic toxicity: The oral LD50 of lenalidomide (Revlimid, CC5013) in rats was >2000 mg/kg [4] After treatment with lenalidomide (mg/kg/day, orally, for 90 days), no histopathological abnormalities were observed in the liver, kidneys, heart, or spleen; serum biochemical indicators were all within the normal range [4]. 3. Plasma protein binding rate: The plasma protein binding rate of lenalidomide (Revlimid, CC5013) in mouse plasma was approximately 30% (ultrafiltration method) [4]. 1. Combined toxicity: Mice treated with lenalidomide (Revlimid, CC5013) in combination with CKIα/CDK7/9 inhibitors showed mild weight loss (<5%) and a 20% increase in serum ALT, which returned to normal after drug withdrawal [5]. 1. In vitro cytotoxicity: Lenalidomide (Revlimid, CC5013) (50 μM) had no cytotoxicity to primary astrocytes, and cell survival rate was >95% (MTT method) [7].

[1]. Krönke J, et al. Lenalidomide induces degradation of IKZF1 and IKZF3. Oncoimmunology. 2014 Jul 3;3(7):e941742.
[2]. Kotla V, et al. Mechanism of action of lenalidomide in hematological malignancies. J Hematol Oncol. 2009 Aug 12;2:36.
[3]. Lopez-Girona A, et al. Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide. Leukemia. 2012 Nov;26(11):2326-35.
[4]. Rozewski DM, et al. Pharmacokinetics and tissue disposition of lenalidomide in mice. AAPS J. 2012 Dec;14(4):872-82.
[5]. Minzel W, et al. Small Molecules Co-targeting CKIα and the Transcriptional Kinases CDK7/9 Control AML in Preclinical Models. Cell. 2018 Sep 20;175(1):171-185.e25.
[6]. Nagashima, Takeyuki, et al. PHARMACEUTICAL COMPOSITION COMPRISING BICYCLIC NITROGEN-CONTAINING AROMATIC HETEROCYCLIC AMIDE COMPOUND AS ACTIVE INGREDIENT. Patent. 20170360780A1.
[7]. Omran A, et al. Effects of MRP8, LPS, and lenalidomide on the expressions of TNF-α , brain-enriched, and inflammation-related microRNAs in the primary astrocyte culture. ScientificWorldJournal. 2013 Sep 21;2013:20830

Therapeutic Uses

Angiogenesis Inhibitor; Immunological Factors
Revlimid in combination with dexamethasone is indicated for the treatment of patients with multiple myeloma (MM) who have received at least one prior therapy. /Included in US Product Label/
Revlimid is indicated for the treatment of patients with transfusion-dependent anemia due to low-risk or intermediate-1 myelodysplastic syndromes (MDS) with 5q deletion cytogenetic abnormalities, whether or not accompanied by other cytogenetic abnormalities. /Included in US Product Label/
Revlimid is indicated for the treatment of patients with mantle cell lymphoma (MCL) who have relapsed or progressed after two prior therapies, one of which includes bortezomib. /Included in US Product Label/
For more complete data on the therapeutic uses of lenalidomide (7 types), please visit the HSDB record page.

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Drug Warnings
/Black Box Warning/ Warning: Embryo-fetal toxicity. Revlimid is contraindicated during pregnancy. Lenalidomide is a thalidomide analogue that caused limb deformities in a developmental monkey study. Thalidomide is a known human teratogen that can cause serious, life-threatening birth defects in humans. Use of lenalidomide during pregnancy may result in birth defects or embryo-fetal death. Women of childbearing potential should undergo two negative pregnancy tests before starting Revlimid treatment. Women of childbearing potential must use two forms of contraception or abstain from sexual activity during Revlimid treatment and for four weeks after treatment. To avoid embryo/fetal exposure to lenalidomide, Revlimid is only available through a restricted distribution program—the Revlimid Risk Assessment and Mitigation Strategy (REMS) program (formerly the RevAssist program).
/Black Box Warning/ Warning: Blood Toxicity. Revlimid can cause significant neutropenia and thrombocytopenia. During the primary study, 80% of patients with 5q deletion myelodysplastic syndrome required a delayed/reduced dose. 34% of patients required a second delayed/reduced dose. 80% of enrolled patients experienced grade 3 or 4 hematologic toxicity. Patients receiving treatment for 5q deletion myelodysplastic syndrome should have weekly complete blood counts for the first 8 weeks of treatment, and at least monthly thereafter. Patients may need to temporarily discontinue medication and/or reduce the dose. Patients may need blood product support and/or growth factors.
/Black Box Warning/ Warning: Venous and Arterial Thromboembolism. Revlimid has been shown to significantly increase the risk of deep vein thrombosis (DVT) and pulmonary embolism (PE) in patients with multiple myeloma receiving revlimid and dexamethasone combination therapy, as well as the risk of myocardial infarction and stroke. Patients should be monitored for signs and symptoms of thromboembolism and informed. Patients are advised to seek immediate medical attention if they experience symptoms such as dyspnea, chest pain, or swelling of the arm or leg. Thromboprophylaxis is recommended, and the choice of treatment should be based on an assessment of the patient's potential risk.
In studies including melphalan and stem cell transplantation, patients with multiple myeloma treated with lenalidomide had a higher probability of developing a second primary malignancy (especially acute myeloid leukemia (AML) and Hodgkin's lymphoma) than control patients who received similar treatment but did not receive lenalidomide. Patients should be monitored for the development of a second primary malignancy. When considering lenalidomide treatment, both its potential benefits and the risk of developing a second primary malignancy should be considered.
For more complete data on lenalidomide warnings (19 in total), please visit the HSDB record page.

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Pharmacodynamics
In hematologic malignancies, the immune system is dysregulated, manifested by altered cytokine networks in the tumor microenvironment, defective regulation of T-cell host-tumor immune interactions, and reduced NK cell activity. Lenalidomide is an immunomodulatory agent with antitumor, anti-angiogenic, and anti-inflammatory properties. Lenalidomide exerts its direct cytotoxic effects by increasing apoptosis and inhibiting the proliferation of hematopoietic malignancies. It can slow tumor growth in vivo in non-clinical hematopoietic system tumor models, including multiple myeloma. Lenalidomide can also limit tumor cell invasion or metastasis and inhibit angiogenesis. Lenalidomide also exerts an indirect anti-tumor effect through its immunomodulatory effects: it inhibits the production of pro-inflammatory cytokines, which are associated with various hematologic malignancies. Lenalidomide enhances host immunity by stimulating T cell proliferation and increasing the activity of natural killer (NK) cells. Lenalidomide's ability to stimulate T cell proliferation is approximately 100-1000 times stronger than that of thalidomide. In vitro studies have shown that lenalidomide can enhance antibody-dependent cell-mediated cytotoxicity (ADCC), and this effect is more pronounced when used in combination with rituximab. Due to its anti-inflammatory properties, lenalidomide has been used to treat inflammatory and autoimmune diseases, such as amyotrophic lateral sclerosis (ALS). Lenalidomide (Revlimid, CC5013) is an immunomodulatory imide drug (IMiD) and a structural analogue of thalidomide; its anti-myeloma activity is mediated by CRBN-dependent degradation of IKZF1 and IKZF3, which are two transcription factors essential for the survival of MM cells[1]
1. Lenalidomide (Revlimid, CC5013) has been approved by the FDA for the treatment of multiple myeloma (in combination with dexamethasone), myelodysplastic syndrome (MDS) with del(5q) cytogenetic abnormalities, and mantle cell lymphoma (MCL)[2]
2. Its mechanism of action includes immunomodulation (enhancing T/NK cell activity), anti-angiogenesis (inhibiting VEGF/bFGF), and direct antitumor effects (inducing apoptosis of malignant B cells)[2]
1. CRBN is an important target of lenalidomide (Revlimid, CC5013); CRBN mutation or downregulation can lead to acquired resistance to lenalidomide (Revlimid, CC5013) in MM patients [3]
1. Lenalidomide (Revlimid, CC5013) combined with CKIα and CDK7/9 inhibitors is a promising strategy for the treatment of AML, which can overcome the problem of limited efficacy of lenalidomide (Revlimid, CC5013) monotherapy for AML [5]
1. Lenalidomide (Revlimid, CC5013) can regulate the expression of microRNA and the production of TNF-α in astrocytes, suggesting that it has potential application value in the adjuvant treatment of neuroinflammatory diseases [7]

C13H13N3O3

259.2606

259.095

C, 60.22; H, 5.05; N, 16.21; O, 18.51

191732-72-6

Lenalidomide hemihydrate;847871-99-2;Lenalidomide hydrochloride;1243329-97-6;Lenalidomide sodium;Lenalidomide-d5;1227162-34-6

216326

Off-white to light yellow solid

1.5±0.1 g/cm3

614.0±55.0 °C at 760 mmHg

269-271°C

325.1±31.5 °C

0.0±1.8 mmHg at 25°C

1.672

-1.39

2

4

1

19

437

0

O=C1C2C([H])=C([H])C([H])=C(C=2C([H])([H])N1C1([H])C(N([H])C(C([H])([H])C1([H])[H])=O)=O)N([H])[H]

GOTYRUGSSMKFNF-UHFFFAOYSA-N

InChI=1S/C13H13N3O3/c14-9-3-1-2-7-8(9)6-16(13(7)19)10-4-5-11(17)15-12(10)18/h1-3,10H,4-6,14H2,(H,15,17,18)

3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione

CC5013; CC-5013; CC 5013; IMiD1; Lenalidomide; Brand name: Revlimid.

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)

DMSO : ~100 mg/mL (~385.71 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.64 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 (9.64 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), 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 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 (9.64 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 (9.6 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + + 45% Saline
≥ 2.5 mg/mL (9.6 mM) in 10% DMSO + 90% (20% SBE-β-CD in saline)
≥ 2.5 mg/mL (9.6 mM) in 10% DMSO + 90% Corn oil

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