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| Targets |
Cereblon
Lenalidomide hemihydrate targets the ubiquitin E3 ligase cereblon (CRBN). By binding to CRBN, it induces the selective ubiquitination and degradation of the lymphoid transcription factors IKZF1 and IKZF3. It is also a potent inhibitor of TNF-alpha with an IC50 of 13 nM. Additionally, it induces tyrosine phosphorylation of CD28 on T cells, which subsequently activates NF-κB. |
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| ln Vitro |
Lenalidomide is effective at promoting T cell proliferation and the production of IFN-γ and IL-2. The anti-inflammatory cytokine IL-10 is produced more abundantly by human PBMCs when lenalidomide is administered, as opposed to pro-inflammatory cytokines TNF-α, IL-1, IL-6, IL-12. In addition to directly inhibiting the production of IL-6, lenalidomide also prevents the interaction between multiple myeloma (MM) cells and bone marrow stromal cells (BMSC), which increases the apoptosis of myeloma cells[2]. Thalidomide, Lenalidomide, and Pomalidomide have dose-dependent interactions with the CRBN-DDB1 complex, with respective IC50 values of ~30 μM, ~3 μM and ~3 μM. Lenalidomide has an antiproliferative effect on these cells with reduced CRBN expression (U266-CRBN60 and U266-CRBN75), but they are less sensitive to it than the parental cells are over a range of dose responses from 0.01 to 10 μM[3]. Lenalidomide, a thalidomide analog, connects the human E3 ubiquitin ligase cereblon and CKIα , causing the latter to be ubiquitinated and degraded and thereby killing leukemic cells by activating p53[5].
In vitro, lenalidomide inhibited autoubiquitination of CRBN in HEK293T cells expressing thalidomide-binding competent wild-type CRBN, but not in cells expressing a thalidomide-binding defective CRBN mutant. Overexpression of wild-type CRBN protein in KMS12 myeloma cells amplified pomalidomide-mediated reductions in c-myc and IRF4 expression and increases in p21(WAF-1) expression. Long-term selection for lenalidomide resistance in H929 myeloma cell lines was accompanied by a reduction in CRBN, while in DF15R myeloma cells resistant to lenalidomide, CRBN protein was undetectable. |
| ln Vivo |
Lenalidomide is toxic at doses of up to 15, 22.5, and 45 mg/kg when administered intravenously, intraperitoneally, or orally. With the exception of one mouse death (out of four dosed), these maximum achievable Lenalidomide doses are well tolerated due to the drug's solubility in our PBS dosing vehicle. Notably, at IV doses of 15 mg/kg (n=3) or 10 mg/kg (n=45) or at any other dose level administered via IV, IP, or PO routes[4], no additional toxicities were seen in the study.
In vivo range-finding studies used lenalidomide concentrations up to 15 mg/kg IV, 22.5 mg/kg intraperitoneally (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 for IP and oral routes. Doses of 15 mg/kg IV, 22.5 mg/kg IP, and 45 mg/kg PO caused no observable toxicity up to 24 hours post-dose. Dose-dependent kinetics were observed over the evaluated dosing range, with systemic bioavailability of 90-105% and 60-75% via IP and oral routes, respectively. |
| Enzyme Assay |
Ficoll-Hypaque density centrifugation is used to isolate human PBMCs from healthy donors. In RPMI supplemented with 10 AB+ serum, 2 mM l-glutamine, 100 μg/mL penicillin, and 100 μg/mL streptomycin, cells are cultured at a density of 106 cells per mL. Lenalidomide is dissolved in DMSO at a concentration of 20 mg/mL before being further diluted with culture medium. All assays, including the controls, had a final DMSO concentration of 0.25 %. One hour before LPS is added to cells, lanalidomide is added. LPS from Salmonella Minnesota R595 is used to stimulate PBMCs (106 cells/mL). Cells are incubated with LPS in triplicate for 18–20 hours at 37 °C with 5% CO2. Supernatants are then harvested and assayed for cytokine levels. Supernatants are stored frozen at 70 °C until use in some experiments. Using the Trypan blue exclusion dye method, cell viability is assessed. ELISA is used to measure the amount of TNFα present in the culture supernatants. At least three different experiments must be performed to evaluate lenalidomide. The formula for percent inhibition is 100 × [1 − (cytokine(experimental)/cytokine(control))].
For cell-free assays, the binding of lenalidomide to CRBN can be assessed using techniques such as surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). Its inhibitory effect on TNF-alpha can be measured using standard ELISA-based assays with purified TNF-alpha protein. The compound's activity as a molecular glue can be studied in ubiquitination assays using purified CRBN-CRL4 E3 ligase complex, IKZF1/IKZF3 substrates, and ubiquitin, monitoring the formation of polyubiquitinated products by Western blot. |
| Cell Assay |
In RPMI-I640 medium with 10% (V/V) heat-inactivated fetal bovine serum supplemented with 2 mM glutamine, cell lines NCI-H929 and U266, as well as DF15 cells, are cultured. NCI-H929 cells are continuously treated for two months with either control (final 0.1% DMSO) or low-dose Lenalidomide (1 μM) until the proliferation of the cells is no longer inhibited by Lenalidomide (1 μM), as determined by cell viability (Vi-cell XR cell viability analyzer), cell proliferation by flow cytometry, and cell cycle analysis (propidium iodide staining). Lenalidomide (10 μM) is administered to the resistant H929 cell lines for an additional 4 months after they develop resistance to 1 μM. After this time, the cell cultures completely established resistance up to high doses of lenalidomide (30 μM). H929 Lenalidomide-resistant cells are removed from compound-culture for 5-7 days before use[3] in order to prepare them for the experiments described here.
For in vitro cellular assays, the activity of lenalidomide is typically assessed in multiple myeloma or other B-cell lymphoma cell lines. Cells are cultured in multi-well plates and treated with various concentrations of lenalidomide for 48-72 hours. Cell viability is measured using MTT, CellTiter-Glo, or similar assays to determine IC50 values. The degradation of IKZF1 and IKZF3 can be confirmed by Western blot analysis. The effect on IL-2 release from T cells can be measured in the culture supernatant by ELISA. |
| Animal Protocol |
In vivo efficacy studies are typically conducted in mouse xenograft models of multiple myeloma. Lenalidomide is administered via oral gavage at doses ranging from 5 to 45 mg/kg. Tumor volume is measured periodically using calipers. At the end of the study, tumors are harvested for analysis of IKZF1/IKZF3 degradation and other biomarkers. Pharmacokinetic parameters are estimated using noncompartmental and compartmental methods from plasma and tissue samples analyzed by liquid chromatography-tandem mass spectrometry.
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| ADME/Pharmacokinetics |
Lenalidomide hemihydrate has a molecular weight of 268.28 g/mol and a molecular formula of C13H13N3O3·½H2O. Its CAS number is 847871-99-2. Purity is typically >98%. Solubility: DMSO 100 mg/mL (186.37 mM); insoluble in water and ethanol. For in vivo oral administration, a homogeneous suspension can be prepared in CMC-Na at ≥5 mg/mL. The powder is stable for 3 years at -20°C and 2 years at 4°C; solutions are stable for 6 months at -80°C and 1 month at -20°C.
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| Toxicity/Toxicokinetics |
Doses of 15 mg/kg IV, 22.5 mg/kg IP, and 45 mg/kg PO caused no observable toxicity up to 24 hours post-dose in mice. However, the compound has a well-established clinical safety profile. Common side effects include myelosuppression (neutropenia, thrombocytopenia), diarrhea, and fatigue. More serious toxicities include an increased risk of secondary malignancies and venous thromboembolism. The compound is contraindicated during pregnancy due to teratogenic effects.
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| References | |
| Additional Infomation |
Lenalidomide hemihydrate is an FDA-approved prescription drug for the treatment of multiple myeloma, myelodysplastic syndromes, and mantle cell lymphoma. It is marketed under the brand name Revlimid. Its mechanism of action involves immunomodulation through cereblon-dependent degradation of IKZF1 and IKZF3 transcription factors, leading to anti-proliferative and pro-apoptotic effects in malignant cells, as well as T-cell activation. It is available in oral capsule formulations.
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| Molecular Formula |
2[C13H13N3O3].H2O
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|---|---|
| Molecular Weight |
536.537
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| Exact Mass |
536.20194725
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| CAS # |
847871-99-2
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| Related CAS # |
Lenalidomide;191732-72-6;Lenalidomide hydrochloride;1243329-97-6;Lenalidomide sodium
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| PubChem CID |
46220375
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| Appearance |
Off-white to pink solid
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| LogP |
1.585
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
39
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| Complexity |
437
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C1CCC(N2CC3C(=CC=CC=3N)C2=O)C(=O)N1.O.O=C1CCC(N2CC3C(=CC=CC=3N)C2=O)C(=O)N1
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| InChi Key |
OTJHSDXKMBRCMM-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/2C13H13N3O3.H2O/c2*14-9-3-1-2-7-8(9)6-16(13(7)19)10-4-5-11(17)15-12(10)18;/h2*1-3,10H,4-6,14H2,(H,15,17,18);1H2
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| Chemical Name |
3-(7-amino-3-oxo-1H-isoindol-2-yl)piperidine-2,6-dione;hydrate
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| Synonyms |
CC-5013 hemihydrate; Lenalidomide hemihydrate
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: ~51 mg/mL (~196.7 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (9.32 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (9.32 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (9.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 5% DMSO+40%PEG 300+5%Tween80+ddH2O: 10mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8638 mL | 9.3190 mL | 18.6379 mL | |
| 5 mM | 0.3728 mL | 1.8638 mL | 3.7276 mL | |
| 10 mM | 0.1864 mL | 0.9319 mL | 1.8638 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT01178814 | Active Recruiting |
Drug: Revlimid (Lenalidomide) |
Myelodysplastic Syndrome | Columbia University | December 2012 | Phase 2 |
| NCT01996865 | Active Recruiting |
Drug: Lenalidomide Drug: Rituximab |
Lymphoma, Non-Hodgkin | Celgene | April 1, 2014 | Phase 3 |
| NCT02523040 | Active Recruiting |
Drug: Lenalidomide | Langerhans Cell Histiocytosis (LCH) Histiocytic Sarcoma (HS) |
Dana-Farber Cancer Institute | August 2015 | Phase 2 |
| NCT01316523 | Active Recruiting |
Drug: Rituximab Drug: Lenalidomide |
Non Hodgkin's Lymphoma | University of California, Davis | December 2010 | Phase 2 |
| NCT01054196 | Active Recruiting |
Drug: lenalidomide Drug: melphalan |
Multiple Myeloma | Weill Medical College of Cornell University |
August 2010 | Phase 1 Phase 2 |
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