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Lenalidomide hemihydrate (Revlimid, CC-5013) is a derivative of thalidomide approved in the United States in 2005 for the treatment for myeloma and blood disorders called myelodysplastic syndromes. In the past ten years, lenalidomide has been used to successfully treat both cancers and inflammatory diseases. There are numerous mechanisms of action, but they can be categorized as mechanisms of action in vitro and in vivo to make things easier to understand. Lenalidomide has three main effects in vitro: direct anti-tumor effect, angiogenesis inhibition, and immunomodulation. Lenalidomide inhibits the growth of stromal cells in the bone marrow, has anti-angiogenic and anti-osteoclastogenic properties, and has immunomodulatory activity in vivo. These actions both directly and indirectly cause tumor cell apoptosis. Numerous hematologic and solid cancers can be treated using a wide range of lenalidomide-related activities.
Physicochemical Properties
| Molecular Formula | 2[C13H13N3O3].H2O |
| Molecular Weight | 536.537 |
| Exact Mass | 536.20194725 |
| CAS # | 847871-99-2 |
| Related CAS # | Lenalidomide;191732-72-6;Lenalidomide hydrochloride;1243329-97-6;Lenalidomide sodium |
| PubChem CID | 46220375 |
| Appearance | Off-white to pink solid |
| LogP | 1.585 |
| Hydrogen Bond Donor Count | 5 |
| Hydrogen Bond Acceptor Count | 9 |
| Rotatable Bond Count | 2 |
| Heavy Atom Count | 39 |
| Complexity | 437 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O=C1CCC(N2CC3C(=CC=CC=3N)C2=O)C(=O)N1.O.O=C1CCC(N2CC3C(=CC=CC=3N)C2=O)C(=O)N1 |
| InChi Key | OTJHSDXKMBRCMM-UHFFFAOYSA-N |
| 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 |
| Chemical Name | 3-(7-amino-3-oxo-1H-isoindol-2-yl)piperidine-2,6-dione;hydrate |
| Synonyms | CC-5013 hemihydrate; Lenalidomide hemihydrate |
| HS Tariff Code | 2934.99.9001 |
| 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) |
Biological Activity
| Targets | Cereblon |
| 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]. |
| 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. |
| 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))]. |
| 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. |
| References |
[1]. 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;2013:208309. [2]. 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. [3]. Mechanism of action of lenalidomide in hematological malignancies. J Hematol Oncol. 2009 Aug 12;2:36. [4]. Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide. Leukemia. 2012 Nov;26(11):2326-35. [5]. Pharmacokinetics and tissue disposition of lenalidomide in mice. AAPS J. 2012 Dec;14(4):872-82. |
Solubility Data
| Solubility (In Vitro) | DMSO: ~51 mg/mL (~196.7 mM) |
| 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. 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. 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. Solubility in Formulation 4: 5% DMSO+40%PEG 300+5%Tween80+ddH2O: 10mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| 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 |