Immunomodulation; Cereblon E3 ligase

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].

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].

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).

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.

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]

Absorption, Distribution and Excretion
Following oral administration, lenalidomide is rapidly absorbed with high bioavailability. It has a Tmax ranging from 0.5 to six hours. Lenalidomide exhibits a linear pharmacokinetic profile, with its AUC and Cmax increasing proportionally with dose. Multiple dosing does not result in drug accumulation. In healthy male subjects, the Cmax was 413 ± 77 ng/ml and the AUCinfinity was 1319 ± 162 h x ng/ml.
Lenalidomide is eliminated predominantly via urinary excretion in the unchanged form. Following oral administration of 25 mg of radiolabeled lenalidomide in healthy subjects, about 90% of the dose (4.59% as metabolites) was eliminated in urine and 4% of the dose (1.83% as metabolites) was eliminated in feces within ten days post-dose. Approximately 85% of the dose was excreted as lenalidomide in the urine within 24 hours.
In healthy male subjects, the apparent volume of distribution was 75.8 ± 7.3 L.
The renal clearance of lenalidomide exceeds the glomerular filtration rate. In healthy male subjects, the oral clearance was 318 ± 41 mL/min.
In vitro (14)C-lenalidomide binding to plasma proteins is approximately 30%.
Administration of a single 25 mg dose of Revlimid with a high-fat meal in healthy subjects reduces the extent of absorption, with an approximate 20% decrease in AUC and 50% decrease in Cmax. In the trials where the efficacy and safety were established for Revlimid, the drug was administered without regard to food intake. Revlimid can be administered with or without food.
Systemic exposure (AUC) of lenalidomide in multiple myeloma (MM) and myelodysplastic syndromes (MDS) patients with normal or mild renal function (CLcr = 60 mL/min) is approximately 60% higher as compared to young healthy male subjects.
Lenalidomide is rapidly absorbed following oral administration. Following single and multiple doses of Revlimid in patients with multiple myeloma (MM) or myelodysplastic syndromes (MDS) the maximum plasma concentrations occurred between 0.5 and 6 hours post-dose. The single and multiple dose pharmacokinetic disposition of lenalidomide is linear with AUC and Cmax values increasing proportionally with dose. Multiple dosing at the recommended dose-regimen does not result in drug accumulation.
For more Absorption, Distribution and Excretion (Complete) data for Lenalidomide (9 total), please visit the HSDB record page.

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Metabolism / Metabolites
Lenalidomide is not subject to extensive hepatic metabolism involving CYP enzymes and metabolism contributes to a very minor extent to the clearance of lenalidomide in humans. Lenalidomide undergoes hydrolysis in human plasma to form 5-hydroxy-lenalidomide and N-acetyl-lenalidomide. Unchanged lenalidomide is the predominant circulating drug form, with metabolites accounting for less than five percent of the parent drug levels in the circulation.
Lenalidomide -undergoes limited metabolism. Unchanged lenalidomide is the predominant circulating component in humans. Two identified metabolites are hydroxy-lenalidomide and N-acetyl-lenalidomide; each constitutes less than 5% of parent levels in circulation.

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Biological Half-Life
In healthy subjects, the mean half-life of lenalidomide is three hours in the clinically relevant dose range (5–50 mg). Half-life can range from three to five hours in patients with multiple myeloma, myelodysplastic syndromes, or mantle cell lymphoma.
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).

Toxicity Summary
IDENTIFICATION AND USE: Lenalidomide is off-white to pale-yellow solid powder. Lenalidomide, a thalidomide analog, is an immunomodulatory agent with antineoplastic and antiangiogenic activity. Lenalidomide is used for the treatment of patients with transfusion-dependent anemia due to low- or intermediate-1-risk myelodysplastic syndromes (MDS) associated with a deletion 5q cytogenetic abnormality with or without additional cytogenetic abnormalities. It is also used for the treatment of patients with mantle cell lymphoma (MCL) whose disease has relapsed or progressed after two prior therapies, one of which included bortezomib. It is used in combination with dexamethasone for the treatment of patients with multiple myeloma (MM) who have received at least one prior therapy. HUMAN EXPOSURE AND TOXICITY: Lenalidomide can cause fetal harm when administered to a pregnant female. Limb abnormalities were seen in the offspring of monkeys that were dosed with lenalidomide during organogenesis. This effect was seen at all doses tested. Due to the results of this developmental monkey study, and lenalidomide's structural similarities to thalidomide, a known human teratogen, lenalidomide is contraindicated in females who are pregnant. Lenalidomide has demonstrated a significantly increased risk of deep vein thrombosis (DVT) and pulmonary embolism (PE), as well as risk of myocardial infarction and stroke in patients with multiple myeloma who were treated with lenalidomide and dexamethasone therapy. Hepatic failure, including fatal cases, has occurred in patients treated with lenalidomide in combination with dexamethasone. Patients with multiple myeloma treated with lenalidomide in studies including melphalan and stem cell transplantation had a higher incidence of second primary malignancies, particularly acute myelogenous leukemia (AML) and Hodgkin lymphoma, compared to patients in the control arms who received similar therapy but did not receive lenalidomide. Lenalidomide can cause significant neutropenia and thrombocytopenia. Fatal instances of tumor lysis syndrome have been reported during treatment with lenalidomide. The patients at risk of tumor lysis syndrome are those with high tumor burden prior to treatment. Angioedema and serious dermatologic reactions, including Stevens-Johnson syndrome and toxic epidermal necrolysis, have been reported in lenalidomide-treated patients. These reactions can be fatal. ANIMAL STUDIES: Acute administration of lenalidomide to rats and mice via intravenous and oral administration did not result in mortality. However, repeated oral administration of 4 and 6 mg/kg/day to monkeys for up to 20 weeks produced mortality and significant toxicity. The embryotoxic and teratogenic potential of lenalidomide was studied in rats, rabbits and monkeys. A pre- and post-natal development study in rats revealed few adverse effects in the offspring of female rats treated with lenalidomide at doses up to 500 mg/kg. In rabbits, no limb abnormalities were attributable to lenalidomide in the offspring of females administered 3, 10 and 20 mg/kg/day orally. Developmental toxicity at the 10 and 20 mg/kg/day dose levels was characterized by slightly reduced fetal body weights, increased incidences of post implantation loss (early and late resorptions and intrauterine deaths), and gross external findings in the fetuses associated with morbidity and pharmacotoxic effects of lenalidomide (purple discoloration of the skin on the entire body). An absence of the intermediate lobe of the lung was observed at 10 and 20 mg/kg/day with dose dependence and displaced kidneys were observed at 20 mg/kg/day. Soft tissue and skeletal variations in the fetuses were also observed at 10 and 20 mg/kg/day. These included minor variations in skull ossification (irregular nasal-frontal suture) and small delays in ossification of the metacarpals, associated with the reduced fetal body weights. In monkeys, malformations were observed in the offspring of female monkeys who received lenalidomide doses as low as 0.5 mg/kg/day during pregnancy. The observed malformations ranged from stiff and slightly malrotated hindlimbs at 0.5 mg/kg/day of lenalidomide up to severe external malformations, such as bent, shortened, malformed, malrotated, and/or absent part of the extremities, oligo- or polydactyly at 4 mg/kg/day of lenalidomide. Lenalidomide was not mutagenic in the bacterial reverse mutation assay (Ames test) and did not induce chromosome aberrations in cultured human peripheral blood lymphocytes, or mutations at the thymidine kinase (tk) locus of mouse lymphoma L5178Y cells. Lenalidomide did not increase morphological transformation in Syrian Hamster Embryo assay or induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats.

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Hepatotoxicity
Serum enzyme elevations occur in 8% to 15% of patients taking lenalidomide and are more frequent with higher doses. The enzyme abnormalities are usually mild and self-limited, and only rarely require drug discontinuation. In addition, lenalidomide has been implicated in rare instances of clinically apparent, acute liver injury which can be severe and has led to deaths from acute liver failure. The onset of injury is typically within 1 to 8 weeks of starting therapy. The pattern of serum enzyme elevation at the time of presentation can be either hepatocellular or cholestatic; however, the injury tends to be cholestatic and can be prolonged. Immunoallergic and autoimmune features are not common. Several instances of acute liver injury associated with lenalidomide therapy have occurred in patients with other apparent causes of liver disease or with preexisting chronic hepatitis B or C. If performed during the acute injury, liver biopsy shows hepatocellular necrosis and inflammatory cell infiltration, consistent with acute drug induced injury. In some instances there is bile duct injury and loss resulting in progressive cholestatic liver injury suggestive of vanishing bile duct syndrome. Lenalidomide has also been shown to increase indirect bilirubin levels in patients with underlying Gilbert syndrome, causing a mild hyperbilirubinemia during therapy that soon resolves with stopping treatment and is otherwise benign.
Thalidomide and its derivatives have also been implicated in causing an increased risk of graft-vs-host disease after autologous or allogeneic hematopoietic stem cell transplantation (HSCT) as well as after liver, kidney and heart transplantation. There appears to be cross reactivity to this complication among lenalidomide, pomalidomide and thalidomide. Therapy usually requires discontinuation of the antineoplastic agent as well as treatment with high doses of corticosteroids and tacrolimus or sirolimus. Furthermore, hepatic graft-vs-host disease can occasionally present with an acute hepatitis that resembles hepatocellular drug induced liver injury.
Reactivation of hepatitis B has been reported in patients receiving thalidomide, lenalidomide and pomalidomide, but generally only after HSCT and the role of these agents in causing reactivation is not always clear. Indeed, in studies of large numbers of patients treated for multiple myeloma, the major risk factor for reactivation was found to be HSCT rather than the specific antineoplastic drugs being used. Indeed, lenalidomide therapy is associated with a reduced risk of reactivation in patients with HSCT (although dexamethasone, thalidomide and bortezomib were not), perhaps because of the immune enhancement typically caused by lenalidomide.
Likelihood score: C (probable rare cause of clinically apparent liver injury).

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Protein Binding
_In vitro_, about 30% of lenalidomide was bound to plasma proteins.

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Interactions
Erythropoietic agents, or other agents that may increase the risk of thrombosis, such as estrogen containing therapies, should be used with caution after making a benefit-risk assessment in patients receiving Revlimid.
Co-administration of multiple dose Revlimid (10 mg) with single dose warfarin (25 mg) had no effect on the pharmacokinetics of total lenalidomide or R- and S-warfarin. Expected changes in laboratory assessments of PT and INR were observed after warfarin administration, but these changes were not affected by concomitant Revlimid administration. It is not known whether there is an interaction between dexamethasone and warfarin. Close monitoring of PT and INR is recommended in multiple myeloma patients taking concomitant warfarin.
When digoxin was co-administered with multiple doses of Revlimid (10 mg/day) the digoxin Cmax and AUC0-8 were increased by 14%. Periodic monitoring of digoxin plasma levels, in accordance with clinical judgment and based on standard clinical practice in patients receiving this medication, is recommended during administration of Revlimid.

[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 Inhibitors; Immunologic 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- or intermediate-1-risk myelodysplastic syndromes (MDS) associated with a deletion 5q cytogenetic abnormality with or without additional cytogenetic abnormalities. /Included in US product label/
Revlimid is indicated for the treatment of patients with mantle cell lymphoma (MCL) whose disease has relapsed or progressed after two prior therapies, one of which included bortezomib. /Included in US product label/
For more Therapeutic Uses (Complete) data for Lenalidomide (7 total), please visit the HSDB record page.

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Drug Warnings
/BOXED WARNING/ WARNING: EMBRYO-FETAL TOXICITY. Do not use Revlimid during pregnancy. Lenalidomide, a thalidomide analogue, caused limb abnormalities in a developmental monkey study. Thalidomide is a known human teratogen that causes severe life-threatening human birth defects. If lenalidomide is used during pregnancy, it may cause birth defects or embryo-fetal death. In females of reproductive potential, obtain 2 negative pregnancy tests before starting Revlimid treatment. Females of reproductive potential must use 2 forms of contraception or continuously abstain from heterosexual sex during and for 4 weeks after Revlimid treatment. To avoid embryo-fetal exposure to lenalidomide, Revlimid is only available through a restricted distribution program, the Revlimid REMS program (formerly known as the RevAssist program).
/BOXED WARNING/ WARNING: HEMATOLOGIC TOXICITY. Revlimid can cause significant neutropenia and thrombocytopenia. Eighty percent of patients with del 5q myelodysplastic syndromes had to have a dose delay/reduction during the major study. Thirty-four percent of patients had to have a second dose delay/reduction. Grade 3 or 4 hematologic toxicity was seen in 80% of patients enrolled in the study. Patients on therapy for del 5q myelodysplastic syndromes should have their complete blood counts monitored weekly for the first 8 weeks of therapy and at least monthly thereafter. Patients may require dose interruption and/or reduction. Patients may require use of blood product support and/or growth factors
/BOXED WARNING/ WARNING: VENOUS AND ARTERIAL THROMBOEMBOLISM. Revlimid has demonstrated a significantly increased risk of deep vein thrombosis (DVT) and pulmonary embolism (PE), as well as risk of myocardial infarction and stroke in patients with multiple myeloma who were treated with Revlimid and dexamethasone therapy. Monitor for and advise patients about signs and symptoms of thromboembolism. Advise patients to seek immediate medical care if they develop symptoms such as shortness of breath, chest pain, or arm or leg swelling. Thromboprophylaxis is recommended and the choice of regimen should be based on an assessment of the patient's underlying risks.
Patients with multiple myeloma treated with lenalidomide in studies including melphalan and stem cell transplantation had a higher incidence of second primary malignancies, particularly acute myelogenous leukemia (AML) and Hodgkin lymphoma, compared to patients in the control arms who received similar therapy but did not receive lenalidomide. Monitor patients for the development of second malignancies. Take into account both the potential benefit of lenalidomide and the risk of second primary malignancies when considering treatment with lenalidomide.
For more Drug Warnings (Complete) data for Lenalidomide (19 total), please visit the HSDB record page.

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Pharmacodynamics
In hematological malignancies, the immune system is deregulated in the form of altered cytokine networks in the tumour microenvironment, defective T cell regulation of host-tumour immune interactions, and diminished NK cell activity. Lenalidomide is an immunomodulatory agent with antineoplastic, antiangiogenic, and anti-inflammatory properties. Lenalidomide exerts direct cytotoxicity by increasing apoptosis and inhibiting the proliferation of hematopoietic malignant cells. It delays tumour growth in nonclinical hematopoietic tumour models _in vivo_, including multiple myeloma. Lenalidomide also works to limit the invasion or metastasis of tumour cells and inhibits angiogenesis. Lenalidomide also mediates indirect antitumour effects via its immunomodulatory actions: it inhibits the production of pro-inflammatory cytokines, which are implicated in various hematologic malignancies. Lenalidomide enhances the host immunity by stimulating T cell proliferation and enhancing the activity of natural killer (NK) cells. Lenalidomide is about 100–1000 times more potent in stimulating T cell proliferation than [thalidomide]. _In vitro_, it enhances antibody-dependent cell-mediated cytotoxicity (ADCC), which is even more pronounced when used in combination with rituximab. Due to its anti-inflammatory properties, lenalidomide has been investigated in the context of inflammatory and autoimmune diseases, such as amyotrophic lateral sclerosis.

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.)