Immunomodulation; Cereblon E3 ligase

Lenalidomide has a strong effect on T cell proliferation, IFN-γ production, and IL-2 production. It has been demonstrated that lenalidomide increases the synthesis of the anti-inflammatory cytokine IL-10 from human PBMCs while inhibiting the production of pro-inflammatory cytokines TNF-α, IL-1, IL-6, and IL-12. Lenalidomide both directly and indirectly reduces the production of IL-6 by preventing the contact between bone marrow stromal cells (BMSC) and multiple myeloma (MM) cells, which increases the myeloma cells' apoptosis[2]. Thalidomide, Lenalidomide, and Pomalidomide all exhibit dose-dependent interaction 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 reduced CRBN expression cells (U266-CRBN60 and U266-CRBN75) exhibit lower sensitivity to the antiproliferative effects of lenalidomide than the original cells[3]. Lenalidomide, an analog of thalidomide, acts as a molecular glue between the human E3 ubiquitin ligase cereblon and CKIα, causing this kinase to become ubiquitinated and degraded and likely causing leukemic cells to die through p53 activation[5].

The toxicity of lenalidomide administered by IV, IP, and PO at dosages of 15, 22.5, and 45 mg/kg. These highest feasible Lenalidomide doses, which are limited by solubility in our PBS dosing medium, are well tolerated, with the exception of one mouse mortality (out of four total dosed) at the 15 mg/kg IV dose. Notably, at IV doses of 15 mg/kg (n = 3) or 10 mg/kg (n = 45) or at any other dose level through IV, IP, and PO routes, no further toxicities are seen in the study[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]

[1]. 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;2013:208309.
[2]. Krönke J, et al. Lenalidomide induces degradation of IKZF1 and IKZF3. Oncoimmunology. 2014 Jul 3;3(7):e941742.
[3]. 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.
[4]. Kotla V, et al. Mechanism of action of lenalidomide in hematological malignancies. J Hematol Oncol. 2009 Aug 12;2:36.
[5]. 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.
[6]. Rozewski DM, et al. Pharmacokinetics and tissue disposition of lenalidomide in mice. AAPS J. 2012 Dec;14(4):872-8

C13H14CLN3O3

295.72

295.07236

1243329-97-6

191732-72-6; 847871-99-2 (Lenalidomide hemihydrate)

Off-white to light grey solid

92.5Ų

O=C1C2=CC=CC(N)=C2CN1C(C(N3)=O)CCC3=O.[H]Cl

RYWZLJSDFZVVTD-UHFFFAOYSA-N

InChI=1S/C13H13N3O3.ClH/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);1H

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

CC-5013; CC 5013;CC-5013 hydrochloride; CC5013; IMiD1; trade name: Revlimid.

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)
H2O : >5 mg/mL (~16.5 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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