CRBN; TNF-α (IC50 = 13 nM)

(magnification, ×200).Pomalidomide has an IC50 of 13 nM and a 25 nM, respectively, for inhibiting lipopolysaccharide (LPS) stimulated TNF-alpha release in human PBMC and human whole blood. [1] Pomalidomide has an IC50 of 1 μM and inhibits T regulatory cell growth that is induced by IL-2. [2] Pomalidomide (6.4 nM–10 M) treatment causes an increase in IL-2 production in human peripheral blood T cells; this effect is marginally more pronounced in the CD4+ subset than in the CD8+ subset. Pomalidomide has a much greater ability to increase IL-2, IL-5, and IL-10 levels than CC-5013, but it has only a marginally greater ability to increase IFN-γ levels. Pomalidomide enhances SEE and Raji cells induced AP-1 transcriptional activity in Jurkat cells in a dose-dependent manner, with a maximal enhancement of 4-fold at 1 μM. [3] When Raji cells are exposed to different Pomalidomide concentrations (2.5–40 μg/mL) for 48 hours, cell proliferation and DNA synthesis are significantly reduced. In comparison to controls treated with the vehicle, there is a 40% reduction. [4]

In mice with severe combined immunodeficiency, pomalidomide improves rituximab's ability to treat B-cell lymphomas. In contrast to the 58 days of CC5013/rituximab treatment and the 45 days of rituximab nonotherapy, the mice with the combination of pomalidomide and rituximab have a median survival period of 74 days. Pomalidomide and rituximab have a synergistic effect, but this effect can be completely reversed by NK cell depletion, which lends support to the idea that one way Pomalidomide may increase rituximab antitumor activity is by promoting NK cell expansion. [4]

TNF-α inhibitory activity is measured in lipopolysacharide (LPS) stimulated PBMC. Pomalidomide is added to human PBMCs one hour before LPS (1 μg/mL) is added, and incubation is then continued for an additional 18 to 20 hours. The concentration of TNF-α is then measured in the supernatants using an ELISA after they are harvested. Nonlinear regression analysis is used to determine the amount of pomalidomide (IC50) required to reduce TNF-production by 50%. Similar to the PBMC assay, the human whole blood TNF-inhibition assay is carried out except that fresh human whole blood that has been heparinized is directly plated onto microtiter plates.

Pomalidomide (5 μg/mL) is applied to Lymphoma cell lines for 24 or 48 hours in order to measure cell apoptosis. Propidium iodine and FITC-labeled Annexin V are used to stain the cells. Fluorescence-activated cell sorter/FACStar Plus flow cytometer multicolor flow cytometric analysis is used to examine cell apoptosis. When cells show signs of early or late apoptosis (Annexin V positivity and propidium iodine negativity or positivity, respectively), they are considered to be apoptotic. The Lymphoma cell lines are exposed to Pomalidomide (2.5, 5, 10, 20, and 40 μg/mL) for 24 or 48 hours to measure cell proliferation. After adding 1 μCi of [3H]-thymidine per well (in a 96-well plate), the cells are given another 18 hours of incubation. The [3H]-thymidine uptake is then determined using an automated scintillation counter after cells are harvested using the Harvest system and placed into 96-well glass filters.

Mice: SCID mice aged six to eight weeks are used for this. All of the animals are injected with 1×106 Raji cells through their tail veins on day 0. The animals are divided into seven cohorts following 72 hours of tumor engraftment. The first cohort (group A) serves as the control and is not given any medication. Animals in Groups B and C were given either CC-5013 (0.5 mg/kg) or Pomalidomide (0.5 mg/kg) intravenously on Days +3, +4, +8, +9, +13, +14, +18, and +19. Rituximab or Trastuzumab (isotype control) monotherapy is administered to Groups D and E on Days +5, +10, +15, and +20 by tail vein injection at a dose of 10 mg/kg. Animals treated with Rituximab and CC-5013 (group E) or Pomalidomide (group G) make up groups F and G, respectively. Prior to each dose of Rituximab, IMiDs are administered intravenously for two consecutive days. Animals are monitored for 90 days after therapy is over. The study's primary outcome is survival, which is measured as the amount of time before limb paralysis sets in. Cervical dislocation is used to kill any animals that reach the end point or remain alive after three months of observation. To find any remaining disease, a pathologic examination of all organs is conducted, including the liver, lungs, and brain. Three different times, the experiments are repeated.
Rats: Three male CD-IGS rats in total are used. Pomalidomide is given as a single PO administration through the stomach cannula at a dose of 50 mg/kg (5 mL/kg) in a suspension formulation of 0.5% carboxymethylcellulose/0.25% Tween 80. Ten hours after dosing, microdialysate is collected in a cooling fraction collector set to 4°C at intervals of 25 minutes. Each sample's corrected concentration is multiplied by the sampling interval, in this case 25 minutes, and divided by the number of hours in a day to obtain the AUC. These values added together represented the overall AUC value for the given time frame. The concentration is plotted at each time point at the halfway point of each collection interval in order to create graphs. Within 12 hours of the specified time points, microdialysates are collected and analyzed for the presence of pomalidomide using a LC-MS/MS assay.

[1]. Molecular mechanism of action of the immune-modulatory drugs, thalidomide, lenalidomide and pomalidomide in multiple myeloma. Leuk Lymphoma. 2013 Apr;54(4):683-7.

[2]. Immunomodulatory drug CC-5013 or CC-4047 and rituximab enhance antitumor activity in a severe combined immunodeficient mouse lymphoma model. Clin Cancer Res. 2005 Aug 15;11(16):5984-92.

[3]. Enhancement of cytokine production and AP-1 transcriptional activity in T cells by thalidomide-related immunomodulatory drugs. J Pharmacol Exp Ther. 2003 Jun;305(3):1222-32.

[4]. Pomalidomide shows significant therapeutic activity against CNS lymphoma with a major impact on the tumor microenvironment in murine models. PLoS One. 2013 Aug 5;8(8):e71754.

[5]. Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4. Chem Biol. 2015 Jun 18;22(6):755-63.

[6]. Tumour necrosis factor-α inhibits hepatic lipid deposition through GSK-3β/β-catenin signaling in juvenile turbot (Scophthalmus maximus L.). Gen Comp Endocrinol. 2016 Mar 1;228:1-8.

C13H11N3O4

273.24

273.07496

C, 57.14; H, 4.06; N, 15.38; O, 23.42

19171-19-8

Pomalidomide-d3;2093128-28-8;Pomalidomide-d5;1377838-49-7;Pomalidomide-d4;1416575-78-4

white solid powder

C1CC(=O)NC(=O)C1N2C(=O)C3=C(C2=O)C(=CC=C3)N

UVSMNLNDYGZFPF-UHFFFAOYSA-N

InChI=1S/C13H11N3O4/c14-7-3-1-2-6-10(7)13(20)16(12(6)19)8-4-5-9(17)15-11(8)18/h1-3,8H,4-5,14H2,(H,15,17,18)

4-amino-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione

CC4047; CC-4047; CC 4047; Pomalidomide. Brand name: Pomalyst

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.15 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 (9.15 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 3: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 15mg/mL

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Solubility in Formulation 4: 10 mg/mL (36.60 mM) in 0.5% CMC-Na 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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