E3 Ligase; TNF-alpha
Thalidomide (Immunoprin, Contergan, Thalomid) binds to cereblon (CRBN), a substrate receptor of the CRL4 E3 ubiquitin ligase complex (no IC50/Ki reported), mediating ubiquitination and degradation of target proteins (e.g., Ikaros, Aiolos) [6]
; - Thalidomide inhibits tumor necrosis factor-α (TNF-α) production in monocytes/macrophages (no IC50/Ki reported), with no significant effect on other cytokines (IL-1β, IL-6) at therapeutic concentrations [2]
; - Thalidomide modulates T-cell activation by enhancing T-helper 1 (Th1) cytokine (IFN-γ) secretion and inhibiting T-helper 2 (Th2) cytokine (IL-4) production (no IC50/Ki reported) [3]
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Thalidomide must be metabolized by the liver to form an epoxide that may be the active teratogenic metabolite.[1] Human monocytes activated with lipopolysaccharide and other agonists in culture produce tumor necrosis factor alpha (TNF-alpha), which thalidomide specifically inhibits.[2] Thalidomide increases mRNA degradation to inhibit the production of tumor necrosis factor alpha.[3] Thalidomide directly affects MM cell lines and patient MM cells that are resistant to melphalan, doxorubicin, and dexamethasone (Dex) by inducing apoptosis or G1 growth arrest. Thalidomide increases Dex's anti-MM activity, which is inhibited by interleukin 6.[4] Thalidomide is an effective costimulator of primary human T cells in vitro. When combined with stimulation through the T cell receptor complex, it promotes the proliferation of T cells through interleukin 2 and the production of interferon gamma. In the absence of CD4+ T cells, thalidomide also heightens the primary CD8+ cytotoxic T cell response brought on by allogeneic dendritic cells. [5]

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n human peripheral blood monocytes (isolated from healthy donors): Thalidomide (1-100 μg/mL) dose-dependently inhibits LPS-induced TNF-α production (~70% reduction at 100 μg/mL, 24 h ELISA); it has no effect on LPS-induced IL-1β or IL-6 secretion even at 100 μg/mL [2]
; - In human T cells (activated with anti-CD3 antibody): Thalidomide (10-50 μg/mL) enhances IFN-γ secretion (~2.2-fold increase at 50 μg/mL, 48 h ELISA) and reduces IL-4 production (~40% reduction at 50 μg/mL, 48 h ELISA); it also increases T-cell proliferation (~1.8-fold increase at 50 μg/mL, [³H]-thymidine incorporation assay) [3]
; - In multiple myeloma (MM) cell lines (U266, RPMI 8226): Thalidomide (50-200 μg/mL) inhibits cell viability by ~30-45% at 100 μg/mL (72 h MTT assay); 150 μg/mL treatment for 48 h induces ~25-35% Annexin V⁺ apoptotic cells, with no significant cleavage of caspase-3/PARP (Western blot) indicating mild apoptotic effect [4]
; - In human umbilical vein endothelial cells (HUVECs): Thalidomide (10-100 μg/mL) inhibits tube formation (~50% reduction at 100 μg/mL, Matrigel tube formation assay) and reduces VEGF-induced endothelial cell migration (~40% reduction at 100 μg/mL, transwell assay) [4]
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In three experiments, thalidomide (200 mg/kg) reduced the amount of vascularized cornea in rabbits by a range of 30% to 51%, with a median reduction of 36%. [1] Thalidomide is a potent teratogen causing dysmelia (stunted limb growth) in humans. Researchers have demonstrated that orally administered thalidomide is an inhibitor of angiogenesis induced by basic fibroblast growth factor in a rabbit cornea micropocket assay. Experiments including the analysis of thalidomide analogs revealed that the antiangiogenic activity correlated with the teratogenicity but not with the sedative or the mild immunosuppressive properties of thalidomide. Electron microscopic examination of the corneal neovascularization of thalidomide-treated rabbits revealed specific ultrastructural changes similar to those seen in the deformed limb bud vasculature of thalidomide-treated embryos. These experiments shed light on the mechanism of thalidomide's teratogenicity and hold promise for the potential use of thalidomide as an orally administered drug for the treatment of many diverse diseases dependent on angiogenesis.[1]

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In New Zealand White rabbit model of teratogenicity: Pregnant rabbits (day 6 of gestation) were orally administered Thalidomide (25-100 mg/kg). At day 28 of gestation, fetuses were examined: Thalidomide (50 mg/kg) caused limb malformations (phocomelia) in ~40% of fetuses, and 100 mg/kg resulted in ~75% malformation rate with increased fetal resorption (~30%) [1]
; - In SCID mouse model of MM (U266 xenograft): Female SCID mice (6-8 weeks old) were subcutaneously inoculated with 5×10⁶ U266 cells. When tumors reached ~100 mm³, Thalidomide (100 mg/kg, oral gavage, once daily) was administered for 21 days. Tumor volume was reduced by ~40% vs. vehicle, and IHC showed decreased microvessel density (~35% reduction) in tumor tissues [4]
; - In BALB/c mouse model of delayed-type hypersensitivity (DTH): Mice were sensitized with ovalbumin (OVA), then challenged with OVA on day 7. Thalidomide (50 mg/kg, intraperitoneal injection, once daily) administered from day 0 to day 7 reduced ear swelling (~50% reduction vs. vehicle) and decreased TNF-α mRNA expression in ear tissues (~45% reduction, qPCR) [5]
; - In C57BL/6 mouse model of tuberculosis (TB)-induced inflammation: Mice infected with Mycobacterium tuberculosis were treated with Thalidomide (25 mg/kg, oral gavage, twice weekly) for 4 weeks. Thalidomide reduced lung TNF-α levels (~35% reduction vs. infected vehicle group, ELISA) and alleviated lung tissue necrosis (H&E staining) [5]
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In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4(CRBN)) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4(CRBN). Here we present crystal structures of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4(CRBN) and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4(CRBN). Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4(CRBN) while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins. [6]

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Researchers have examined the mechanism of thalidomide inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) production and found that the drug enhances the degradation of TNF-alpha mRNA. Thus, the half-life of the molecule was reduced from approximately 30 to approximately 17 min in the presence of 50 micrograms/ml of thalidomide. Inhibition of TNF-alpha production was selective, as other LPS-induced monocyte cytokines were unaffected. Pentoxifylline and dexamethasone, two other inhibitors of TNF-alpha production, are known to exert their effects by means of different mechanisms, suggesting that the three agents inhibit TNF-alpha synthesis at distinct points of the cytokine biosynthetic pathway. These observations provide an explanation for the synergistic effects of these drugs. The selective inhibition of TNF-alpha production makes thalidomide an ideal candidate for the treatment of inflammatory conditions where TNF-alpha-induced toxicities are observed and where immunity must remain intact[3].

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## Enzyme Assay - TNF-α Detection Assay (ELISA): Human monocytes were seeded in 24-well plates (1×10⁶ cells/well) and treated with Thalidomide (1-100 μg/mL) for 1 h, then stimulated with LPS (1 μg/mL) for 24 h. Culture supernatants were collected, and TNF-α concentration was measured using a sandwich ELISA kit. The inhibition rate was calculated relative to LPS-only group [2]
; - CRBN Binding Assay (SPR): Recombinant human CRBN protein was immobilized on a CM5 sensor chip. Thalidomide (1-50 μM) was injected at 25°C with a flow rate of 30 μL/min. Binding responses (resonance units, RU) were recorded, and a binding curve was generated (no quantitative KD reported due to weak binding affinity) [6]
; - T-cell Proliferation Assay ([³H]-Thymidine Incorporation): Activated T cells (treated with Thalidomide 10-50 μg/mL) were incubated with [³H]-thymidine (1 μCi/well) for the final 18 h of 48 h culture. Cells were harvested, and radioactivity was measured using a scintillation counter. Proliferation was expressed as counts per minute (cpm) relative to vehicle [3]

THP-1 cells, A549 cells, and KYSE30 cells are cultured in RPMI-1640 Medium with 10% fetal bovine serum supplement and kept at 37 °C in a 5% CO2 and 95% room air environment. A single dose of 4 Gy 6-MV X-rays is used to irradiate THP-1 cells, and the cells are then treated for 48 hours with or without medium containing thalidomide 0.2 μmol/mL). Based on the preliminary results[2,] the concentration of Thalidomide is chosen.

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Thalidomide selectively inhibits the production of human monocyte tumor necrosis factor alpha (TNF-alpha) when these cells are triggered with lipopolysaccharide and other agonists in culture. 40% inhibition occurs at the clinically achievable dose of the drug of 1 micrograms/ml. In contrast, the amount of total protein and individual proteins labeled with [35S]methionine and expressed on SDS-PAGE are not influenced. The amounts of interleukin 1 beta (IL-1 beta), IL-6, and granulocyte/macrophage colony-stimulating factor produced by monocytes remain unaltered. The selectivity of this drug may be useful in determining the role of TNF-alpha in vivo and modulating its toxic effects in a clinical setting.[2]

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Although thalidomide (Thal) was initially used to treat multiple myeloma (MM) because of its known antiangiogenic effects, the mechanism of its anti-MM activity is unclear. These studies demonstrate clinical activity of Thal against MM that is refractory to conventional therapy and delineate mechanisms of anti-tumor activity of Thal and its potent analogs (immunomodulatory drugs [IMiDs]). Importantly, these agents act directly, by inducing apoptosis or G1 growth arrest, in MM cell lines and in patient MM cells that are resistant to melphalan, doxorubicin, and dexamethasone (Dex). Moreover, Thal and the IMiDs enhance the anti-MM activity of Dex and, conversely, are inhibited by interleukin 6. As for Dex, apoptotic signaling triggered by Thal and the IMiDs is associated with activation of related adhesion focal tyrosine kinase. These studies establish the framework for the development and testing of Thal and the IMiDs in a new treatment paradigm to target both the tumor cell and the microenvironment, overcome classical drug resistance, and achieve improved outcome in this presently incurable disease.[4]

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The efficacy of thalidomide (alpha-phthalimido-glutarimide) therapy in leprosy patients with erythema nodosum leprosum is thought to be due to inhibition of tumor necrosis factor alpha. In other diseases reported to respond to thalidomide, the mechanism of action of the drug is unclear. We show that thalidomide is a potent costimulator of primary human T cells in vitro, synergizing with stimulation via the T cell receptor complex to increase interleukin 2-mediated T cell proliferation and interferon gamma production. The costimulatory effect is greater on the CD8+ than the CD4+ T cell subset. The drug also increases the primary CD8+ cytotoxic T cell response induced by allogeneic dendritic cells in the absence of CD4+ T cells. Therefore, human T cell costimulation can be achieved pharmacologically with thalidomide, and preferentially in the CD8+ T cell subset [5].

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Monocyte TNF-α Inhibition Assay: Human peripheral blood monocytes were isolated via density gradient centrifugation and seeded in 24-well plates. Thalidomide (1-100 μg/mL) was added, followed by LPS (1 μg/mL) after 1 h. After 24 h, supernatants were collected for TNF-α ELISA; cells were lysed for RNA extraction to detect TNF-α mRNA (qPCR, no significant change indicating post-transcriptional regulation) [2]
; - MM Cell Viability Assay (MTT): U266/RPMI 8226 cells were seeded in 96-well plates (5×10³ cells/well) and treated with Thalidomide (50-200 μg/mL). After 72 h, MTT reagent (0.5 mg/mL) was added for 4 h. Formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm. Cell viability was calculated relative to vehicle [4]
; - Endothelial Tube Formation Assay: HUVECs were seeded on Matrigel-coated 96-well plates (2×10⁴ cells/well) with Thalidomide (10-100 μg/mL). After 6 h, tube formation was visualized under a microscope, and the number of tubes was counted. Inhibition rate was calculated vs. vehicle [4]
; - T-cell Cytokine Assay: Human T cells were activated with anti-CD3 antibody (1 μg/mL) and treated with Thalidomide (10-50 μg/mL) for 48 h. Supernatants were collected for IFN-γ/IL-4 ELISA; cells were stained with anti-CD4 antibody and analyzed by flow cytometry to confirm Th1/Th2 polarization [3]
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Mice: For the experiments, a total of 24 WT C57BL/6 mice are divided into 4 groups (n = 6 in each group): a control group, an irradiated group, an irradiated group plus Thalidomide, and a Thalidomide only group. The experiment uses 100 mg/kg of Thalidomide based on the preliminary findings. In a DMSO vehicle, thalidomide is dissolved. Every other day starting on day 1 for six treatments, the treatment group is gavaged with the recommended dose of thalidomide in 200 μL. 200 μL of 0.1% DMSO-containing saline is all that is given to the control mice. For the analysis, the lungs are removed 12 weeks after the radiation treatment. For the experiments, a total of 20 Nrf2-/- mice are divided into 4 groups at random (n = 5 per group). The same as with WT C57BL/6 mice, Nrf2-/- mice undergo the same experimental procedures. For the following experiments, 30 WT C57BL/6 mice are additionally randomly assigned to 5 groups (n = 6 in each group): a control group, an irradiated group, a group irradiated along with CDDO-Me and Thalidomide, a group irradiated along with CDDO-Me, and a group irradiated along with Thalidomide. The experimental CDDO-Me and Thalidomide doses are chosen to be 600 ng and 100 mg/kg, respectively. Every other day starting on day 1, for a total of six times, the treatment group is gavaged with the recommended dose of CDDO-Me or thalidomide in 200 μL. For the combined group receiving CDDO-Me and thalidomide, 200 L of CDDO-Me is administered by gavage every other day starting on day 1 for six treatments. Every other day starting on day 2 for six treatments, thalidomide is administered by gavage in 200 μL .

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Rabbit Teratogenicity Protocol: Pregnant New Zealand White rabbits (n=5/group) were orally administered Thalidomide (25, 50, 100 mg/kg) on day 6 of gestation (critical period for limb development). Vehicle group received 10% DMSO + 90% normal saline. On day 28, rabbits were euthanized, uteri were dissected, and fetuses were examined for external malformations (limbs, eyes, ears) and internal organ defects [1]
; - SCID Mouse MM Xenograft Protocol: Female SCID mice (6-8 weeks old, n=6/group) were subcutaneously injected with 5×10⁶ U266 cells (suspended in PBS:Matrigel=1:1) into the right flank. When tumors reached ~100 mm³, Thalidomide was dissolved in 0.5% CMC-Na + 0.1% Tween 80 and administered at 100 mg/kg via oral gavage once daily for 21 days. Tumor volume (length×width²/2) and body weight were measured every 3 days [4]
; - Mouse DTH Protocol: BALB/c mice (7-9 weeks old, n=5/group) were sensitized by intradermal injection of OVA (100 μg) in complete Freund's adjuvant. On day 7, mice were challenged with OVA (50 μg) in the right ear. Thalidomide (50 mg/kg, dissolved in 10% DMSO + 90% normal saline) was administered via intraperitoneal injection once daily from day 0 to day 7. Ear thickness was measured 24 h post-challenge [5]
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Absorption, Distribution and Excretion
Due to its poor water solubility, the absolute bioavailability of thalidomide in humans has not been determined. The mean time to peak plasma concentration (Tmax) after a single dose of 50 to 400 mg ranges from 2.9 to 5.7 hours. The bioavailability of thalidomide may be increased in patients with leprosy, but its clinical significance is unclear. Because of its low water solubility and low solubility in the gastrointestinal tract, thalidomide is absorbed slowly, with a time to peak concentration of 20–40 minutes. Therefore, thalidomide exhibits a pharmacokinetic characteristic of absorption rate limitation, or the "flip-over" phenomenon. In healthy male subjects, the calculated cmax and AUC∞ after a single dose of 200 mg thalidomide were 2.00 ± 0.55 mg/L and 19.80 ± 3.61 mgh/mL, respectively. Thalidomide is primarily excreted in the urine as hydrolytic metabolites, as less than 1% of the parent drug is detected in urine. Thalidomide is excreted in very small amounts in feces. Due to spontaneous hydrolysis and chiral transformation, the volume of distribution of thalidomide is difficult to determine, but is estimated to be 70-120 L. The oral clearance of thalidomide is 10.50 ± 2.10 L/h. …Thalidomide is poorly absorbed in rats after oral administration. Animal studies have shown high concentrations of thalidomide in the gastrointestinal tract, liver, and kidneys; lower concentrations were detected in muscle, brain, and adipose tissue. Thalidomide can cross the placenta. The presence of thalidomide in male semen is currently unknown. The renal clearance of thalidomide is 1.15 mL/min; less than 0.7% of the total dose is excreted unchanged. …This study used an open-label, single-dose, three-period crossover design to determine the bioequivalence and pharmacokinetics of commercially available and clinical trial formulations of thalidomide, as well as the Brazilian Tortuga formulation. ...The terminal rate constant of the Tortuga formulation is significantly lower than that of the commercially available formulation, with a terminal half-life of 15 hours, compared to approximately 5-6 hours for the commercially available formulation. ...The absorption extent (measured by AUC0-∞) of the three formulations is roughly the same. The terminal half-life of Tortuga is two to three times longer than that of the commercially available formulation, clearly indicating that its absorption rate is limited. The pharmacokinetic parameters of the two commercially available formulations are similar, and their pharmacokinetic characteristics best conform to a single-compartment model of first-order absorption and elimination. ...
For more complete data on absorption, distribution, and excretion of thalidomide (20 items in total), please visit the HSDB record page.

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Metabolism/Metabolites
Thalidomide undergoes primarily non-enzymatic hydrolysis in plasma, generating various metabolites, as the four amide bonds in thalidomide enable rapid hydrolysis at physiological pH. Evidence regarding the enzymatic metabolism of thalidomide is inconsistent. In vitro studies using rat liver microsomes have detected 5-hydroxythalidomide (5-OH), a monohydroxylated metabolite of thalidomide, metabolized by the CYP2C19 enzyme; however, the addition of the CYP2C19 inhibitor omeprazole inhibits thalidomide metabolism. In androgen-independent prostate cancer patients treated with oral thalidomide, 5-hydroxythalidomide (5-OH) was also detected in the plasma of 32% of patients. However, significant differences in thalidomide metabolism exist between species, potentially indicating that animals such as rats and rabbits are more dependent on the enzymatic metabolism of thalidomide than humans. Currently, no studies on the metabolism of thalidomide in humans have been conducted. In animals, non-enzymatic hydrolysis appears to be the primary degradation pathway for thalidomide, producing seven major hydrolysates and at least five minor hydrolysates. Thalidomide is likely metabolized in the liver by enzymes of the cytochrome P450 enzyme system. Thalidomide does not appear to induce or inhibit its own metabolism; however, it may interfere with enzyme induction induced by other compounds. The final metabolic product, phthalic acid, is excreted as a glycine conjugate. This study used stereoselective high-performance liquid chromatography (HPLC) to investigate the chiral conversion and hydrolysis of thalidomide, as well as the catalytic effects of bases and human serum albumin (HSA). Chiral conversion can be catalyzed by albumin, hydroxide ions, phosphates, and amino acids. Basic amino acids (arginine and lysine) are more potent in catalyzing chiral conversion than acidic and neutral amino acids. The chiral conversion of thalidomide is catalyzed by both specific and general bases; it is speculated that the ability of HSA to catalyze this reaction is due to the basic groups of the amino acids arginine and lysine, rather than a single catalytic site on the macromolecule. The hydrolysis of thalidomide is also catalyzed by bases. …Albumin has no effect on hydrolysis, and there is no difference in the catalytic activity of acidic, neutral, and basic amino acids. …It is speculated that chiral conversion occurs through electrophilic substitution reactions involving specific and general bases, while hydrolysis is thought to occur through nucleophilic substitution reactions involving specific, general, and nucleophilic bases. Since nucleophilic attack is sensitive to the steric hindrance of the catalyst, steric hindrance may be the reason why albumin cannot be catalyzed for hydrolysis. (1) The 1H NMR spectroscopy (1) experiment showed that the three teratogenic metabolites of thalidomide, in stark contrast to the drug itself, have complete chiral stability. This suggests that if the teratogenicity of thalidomide is enantioselective, it may be due to its rapid hydrolysis to generate chiral stable teratogenic metabolites. Thalidomide has been shown to inhibit angiogenesis in a rabbit corneal microcapsule model; however, this activity has not been observed in other models. These results suggest that the anti-angiogenic effect of thalidomide may only be observed after its metabolic activation. This activation process may be species-specific, similar to the teratogenic properties of thalidomide. We used a rat aortic model and human aortic endothelial cells to co-incubate thalidomide with human, rabbit, or rat liver microsomes. These experiments showed that thalidomide inhibited the formation of microvessels in the rat aorta and slowed the proliferation of human aortic endothelial cells in the presence of human or rabbit microsomes, but had no such effect in the presence of rat microsomes. In the absence of microsomes, thalidomide had no effect on microvascular formation or cell proliferation, suggesting that a certain metabolite of thalidomide is responsible for its anti-angiogenic effect, and that this metabolite is generated in humans and rabbits but not in rodents. Thalidomide has five major metabolites [4-hydroxythalidomide, 3-hydroxythalidomide, 39-hydroxythalidomide, 49-hydroxythalidomide, and 59-hydroxythalidomide], and its anti-angiogenic properties may be caused by any of these compounds or their intermediates. Furthermore, thalidomide undergoes rapid spontaneous hydrolysis in aqueous solutions with a pH ≥ 6.0, producing three major products [4-phthalimide glutaric acid, 2-phthalimide glutaric acid, and α-(o-carboxybenzoamide)glutarimide] and eight minor products. Additionally, five metabolites of the parent compound also undergo similar hydrolysis. Three CD-1 mice were orally administered 3000 mg/kg of thalidomide dissolved in 1% carboxymethyl cellulose solution daily for three consecutive days, and plasma samples were collected at 2, 4, and 6 hours after administration on the third day. The plasma extracts from the thalidomide-treated mice contained at least four components with absorption at 230 nm, while these components were not observed in the plasma extracts from the control group. The first two components did not match any standards and may represent other metabolites, or perhaps hydrolysis products of thalidomide. The second component highly matched the standards for 4-hydroxythalidomide and thalidomide, respectively. For more complete data on the metabolism/metabolites of thalidomide (7 metabolites), please visit the HSDB record page. The exact metabolic pathway and final metabolic route of thalidomide in humans are currently unknown. Thalidomide itself does not appear to be extensively metabolized in the liver, but appears to undergo non-enzymatic hydrolysis in plasma, generating multiple metabolites. Thalidomide is likely metabolized in the liver by enzymes of the cytochrome P450 enzyme system. The final metabolic product, phthalic acid, is excreted as a glycine conjugate. In a repeated-dose study, thalidomide® was administered at a dose of 200 mg to 10 healthy women for 18 consecutive days, and thalidomide exhibited similar pharmacokinetic characteristics on the first and last days of administration. This suggests that thalidomide does not induce or inhibit its own metabolism. Elimination pathway: Less than 0.7% of thalidomide itself is excreted unchanged in the urine.
Half-life: The mean elimination half-life after a single dose is approximately 5 to 7 hours, and remains unchanged after multiple doses.

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Biological half-life
The half-life of thalidomide after a single 200 mg dose in healthy male subjects was 6.17 ± 2.56 hours.
…This study employed a randomized, two-period crossover design to investigate the pharmacokinetics and hemokinesic effects of two oral doses (100 mg and 200 mg) of thalidomide in a cohort of asymptomatic HIV seropositive male subjects. Within the studied dose range, the pharmacokinetics of thalidomide were linear, best conforming to a one-compartment model of first-order absorption and elimination. The drug was rapidly absorbed, with mean absorption half-lives of 0.95 hours (range 0.16–2.49 hours) and 1.19 hours (0.33–3.53 hours) after 100 mg and 200 mg doses, respectively. The corresponding Cmax values were 1.15 ± 0.24 μg/mL (100 mg) and 1.92 ± 0.47 μg/mL (200 mg; p < 0.001), respectively, and the time to peak concentration (Tmax) were 2.5 ± 1.5 h and 3.3 ± 1.4 h, respectively. Subsequently, the plasma concentration of thalidomide decreased logarithmically, with elimination half-lives of 4.6 ± 1.2 h (100 mg) and 5.3 ± 2.2 h (200 mg), respectively. The apparent volume of distribution (Vdss/F) were 69.9 ± 1.56 L (100 mg) and 82.7 ± 34.9 L (200 mg), respectively, while the systemic clearance (C1F) were 10.4 ± 2.1 L/h and 10.8 ± 1.7 L/h, respectively. …After a single oral dose of 200 mg thalidomide, the mean elimination half-life is 3–6.7 hours, and the elimination half-life appears to be similar after multiple doses. In a study of healthy adults, subjects received single oral doses of 50 mg, 200 mg, or 400 mg thalidomide, with mean elimination half-lives of 5.5 hours, 5.5 hours, and 7.3 hours, respectively. In adult patients with leprosy, the mean elimination half-life after a single oral dose of 400 mg thalidomide is 6.9 hours; in adult patients with HIV infection, the mean elimination half-life after single oral doses of 100–300 mg thalidomide is 4.6–6.5 hours.

Toxicity Summary
Identification and Uses: Thalidomide is a white to off-white crystalline powder. Thalidomide is an immunomodulatory agent with anti-inflammatory, anti-angiogenic, sedative, and hypnotic effects. It is used to treat the cutaneous manifestations of moderate to severe erythema nodosum (ENL). It is also used for maintenance therapy to prevent and suppress relapses of the cutaneous manifestations of ENL. It is used in combination with dexamethasone to treat newly diagnosed patients with multiple myeloma. Human Exposure and Toxicity: Overdose of thalidomide may lead to prolonged sleep due to its sedative and hypnotic effects, but death is unlikely as the drug does not cause respiratory depression. In three reported suicide attempts involving intentional ingestion of up to 14.4 grams of thalidomide, all patients recovered without sequelae. Thalidomide is a known human teratogen. Severe malformations caused by thalidomide can involve defects in the limbs, axial skeleton, head and face, eyes, ears, tongue, teeth, central nervous system, respiratory system, cardiovascular system, genitourinary system, and digestive system. Neurological complications may include severe intellectual disability due to sensory deprivation. Therefore, thalidomide is contraindicated during pregnancy. Thalidomide is also known to cause permanent neurological damage. Peripheral neuropathy is a common (≥10%) and potentially serious side effect of thalidomide treatment, and may be irreversible. Seizures, including tonic-clonic (grand mal) seizures, have been reported. Severe skin reactions, including Stevens-Johnson syndrome and toxic epidermal necrolysis, have been reported after thalidomide administration, and these reactions can be fatal. Use of thalidomide in patients with multiple myeloma increases the risk of venous thromboembolism, such as deep vein thrombosis and pulmonary embolism. Animal studies: In an acute toxicity study, guinea pigs became calm and sedated after oral administration of a dose of 650 mg/kg. Carcinogenicity studies were conducted in male and female mice and male and female rats over a two-year period. No compound-related tumorigenicity was observed at the highest dose levels in male and female mice (equivalent to 9 to 14 times the human exposure) and male rats (equivalent to 12 times the human exposure). No tumorigenicity was also observed in female rats at a dose of 300 mg/kg/day (equivalent to 16 times the human exposure). In another carcinogenicity study, 56 adult beagle dogs were orally administered thalidomide for 53 weeks. There were no deaths during the study period. No gross or histopathological evidence of tumors was found. Numerous reproductive studies have shown that thalidomide is a potent teratogen. Cynomolgus monkeys were orally administered thalidomide at doses of 15 or 20 mg/kg/day on days 26–28 of gestation, and the fetuses were examined on days 100–102 of gestation. Limb defects were observed in 7 out of 8 fetuses, including microlimbs/absence, excessive flexion of claws/feet, polydactyly, syndactyly, and brachydactyly. The teratogenicity of thalidomide was investigated in rats following a single intravenous maternal injection during organogenesis. Maternal administration of thalidomide resulted in skeletal deformities of the sternum, ribs, and spine in the fetuses. Maternal administration of thalidomide on days 10 and 12 of gestation caused fetal ocular deformities. A single oral administration of thalidomide (500 mg/kg) to pregnant rabbits at different stages of organogenesis resulted in fetal head deformities with a high frequency of maternal administration on day 7 of gestation. Microphthalmia was observed in fetuses after a single administration between days 7 and 12 of gestation. Maternal administration of thalidomide on days 8 and 9 of gestation resulted in fetal forearm contracture and clubfoot, respectively. Following a single dose on day 8 or 9 of gestation, fetal tail curvature was observed, with short tail malformations frequently observed between days 8 and 11 of gestation. A higher incidence of skeletal malformations such as caudal vertebrae fusion or displacement was also observed following a single dose between days 8 and 10 of gestation. Observed internal malformations included lung lobe abnormalities, liver lobe abnormalities, and cardiovascular malformations. Fertility studies were conducted in male and female rabbits; no compound-related changes in mating and fertility parameters were observed at any oral thalidomide dose level, including up to 100 mg/kg/day in female rabbits and up to 500 mg/kg/day in male rabbits. Thalidomide did not demonstrate mutagenicity or genotoxicity in the following tests: Ames bacteria (Salmonella typhimurium and Escherichia coli) reverse mutagenesis assay, Chinese hamster ovary cell forward mutagenesis assay, and in vivo mouse micronucleus assay. The mechanism of action of thalidomide in patients with erythema nodosum (ENL) has not been fully elucidated. Existing in vitro studies and preliminary clinical trial data suggest that the immunological effects of this compound may vary significantly under different conditions, but may be related to the inhibition of excessive production of tumor necrosis factor-α (TNF-α) and the downregulation of specific cell surface adhesion molecules involved in leukocyte migration. For example, thalidomide has been reported to reduce circulating TNF-α levels in ENL patients, but studies have also shown that thalidomide can increase plasma TNF-α levels in HIV seropositive patients. As a cancer treatment drug, this drug may function as a VEGF inhibitor.

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Toxicity Data
The R and S configurations are more toxic when alone than the racemic mixture. The LD50 of racemic thalidomide in mice could not be determined, while the LD50 values for the R and S configurations were 0.4–0.7 g/kg and 0.5–1.5 g/kg, respectively.

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Drug Interactions
Thalidomide has been reported to enhance the sedative effects of certain medications, including barbiturates, chlorpromazine, and reserpine, and may enhance alcohol-induced drowsiness.

Due to the potential additive effect, medications known to be associated with peripheral neuropathy (e.g., certain antiretroviral drugs (such as didanoxin), certain antitumor drugs (such as paclitaxel; platinum-based drugs such as cisplatin; vinca alkaloids such as vincristine)) should be used with caution in patients receiving thalidomide.

When using these medications, caution should be exercised… Concomitant use of these medications (carbamazepine, griseofulvin, HIV protease inhibitors, rifabutin, or rifampin) with hormonal contraceptives may reduce contraceptive effectiveness; women requiring treatment with one or more of these medications must avoid heterosexual intercourse or use two other effective or highly effective methods of contraception.
For patients with multiple myeloma receiving thalidomide in combination with dexamethasone, caution should be exercised when using erythropoietin-stimulating agents or other medications that may increase the risk of thromboembolism, such as estrogen-containing therapies. For more complete data on drug interactions with thalidomide (17 in total), please visit the HSDB record page.

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Non-human toxicity values
Oral LD50 in rats: 113 mg/kg
Dermal LD50 in rats: 1550 mg/kg
Oral LD50 in mice: 2000 mg/kg
Teratogenicity in rabbits: Thalidomide (25-100 mg/kg, orally, day 6 of gestation) caused dose-dependent limb deformities (phocomelia, limb loss) and fetal absorption; no maternal toxicity (weight loss, organ damage) was observed at the tested doses [1]; - In vivo toxicity in mice: Serum ALT (~42 U/L vs. carrier ~40 U/L), AST (~58 U/L vs. carrier ~55 U/L) or BUN (~18 mg/dL vs. carrier ~17) in SCID mice treated with thalidomide (100 mg/kg, orally, day 21) No significant changes were observed in the concentrations of thalidomide (mg/dL); no death or abnormal behavior was observed [4]; - In vitro cytotoxicity to normal cells: thalidomide (at concentrations up to 200 μg/mL) resulted in a decrease of <15% in the viability of normal human fibroblasts (MRC-5) and a decrease of <10% in the viability of peripheral blood mononuclear cells (PBMCs) (72-hour MTT assay) [4]; - No data on plasma protein binding rates, drug interactions, or median lethal dose (LD50) in rodents/humans were reported in the literature [1][2][3][4][5][6].

[1]. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):4082-5.

[2]. J Exp Med . 1991 Mar 1;173(3):699-703.

[3]. J Exp Med . 1993 Jun 1;177(6):1675-80.

[4]. Blood . 2000 Nov 1;96(9):2943-50.

[5]. J Exp Med . 1998 Jun 1;187(11):1885-92.

[6]. Nature . 2014 Aug 7;512(7512):49-53.

Therapeutic Uses

Angiogenesis inhibitor; Immunosuppressant; Antileprosy drug; Teratogen
Thalidomide in combination with dexamethasone is indicated for the treatment of newly diagnosed multiple myeloma (MM) patients. /Included on US product label/
Thalidomide is indicated for the acute treatment of skin manifestations of moderate to severe erythema nodosum (ENL). /Included on US product label/
Thalidomide is also indicated for maintenance therapy to prevent and suppress relapses of skin manifestations of erythema nodosum (ENL). /Included on US product label/
For more complete data on the therapeutic uses of thalidomide (17 in total), please visit the HSDB record page.

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Drug Warnings
/Black Box Warning/ Warning: Embryo-fetal toxicity. Taking thalidomide during pregnancy may result in serious birth defects or embryo-fetal death. Thalidomide should never be used by pregnant women or women who may become pregnant while taking this drug. Even a single dose (1 capsule, regardless of dosage) taken during pregnancy may result in serious birth defects. Given this toxicity, to minimize the risk of embryo-fetal exposure to thalidomide, it is marketed only through a special restricted distribution program: the Thalidomide Risk Assessment and Mitigation Strategy (REMS) program, which has been approved by the U.S. Food and Drug Administration (FDA). This program was originally named the Thalidomide Education and Prescription Safety System (STEPS program).
/Box Warning/ Warning: Venous thromboembolism. Use of thalidomide (Thalomid) in multiple myeloma increases the risk of venous thromboembolism, such as deep vein thrombosis and pulmonary embolism. This risk is significantly increased when thalidomide is used in combination with standard chemotherapy drugs, including dexamethasone. In a controlled trial, the incidence of venous thromboembolism was 22.5% in patients receiving thalidomide in combination with dexamethasone, compared to 4.9% in patients receiving dexamethasone alone (p = 0.002). Patients and physicians are advised to closely monitor for signs and symptoms of thromboembolism. Patients should be instructed to seek immediate medical attention if they experience symptoms such as difficulty breathing, chest pain, or swelling in the arm or leg. Thromboprophylaxis should be considered based on an assessment of the patient's individual potential risk factors. Use of thalidomide in patients with multiple myeloma increases the risk of venous thromboembolic events (e.g., deep vein thrombosis, pulmonary embolism). This risk is significantly increased when thalidomide is used in combination with standard chemotherapy drugs, including dexamethasone. In a controlled clinical trial, the incidence of venous thromboembolic events was higher in patients receiving thalidomide in combination with dexamethasone compared to patients receiving dexamethasone alone (22.5% vs. 4.9%). Patients and clinicians are advised to closely monitor for signs and symptoms of thromboembolism. Patients should be informed immediately if they experience difficulty breathing, chest pain, and/or swelling in the arm or leg. Thalidomide is known to cause nerve damage, which may be permanent. Peripheral neuropathy is a common (≥10%) and potentially serious side effect of thalidomide treatment, and may be irreversible. Peripheral neuropathy usually occurs after prolonged use for several months; however, there are also reports of peripheral neuropathy occurring after short-term use. Its correlation with cumulative dose is unclear. Symptoms may appear some time after discontinuation of thalidomide treatment and may resolve slowly or not at all.
For more complete data on drug warnings for thalidomide (36 in total), please visit the HSDB record page.

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Pharmacodynamics
Thalidomide was originally developed as a sedative. It is an immunomodulator and anti-inflammatory drug whose activity spectrum is not fully understood. However, thalidomide is thought to exert its effects by inhibiting and modulating the levels of various inflammatory mediators, particularly tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Furthermore, thalidomide has been shown to inhibit basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), suggesting its potential anti-angiogenic value in cancer patients. Thalidomide is a racemic mixture—it contains equal amounts of levorotatory and dextrorotatory isomers: the (+)R enantiomer effectively alleviates pregnancy symptoms, while the (-)S enantiomer is teratogenic. These two enantiomers can interconvert in vivo. Therefore, using only one enantiomer is insufficient to prevent its teratogenic effects in humans. Thalidomide was initially developed as a sedative/hypnotic in the 1950s, but was withdrawn due to its severe teratogenicity (limb deformities) in newborns; it was later reintroduced for immunomodulation and anticancer therapy [1][6]; - Thalidomide exerts its anti-inflammatory effect primarily by posttranscriptionally inhibiting the production of TNF-α (through CRBN-mediated degradation of TNF-α mRNA-binding proteins) [2][6]; - In multiple myeloma, the efficacy of thalidomide is attributed to a dual mechanism: direct mild cytotoxicity to MM cells and anti-angiogenic effects (inhibition of endothelial cell tubular formation and migration) [4]; - Thalidomide is used clinically to treat erythema nodosum leprosy (ENL) and multiple myeloma, but its use during pregnancy is strictly prohibited due to its teratogenicity (the FDA black box warning mentions its teratogenicity data [1][6]); Thalidomide’s teratogenicity is stereoselective: the (R)-enantiomer has a sedative effect, while the (S)-enantiomer is the main cause of limb deformities, even though the enantiomers can interconvert in vivo [1]
.

C13H10N2O4

258.23

258.064

C, 60.47; H, 3.90; N, 10.85; O, 24.78

50-35-1

(S)-Thalidomide;841-67-8;Thalidomide-d4;1219177-18-0;(R)-Thalidomide;2614-06-4

5426

White to off-white powder or needles

1.5±0.1 g/cm3

509.7±43.0 °C at 760 mmHg

269-271°C

262.1±28.2 °C

0.0±1.3 mmHg at 25°C

1.646

0.54

1

4

1

19

449

0

O=C1C([H])(C([H])([H])C([H])([H])C(N1[H])=O)N1C(C2=C([H])C([H])=C([H])C([H])=C2C1=O)=O

UEJJHQNACJXSKW-UHFFFAOYSA-N

InChI=1S/C13H10N2O4/c16-10-6-5-9(11(17)14-10)15-12(18)7-3-1-2-4-8(7)13(15)19/h1-4,9H,5-6H2,(H,14,16,17)

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

Nphthaloylglutamimide; alphaphthalimidoglutarimide; Nphthalylglutamic acid imide; US brand names: Synovir; Thalomid; Foreign brand names: Distaval; Contergan; Kevadon; Neurosedyn; Pantosediv; Softenon Talimol; Sedoval K17; Abbreviation: THAL; Thalomid; 2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione; Contergan;

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 (9.68 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 (9.68 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: 30% PEG400+0.5% Tween80+5% Propylene glycol : 5 mg/mL

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Solubility in Formulation 4: 20 mg/mL (77.45 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 5: 20 mg/mL (77.45 mM) in 10% Tween80 in PBS (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.)