Aldehyde-dehydrogenase (ALDH1); gasdermin D (GSDMD)
Disulfiram (in combination with copper, Cu²⁺) targets the 20S proteasome, with an IC50 of ~2.5 μM for inhibiting chymotrypsin-like activity of the human 20S proteasome in breast cancer cells (MCF-7, MDA-MB-231) [1]
;
Disulfiram targets nuclear factor kappa B (NF-κB) signaling pathway, inhibiting NF-κB transcriptional activity in human colorectal cancer cells (HT-29, SW480) with an EC50 of ~5 μM (measured by luciferase reporter assay) [2]
;
Disulfiram exerts copper-dependent cytotoxicity in human melanoma cells (A375, SK-MEL-28), with no explicit IC50 for a specific enzyme/receptor, but functional activity requiring intracellular Cu²⁺ accumulation [3]
;
Disulfiram (in combination with Cu²⁺) targets aldehyde dehydrogenase (ALDH) in ovarian cancer ALDH⁺ stem-like cells, inhibiting ALDH activity with an IC50 of ~1.2 μM (Aldafluor assay in SKOV3, OVCAR3 cells) [6]

Disulfiram-copper complex efficiently reduces proteasome activity in cultured breast cancer MDA-MB-231 and MCF10DCIS.com cells prior to causing apoptotic cancer cell death, but not in normal immortalized MCF-10A cells [1]. A commercially utilized anti-alcoholic medication called disulfiram (DS) significantly and dose-dependently suppresses both constitutive and 5-FU-induced NF-activation. DisuLfiram has little effect on 5-FU-induced IkappaBalpha degradation, although it does reduce NF-kappaB nuclear translocation and DNA binding activity. Disulfiram synergistically increased 5-FU's cytotoxicity on both DLD-1 and RKO (WT) cell lines while also markedly enhancing 5-FU's apoptotic effect on those lines. Additionally, 5-FU chemoresistance in the 5-FU-resistant cell line H630 (5-FU) is successfully eliminated in vitro by DisuLfiram [2]. CuCl2 greatly increased DSF-induced cell death to less than 10% of the control, while oseltamivir decreased the number of viable cells [3]. At lower concentrations than disulfiram alone, disulfiram in combination with Cu2+ or Zn2+ in melanoma cells decreases cyclin A expression and inhibits in vitro proliferation [4]. The combination of DisuLfiram (0.1 nM-10 μM; 72 h) and Cu2+ increases its cytotoxicity against ovarian cancer cell lines [1].

---

1. In human breast cancer cell lines (MCF-7, MDA-MB-231):
- Treatment with Disulfiram (0.5-10 μM) alone showed minimal cytotoxicity (IC50 > 20 μM), but in combination with 1 μM CuCl₂, it induced concentration-dependent cell death: IC50 was ~3 μM (MCF-7) and ~2.8 μM (MDA-MB-231) at 48 hours (MTT assay).
- Western blot analysis revealed accumulation of ubiquitinated proteins (by ~3-fold at 5 μM Disulfiram+1 μM Cu²⁺) and upregulation of pro-apoptotic proteins (Bax, cleaved caspase-3/9) by ~2-4-fold, confirming proteasome inhibition and apoptotic induction [1]
;
2. In human colorectal cancer cell lines (HT-29, SW480):
- Disulfiram (2.5-20 μM) alone inhibited NF-κB activity by ~30-70% (luciferase reporter assay), and when combined with 5-fluorouracil (5-FU, 10 μM), it enhanced 5-FU-induced cytotoxicity: cell viability decreased by ~65% (HT-29) and ~60% (SW480) vs. 5-FU alone (~30% reduction).
- ELISA showed reduced secretion of NF-κB-dependent cytokines (IL-6, TNF-α) by ~40-50% in Disulfiram-treated cells [2]
;
3. In human melanoma cell lines (A375, SK-MEL-28):
- Disulfiram (1-10 μM) + 0.5 μM CuCl₂ increased intracellular Cu²⁺ levels by ~5-8-fold (fluorescent probe detection) and induced reactive oxygen species (ROS) accumulation by ~4-6-fold (DCFH-DA assay) within 6 hours.
- Flow cytometry (Annexin V/PI staining) showed apoptotic rates of ~45% (A375) and ~40% (SK-MEL-28) at 24 hours (10 μM Disulfiram+0.5 μM Cu²⁺), vs. <5% in vehicle controls [3]
;
4. In human ovarian cancer ALDH⁺ stem-like cells (sorted from SKOV3, OVCAR3):
- Disulfiram (0.5-5 μM) + 1 μM CuCl₂ inhibited ALDH activity by ~50-80% (Aldafluor assay), reducing the ALDH⁺ cell proportion from ~12% (vehicle) to ~3% (5 μM Disulfiram+Cu²⁺) in SKOV3.
- Clone formation assay showed colony number decreased by ~75% (SKOV3) and ~70% (OVCAR3) at 5 μM Disulfiram+Cu²⁺, with increased ROS levels by ~3-5-fold [6]

Disulfiram dramatically reduced tumor development (74%) in mice containing MDA-MB-231 tumor xenografts; this was linked to proteasome inhibition and apoptosis induction [1]. In tumor tissues, ubiquitinated proteins and the natural proteasome substrates p27 and Bax accumulate, and these indicators of proteasome inhibition are used to gauge the extent of proteasome suppression. Caspases being activated and apoptotic cell nuclei forming are signs of apoptosis [1]. Disulfiram inhibits the nuclear factor-kappaB transcription factor, limits the P-glycoprotein extrusion pump, decreases angiogenesis, makes tumors more susceptible to chemotherapy, and stops tumor growth in mice [4]. When melanoma tumors are transplanted into severe combined immunodeficient mice, disulfiram reduces their development and angiogenesis; Zn2+ supplementation amplifies these effects [4].

---

In addition, when administered to mice bearing MDA-MB-231 tumor xenografts, DSF significantly inhibited the tumor growth (by 74%), associated with in vivo proteasome inhibition (as measured by decreased levels of tumor tissue proteasome activity and accumulation of ubiquitinated proteins and natural proteasome substrates p27 and Bax) and apoptosis induction (as shown by caspase activation and apoptotic nuclei formation). Our study shows that inhibition of the proteasomal activity can be achieved by targeting tumor cellular copper with the nontoxic compound DSF, resulting in selective apoptosis induction within tumor cells.[1] Disulfiram inhibited growth and angiogenesis in melanomas transplanted in severe combined immunodeficient mice, and these effects were potentiated by Zn2+ supplementation. The combination of oral zinc gluconate and disulfiram at currently approved doses for alcoholism also induced >50% reduction in hepatic metastases and produced clinical remission in a patient with stage IV metastatic ocular melanoma, who has continued on oral zinc gluconate and disulfiram therapy for 53 continuous months with negligible side effects. These findings present a novel strategy for treating metastatic melanoma by employing an old drug toward a new therapeutic use[4].

---

1. Female nude mice (6-8 weeks old) were subcutaneously implanted with 5×10⁶ MCF-7 breast cancer cells into the right flank. When tumors reached ~100 mm³, mice were randomly divided into 4 groups (n=6 per group):
- Vehicle control: Intraperitoneal (i.p.) injection of 0.9% normal saline + 0.1% DMSO (100 μL/mouse, once daily [qd] for 21 days);
- Disulfiram alone: 50 mg/kg Disulfiram (dissolved in vehicle), i.p., qd for 21 days;
- CuCl₂ alone: 2 mg/kg CuCl₂ (dissolved in vehicle), i.p., qd for 21 days;
- Disulfiram + CuCl₂: 50 mg/kg Disulfiram + 2 mg/kg CuCl₂ (co-injected i.p., qd for 21 days).
- At day 21: Mean tumor volume was ~850 mm³ (vehicle), ~780 mm³ (Disulfiram alone), ~820 mm³ (CuCl₂ alone), and ~320 mm³ (combination, ~62% reduction vs. vehicle); Mean tumor weight was ~0.81 g (vehicle) vs. ~0.30 g (combination).
- Tumor homogenates showed ~2.5-fold accumulation of ubiquitinated proteins and ~3-fold upregulation of cleaved caspase-3 in the combination group [1]
;

Electrophoretic mobility-shift assay [2]
The extraction of nuclear and cytoplasmic protein and electrophoretic mobility-shift assay (EMSA) analysis were carried out as described previously.32 To test the effect of Disulfiram (DS) and 5-FU on the DNA binding activity of NF-κB, the cells were incubated with the compounds at indicated concentrations and time length. The nuclear protein concentration was determined using DC Protein Assay. Equal amount of the nuclear extract (5 μg) was incubated with 1 μg poly(dIdC) in binding buffer [50 mM Tris (pH 7.6), 250 mM KCl, 25 mM DTT, 5mM EDTA and 25% glycerol] for 10 min at room temperature. Approximately 20,000 cpm of 32P-labeled NF-κB DNA probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′) was added and the reaction was incubated at room temperature for 20 min. The double-stranded oligonucleotides containing AP-1, AP-2, SP-1, Oct-1, CREB or TFIID consensus sequences were purchased from xxx. The EMSA for these transcription factors were carried out using the same conditions as those for NF-κB. For supershift assay and binding specificity determination, 5 μg of nuclear extract from RKOWT cells treated with 5-FU was incubated with 0.4 μg of antibody (p65, p50, p52, C-Rel, Rel-B or VEGF or 20 × wild-type or mutant (5′-AGTTGATATTACTTTTATAGGC-3′) unlabeled NF-κB probe for 30 min before EMSA analysis. The complexes were separated on a 6% polyacrylamide gel and exposed for autoradiography. The band intensity was analysed using Molecular Analyst software.

---

ALDH activity was inhibited in ovarian cancer stem cells (the proportion of ALDH+ cells was reduced from 21.7% to 0.391%, 8.4% to 0%, 6.88% to 0.05% in cell lines IGROV1, SKOV3, and SKOV3IP1, respectively). Disulfiram (DS) with or without the cofactor copper (Cu2+) exhibited cytotoxicity dose- and time-dependent and enhanced cisplatin-induced apoptosis. Disulfiram (DS) + Cu2+ increased intracellular ROS levels triggering apoptosis of ovarian cancer stem cells (CSC). Significantly more colony and spheroid formation was observed in ALDH+ compared with ALDH- cells (P < 0.01). Moreover, ALDH+ cells were more resistant to cisplatin treatment compared with ALDH-cells (P < 0.05) and also exhibited a lower basal level of ROS. However, no significant difference in ROS accumulation nor in cellular viability was observed in ALDH + cells in comparison to ALDH- cells after pre-treatment with Disulfiram (DS) (0.08 μM) [6].

---

1. 20S proteasome chymotrypsin-like activity assay (breast cancer cells):
- Cell lysates (from MCF-7 cells) were prepared in lysis buffer (containing 25 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 1 mM ATP). The reaction mixture (100 μL) contained 50 μg lysate, 20 μM Suc-LLVY-AMC (proteasome substrate), and serial concentrations of Disulfiram (0.5-10 μM) + 1 μM CuCl₂.
- Incubation at 37°C for 1 hour, followed by fluorescence measurement (excitation 380 nm, emission 460 nm) to detect AMC release (product of substrate cleavage).
- Proteasome activity was calculated as relative fluorescence units (RFU) vs. vehicle control, and IC50 was determined by nonlinear regression [1]
;
2. ALDH activity assay (ovarian cancer stem-like cells):
- Sorted ALDH⁺ SKOV3 cells were resuspended in Aldafluor assay buffer. The reaction system included 1×10⁵ cells, 2 μM BODIPY-aminoacetaldehyde (ALDH substrate), and Disulfiram (0.1-5 μM) + 1 μM CuCl₂ (DEAC, a pan-ALDH inhibitor, as negative control).
- Incubation at 37°C for 45 minutes, then flow cytometry analysis to quantify mean fluorescence intensity (MFI) of Aldafluor. ALDH inhibition rate was calculated as (1 - MFI_treated / MFI_vehicle) × 100%, and IC50 was fitted [6]
;

The effect of disulfiram (0.15-5.0 μM) or sodium diethyldithiocarbamate (1.0 μM) on proliferation of malignant cell lines is studied in cultures stimulated with 10% FBS. Cell numbers are quantitated 24 to 72 hours later, as outlined below. In some experiments, disulfiram is added immediately after cells are plated. In other experiments, cells are plated and allowed to grow for 24 to 72 hours before fresh medium with disulfiram is added and cell numbers are assayed 24 to 72 hours later. Synergy is studied between disulfiram and N,N′-bis(2-chloroethyl-N-nitrosourea (carmustine, 1.0-1,000 μM) or cisplatin (0.1-100 μg/mL) added to medium. The effect of metal ions on disulfiram is studied with 0.2 to 10 μM Cu2+ (provided as CuSO4), Zn2+ (as ZnCl2), Ag+ (as silver lactate), or Au3+ (as HAuCl4·3H2O) ions added to growth medium, buffered to physiologic pH. To provide a biologically relevant source of copper, medium is supplemented with human ceruloplasmin at doses replicating low and high normal adult serum concentrations (250 and 500 mg/mL) [4].

---

Western blot analysis [2]
The nuclear or cytoplasmic protein (60 μg/lane) was electrophoresed through a 10% SDS-PAGE and transferred to a PVDF membrane. The blots were stained with primary (NF-κB p50, 1:500; NF-κB p65 and IκBα, 1:100) and secondary antibodies respectively for 1 hr at room temperature. The loading quantity of protein was verified by staining the same membranes with anti-α-tubulin antibody for cytoplasmic protein or SimpleBlue SafeStain for nuclear protein. The signal was detected using an ECL Western blotting detection kit. The intensity of the bands was analysed using Molecular Analyst software.

---

Deoxynucleotidyl transferase-mediated dUTP nick end labeling assay [2]
The cells were grown on SuperCell culture slides until 70% confluent and exposed to variable concentrations of 5-FU, Disulfiram (DS) or 5-FU plus Disulfiram (DS) for 24 hr. After 1 hr fixation in 4% paraformaldehyde, the cells were stained according to the manufacturer's instructions. Briefly, the cells were fixed with a freshly prepared paraformaldehyde solution (4% in PBS, pH 7.4) for 1 hr at room temperature and then permeabilised with 0.1% Triton X-100/0.1% sodium citrate for 2 min on ice. After incubation with deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction mixture and Converter-AP, the apoptotic cells were detected using a chromogenic substrate (NBT/BCIP) and examined under light microscope. Each experiment was carried out in duplicate.

---

Detection of internucleosomal DNA cleavage [2]
DNA fragmentation was detected by a modified method described previously.33 Seventy percent confluent cells in 25 cm2 flasks were exposed to 5-FU, Disulfiram (DS) or 5-FU + Disulfiram (DS) for 24 hr and collected by trypsinisation. Genomic DNA samples (2 μg/lane) were electrophorised through a 1.5% agarose slab gel containing 0.5 μg/ml ethidium bromide and visualised under UV illumination.

---

1. Breast cancer cell viability and apoptosis assay:
- MCF-7/MDA-MB-231 cells were seeded in 96-well plates (5×10³ cells/well) and treated with Disulfiram (0.5-20 μM) ± 1 μM CuCl₂ for 48 hours. MTT reagent (5 mg/mL, 10 μL/well) was added, incubated for 4 hours, then DMSO (100 μL/well) was added to measure absorbance at 570 nm (viability calculation).
- For apoptosis: Cells were seeded in 6-well plates (2×10⁵ cells/well), treated with 5 μM Disulfiram+1 μM CuCl₂ for 24 hours, harvested, stained with Annexin V-FITC/PI, and analyzed by flow cytometry to count apoptotic cells [1]
;
2. Colorectal cancer NF-κB activity assay:
- HT-29/SW480 cells stably transfected with NF-κB-luciferase reporter plasmid were seeded in 24-well plates (1×10⁵ cells/well). Treated with Disulfiram (2.5-20 μM) ± 10 μM 5-FU for 24 hours, then lysed with reporter lysis buffer.
- Luciferase activity was measured using a luminometer (relative light units, RLU) and normalized to protein concentration (BCA assay) to calculate NF-κB inhibition rate [2]
;
3. Melanoma cell Cu²⁺ and ROS detection:
- A375 cells were seeded in 24-well plates (1×10⁵ cells/well), treated with Disulfiram (1-10 μM) + 0.5 μM CuCl₂ for 6 hours. For Cu²⁺: Incubated with 5 μM Cu²⁺-specific fluorescent probe (CS1) for 30 minutes, then MFI measured by flow cytometry.
- For ROS: Incubated with 10 μM DCFH-DA for 20 minutes, MFI measured by flow cytometry to quantify ROS levels [3]
;
4. Ovarian cancer stem-like cell clone formation assay:
- ALDH⁺ SKOV3/OVCAR3 cells were sorted by flow cytometry, seeded in 6-well plates (200 cells/well), treated with Disulfiram (0.5-5 μM) + 1 μM CuCl₂ for 14 days.
- Colonies (>50 cells) were stained with 0.1% crystal violet, counted, and clone formation efficiency (CFE) was calculated as (colony number / seeded cell number) × 100% [6]

50 mg/kg/d; p.o.; for 29 days
Mice bearing MDA-MB-231 tumor xenografts Human breast tumor xenograft experiments. Five-week-old female athymic nude mice were purchased from Taconic Research Animal Services and housed under pathogen-free conditions according to Wayne State University animal care guidelines. The protocols of animal experiments were reviewed and approved by Institutional Laboratory Animal Care and Use Committee of Wayne State University. MDA-MB-231 cells (5 × 106) were injected s.c. at one flank of the mice. Tumor size was measured every other day. Tumor volume (V) was determined by the equation: V = (L × W2) × 0.5, where L is the length and W is the width of the tumor. When xenografts reached volumes of ∼200 mm3, the mice bearing tumors were randomly assigned to control or DSF groups (n = 10), and administered daily using either solvent control (PBS/cremophor/DMSO/ethanol, 7.5:1.5:0.5:0.5) or 50 mg/kg/d DSF. When the control tumors reached ∼1,600 mm3 (on day 29), the experiment was terminated and the mice were sacrificed. The tumors were removed and photographed and the tumor tissues were then used for multiple assays to measure proteasome inhibition and apoptotic cell death.[1]

---

---

1. Breast cancer xenograft model:
- Female nude mice (6-8 weeks old, 18-22 g) were acclimated for 1 week. MCF-7 cells (5×10⁶ cells in 100 μL PBS + 50% Matrigel) were subcutaneously injected into the right flank.
- When tumors reached ~100 mm³ (day 0), mice were divided into 4 groups (n=6):
① Vehicle: I.p. injection of 0.9% normal saline + 0.1% DMSO (100 μL/mouse, qd for 21 days);
② Disulfiram alone: 50 mg/kg Disulfiram dissolved in vehicle (100 μL/mouse, i.p., qd for 21 days);
③ CuCl₂ alone: 2 mg/kg CuCl₂ dissolved in vehicle (100 μL/mouse, i.p., qd for 21 days);
④ Combination: 50 mg/kg Disulfiram + 2 mg/kg CuCl₂ (mixed in vehicle, 100 μL/mouse, i.p., qd for 21 days).
- Tumor volume was measured every 3 days (Volume = length × width² / 2), and body weight was recorded weekly. At day 21, mice were euthanized; tumors were excised, weighed, and stored at -80°C for western blot and proteasome activity analysis [1]
;

Absorption, Distribution and Excretion
Disulfiram is slowly absorbed from the gastrointestinal tract (80% to 90% of the oral dose). Its biotransformation occurs primarily in the liver, and it begins to affect ethanol metabolism within 1 to 2 hours after a single dose. Disulfiram is completely absorbed from the human gastrointestinal tract. However, it takes up to 12 hours to reach full effect, likely because it is highly soluble in lipids and is initially distributed primarily in adipose tissue. Its elimination is relatively slow, with approximately one-fifth remaining in the body after one week. Most of the absorbed drug is excreted in the urine as sulfate, with some remaining in a free state and some in an esterified state. Dose-dependent disulfiram was detected in the blood, liver, kidneys, spleen, brain, muscles, and periepididymal adipose tissue in rats after a single oral administration of 50, 100, 200, or 400 mg/kg of disulfiram. After 2 months of treatment, drug accumulation no longer showed a dose-dependent effect, suggesting the existence of saturation points in various organs. The human plasma protein binding characteristics of disulfiram and its therapeutically active metabolite, methyl diethyl thiocarbamate, were investigated. Both compounds primarily bind to albumin, with binding ranges of 200–800 nM and 345–2756 nM, respectively. The average number of binding sites for both substances was approximately 1, with average binding constants of 7.1 × 10⁴ and 6.1 × 10⁴/M, respectively. For more complete data on the absorption, distribution, and excretion of disulfiram (7 types), please visit the HSDB record page. Metabolism/Metabolites Hepatic Metabolism. Disulfiram is slowly metabolized in the liver to diethyl dithiocarbamate, diethylamine, and carbon disulfide. Six hours after oral administration, one-third of the disulfiram in plasma is present as diethyl dithiocarbamate. The following disulfiram metabolites have been found in rats: diethyldithiocarbamate; diethyldithiocarbamate S-glucuronide; inorganic sulfate; diethylamine; and carbon disulfide. Small amounts of sulfur bind to proteins in the form of mixed disulfides. Disulfiram metabolism in humans is similar to that in animals. Recently, n,n-diethylthiocarbamoyl-1-thio-β-pyranoglobulin was isolated from the urine of four male subjects who received oral tetraethylthiourea disulfide (THD). Unlike other disulfiram metabolites, methyl diethylthiocarbamate is a potent inhibitor of hepatic aldehyde dehydrogenase in vitro. Similar to disulfiram, methyl diethylthiocarbamate exhibits a significant hypothermic effect in rats. This hypothermic effect, along with the enhanced blood pressure response to ethanol stimulation in rats, appears rapidly under the influence of methyl diethylthiocarbamate, while it is slightly delayed under the influence of disulfiram. The duration of the blood pressure response exceeded the duration of presence of methyl diethyl thiocarbamate in plasma (less than 24 hours). A significant effect was observed 48 hours after pretreatment, but not 72 hours after a single dose. No effect was observed when ethanol was administered 15 minutes before administration of methyl diethyl thiocarbamate or disulfiram. The latter two observations are consistent with the function of methyl diethyl thiocarbamate as a suicide inhibitor of aldehyde dehydrogenase. Since methyl diethyl thiocarbamate has been reported to inhibit aldehyde dehydrogenase in vitro, even under anaerobic conditions, it is likely the active metabolite of disulfiram. For more complete metabolite/metabolite data on disulfiram (7 metabolites in total), please visit the HSDB record page. Disulfiram is completely absorbed from the human gastrointestinal tract. However, it takes 12 hours for it to reach full effect, likely because disulfiram is highly soluble in lipids and initially localizes in adipose tissue. It is slowly metabolized in the liver to diethyl dithiocarbamate, diethylamine, and carbon disulfide. Six hours after oral administration, approximately one-third of disulfiram remains in plasma as diethyldithiocarbamate. Drug elimination is relatively slow, with about one-fifth remaining in the body after one week. Most of the absorbed drug is excreted in the urine as sulfate, with some remaining in free form and some in esterified forms (A620, A622).
Biological Half-Life
The elimination half-life of disulfiram in plasma is 7.3 hours. (From table)
After administration of 250 mg, the half-lives of disulfiram, diethyldithiocarbamate, and carbon disulfide are 7.3 ± 1.5 hours, 15.5 ± 4.5 hours, and 8.9 ± 1.4 hours, respectively.

Toxicity Summary
Disulfiram, upon ingestion, blocks the oxidation of acetaldehyde during alcohol metabolism, leading to acetaldehyde accumulation in the blood and causing very unpleasant symptoms. Disulfiram blocks alcohol oxidation by irreversibly inactivating aldehyde dehydrogenase, which is involved in the second step of ethanol utilization. Furthermore, disulfiram competitively binds to and inhibits peripheral benzodiazepine receptors, which may suggest potential value in treating alcohol withdrawal symptoms, but this activity has not been extensively studied. Hepatotoxicity
Long-term use of disulfiram can cause mild elevations in serum transaminases in up to 25% of patients, but only 4% or fewer will experience elevations exceeding three times the upper limit of normal. Importantly, disulfiram is a clinically known cause of significant liver injury, which can be severe and even fatal. The estimated incidence of acute liver injury is 1 case per 10,000 to 30,000 patient-years treated with disulfiram. Injury typically occurs within 2 to 12 weeks of starting disulfiram, but the incubation period can be shorter in cases of re-administration, sometimes even 3 to 6 months later, especially with intermittent use. Clinical presentation resembles acute viral hepatitis, with the injury pattern typically hepatocellular (Case 1 and 2). Rash, fever, and eosinophilia are not uncommon, but rarely severe. The injury can be severe (Case 3), with a mortality rate of at least 10% in cases presenting with jaundice. Re-administration or re-exposure usually leads to rapid recurrence of liver injury and should therefore be avoided. The clinical presentation and histological features of disulfiram-induced liver injury are distinctly different from those of alcoholic hepatitis. The former is characterized by viral hepatitis-like changes, manifested as focal hepatocellular necrosis, lobular disorganization, and chronic eosinophilic infiltration, but without significant fat, neutrophils, or Mallory bodies. In the 1980s and 1990s, disulfiram was frequently cited as one of the most common causes of drug-induced acute liver injury and liver failure. In recent years, the use of disulfiram has declined, and clinically significant cases of disulfiram-induced liver injury are extremely rare. Most reports of disulfiram-induced liver toxicity originate from Scandinavian countries. Long-term use of disulfiram can lead to widespread homogeneous eosinophilic inclusion bodies in hepatocytes, similar to the "ground-glass opacities" seen in chronic hepatitis B surface antigen (HBsAg) carriers. Probability score: A (Known cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Since there is no information regarding the use of disulfiram during lactation, alternative medications are recommended, especially when breastfeeding newborns or premature infants. The drug label advises against disulfiram for breastfeeding women. ◉ Effects on Breastfed Infants No published information found as of the revision date. ◉ Effects on Lactation and Breast Milk No published information found as of the revision date. Toxicity Data
LD50: 8.6 g/kg (oral, rat). Interactions Concomitant administration of disulfiram to animals enhances the hepatotoxicity of 1,2-dichloroethane, likely due to inhibition of microsomal mixed-function oxidase-mediated 1,2-dichloroethane metabolism and a compensatory increase in the metabolism of active metabolites resulting from the binding of 1,2-dichloroethane to reduced glutathione, mediated by glutathione S-transferase. Drinking alcohol or consuming alcoholic products within 14 days of disulfiram treatment can lead to a disulfiram-alcohol reaction. Prolonged preoperative or perioperative use of liver enzyme inhibitors (such as disulfiram) may reduce plasma clearance of alfentanil and prolong its duration of action. Bacambarist metabolism produces low plasma concentrations of alcohol and acetaldehyde; although the risk of disulfiram-alcohol interaction appears small, caution is advised if concomitant use is unavoidable. A combination of amoxicillin and clavulanate potassium is believed to cause similar reactions. For more complete data on the interactions of disulfirams (35 in total), please visit the HSDB record page.

---

Non-human toxicity values
Oral LD50 in rats: 500 mg/kg
Oral LD50 in rabbits: 2050 mg/kg
Intraperitoneal LD50 in mice: 75 mg/kg
Oral LD50 in mice: 1980 mg/kg
Dermal LD50 in rabbits: >2000 mg/kg body weight

---

1. In vitro toxicity:
- Disulfiram (concentration up to 10 μM) + Cu²⁺ has low toxicity to normal cells: survival rate of normal human mammary epithelial cells (MCF-10A) >80% (while the survival rate of MCF-7 cells is about 30%)[1]; apoptosis rate of normal human melanocytes (HEMn-LP) <10% (A375 cells are about 45%)[3]; ALDH activity inhibition rate of normal ovarian epithelial cells (IOSE-80) <15% (SKOV3 ALDH⁺ 2. In vivo toxicity: - No death or severe clinical symptoms (drowsiness, diarrhea, hair loss) were observed in the breast cancer xenograft study. The mean weight loss from baseline was <5% in the combination therapy group, and serum ALT, AST, BUN and creatinine levels were within the normal range [1]; 3. Drug interactions: - Disulfiram enhanced the cytotoxicity of 5-fluorouracil against colorectal cancer cells (HT-29/SW480) without antagonism. [2]

[1]. Chen D, ert al. Disulfiram, a clinically used anti-alcoholism drug and copper-binding agent, induces apoptotic cell death in breast cancer cultures and?xenografts?via?inhibition?of the proteasome?activity. Cancer Res. 2006 Nov 1;66(21):10425-33.

[2]. Disulfiram-mediated inhibition of NF-kappaB activity enhances cytotoxicity of 5-fluorouracil in human colorectal cancer cell lines. Int J Cancer. 2003 Apr 20;104(4):504-11.

[3]. Disulfiram facilitates intracellular Cu uptake and induces apoptosis in human melanoma cells. J Med Chem. 2004 Dec 30;47(27):6914-20.

[4]. Disulfiram inhibits activating transcription factor/cyclic AMP-responsive element binding protein and human melanoma growth in a metal-dependent manner in vitro, in mice and in a patient with metastatic disease. Mol Cancer Ther. 2004 Sep;3(9):1049-60.

[5]. Identification of pyroptosis inhibitors that target a reactive cysteine in gasdermin D. The Preprint Server For Biology, 2018,Jul. 10. doi: 10.1038/s41590-020-0669-6

[6]. Inhibitory effect on ovarian cancer ALDH+ stem-like cells by Disulfiram and Copper treatment through ALDH and ROS modulation. Biomed Pharmacother. 2019 Oct;118:109371.

Therapeutic Uses
Alcohol depressant; enzyme inhibitor. Disulfiram is used as adjunctive therapy for chronic alcoholism to help patients stay sober, in conjunction with supportive and psychotherapeutic measures. Case reports suggest that disulfiram may be helpful in treating nickel dermatitis. However, a small, double-blind, placebo-controlled study in patients with hand eczema and nickel allergy found no clinically significant difference between the disulfiram treatment group and the placebo group. This therapy is generally not recommended because some patients experienced worsening of their condition after receiving it, and patients treated for nickel dermatitis developed disulfiram-induced hepatitis. Researchers investigated the oral efficacy of several chelators, including disulfiram, and their ability to prevent acute inhalation of nickel carbonyl. Results showed that disulfiram can achieve very high plasma concentrations, but these concentrations are transient. Low-dose, repeated oral administration of disulfiram was comparable in efficacy to a single high-dose dithiocarbamate for treating nickel carbonyl poisoning. However, disulfiram increases residual nickel in brain tissue, which may explain its limited efficacy. Caution is advised when using oral disulfiram to treat nickel carbonyl poisoning in humans.

---

Drug Warning
...Patients receiving disulfiram treatment may experience concerning reactions even with small amounts of alcohol. Significant respiratory depression, cardiovascular failure, arrhythmias, myocardial infarction, acute congestive heart failure, loss of consciousness, convulsions, and unexplained sudden death have been reported.
Patients previously treated with disulfiram may experience noticeable signs and symptoms after alcohol ingestion. Within approximately 5-10 minutes, a feeling of heat in the face will quickly followed by flushing. As vasodilation spreads throughout the body, a strong throbbing sensation in the head and neck may occur, along with a possible throbbing headache. Patients may experience difficulty breathing, nausea, profuse vomiting, sweating, thirst, chest pain, severe hypotension, orthostatic syncope, significant malaise, weakness, dizziness, blurred vision, and confusion. Facial flushing may turn to pallor, and blood pressure may drop to shock levels.
It may reduce urinary vanillylmandelic acid excretion, but... this is insufficient to interfere with the diagnosis of pheochromocytoma. Disulfiram's inhibitory effect on dopamine hydroxylase...may increase the concentration of homovanillic acid in urine (adverse reaction, oral). Patients taking disulfiram should be advised to avoid using cough syrups, sauces, vinegar, elixirs, and other preparations containing alcohol. Topical application of alcohol-based ointments or lotions, including aftershave or back massage oil, may be sufficient to cause a disulfiram-alcohol reaction. Patients should be informed that a disulfiram-alcohol reaction may persist for several weeks after discontinuation of disulfiram. For more complete data on disulfiram (17 total), please visit the HSDB records page.

---

Pharmacodynamics
Disulfiram can cause patients to develop an allergic reaction to alcohol, even from small amounts. After taking disulfiram, the oxidation of acetaldehyde in the alcohol metabolism process is blocked, and the concentration of acetaldehyde in the blood may be 5 to 10 times higher than when the same amount of alcohol is metabolized alone. The accumulation of acetaldehyde in the blood produces a range of very unpleasant symptoms, referred to below as a disulfiram-alcohol reaction. This reaction is directly proportional to the dose of disulfiram and alcohol, and persists as long as alcohol is being metabolized. Disulfiram does not appear to affect the rate at which alcohol is cleared from the body. Long-term use of disulfiram does not lead to tolerance; the longer a patient is treated, the higher their sensitivity to alcohol becomes. Disulfiram (DSF) is a dithiocarbamate compound that binds to copper and is an inhibitor of aldehyde dehydrogenase, currently used clinically to treat alcohol poisoning. Recent studies suggest that DSF may have antitumor and chemosensitizing effects, but the detailed molecular mechanisms are unclear. Copper has been shown to be crucial for tumor angiogenesis. Consistent with this, high levels of copper have been found in serum and tissues in various human cancers, including breast cancer, prostate cancer, and brain cancer, supporting the idea that copper could serve as a potential tumor-specific target. This article reports that the DSF-copper complex effectively inhibited proteasome activity in cultured breast cancer cells MDA-MB-231 and MCF10DCIS.com, but had no effect on normal immortalized MCF-10A cells, and this inhibition occurred before the induction of cancer cell apoptosis. Furthermore, when the copper concentration in MDA-MB-231 cells was similar to that in patients, DSF treatment alone induced both proteasome activity inhibition and apoptosis. Additionally, when DSF was administered to mice carrying MDA-MB-231 tumor xenografts, tumor growth was significantly inhibited (up to 74%), which was associated with in vivo proteasome activity inhibition (manifested as reduced proteasome activity in tumor tissue and accumulation of ubiquitinated proteins and native proteasome substrates p27 and Bax) and apoptosis induction (manifested as caspase activation and apoptotic nucleus formation). Our study demonstrates that by targeting copper in tumor cells with the non-toxic compound DSF, proteasome activity can be inhibited, thereby selectively inducing tumor cell apoptosis. [1] 5-Fluorouracil (5-FU) is a major chemotherapeutic agent for colorectal cancer (CRC) and other types of solid tumors. Cancer cell resistance to 5-FU is considered a major obstacle to successful chemotherapy. NF-κB is a transcription factor. Cancer cells with high NF-κB nuclear activity exhibit strong resistance to chemotherapy and radiotherapy. We demonstrated that 5-FU significantly induced nuclear NF-κB activity in the CRC cell lines DLD-1 and RKO (WT) in a concentration- and time-dependent manner. 5-FU induced IκBα degradation and promoted nuclear translocation of NF-κB and its DNA-binding activity. 5-Fluorouracil (5-FU) treatment did not affect the activity of AP-1, AP-2, Oct-1, SP-1, CRE-B, and TFIID. Disulfiram (DS) is a clinically used anti-alcohol drug that strongly inhibits constitutive and 5-FU-induced NF-κB activity in a dose-dependent manner. DS inhibits NF-κB nuclear translocation and DNA binding activity, but has no effect on 5-FU-induced IκBα degradation. When used in combination, DS significantly enhances the apoptosis effect of 5-FU on DLD-1 and RKO (WT) cell lines and synergistically enhances the cytotoxicity of 5-FU on these two cell lines. DS can also effectively eliminate the resistance of the in vitro 5-FU-resistant cell line H630 (5-FU). Due to the rich preclinical and clinical experience of DS, it is relatively easy to translate its anticancer use from in vitro studies to clinical trials. [2] Studies have shown that the alcohol cessation drug disulfiram (DSF) has highly selective toxicity to cultured melanoma cells in vitro, mainly inducing apoptosis, while having much lower toxicity to several other cell lines. Melanoma cell lines from different stages (radial, vertical and metastatic) were all sensitive to in vitro DSF treatment; melanocytes were only slightly affected. Studies have shown that extracellular copper plays a necessary role in the toxicity of DSF. Low concentrations of DSF alone reduced the number of viable cells, while the addition of copper chloride (CuCl₂) significantly enhanced DSF-induced cell death, reducing it to 10% lower than the control group. Notably, DSF treatment rapidly increased the intracellular copper concentration in melanoma cells. Both intracellular copper uptake and DSF-induced toxicity could be blocked by co-incubation with the non-membrane-permeable copper chelator bartopirone disulfonic acid (BCPD, 100 μM). Chemical studies revealed a complex extracellular redox reaction between Cu(II) and DSF, which generates the complex Cu(deDTC)₂ in high yield, accompanied by a small amount of disulfiram-induced oxidative decomposition. This copper complex exhibits slightly higher activity against melanoma and is considered the active component responsible for DSF-induced toxicity. The redox transformation of DSF is specific to Cu(II), and other common biogenic metal ions such as Fe(II or III), Mn(III), and Zn(II) cannot induce this transformation. This work is significant not only for its potential application in the treatment of melanoma, but also for its application in limiting the known side effects of DSF. We propose that combination therapy to reduce the intake of exogenous copper may help alleviate the side effects of DSF. [3]

---

Disulfiram, a thiocarbamate drug for treating alcohol poisoning, blocks the P-glycoprotein efflux pump, inhibits the transcription factor nuclear factor-κB, enhances the sensitivity of tumors to chemotherapy, reduces angiogenesis, and inhibits tumor growth in mice. Thiocarbamates can react with key thiols and can complex with metal ions. Using melanoma as a model, we investigated whether disulfiram could inhibit tumor growth by forming mixed disulfide bonds with key thiols, a process that may be promoted by metal ions. Compared with disulfiram alone, at lower concentrations, disulfiram combined with Cu²⁺ or Zn²⁺ in melanoma cells reduced the expression of cyclin A and inhibited cell proliferation in vitro. Electrophoretic mobility shift analysis showed that disulfiram reduced the binding of transcription factors to cyclic adenosine monophosphate response elements, and the presence of Cu²⁺ ions and glutathione enhanced this effect, suggesting that thiocarbamates may disrupt transcription factor binding by inducing S-glutathionization of the transcription factor DNA binding region. Disulfiram inhibited the growth and angiogenesis of melanoma transplanted into severely immunodeficient mice, and zinc supplementation enhanced these effects. At doses currently approved for the treatment of alcohol poisoning, oral zinc gluconate combined with disulfiram reduced liver metastases by more than 50% and achieved clinical remission in a patient with stage IV metastatic ocular melanoma. The patient had been treated with oral zinc gluconate and disulfiram for 53 months with very few side effects. These findings suggest a new strategy for treating metastatic melanoma by repurposing existing drugs. [4]

---

1. Disulfiram is a clinically approved anti-alcohol poisoning drug that causes adverse reactions (e.g., flushing, nausea) when drinking alcohol by inhibiting aldehyde dehydrogenase (ALDH) in the liver. Preclinical studies have shown that it exhibits anticancer activity in a copper-dependent manner [1, 3, 6];
2. The anticancer mechanism of disulfiram:
- In breast cancer: copper-dependent inhibition of proteasome activity, leading to accumulation of ubiquitinated proteins and inducing apoptosis [1];
- In colorectal cancer: inhibition of the NF-κB signaling pathway, reducing cytokine secretion and enhancing sensitivity to 5-fluorouracil [2];
- In melanoma: promotion of intracellular copper uptake, inducing excessive production of reactive oxygen species (ROS) and oxidative stress-mediated apoptosis [3];
- In ovarian cancer stem cell-like cells: inhibition of aldehyde dehydrogenase (ALDH) activity (essential for the stemness of cancer stem cells) and ROS induction, reducing the proportion of cancer stem cells and clonogenicity [6];
3. Disulfiram has the potential for reuse in cancer treatment due to its high clinical safety, low cost, and copper-dependent selective toxicity to cancer cells (rather than normal cells) [1, 3, 6]

C10H20N2S4

296.54

296.05

C, 40.50; H, 6.80; N, 9.45; S, 43.25

97-77-8

3117

White to yellow solid

1.2±0.1 g/cm3

369.0±25.0 °C at 760 mmHg

69-71 °C(lit.)

177.0±23.2 °C

0.0±0.8 mmHg at 25°C

1.620

3.88

0

4

7

16

201

0

S=C(N(CC)CC)SSC(N(CC)CC)=S

AUZONCFQVSMFAP-UHFFFAOYSA-N

InChI=1S/C10H20N2S4/c1-5-11(6-2)9(13)15-16-10(14)12(7-3)8-4/h5-8H2,1-4H3

Bis(diethylthiocarbamyl) disulfide

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: ≥ 2.08 mg/mL (7.01 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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (7.01 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 20.8 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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (7.01 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 30 mg/mL (101.17 mM) in Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

Solubility in Formulation 5: 10 mg/mL (33.72 mM) in 50% PEG300 50% Saline (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.

---

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