Disulfiram   中文名称: 双硫仑

Aldehyde-dehydrogenase (ALDH1); gasdermin D (GSDMD)

双硫仑-铜复合物在导致癌细胞凋亡之前，可有效降低培养的乳腺癌 MDA-MB-231 和 MCF10DCIS.com 细胞中的蛋白酶体活性，但不会降低正常永生化 MCF-10A 细胞中的蛋白酶体活性 [1]。商业上使用的抗酒精药物双硫仑 (DS) 可显着且剂量依赖性地抑制结构性和 5-FU 诱导的 NF 激活。 DisuLfiram 对 5-FU 诱导的 IkappaBalpha 降解几乎没有影响，尽管它确实降低了 NF-kappaB 核易位和 DNA 结合活性。双硫仑协同增加 5-FU 对 DLD-1 和 RKO (WT) 细胞系的细胞毒性，同时还显着增强 5-FU 对这些细胞系的凋亡作用。此外，DisuLfiram 在体外成功消除了 5-FU 耐药细胞系 H630 (5-FU) 中的 5-FU 化疗耐药性 [2]。 CuCl2 极大地增加了 DSF 诱导的细胞死亡，使其低于对照的 10%，而奥司他韦则减少了活细胞的数量 [3]。在比单独使用双硫仑更低的浓度下，双硫仑与黑色素瘤细胞中的 Cu2+ 或 Zn2+ 组合可降低细胞周期蛋白 A 的表达并抑制体外增殖 [4]。 DisuLfiram（0.1 nM-10 μM；72 小时）和 Cu2+ 的组合可增加其对卵巢癌细胞系的细胞毒性 [1]。

双硫仑显着减少含有 MDA-MB-231 肿瘤异种移植物的小鼠的肿瘤发展 (74%)；这与蛋白酶体抑制和细胞凋亡诱导有关[1]。在肿瘤组织中，泛素化蛋白和天然蛋白酶体底物p27和Bax积累，这些蛋白酶体抑制指标用于衡量蛋白酶体抑制的程度。 Caspases 被激活和凋亡细胞核形成是细胞凋亡的标志[1]。双硫仑抑制核因子-kappaB 转录因子，限制 P-糖蛋白挤出泵，减少血管生成，使肿瘤对化疗更敏感，并阻止小鼠肿瘤生长 [4]。当黑色素瘤移植到严重联合免疫缺陷小鼠体内时，双硫仑会减少其发育和血管生成；补充 Zn2+ 会放大这些效果 [4]。

ALDH活性在卵巢癌症干细胞中受到抑制（细胞系IGROV1、SKOV3和SKOV3IP1中ALDH+细胞的比例分别从21.7%降低到0.391%、8.4%降低到0%、6.88%降低到0.05%）。添加或不添加辅因子铜（Cu2+）的DSF表现出剂量依赖性和时间依赖性的细胞毒性，并增强顺铂诱导的细胞凋亡。DSF+Cu2+增加细胞内ROS水平，触发卵巢癌症干细胞（CSC）凋亡。与ALDH-细胞相比，ALDH+细胞中观察到明显更多的集落和球体形成（P<0.01）。此外，与ALDH细胞相比，ALDH+细胞对顺铂治疗更有抵抗力（P<0.05），并且还表现出较低的ROS基础水平。然而，在用DSF（0.08μM）预处理后，与ALDH-细胞相比，在ALDH+细胞中没有观察到ROS积累和细胞活力的显著差异[6]。

在10%FBS刺激的培养物中研究了二硫仑（0.15-5.0μM）或二乙基二硫代氨基甲酸钠（1.0μM）对恶性细胞系增殖的影响。24至72小时后对细胞数量进行定量，如下所述。在一些实验中，在细胞接种后立即加入二硫仑。在其他实验中，在加入含有双硫仑的新鲜培养基之前，对细胞进行铺板并使其生长24-72小时，并在24-72小时后测定细胞数。研究了二硫仑与添加到培养基中的N，N′-双（2-氯乙基-N-亚硝基脲（卡莫司汀，1.0-1000μM）或顺铂（0.1-100μg/mL）之间的协同作用。将0.2至10μM的Cu2+（以CuSO4的形式提供）、Zn2+（以ZnCl2的形式）、Ag+（以乳酸银的形式）或Au3+（以HAuCl4·3H2O的形式）离子添加到生长培养基中，缓冲至生理pH，研究金属离子对双硫仑的影响。为了提供与生物学相关的铜来源，在培养基中补充人铜蓝蛋白，剂量复制正常成人血清的低浓度和高浓度（250和500 mg/mL）[4]。

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]

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50 mg/kg/d; p.o.; for 29 days

Mice bearing MDA-MB-231 tumor xenografts

Absorption, Distribution and Excretion
Disulfiram is absorbed slowly from the gastrointestinal tract (80 to 90% of oral dose).
Absorption /of disulfiram is/ slow. Eighty to ninety percent of an oral dose is absorbed. /Its/ biotransformation /is predominately/ hepatic /and/ a single dose will begin to affect ethanol metabolism within 1 to 2 hours.
Disulfiram is ... completely absorbed from the human GI tract. However, a period of 12 hr is required for its full action, perhaps because, being highly sol in lipid, it is initially localized in fat. Elimination is relatively slow, and about 1/5 still remains in body at end of a week. The greater part of the absorbed drug is ... excreted in the urine as the sulfate, partly free and partly esterified.
After a single oral dose of 50, 100, 200, or 400 mg/kg, disulfiram was found in dose-dependent quantities in blood, liver, kidney, spleen, brain, muscle, and peri-epididymal adipose tissue of rats. After a 2-mo treatment, accumulation was not dose-dependent, suggesting a saturation point for various organs.
The human plasma protein binding characteristics of disulfiram and its therapeutically active metabolite, diethylthiocarbamic acid methyl ester were investigated. Both compounds were bound principally to albumin over the ranges 200-800 and 345-2756 nM, respectively. The average number of binding sites was approximately one for both substances, whereas the average association constants were 7.1X10+4 and 6.1X10+3/M, respectively.
For more Absorption, Distribution and Excretion (Complete) data for DISULFIRAM (7 total), please visit the HSDB record page.

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Metabolism / Metabolites
Hepatic.
Disulfiram is slowly metabolized in the liver to diethyldithiocarbamate, diethylamine, and carbon disulfide. Six hr after oral administration of the drug, one third of plasma disulfiram is in the form of diethyldithiocarbamate.
In rats the following metabolites of disulfiram were found: diethyldithiocarbamate; diethyldithiocarbamate s-glucuronide; inorganic sulfate; diethylamine and carbon disulfide. A small amount of S was bound to proteins as mixed disulfides. ... Metabolism of disulfiram in man is similar to that in animals.
Recently, n,n-diethylthiocarbamoyl-1-thio-beta-glucopyranosiduronic acid was isolated from combined urine of 4 men given oral doses of tetraethylthiuram disulfide.
Diethylthiocarbamic acid methyl ester, in contrast to other disulfiram metabolites, is a potent inhibitor of liver aldehyde dehydrogenase in vitro. Like disulfiram, diethylthiocarbamic acid methyl ester had a pronounced hypothermic effect in rats. This hypothermic effect and the augmented blood pressure response to ethanol challenge in rats developed rapidly with diethylthiocarbamic acid methyl ester but were somewhat delayed with disulfiram. The blood pressure response outlasted the presence of diethylthiocarbamic acid methyl ester in plasma (less than 24 hr); a significant effect was found 48 hr after pretreatment but not 72 hr after a single dose. No effect was observed when ethanol was given 15 min before diethylthiocarbamic acid methyl ester or disulfiram. These latter two observations are consistent with a function of diethylthiocarbamic acid methyl ester as a suicide inhibitor of aldehyde dehydrogenase. Since diethylthiocarbamic acid methyl ester has been reported to inhibit aldehyde dehydrogenase in vitro, even under anaerobic conditions, diethylthiocarbamic acid methyl ester may be the active metabolite of disulfiram.
For more Metabolism/Metabolites (Complete) data for DISULFIRAM (7 total), please visit the HSDB record page.
Disulfiram is completely absorbed from the human GI tract. However, a period of 12 hr is required for its full action, perhaps because, being highly sol in lipid, it is initially localized in fat. It is slowly metabolized in the liver to diethyldithiocarbamate, diethylamine, and carbon disulfide. Six hr after oral administration of the drug, one third of plasma disulfiram is in the form of diethyldithiocarbamate. Elimination is relatively slow, and about 1/5 still remains in body at end of a week. The greater part of the absorbed drug is excreted in the urine as the sulfate, partly free and partly esterified (A620, A622).

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Biological Half-Life
The elimination half-life of disulfiram in plasma is 7.3 hr. /From table/
Following a 250 mg dose, 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 blocks the oxidation of alcohol at the acetaldehyde stage during alcohol metabolism following disulfiram intake causing an accumulation of acetaldehyde in the blood producing highly unpleasant symptoms. Disulfiram blocks the oxidation of alcohol through its irreversible inactivation of aldehyde dehydrogenase, which acts in the second step of ethanol utilization. In addition, disulfiram competitively binds and inhibits the peripheral benzodiazepine receptor, which may indicate some value in the treatment of the symptoms of alcohol withdrawal, however this activity has not been extensively studied.

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Hepatotoxicity
Chronic therapy with disulfiram is associated with mild serum aminotransferase elevations in up to 25% of patients, but elevations above 3 times the upper limit of normal (ULN) occur in 4% of patients or less. Importantly, disulfiram is a well established cause of clinically apparent liver injury, which can be severe and even fatal. The estimated incidence of acute liver injury is 1 per 10,000 to 30,000 patient-years of disulfiram treatment. The injury usually arises within 2 to 12 weeks of starting disulfiram, but the latency can be shorter in cases of reexposure and may arise only after 3 to 6 months, particularly with intermittent therapy. The clinical presentation resembles acute viral hepatitis and the pattern of injury is typically hepatocellular (Cases 1 and 2). Rash, fever and eosinophilia are not uncommon, but are rarely severe. The injury can be severe (Case 3) and the fatality rate is at least 10% in cases with jaundice. Rechallenge or reexposure is usually associated with rapid recurrence of liver injury and should be avoided. The clinical presentation and histology differ greatly from alcoholic hepatitis, in that disulfiram liver injury is marked by viral hepatitis-like changes of focal hepatocellular necrosis, lobular disarray and chronic inflammatory cell infiltrates with eosinophils, but without significant fat, neutrophils or Mallory bodies. In the 1980s and 1990s, disulfiram was often listed among the most common causes of acute liver injury and liver failure due to medications. More recently, disulfiram use has decreased and cases of clinically apparent liver injury from disulfiram are now rare. The majority of cases of disulfiram hepatotoxicity have been reported from Scandinavian countries.
Chronic therapy with disulfiram can cause widespread homogenous eosinophilic inclusions in hepatocytes, similar to the “ground glass” changes that can occur in chronic HBsAg carriers.
Likelihood score: A (well known cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Because no information is available on the use of disulfiram during breastfeeding, an alternate drug is preferred, especially while nursing a newborn or preterm infant. Drug labeling recommends that disulfiram not be given to nursing mothers.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Toxicity Data
LD50: 8.6g/kg (oral, rat).

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Interactions
Simultaneously feeding of the animals with disulfiram caused a potentiation of the hepatotoxicity of 1,2-dichloroethane, possibly due to an inhibition of microsomal mixed-function oxidase-mediated metabolism of 1,2-dichloroethane and to a compensatory increase in metabolism to reactive metabolites generated by glutathione-S-transferase-mediated conjugation of 1,2-dichloroethane with reduced glutathione.
Use of alcohol or alcohol containing products within 14 days of disulfiram therapy will result in disulfiram-alcohol reaction.
Chronic preoperative administration or perioperative use of hepatic enzyme inhibitors, such as disulfiram, may decrease the plasma clearance and prolong the duration of action of alfentanil.
Metabolism of bacampicillin produces low plasma concentrations of alcohol and acetaldehyde; although the risk of disulfiram-alcohol interaction appears minimal, caution is recommended if concurrent use is unavoidable. A similar reaction is thought to occur with amoxicillin and clavulanate combination.
For more Interactions (Complete) data for DISULFIRAM (35 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Rat oral 500 mg/kg
LD50 Rabbit oral 2050 mg/kg
LD50 Mouse intraperitoneal 75 mg/kg
LD50 Mouse oral 1980 mg/kg
LD50 Rabbit dermal >2000 mg/kg bw

[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.

[5]. Identification of pyroptosis inhibitors that target a reactive cysteine in gasdermin D. The Preprint Server For Biology, 2018,Jul. 10.

[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 Deterrents; Enzyme Inhibitors
Disulfiram is used to help maintain sobriety in the treatment of chronic alcoholism in conjunction with supportive and psychotherapeutic measures.
Case reports suggest that disulfiram may be useful for the treatment of nickel dermatitis. However, small double-blind placebo-controlled study of patients with hand eczema and nickel allergy did not find a clinically significant difference between those treated with disulfiram and those treated with placebo. Because some patients worsen with this therapy and because patients treated for nickel dermatitis have developed disulfiram induced hepatitis, this therapy is not generally indicated.
The oral efficacy of several chelating drugs, incl disulfiram, was studied in relation to their ability to prevent lethality due to acute inhalation exposure to nickel carbonyl. Disulfiram resulted in very high, but transient, plasma levels. Small, repeated oral doses of disulfiram would be just as effective in nickel carbonyl poisoning as a single large dithiocarb dose. However, disulfiram increase the nickel retained in brain tissue, possibly accounting for its limited efficacy. Caution in the use of oral disulfiram in human nickel carbonyl intoxication is recommended.

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Drug Warnings
... Alarming reactions may result from the ingestion of even small amt of alc in persons being treated with disulfiram. Marked resp depression, cardiovascular collapse, cardiac arrhythmias, myocardial infarction, acute congestive heart failure, unconsciousness, convulsions, and sudden and unexplained fatalities have occurred.
The ingestion of alcohol by individuals previously treated with disulfiram gives rise to marked signs and symptoms. Within about 5-10 min face feels hot, and soon afterwards it is flushed and scarlet in appearance. As vasodilatation spreads over whole body, intense throbbing is felt in head and neck, and a pulsating headache may develop. Respiratory difficulties, nausea, copious vomiting, sweating, thirst, chest pain, considerable hypotension, orthostatic syncope, marked uneasiness, weakness, vertigo, blurred vision, and confusion are observed. Facial flush is replaced by pallor, and blood pressure may fall to shock level.
May decrease urinary vanilmandelic acid excretion, although ... not sufficient to interfere with diagnosis of pheochromocytoma. /Disulfiram's/ inhibition of dopamine hydroxylase ... may increase urinary concn of homovanillic acid /adverse effect, oral/
Patients receiving disulfiram should be warned to avoid cough syrups, sauces, vinegars, elixirs, and other preparations that contain alcohol. External application of alcoholic liniments or lotions, including aftershave or back rub, may be sufficient to produce a disulfiram-alcohol reaction. Patients should be cautioned that disulfiram-alcohol reactions may occur for several weeks after discontinuance of disulfiram.
For more Drug Warnings (Complete) data for DISULFIRAM (17 total), please visit the HSDB record page.

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Pharmacodynamics
Disulfiram produces a sensitivity to alcohol which results in a highly unpleasant reaction when the patient under treatment ingests even small amounts of alcohol. Disulfiram blocks the oxidation of alcohol at the acetaldehyde stage during alcohol metabolism following disulfiram intake, the concentration of acetaldehyde occurring in the blood may be 5 to 10 times higher than that found during metabolism of the same amount of alcohol alone. Accumulation of acetaldehyde in the blood produces a complex of highly unpleasant symptoms referred to hereinafter as the disulfiram-alcohol reaction. This reaction, which is proportional to the dosage of both disulfiram and alcohol, will persist as long as alcohol is being metabolized. Disulfiram does not appear to influence the rate of alcohol elimination from the body. Prolonged administration of disulfiram does not produce tolerance; the longer a patient remains on therapy, the more exquisitely sensitive he becomes to alcohol.

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

121.26

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (7.01 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (7.01 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (7.01 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 30 mg/mL (101.17 mM) in Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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配方 5 中的溶解度: 10 mg/mL (33.72 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。