Alcohol use disorders and current pharmacological therapies: the role of GABAA receptors

Alcohol use disorders (AUD) are defined as alcohol abuse and alcohol dependence, which create large problems both for society and for the drinkers themselves. To date, no therapeutic can effectively solve these problems. Understanding the underlying mechanisms leading to AUD is critically important for developing effective and safe pharmacological therapies. Benzodiazepines (BZs) are used to reduce the symptoms of alcohol withdrawal syndrome. However, frequent use of BZs causes cross-tolerance, dependence, and cross-addiction to alcohol. The FDA-approved naltrexone and acamprosate have shown mixed results in clinical trials. Naltrexone is effective to treat alcohol dependence (decreased length and frequency of drinking bouts), but its severe side effects, including withdrawal symptoms, are difficult to overcome. Acamprosate showed efficacy for treating alcohol dependence in European trials, but two large US trials have failed to confirm the efficacy. Another FDA-approved medication, disulfiram, does not diminish craving, and it causes a peripheral neuropathy. Kudzu is the only natural medication mentioned by the National Institute on Alcohol Abuse and Alcoholism, but its mechanisms of action are not yet established. It has been recently shown that dihydromyricetin, a flavonoid purified from Hovenia, has unique effects on GABA_A receptors and blocks ethanol intoxication and withdrawal in alcoholic animal models. In this article, we review the role of GABA_A receptors in the treatment of AUD and currently available and potentially novel pharmacological agents.     

EFFECTS OF DISULFIRAM AND THREE RELATED COMPOUNDS ON THE ACTIVITY OF ALCOHOL DEHYDROGENASE AND ALDEHYDE DEHYDROGENASE IN RAT LIVER AND GASTRIC MUCOSA

SUMMARY 1. Disulfiram or related compounds were administered to rats by stomach tube and the activities of alcohol dehydrogenase and aldehyde dehydrogenase in the liver and the gastric mucosa were determined 43 h later.
2. In liver, disulfiram did not affect alcohol dehydrogenase activity but reduced aldehyde dehydrogenase activity. The disulfiram metabolite diethyldithiocarbamate was without action.
3. In gastric mucosa, disulfiram had no effect on aldehyde dehydrogenase activity but enhanced alcohol dehydrogenase activity. Diethyldithiocarbamate had a similar effect.
4. The increase in alcohol dehydrogenase activity in the gastric mucosa might explain the hyperacetaldehydaemia produced by ethanol in disulfiram-treated patients.     

双硫仑联合铜离子对人骨肉瘤细胞增殖与凋亡的影响

文题释义：双硫仑：是一种广泛用于治疗慢性酒精上瘾以及古柯碱的戒断药物,其为二硫代氨基甲酸盐,水溶性极差。早在20世纪中期,Jacobsen等发现双硫仑可有效抑制乙醛脱氢酶的活性,造成乙醛的饱和氧化反应,其本身和代谢产物可引起体内相关蛋白失活,使人体产生不适反应,从而达到抗酗酒目的。二乙基二硫代氨基甲酸钠(diethyldithiocarbamate,DDTC)：白色至无色片状结晶,有吸湿性,易溶于水,溶于乙醇、甲醇、丙酮,不溶于乙醚和苯。水溶液呈碱性并逐渐分解,遇酸能分离出二硫化碳而使溶液混浊,熔点 94-102 ℃。最大吸收波长(乙醇中)257、290 nm(摩尔吸光系数1 200、13000)。低毒,半数致死量(大鼠,经口)2 830 mg/kg。研究显示,双硫仑在进入人体后会迅速转化为体内代谢物DDTC,Cu可与DDTC结合形成较稳定的DDTC-Cu复合物。背景：研究表明双硫仑本身具有抗肿瘤活性,可联合铜(Cu)离子在体内外水平对多种肿瘤发挥抑癌作用,但关于双硫仑对骨肉瘤细胞增殖和凋亡作用的影响尚未阐明。目的：探讨双硫仑-Cu在体内外水平对骨肉瘤增殖与凋亡能力的影响以及可能的作用机制。方法：实验方案经山西医科大学动物实验伦理委员会批准(批准号为2017LL077)。①体外实验：配置双硫仑在进入人体后的转换物二乙基二硫代氨基甲酸钠(diethyldithiocarbamate,DDTC)和Cu离子的复合物DDTC-Cu(0.5,1,2,3和5 μmol/L),设置DDTC单药(5 μmol/L)、Cu单药(5 μmol/L)和空白对照组。药物处理人骨肉瘤细胞Saos-2细胞和MG-63细胞,CCK8法检测不同浓度DDTC-Cu对Saos-2细胞和MG-63细胞的增殖抑制作用;采用AnnexinV-FITC/PI双染法检测DDTC-Cu对Saos-2细胞凋亡水平变化;②体内实验：取4周龄BALB/c-nu/nu雌性裸鼠共10只,随机分为DDTC-Cu组和对照组。采用异位移植方法,在裸鼠右侧背部皮下注射Saos-2细胞和Matrigel混悬液(1∶1混合),注射量400 μL/只;接种2周后,对照组裸鼠腹腔注射地塞米松(0.5 mg/kg,隔日1次),DDTC-Cu组裸鼠腹腔注射地塞米松(0.5 mg/kg,隔日1次)和DDTC-Cu复合物(10 nmol/g,隔日1次);观察两组荷瘤小鼠移植瘤生长情况,绘制瘤体生长曲线。接种5周后麻醉下处死动物,完整取出瘤体,免疫组织化学检测瘤体石蜡切片组织中ki67蛋白的表达水平,Western blot检测瘤体组织中细胞增殖和凋亡蛋白的表达及JNK通路蛋白表达的变化。结果与结论：①体外实验结果：DDTC-Cu组对骨肉瘤细胞的增殖抑制作用明显高于其他3组;CCK-8实验结果显示DDTC-Cu对骨肉瘤细胞增殖抑制呈剂量依赖性,两株细胞的24 h药物半抑制浓度分别为0.337 μmol/L和0.487μmol/L;流式细胞学检测结果显示DDTC-Cu可呈剂量依赖性促进Saos-2细胞的凋亡;②体内实验结果：DDTC-Cu组裸鼠移植瘤的体积和质量均小于对照组;免疫组织化学结果显示,DDTC-Cu组的瘤体中ki-67蛋白表达水平较对照组降低;Westernblot检测结果显示,DDTC-Cu组瘤体蛋白中p-JNK和c-jun的表达水平均上调。结果提示,双硫仑联合Cu离子在体内外水平抑制人骨肉瘤细胞增殖并促进骨肉瘤细胞凋亡,其作用机制可能与JNK通路活化有关。ORCID: 0000-0003-4818-901X(徐朝健)中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

High-Throughput Chemical Screens Identify Disulfiram as an Inhibitor of Human Glioblastoma Stem Cells

Glioblastoma Multiforme (GBM) continues to have a poor patient prognosis despite optimal standard of care. Glioma stem cells (GSCs) have been implicated as the presumed cause of tumor recurrence and resistance to therapy. With this in mind, we screened a diverse chemical library of 2,000 compounds to identify therapeutic agents that inhibit GSC proliferation and therefore have the potential to extend patient survival. High-throughput screens (HTS) identified 78 compounds that repeatedly inhibited cellular proliferation, of which 47 are clinically approved for other indications and 31 are experimental drugs. Several compounds (such as digitoxin, deguelin, patulin and phenethyl caffeate) exhibited high cytotoxicity, with half maximal inhibitory concentrations (IC₅₀) in the low nanomolar range. In particular, the FDA approved drug for the treatment of alcoholism, disulfiram (DSF), was significantly potent across multiple patient samples (IC₅₀ of 31.1 nM). The activity of DSF was potentiated by copper (Cu), which markedly increased GSC death. DSF–Cu inhibited the chymotrypsin-like proteasomal activity in cultured GSCs, consistent with inactivation of the ubiquitin-proteasome pathway and the subsequent induction of tumor cell death. Given that DSF is a relatively non-toxic drug that can penetrate the blood-brain barrier, we suggest that DSF should be tested (as either a monotherapy or as an adjuvant) in pre-clinical models of human GBM. Data also support targeting of the ubiquitin-proteasome pathway as a therapeutic approach in the treatment of GBM.     

Cytotoxicity of diethyldithiocarbamate in human versus rodent cell lines

Summary Diethyldithiocarbamate (DDTC) and other dithiocarbamates are currently receiving attention as potential adjuncts to traditional chemotherapy. In vitro studies with rodent cancer cell lines have consistently shown that DDTC concentrations of 0.1–1.0 g/ml are highly cytotoxic. Paradoxically, however, concentrations of 10–100 g/ml have been significantly less toxic.In the present study, such a biphasic pattern was reproduced when 3 rodent cell lines were exposed for 1 hour to 0.001 to 1000 g DDTC/ml. In contrast, in 7 human cell lines survival decreased steadily with increasing DDTC concentration (in the same dose range) without evidence of a biphasic pattern. These data might have implications for studies in which rodent cell lines are used to model the effects of dithiocarbamates in human tissues.     

Effects of disulfiram on mixed function oxidase system and trace element concentration in the liver of rats

Disulfiram (DSF), an inhibitor of chemically induced carcinogenesis, and its metabolite diethyldithiocarbamate (DDTC) have been investigated for their influence on trace element distribution and on certain enzymes of the drug metabolizing system in the livers of phenobarbital (PB) treated rats. Both substances diminished the PB induced enzyme response in liver microsomes, DDTC being more effective (?85%) than DSF (?60%). The copper, cobalt and zinc content of the livers of DSF treated animals were increased by factors of 6, 3 and 1.5 respectively as compared to controls, while DDTC treatment had no influence on liver trace element content. A correlation between enzyme inhibition and enhanced trace element uptake of the liver after DSF administration could not be observed. The change of trace element transport into the liver during DSF treatment is discussed.     

Effects of the Dopamine-β-hydroxylase Inhibitors FLA-57 and FLA-63 on Ethanol Metabolism and Aldehyde Dehydrogenase Activity in Rats

Abstract: In rats pretreated with the dopamine-β-hydroxylase (DBH)-inhibitors FLA-57 and FLA-63 (60 mg/kg, intraperitoneally, for 4 and 18 hrs), no effects on the blood acetaldehyde level after ethanol administration or on the activity of the low-K_m aldehyde dehydrogenase (ALDH) in the liver were found. FLA-63 but not FLA-57 decreased the rate of ethanol elimination. FLA-63 inhibited the low-K_m enzyme in vitro, but much less than the ALDH-inhibitors disulfiram and cyanamide. FLA-57 caused no inhibition in vitro. The results show that the previously observed suppression of ethanol intake in rats by FLA-57 and FLA-63 was not caused by an acetaldehyde-mediated aversion such as during the disulfiram-ethanol reaction.     

Pharmacological treatment of alcoholism

This paper presents an overview on pharmacologic treatment of alcoholism. A wide range of drugs have been tested over the years, some showing a reduction of alcohol drinking in alcoholics. However, at present, there is little evidence that phnrmacotherapy is effective in the rehabilitation of alcoholics. Although research in this area still has several methodological problems and much needs to be done, a greater understanding of the neuropharmacoiogy and neurobiology of alcohol use and abuse will certainly open new avenues for investigation and drug development. Patient-treatment matching and the combination of two or more modalities of intervention may also help increase overall effectiveness of alcohol treatment. (Aust NZ J Med 1992; 22: 220–223.)