Effects and Mode of Action of Oleic Acid and Tween 80 on Skin Permeation of Disulfiram.

Oral disulfiram (DSF) has been used clinically for alcohol dependence and recently has been found to have antitumor activity. A transdermal delivery system would be useful for maintaining drug concentration and reducing the frequency of administration of DSF for cancer treatment. Penetrating the stratum corneum (SC) barrier is a challenge to the transdermal delivery of DSF. Therefore, we investigated the promoting effects and mechanism of action of the combination of oleic acid (OA) and Tween 80 on the skin permeation of DSF. Hairless mouse skin was exposed to OA and Tween 80, combined in various ratios (1 : 0, 2 : 1, 1 : 1, 1 : 2, and 0 : 1). A permeation experiment was performed, and total internal reflection IR spectroscopic measurements, differential scanning calorimetry, and synchrotron radiation X-ray diffraction measurements were taken of the SC with each applied formulation. The combination of OA and Tween 80 further enhanced the absorption-promoting effect of DSF, compared with individual application. The peak of the CH2 inverse symmetric stretching vibration near the skin surface temperature was shifted by a high frequency due to the application of OA, and DSF solubility increased in response to Tween 80. We believe that the increased fluidity of the intercellular lipids due to OA and the increased solubility of DSF due to Tween 80 promoted the absorption of DSF. Our study clarifies the detailed mechanism of action of the skin permeation and promoting effect of DSF through the combined use of OA and Tween 80, contributing to the development of a transdermal preparation of DSF.

[Liquid chromatography for determination of disulfiramin workplace atmosphere].

OBJECTIVE: To establish a method for determination of disulfiram in the workplace atmosphere by liquid chromatography. METHODS: Sampling with glass fiber filter, eluting with methanol, separating with C18 column, and determination with liquid chromatography. RESULTS: The bearing capacity of glass fiber filter exceeded 3.45 mg per piece. The elution efficiency was 97.8%∼101.0% The relative standard deviation varied from 1.09% to 1.44%. The limit of detection was 0.1 µg/ml. The minimum detectable concentration was 0.011 mg/m³ (with sampled air volume of 45 L). CONCLUSION: The method has high selectivity, accuracy, and precision and strong applicability.

Development of gold nanoparticles modified screen-printed carbon electrode for the analysis of thiram, disulfiram and their derivative in food using ultra-high performance liquid chromatography.

For the first time, gold nanoparticles (AuNPs) modified screen-printed carbon electrode (SPCE) was developed as working electrode in ultra-high performance liquid chromatography (UHPLC) coupled with electrochemical detection (UHPLC-ED) for simultaneous determination of thiram, disulfiram, and N,N-diethyl-N',N'-dimethylthiuram disulfide, their derivative compound. The separation was performed in reversed-phase mode using C18 column, mobile phase consisting of 55:45 (v/v) ratio of 0.05 M phosphate buffer solution (pH 5) and acetonitrile at a flow rate of 1.5 mL min(-1). For the detection part, the amperometric detection was chosen with a detection potential of 1.2 V vs. Ag/AgCl. Under the optimal conditions, the good linear relationship was obtained in the range of 0.07-15, 0.07-12, and 0.5-15 µg mL(-1) (correlation coefficient more than 0.9900) for thiram, N,N-diethyl-N',N'-dimethylthiuram disulfide, and disulfiram, respectively. The limits of detection (LODs) of thiram, N,N-diethyl-N',N'-dimethylthiuram disulfide, and disulfiram were 0.022, 0.023, and 0.165 µg mL(-1), respectively. Moreover, this method was successfully applied for the detection of these compounds in real samples (apple, grape and lettuce) with the recoveries ranging from 94.3% to 108.8%. To validate this developed method, a highly quantitative agreement was clearly observed compared to standard UHPLC-UV system. Therefore, the proposed electrode can be effectively used as an alternative electrode in UHPLC-ED for rapid, selective, highly sensitive, and simultaneous determination of thiram, disulfiram, and N,N-diethyl-N',N'-dimethylthiuram disulfide.

[Peculiarities of detection and evaluation of the persistence of tetraethyltiuram disulfide in the biological material].

Optimal conditions for the identification and quantitative determination of tetraethyltiuram disulfide in "fresh" and putrefactive tissues of cadaverous liver are described for the purpose of TLC, HPLC, and IK-spectrophotometry following extraction of the compound of interest with ethyl acetate and its purification on a silicagel L column, 40/100 mcm. The persistence of tetraethyltiuram disulfide in the cadaverous tissues was evaluated. It was shown that the period during which tetraethyltiuram disulfide can be detected in the autopsied tissues decreases from 203 to 28 days with a rise in temperature from -15 degrees C to +36 degrees C.

Determination of in vivo adducts of disulfiram with mitochondrial aldehyde dehydrogenase.

Extensive use for disulfiram (DSF) has been found in the aversion therapy treatment of recovering alcoholics. Although it is known to irreversibly inhibit hepatic aldehyde dehydrogenase (ALDH), the specific mechanism of in vivo inhibition of the enzyme by the drug has not been determined yet. We have demonstrated in this report a novel, but simple and rapid method for structurally characterizing in vivo derived protein-drug adducts by linking on-line sample processing to HPLC-electrospray ionization mass spectrometry (HPLC-MS) and HPLC-tandem mass spectrometry (HPLC-MS/MS). Employing this approach, rats were administered DSF, and their liver mitochondria were isolated and solubilized. Both native and in vivo DSF-treated mitochondrial ALDH (mALDH) were purified in one step with an affinity cartridge. The in vivo DSF-treated mALDH showed 77% inhibition in enzyme activity as compared with that of the control. Subsequently, the control and DSF-inhibited mALDH were both subjected to HPLC-MS analyses. We were able to detect two adducts on DSF-inhibited mALDH, as indicated by the mass increases of approximately 71 and approximately 100 Da. To unequivocally determine the site and structure of these adducts, on-line pepsin digestion-HPLC-MS and HPLC-MS/MS were performed. We observed two new peptides at MH(+) = 973.7 and MH(+) = 1001.8 in the pepsin digestion of DSF-inhibited enzyme. These two peptides were subsequently subjected to HPLC-MS/MS for sequence determination. Both peptides possessed the sequence FNQGQC(301)C(302)C(303), derived from the enzyme active site region, and were modified at Cys(302) by N-ethylcarbamoyl (+71 Da) and N-diethylcarbamoyl (+99 Da) adducts. These findings indicated that N-dealkylation may be an important step in DSF metabolism, and that the inhibition of ALDH occurred by carbamoylation caused by one of the DSF metabolites, most likely S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO). Finally, there was no evidence of the presence of an intramolecule disulfide bridge modification on the peptide FNQGQCCC.

Quantitative determination of isothiocyanates, dithiocarbamates, carbon disulfide, and related thiocarbonyl compounds by cyclocondensation with 1,2-benzenedithiol.

A recently developed UV spectroscopic method for quantitating isothiocyanates (R-N=C=S) at the nanomole level is based on the observation that the highly electrophilic central carbon atom of the -N=C=S group can undergo successive nucleophilic additions with reagents containing two sulfhydryl groups on adjacent carbon atoms to form a cyclic thiocarbonyl product and release the nitrogen atom as a primary amine (Y. Zhang, C.-G. Cho, G. H. Posner, and P. Talalay, Anal. Biochem. 205, 100-107, 1992). The assay utilizes 1, 2-benzenedithiol as the vicinal dithiol reagent and measures the reaction product, 1,3-benzodithiole-2-thione, spectroscopically (am of 23,000 M-1 cm-1 at lambdamax = 365 nm). This paper reports a dramatic improvement in the analytical sensitivity of this method. By separating the cyclocondensation product by a simple isocratic HPLC method and using an automatic integrator, the sensitivity of detection has been lowered to a few picomoles of isothiocyanate. Furthermore, we now find that the chemical specificity of the cyclocondensation reaction is not restricted to isothiocyanates, but includes dithiocarbamates, and related thiocarbonyl compounds such as carbon disulfide, certain substituted thiourea derivatives, and xanthates. The availability of such analytical methods is important not only because isothiocyanates (and their glucosinolate precursors) are present in edible plants and are consumed by humans in substantial quantities, but also because some dithiocarbamates are toxic, and are widely used in the rubber industry as vulcanization accelerators and in agriculture as fungicides, insecticides, and herbicides. The analysis of many isothiocyanates is complicated by their extreme volatility. This difficulty can be circumvented by converting isothiocyanates quantitatively into dithiocarbamates (by facile addition of a mercaptan such as N-acetylcysteine) and quantitating the nonvolatile dithiocarbamate by the cyclocondensation reaction.

Uso do monossulfeto de tetraetiltiuram no tratamento de ectoparasitas em camundongos / Use of tetraethylthiuram monosulfide in the treatment of ectoparasites in mice

Casais de camundongos isogênicos das raças CBA, BALB/c e machos e fêmeas de Swiss heterogenéticos criados no sistema convencional, que se encontravam infectados em diferentes graus por Myobia musculi e Myocoptes musculinus, foram submetidos a quatro esquemas de tratamento com Tetmosol. A incidência de ectoparasitas na raça C57BL/10 foi apenas 1,2%. Todos os animais da raça CBA e Swiss encontravam-se parasitados, enquanto que a infestaçäo do BALB/c foi de 50 a 90%. Os melhores resultados dos 4 esquemas de tratamento testados foram observados utilizando-se Tetmosol na concentraçäo 2,5% em banhos seriados com intervalos alternados de 3 e 4 dias, num período de 21 dias. A incidência de ectoparasitas adultos em CBA, BALB/c e Swiss após o tratamento foi de respectivamente 0%, 40% e 0%, enquanto que para ovos viáveis os repectivos resultados foram de 20%, 50% e 0%. Após a introduçäo de matrizes tratadas num sistema de criaçäo sob barreiras, a colônia tem sido examinada sistematicamente há 24 meses e encontra-se isenta de ectoparasitas

Metallic copper-containing post-column reactor for the detection of thiram and disulfiram in liquid chromatography.

A reaction detector has been developed for the selective detection of thiram and disulfiram. The detection is based on the post-column complexation of these analytes on a solid-state reactor packed with finely divided metallic copper to form a coloured copper complex, copper(II) N,N-dimethyldithiocarbamate, with an absorption maximum at 435 nm. The method is combined with a pre-concentration and clean-up step on a pre-column to permit the sub-ppb determination of, e.g., thiram in surface water samples or disulfiram in urine. Separation is achieved by reversed-phase liquid chromatography.

Rapid and sensitive on-line precolumn purification and high-performance liquid chromatographic assay for disulfiram and its metabolites.

The disulfiram (Antabus) metabolites diethyldithiocarbamate, diethyldithiocarbamate methyl ester, carbon disulphide and bis(diethyldithiocarbamato) copper complex were quantitatively analysed from directly injected heparin plasma by reversed-phase high-performance liquid chromatography. The highly volatile metabolite, carbon disulphide, was converted to the methyl ester of dimethyldithiocarbamate before chromatography. The analytical procedure is simple and does not require sample preparation or addition of an internal standard, and the compounds are eluted from the columns in 15 min. After automated on-line precolumn enrichment, the parent compound and biotransformation products could be back-flushed and chromatographed on an ordinary reversed-phase column. The influence of plasma protein binding on the constituents in the precolumn enrichment step was also investigated. Dissociation and partition of constituents from plasma proteins gave a complete retardation on the precolumn. The time courses of diethyldithiocarbamate and its methyl ester were followed in patients receiving therapeutic doses of disulfiram.

Colorimetric determination of disulfiram in tablets: collaborative study.

The colorimetric reaction of cuprous iodide and disulfiram has been collaboratively studied in 8 laboratories to determine the drug in tablet form at 2 dosage levels. The disulfiram was extracted from the tablet matrix with methylene chloride. After a basic wash with 1 M NaOH, an aliquot of the methylene chloride solution was reacted with solid cuprous iodide, and the absorbance of the resulting color was measured. Single assays on 2 synthetic preparations of known disulfiram content were performed with average recoveries of 98.4 and 99.2% for 93.3 and 105.3 mg, respectively. Duplicate determinations on 2 commercial tablet preparations declared at 250 and 500 mg gave mean and standard deviation values of 246.6 +/- 6.40 and 493.6 +/- 12.0 mg, respectively. The results agreed closely with those obtained by the author using the NF iodometric titration procedure. The method has been adopted official first action.

Stability of aqueous suspensions of disulfiram.

The stability of disulfiram in aqueous suspension is reported. Aqueous suspensions of disulfiram were prepared from bulk powder and from disulfiram tablets. Acacia and sodium benzoate were present in the suspension, the final concentration of which was 25 mg/ml disulfiram. Suspensions were stored in amber bottles under fluorescent light at 24 degrees C for 178 days (tablets) and 295 days (bulk powder). Samples were taken at various intervals during storage and assayed by high-pressure liquid chromatography (HPLC) and a modified NF method. Both types of suspension were stable. Potency remained constant, and no new peaks appeared in the chromatograph. Physical integrity of the suspensions was maintained. The suspension made from tablets contained less foam and air than the one made from bulk powder; both types were easily dispersible. A degradation product of disulfiram, diethyldithiocarbamic acid, did not interfere with the assay. The HPLC assay method was more reproducible than the modified NF method. Aqueous suspensions of disulfiram are stable at room temperature for the indicated time periods, and the HPLC assay method for disulfiram is reproducible and accurate.

Radioactive and nonradioactive methods for the in vivo determination of disulfiram, diethyldithiocarbamate, and diethyldithiocarbamate-methyl ester.

Although disulfiram (tetraethylthiuram disulfide; DSF) has been used in the treatment of alcoholism for almost a quarter of a century, little is known about its in vivo metabolism. One reason for this is that few analytical methods are available that can determine DSF and its various metabolites in biologic fluids and tissues. This article describes two simple procedures for the determination of these substances.

Methods for detecting disulfiram in biologic fluids: application in studies of compliance and effect of divalent cations on bioavailability.

Studies regarding the efficacy of disulfiram in the treatment of alcoholism have not included a method of evaluating compliance to the prescribed regimen. This article outlines current methods used to detect disulfiram in blood, breath, and urine.

Proton magnetic resonance spectroscopic determination of disulfiram: collaborative study.

A proton magnetic resonance spectroscopic method for determining disulfiram in the bulk drug product and in the formulated material was collaboratively studied. The method depends on the use of chloroform-d as a solvent and hexamethylcyclotrisiloxane as the internal standard. No interference from tablet excipients was observed. The method is rapid and specific. Eighteen laboratories analyzed duplicate samples of a bulk drug product, a 250 mg tablet composite, and a 500 mg tablet composite. The average per cent results and standard deviations were 99.7 +/- 1.4, 100.9 +/- 2.0, and 99.9 +/- 2.2, respectively.