In vitro characterization and in vivo performance of mefenamic acid-sodium diethyldithiocarbamate based liposomes

Mefenamic acid (MFA) is a hydrophobic drug with low dissolution rate. This study aimed to develop stable and reproducible aqueous formulations of MFA using liposomes as drug carriers. The drug entrapment, particles size and drug release profiles, and stability and reproducibility of the liposomes were determined. In addition, the maximum tolerated dose (MTD) was determined in rats via the oral and intraperitoneal routes of administration. Also, the anti-inflammatory efficacy of these liposomes was evaluated using carrageenan-induced paw edema model in rats. MFA-DDC based liposomes demonstrated a drug entrapment efficacy of 93.6%, particles size of 170.9 nm, and polydispersity index of 0.24 which were not statistically affected when stored in room and refrigerated temperatures for at least 4 weeks. The MTD of the intraperitoneally administrated MFA-loaded liposomes was 20 mg MFA/kg, whereas for those of oral administrations, it was up to 80 mg MFA/kg. Intraperitoneal dose (80 mg MFA/kg) of MFA-DDC liposomes induced extrapyramidal symptoms associated with significant elevation in serum potassium and muscle enzymes. Moreover, significant inhibition of paw edema was demonstrated by the oral and intraperitoneal routes. These findings suggest that MFA-DDC based liposomes are an effective formulation of MFA and recommend the use of bioequivalence assessments with commercial formulations.

Novel pollutants in the Moscow atmosphere in winter period: Gas chromatography-high resolution time-of-flight mass spectrometry study.

The most common mass spectrometry approach analyzing contamination of the environment deals with targeted analysis, i.e. detection and quantification of the selected (priority) pollutants. However non-targeted analysis is becoming more often the method of choice for environmental chemists. It involves implementation of modern analytical instrumentation allowing for comprehensive detection and identification of the wide variety of compounds of the environmental interest present in the sample, such as pharmaceuticals and their metabolites, musks, nanomaterials, perfluorinated compounds, hormones, disinfection by-products, flame retardants, personal care products, and many others emerging contaminants. The paper presents the results of detection and identification of previously unreported organic compounds in snow samples collected in Moscow in March 2016. The snow analysis allows evaluation of long-term air pollution in the winter period. Gas chromatography coupled to a high resolution time-of-flight mass spectrometer has enabled us with capability to detect and identify such novel analytes as iodinated compounds, polychlorinated anisoles and even Ni-containing organic complex, which are unexpected in environmental samples. Some considerations concerning the possible sources of origin of these compounds in the environment are discussed.

Dissipation pattern, safety evaluation, and generation of processing factor (PF) for pyraclostrobin and metiram residues in grapes during raisin preparation.

A residue analysis method was validated for trace level estimation of pyraclostrobin by liquid chromatography-mass spectrometry and metiram (analyzed as CS2) by gas chromatography mass spectrometry in grapes and raisin matrix. Dissipation of their residues and processing factors (PFs) during raisin making were evaluated through field studies with applications at single dose (SD) and double dose (DD). Residue data during drying process were best fitted to first + first-order kinetics model giving half-life ranging between 6 and 7 days for pyroclostrobin and 4 days for metiram. PFs for metiram and pyraclostrobin related to washing and oil dipping were 0.47 and 0.41, and 0.78 and 0.63 at single dose (SD) and double dose (DD), respectively. PF value of >1 for drying (1.01 and 1.31 for metiram and 1.34 and 1.10 for pyraclostrobin) indicates concentration of the residues during the drying process. The dietary exposure corresponding to average daily consumption of 0.0043 kg raisin per day on each sampling day was less than the respective maximum permissible intake at both the doses.

Silver-gelatine bionanocomposites for qualitative detection of a pesticide by SERS.

The controlled release of pesticides using hydrogel vehicles is an important procedure to limit the amount of these compounds in the environment, providing an effective way for crop protection. A key-step in the formulation of new materials for these purposes encompasses the monitoring of available pesticides in the gel matrix under variable working conditions. In this work, we report a series of bionanocomposites made of Ag nanoparticles (NPs) and gelatine A for the surface enhanced Raman scattering (SERS) detection of sodium diethyldithiocarbamate (EtDTC) as a pesticide model. These studies demonstrate the effectiveness of these substrates for the detection of EtDTC in aqueous solutions in a concentration as low as 10(-5) M. We have monitored the Raman signal enhancement of this analyte in bionanocomposites having an increasing amount of gelatine due to their relevance in formulating hydrogels of variable gel strengths. Under these conditions, the bionanocomposites have shown an effective SERS activity using EtDTC, demonstrating their effectiveness in the qualitative detection of this analyte. Finally, experiments involving the release of EtDTC from Ag/gelatine samples have been monitored by SERS, which attest the potential of this spectroscopic method in the laboratorial monitoring of hydrogels for pesticide release.

Feasibility of using terahertz spectroscopy to detect seven different pesticides in wheat flour.

This study investigated the feasibility of detecting pesticides using terahertz (THz) spectroscopy in high-density polyethylene and/or wheat flour mixtures. The absorption spectra of seven pesticides (dicofol, chlorpyrifos, chlorpyrifos-methyl, daminozide, imidacloprid, diethyldithiocarbamate, and dimethyldithiocarbamate) were measured in the frequency range 0.1 to 3 THz at room temperature. Five of the seven pesticides exhibited specific absorption peaks in the low-energy THz range. The two remaining pesticides had no specific absorption peaks in this frequency range, but they exhibited different frequency-dependent refractive indices. The absorption coefficients of imidacloprid increased with its increasing weight ratio in high-density polyethylene, and the fitted power absorptions and refractive indices using a Maxwell-Garnett effective medium model were comparable to the measured data. Imidacloprid was also identified from its characteristic absorption peaks in wheat flour mixtures, and a linear relationship between the absorption coefficient and the weight ratio was observed. Our results show the potential of detection of selected pesticides in foods, such as wheat flour, using THz spectroscopy.

Effects of the fungicide metiram in outdoor freshwater microcosms: responses of invertebrates, primary producers and microbes.

The ecological impact of the dithiocarbamate fungicide metiram was studied in outdoor freshwater microcosms, consisting of 14 enclosures placed in an experimental ditch. The microcosms were treated three times (interval 7 days) with the formulated product BAS 222 28F (Polyram®). Intended metiram concentrations in the overlying water were 0, 4, 12, 36, 108 and 324 µg a.i./L. Responses of zooplankton, macroinvertebrates, phytoplankton, macrophytes, microbes and community metabolism endpoints were investigated. Dissipation half-life (DT50) of metiram was approximately 1-6 h in the water column of the microcosm test system and the metabolites formed were not persistent. Multivariate analysis indicated treatment-related effects on the zooplankton (NOEC(community) = 36 µg a.i./L). Consistent treatment-related effects on the phytoplankton and macroinvertebrate communities and on the sediment microbial community could not be demonstrated or were minor. There was no evidence that metiram affected the biomass, abundance or functioning of aquatic hyphomycetes on decomposing alder leaves. The most sensitive populations in the microcosms comprised representatives of Rotifera with a NOEC of 12 µg a.i./L on isolated sampling days and a NOEC of 36 µg a.i./L on consecutive samplings. At the highest treatment-level populations of Copepoda (zooplankton) and the blue-green alga Anabaena (phytoplankton) also showed a short-term decline on consecutive sampling days (NOEC = 108 µg a.i./L). Indirect effects in the form of short-term increases in the abundance of a few macroinvertebrate and several phytoplankton taxa were also observed. The overall community and population level no-observed-effect concentration (NOEC(microcosm)) was 12-36 µg a.i./L. At higher treatment levels, including the test systems that received the highest dose, ecological recovery of affected measurement endpoints was fast (effect period < 8 weeks).

Cloud point extraction for determination of lead in blood samples of children, using different ligands prior to analysis by flame atomic absorption spectrometry: a multivariate study.

The phase-separation phenomenon of non-ionic surfactants occurring in aqueous solution was used for the extraction of lead (Pb(2+)) from digested blood samples after simultaneous complexation with ammonium pyrrolidinedithiocarbamate (APDC) and diethyldithiocarbamate (DDTC) separately. The complexed analyte was quantitatively extracted with octylphenoxypolyethoxyethanol (Triton X-114). The multivariate strategy was applied to estimate the optimum values of experimental factors. Acidic ethanol was added to the surfactant-rich phase prior to its analysis by flame atomic absorption spectrometer (FAAS). The detection limit value of Pb(2+) for the preconcentration of 10 mL of acid digested blood sample was 1.14 µg L(-1). The accuracy of the proposed methods was assessed by analyzing certified reference material (whole blood). Under the optimized conditions of both CPE methods, 10 mL of Pb(2+) standards (10 µg L(-1)) complexed with APDC and DDTC, permitted the enhancement factors of 56 and 42, respectively. The proposed method was used for determination of Pb(2+) in blood samples of children with kidney disorders and healthy controls.

[Determination of 4 dithiocarbamate residues in 22 matrices by gas chromatography].

A gas chromatographic method with flame photometric detection (FPD (sulfur filter)) for analyzing the residues of 4 dithiocarbamates in 22 matrices in a heated closed vial has been established. The dithiocarbamate residues were decomposed to carbon disulfide (CS2) by SnCl2-HCl solution. The liberated CS2 was absorbed by hexane in the vial, and then determined by gas chromatography with FPD (sulfur filter). The results were expressed as the residues of dithiocabamates. The residue analysis method was validated by mancozeb, metiram, propineb and thiram in 22 matrices (apple, grape, etc). The average recovery ranges were from 72% to 110% with the relative standard deviations (RSD) between 0.8% and 22.0% when the fortified concentrations were between 0.06 and 3.0 mg/kg, and the quantitative analysis was performed by using external standard method. The limits of detection were in the range of 0.01-0.1 mg/kg (S/N = 3), and the limits of quantification were between 0.02 and 0.2 mg/kg (S/N = 10) for the 4 dithiocarbamate residues in the 22 matrices. This method is simple, fast, accurate, reproducible, and suitable for the determination of the 4 dithiocarbamate residues in the matrices mentioned in this paper.

Capillary electrophoretic analysis of organic pollutants.

Environmental pollutants comprise a variety of compounds from inorganic anions, cations, ionizable organic compounds and moderately hydrophobic organic compounds to highly hydrophobic organic compounds. Correspondingly different separation strategies are required for their separation. In this chapter, we have presented some methods for the separation and the analysis of the organic pollutants such as polycyclic aromatic hydrocarbons, phenoxy acids, dithiocarbamates, paraquat and diquat, endocrine disruptors, toxins and explosives.

Determination of zinc diethyldithiocarbamate released into artificial sweat from natural rubber latex vulcanizates by HPLC.

A simple high-performance liquid chromatography method is adopted in order to quantitate the amount of zinc diethyldithiocarbamate (ZDEC) released into artificial sweat from natural rubber latex vulcanizates. The artificial sweat is extracted with dichloromethane, and the residue is recovered and re-dissolved in a known quantity of dichloromethane. ZDEC is quantitated as its copper complex by reacting with copper(II) sulphate. A reversed-phase C18 column and detection wavelength of 435 nm are used to measure the copper-dithiocarbamate complex. The procedure is repeated with cobalt(II) chloride, and the amount of ZDEC obtained by both the methods is compared. It is found that the recovery of ZDEC from the artificial sweat is high when copper(II) sulphate is used, indicating that the copper(II) sulphate is a better complexing agent than cobalt(II) chloride under the conditions used in the present study. The limits of detection and the quantitation of ZDEC are found to be 0.25 and 0.86 microg/mL, respectively. The present method, based on precolumn derivatization using copper(II) sulphate, facilitates the quantitation of ZDEC in latex products.

Extraction of haptens from solid products and their delivery to the skin, exemplified by dithiocarbamates from rubber gloves.

Allergic contact dermatitis is often caused by solid products such as rubber gloves. Patch testing with the product as is often gives negative results. Extraction of the haptens into an organic solvent is commonly performed to achieve a more correct investigation. The technique used for extraction of haptens from solid materials is only sporadically described. In this study, we investigated and optimized the yields of dithiocarbamates obtained by extraction from rubber gloves. The influence of solvent, extraction time and the procedures for extraction are evaluated. The delivery of zinc dibutyldithiocarbamate from the patch test preparation to the skin is determined.

EPR investigation of in vivo inhibitory effect of guanidine compounds on nitric oxide production in rat tissues.

The aim of the present study was to evaluate in vivo effects on NO production of pharmacologically widely used, commercially available NOS inhibitors, structurally related to guanidine. We compared the NO inhibitory potency and selectivity of L-NAME, aminoguanidine and guanabenz in tissues of normal and LPS-stimulated rats using ex vivo EPR measurements of the NO radical in its complex with dithiocarbamate-Fe(II). The tissues studied were the brain cortex, kidney, liver, heart and testis. Differential inhibitory effects were seen for L-NAME, aminoguanidine and guanabenz when applied during basal or LPS-stimulated conditions. Aminoguanidine exerted inhibition of NO only after stimulation with LPS. Guanabenz had little effect on NO in liver, kidney, testis and heart under normal conditions, while it reduced the basal NO in brain cortex. After stimulation with LPS guanabenz afforded a partial inhibition of the NO formation in all tissues studied. L-NAME was a potent inhibitor of NO synthesis in all tested tissues, both during basal and LPS stimulated conditions. Our results show that compounds containing a guanidine moiety might possess different NOS inhibitory profiles in vivo.

Allergologically relevant rubber accelerators in single-use medical gloves.

To prevent contact with specific rubber accelerators, sensitized patients have to know in which glove brands these accelerators are present. Additionally, quantitative measurements of the bioavailable amounts of accelerators are needed to assist consumers in selecting gloves with the lowest possible amount of residual rubber accelerators. The aim of the study was to develop an analytical method by which residuals of rubber accelerators in single-use medical gloves could be determined qualitatively and quantitatively. 19 different glove brands were analysed for content of accelerators, and the results were compared to manufacturers' ingredient claims of the identical gloves. ZDEC, ZDBC, ZMBT and ZPC were the most frequently detected chemicals. In 9 of 15 gloves discrepancies were found, usually minor, between content of accelerators declared by manufacturers to be present and accelerators detected by analysis. Both phosphate buffer and acetone were tested as extraction media. No accelerators were detectable with the described chemical analysis in phosphate extracts, whereas acetone was demonstrated to be a technically suitable medium for extraction. However, more kinetic studies of the extraction procedure and studies of skin penetration are needed to document that the extraction procedure simulates the clinical situation.

Identification of the protein-drug adduct formed between aldehyde dehydrogenase and S-methyl-N,N-diethylthiocarbamoyl sulfoxide by on-line proteolytic digestion high performance liquid chromatography electrospray ionization mass spectrometry.

Disulfiram has been used clinically as an aversion therapy treatment for recovering alcoholics. One of its metabolites, S-methyl-N, N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), is currently believed by some to be the active metabolite in vivo. We demonstrate in this report that MeDTC-SO is a potent irreversible inhibitor of recombinant rat liver mitochondrial aldehyde dehydrogenase (rlmALDH), the enzyme responsible for oxidizing acetaldehyde formed during ethanol metabolism. Recombinant rlmALDH was inhibited by MeDTC-SO after in vitro incubation with an IC(50) = 4.62 microM. The inhibition of rlmALDH was found to be accompanied by a concomitant increase of approximately 100 Da to the molecular mass of the native enzyme as determined by on-line high performance liquid chromatography (HPLC) electrospray ionization mass spectrometry (LC/MS), indicating that a covalent modification has occurred. To determine the site and structure of this covalent adduct, we developed a novel approach to characterize specific protein-drug interactions by linking a proteolytic enzyme digestion cartridge on-line with LC/MS. The on-line pepsin digestion LC/MS of MeDTC-SO-inhibited rlmALDH revealed an ion at MH(2)(2+) = 500.9, which was not present in the pepsin digestion of the non-inhibited enzyme. This peptide was tentatively attributed to the putative active site peptide (FNQGQC(301)C(302)C(303)) plus the adduct. This peptide was subjected to analysis by LC/MS/MS, which allowed us to determine that the covalent modification was associated with a single carbamoyl adduct at Cys-302, which has been shown to be the active site nucleophile of the enzyme.

Correlation between prevention of cataract development by disulfiram and fates of selenium in selenite-treated rats.

PURPOSE: We found a new pharmacological effect of disulfiram (DSF) against rat pups with cataract induced by selenite injection. The possible reactive mechanism is discussed in this present paper. METHODS: Wistar male and female rats aged 13 and 30 days, and male rats aged 7 weeks were used this present study. Cataract was induced by injection of selenite (19 micromol/kg, s.c.) to 13-day old rats. The lens opacification was monitored by using the slit lamp equipped with an anterior eye segment analysis system (EAS-1000, Nidek). The selenium contents in rat organs were detected fluorimetrically. Liposomes containing DSF (DSF-liposomes) were prepared by the reverse-phase evaporation method. Rat pups were instilled 5 microl of DSF-liposomes into both eyes 4 times per day. Adult rats were administered with DSF suspensions (100 mg/kg) by nasal cannulation. The changes of plasma concentration of diethyldithiocarbamate (DDC), which was a metabolite of disulfiram, were determined by HPLC method. RESULTS: Intraocular treatment with DSF-liposomes prevented the onset of cataract development in rat pups injected with sodium selenite. Treatment with DSF also significantly reduced the selenium contents in plasma at 1 h post-treatment and in the eye at 96 h post-treatment. No significant differences of selenium content in liver and kidney were observed in 13-day old rats instilled with DSF-liposomes or DSF free liposomes. Cataract could not be induced in the 30-days-old rats with the same dose of selenite (19 micromol/kg) and the liver, kidney, and especially eye of the older rats had lower levels of selenium than 13-day old rats. Diethyldithiocarbamate (DDC), an active metabolite of DSF, was decreased in the plasma following a subcutaneous injection of sodium selenite. The selenium concentration in the plasma was decreased by the intranasal administration of DSF suspensions. CONCLUSIONS: Instillation of DSF-liposomes into the eyes of rat pups given a subcutaneous injection of sodium selenite to induce cataracts prevented the formation of cataracts. The mechanism of inhibition may have resulted from a decreased level of selenium in the eyes following the treatment with DSF. On the other hand, as it is known that the cataracts may result from selenite-induced oxidative stress in the lens, DSF and DDC may react as anti-oxidants.

Effects of EGb 761 on nitric oxide and oxygen free radicals, myocardial damage and arrhythmia in ischemia-reperfusion injury in vivo.

The cardioprotective effects of EGb 761 on the release of nitric oxide (NO), the concentration of serum thiobarbituric acid reaction substance (TBARS), the activity of creatine kinase (CK) and the incidence of ventricular arrhythmias were investigated in myocardial ischemia-reperfusion injury in vivo. Using sodium nitrite (NaNO2) as standard source of nitric oxide (NO), we compared the correlation coefficients of the three measuring methods used currently in the determination of NOFe2+(DETC)2 complex with that of the measuring method suggested in this study. The result showed that measuring the whole height of three splitting signals is the best linear correlation to the concentration of NO comparing with other methods in this system. Using this method, we observed the effects of EGb 761 on NOFe2+(DETC)2 complex in myocardial ischemia-reperfusion injury in vivo. The hearts of the Wistar rats were subjected to 30 min of ischemia and 10 min of reperfusion in vivo. Different doses of EGb 761 (25, 50, 100, 200 mg/kg i.p.), superoxide dismutase (SOD, 10(4) U/kg), l-arginine (50 mg/kg i.p.) and nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine (NNA, 50 mg/kg i.p.) were administered to the ischemia-reperfusion rats. EGb 761 under the dose of 100 mg/kg increased the signal intensity of NOFe2+(DETC)2 complex, while EGb 761 at 200 mg/kg showed an effect of decreasing the signal intensity of NOFe2+(DETC)2 complex. EGb 761 inhibited the formation of TBARS, the release of CK, and mitigated the incidence of ventricular arrhythmias in a dose dependent way. Both l-arginine and SOD increased the signal intensity of NOFe2+(DETC)2 complex and inhibited the formation of TBARS, the leakage of CK and the incidence of ventricular arrhythmia. NNA not only had no protective effects on myocardial injury, but also increased the incidence of reperfusion-induced arrhythmia. In conclusion, EGb 761 has cardiovascular protective effects by means of adjusting the level of NO and inhibiting oxygen free radicals induced lipid peroxidation in myocardial ischemia-reperfusion injury in vivo.

Identification of causative chemicals of allergic contact dermatitis using a combination of patch testing in patients and chemical analysis. Application to cases from industrial rubber products.

3 cases of allergic contact dermatitis from industrial rubber products were investigated using a combination of patch testing in patients and chemical analysis of causative rubber products by gas chromatography (GC), GC-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). Our studies revealed N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD), a typical rubber allergen, as a causative chemical in a case from a brand of heavy-duty rubber gloves and a case from a black rubber ring for car manufacture, and zinc ethylphenyldithiocarbamate (ZEPC), a dithiocarbamate (DTC)-type accelerator, as a causative chemical in a case from a brand of rubber work gloves: both IPPD and ZEPC, which showed positive patch test reactions, were confirmed in the extracts of the causative rubber products by GC, GC-MS and/or HPLC.

Evaluation of contact sensitivity of rubber chemicals using the murine local lymph node assay.

The sensitizing abilities of 4 rubber additives, tetramethylthiuram disulfide (TMTD). 2-mercaptobenzothiazole (MBT), N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD) and zinc diethyldithiocarbamate (ZDEC), were evaluated using the murine local lymph node assay. Exposure to IPPD induced a significant increase of lymph node cell proliferation in the draining lymph nodes even at a low concentration. Exposure to TMTD and MBT induced moderate proliferative responses, while ZDEC induced a weak proliferation even at the higher concentrations. The sensitizing potency of each chemical was described in terms of the concentration that increased lymph node cell proliferation by a factor of 2 over that in the vehicle-treated control group. The concentrations of IPPD, TMTD, MBT and ZDEC were 0.14%, 0.35%, 4.2% and > 10%, respectively.